Patent application title: FUSION PROTEINS
Inventors:
Keith Foster (Salisbury, GB)
Keith Foster (Salisbury, GB)
John Chaddock (Salisbury, GB)
Philip Marks (Salisbury, GB)
Patrick Stancombe (Salisbury, GB)
Patrick Stancombe (Salisbury, GB)
Kei Roger Aoki (Irvine, CA, US)
Kei Roger Aoki (Irvine, CA, US)
Joseph Francis (Irvine, CA, US)
Joseph Francis (Irvine, CA, US)
Lance Steward (Irvine, CA, US)
Assignees:
SYNTAXIN LIMITED
Allergan, Inc.
IPC8 Class: AA61K3848FI
USPC Class:
424 943
Class name: Drug, bio-affecting and body treating compositions enzyme or coenzyme containing stabilized enzymes or enzymes complexed with nonenzyme (e.g., liposomes, etc.)
Publication date: 2011-04-21
Patent application number: 20110091437
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Patent application title: FUSION PROTEINS
Inventors:
John Chaddock
Lance Steward
Keith Foster
Patrick Stancombe
Kei Roger Aoki
Joseph Francis
Philip Marks
Agents:
Assignees:
Origin: ,
IPC8 Class: AA61K3848FI
USPC Class:
Publication date: 04/21/2011
Patent application number: 20110091437
Abstract:
A single chain, polypeptide fusion protein, comprising: a non-cytotoxic
protease, or a fragment thereof, which protease or protease fragment is
capable of cleaving a protein of the exocytic fusion apparatus of a
nociceptive sensory afferent; a dynorphin Targeting Moiety that is
capable of binding to a Binding Site on the nociceptive sensory afferent,
which Binding Site is capable of undergoing endocytosis to be
incorporated into an endosome within the nociceptive sensory afferent; a
protease cleavage site at which site the fusion protein is cleavable by a
protease, wherein the protease cleavage site is located between the
non-cytotoxic protease or fragment thereof and the dynorphin Targeting
Moiety; and a translocation domain that is capable of translocating the
protease or protease fragment from within an endosome, across the
endosomal membrane and into the cytosol of the nociceptive sensory
afferent. Nucleic acid sequences encoding the polypeptide fusion
proteins, methods of preparing same and uses thereof are also described.Claims:
1. A single chain, polypeptide fusion protein, comprising: a. a
non-cytotoxic protease, or a fragment thereof, which protease or protease
fragment cleaves a protein of the exocytic fusion apparatus of a
nociceptive sensory afferent; b. a dynorphin Targeting Moiety that binds
to a Binding Site on the nociceptive sensory afferent, which Binding Site
endocytoses to be incorporated into an endosome within the nociceptive
sensory afferent; c. a protease cleavage site at which site the fusion
protein is cleavable by a protease, wherein the protease cleavage site is
located between the non-cytotoxic protease or fragment thereof and the
Targeting Moiety; d. a translocation domain that translocates the
protease or protease fragment from within an endosome, across the
endosomal membrane and into the cytosol of the nociceptive sensory
afferent; and wherein the Targeting Moiety is located between the
protease cleavage site and the translocation domain.
2. The fusion protein according to claim 1, wherein the Targeting Moiety and the protease cleavage site are separated by at most 10 amino acid residues, or by at most 5 amino acid residues, or by at most zero amino acid residues.
3. The fusion protein according to claim 1, wherein the non-cytotoxic protease is a clostridial neurotoxin L-chain or an IgA protease.
4. The fusion protein according to claim 1, wherein the translocation domain is the HN domain of a clostridial neurotoxin.
5. The fusion protein according to claim 1, wherein the Targeting Moiety binds specifically to the ORL1 receptor.
6. The fusion protein according to claim 1, wherein the Targeting Moiety comprises or consists of an amino acid sequence according to SEQ ID NO. 89 or a fragment comprising or consisting of at least 14 or 16 contiguous amino acid residues thereof, or a variant amino acid sequence of said SEQ ID NO: 89 or said fragment having a maximum of 5 or 6 conservative amino acid substitutions.
7. The fusion protein according to claim 1, wherein the Targeting Moiety comprises or consists of an amino acid sequence according to SEQ ID NO. 89 or a fragment comprising or consisting of at least 14 or 16 contiguous amino acid residues thereof, or a variant amino acid sequence of said SEQ ID NO: 89 or said fragment having a maximum of 3 or 4 conservative amino acid substitutions.
8. The fusion protein according to claim 1, wherein the Targeting Moiety comprises or consists of an amino acid sequence according to SEQ ID NO. 89 or a fragment comprising or consisting of at least 14 or 16 contiguous amino acid residues thereof, or a variant amino acid sequence of said SEQ ID NO: 89 or said fragment having a maximum of 1 or 2 conservative amino acid substitutions.
9. The fusion protein according to claim 1, wherein the Targeting Moiety comprises or consists of an amino acid sequence according to SEQ ID NO. 89 or a fragment comprising or consisting of at least 10 or 12 contiguous amino acid residues thereof, or a variant amino acid sequence of said SEQ ID NO: 89 or said fragment having a maximum of 5 or 6 conservative amino acid substitutions.
10. The fusion protein according to claim 1, wherein the Targeting Moiety comprises or consists of an amino acid sequence according to SEQ ID NO. 89 or a fragment comprising or consisting of at least 10 or 12 contiguous amino acid residues thereof, or a variant amino acid sequence of said SEQ ID NO: 89 or said fragment having a maximum of 3 or 4 conservative amino acid substitutions.
11. The fusion protein according to claim 1, wherein the Targeting Moiety comprises or consists of an amino acid sequence according to SEQ ID NO. 89 or a fragment comprising or consisting of at least 10 or 12 contiguous amino acid residues thereof, or a variant amino acid sequence of said SEQ ID NO: 89 or said fragment having a maximum of 1 or 2 conservative amino acid substitutions.
12. A polypeptide fusion protein comprising any one of SEQ ID NOs: 91, 92, 93, 94, 95, or 96, or a fusion protein having at least 90% or 95% sequence identity therewith.
13. A polynucleotide molecule comprising a nucleic acid sequence encoding the polypeptide fusion protein according to claim 1.
14. An expression vector, which comprises a promoter, the polynucleotide molecule according to claim 13, wherein said polynucleotide molecule is located downstream of the promoter, and a terminator located downstream of the polynucleotide molecule.
15. A polynucleotide molecule comprising a nucleic acid sequence that is the complement of the nucleic acid sequence according to claim 13.
16. A method for preparing a single-chain polypeptide fusion protein, comprising: a. transfecting a host cell with the expression vector of claim 14, and b. culturing said host cell under conditions promoting expressing of the polypeptide fusion protein by the expression vector.
17. A method of preparing a non-cytotoxic agent, comprising: a. contacting a single-chain polypeptide fusion protein according to claim 1 with a protease capable of cleaving the protease cleavage site; b. cleaving the protease cleavage site; and thereby forming a di-chain fusion protein.
18. A non-cytotoxic polypeptide, obtained by the method of claim 17, wherein the polypeptide is a di-chain polypeptide, and wherein: a. the first chain comprises the non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent; b. the second chain comprises the dynorphin TM and the translocation domain that is capable of translocating the protease or protease fragment from within an endosome, across the endosomal membrane and into the cytosol of the nociceptive sensory afferent; and the first and second chains are disulphide linked together.
19. A method of treating, preventing or ameliorating pain in a subject, comprising administering to said patient a therapeutically effective amount of the fusion protein according to claim 1.
20. A method according to claim 19, wherein the pain is chronic pain selected from neuropathic pain, inflammatory pain, headache pain, somatic pain, visceral pain, and referred pain.
21. A method of treating, preventing or ameliorating pain in a subject, comprising administering to said patient a therapeutically effective amount of a polypeptide according to claim 18.
22. A method according to claim 21, wherein the pain is chronic pain selected from neuropathic pain, inflammatory pain, headache pain, somatic pain, visceral pain, and referred pain.
Description:
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. application Ser. No. 12/303,078, filed Dec. 1, 2008, which is the National Stage of International Application No. PCT/GB/2007/02049, filed Jun. 1, 2007, which claims priority from Great Britain Application No. 0610867.4, filed Jun. 1, 2006. Each application is expressly incorporated herein by referenced in its entirety.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is 35492_SEQ_FINAL.txt. The text file is 372 KB; was created on Aug. 23, 2010; and is being submitted via EFS-Web with the filing of the specification.
FIELD OF THE INVENTION
[0003] This invention relates to non-cytotoxic fusion proteins, and to the therapeutic application thereof as analgesic molecules.
BACKGROUND OF THE INVENTION
[0004] Toxins may be generally divided into two groups according to the type of effect that they have on a target cell. In more detail, the first group of toxins kill their natural target cells, and are therefore known as cytotoxic toxin molecules. This group of toxins is exemplified inter alia by plant toxins such as ricin, and abrin, and by bacterial toxins such as diphtheria toxin, and Pseudomonas exotoxin A. Cytotoxic toxins have attracted much interest in the design of "magic bullets" (e.g. immunoconjugates, which comprise a cytotoxic toxin component and an antibody that binds to a specific marker on a target cell) for the treatment of cellular disorders and conditions such as cancer. Cytotoxic toxins typically kill their target cells by inhibiting the cellular process of protein synthesis.
[0005] The second group of toxins, which are known as non-cytotoxic toxins, do not (as their name confirms) kill their natural target cells. Non-cytotoxic toxins have attracted much less commercial interest than have their cytotoxic counterparts, and exert their effects on a target cell by inhibiting cellular processes other than protein synthesis. Non-cytotoxic toxins are produced by a variety of plants, and by a variety of microorganisms such as Clostridium sp. and Neisseria sp.
[0006] Clostridial neurotoxins are proteins that typically have a molecular mass of the order of 150 kDa. They are produced by various species of bacteria, especially of the genus Clostridium, most importantly C. tetani and several strains of C. botulinum, C. butyricum and C. argentinense. There are at present eight different classes of the clostridial neurotoxin, namely: tetanus toxin, and botulinum neurotoxin in its serotypes A, B, C1, D, E, F and G, and they all share similar structures and modes of action.
[0007] Clostridial neurotoxins represent a major group of non-cytotoxic toxin molecules, and are synthesised by the host bacterium as single polypeptides that are modified post-translationally by a proteolytic cleavage event to form two polypeptide chains joined together by a disulphide bond. The two chains are termed the heavy chain (H-chain), which has a molecular mass of approximately 100 kDa, and the light chain (L-chain), which has a molecular mass of approximately 50 kDa.
[0008] L-chains possess a protease function (zinc-dependent endopeptidase activity) and exhibit a high substrate specificity for vesicle and/or plasma membrane associated proteins involved in the exocytic process. L-chains from different clostridial species or serotypes may hydrolyse different but specific peptide bonds in one of three substrate proteins, namely synaptobrevin, syntaxin or SNAP-25. These substrates are important components of the neurosecretory machinery. Neisseria sp., most importantly from the species N. gonorrhoeae, produce functionally similar non-cytotoxic proteases. An example of such a protease is IgA protease (see WO99/58571).
[0009] It has been well documented in the art that toxin molecules may be re-targeted to a cell that is not the toxin's natural target cell. When so re-targeted, the modified toxin is capable of binding to a desired target cell and, following subsequent translocation into the cytosol, is capable of exerting its effect on the target cell. Said re-targeting is achieved by replacing the natural Targeting Moiety (TM) of the toxin with a different TM. In this regard, the TM is selected so that it will bind to a desired target cell, and allow subsequent passage of the modified toxin into an endosome within the target cell. The modified toxin also comprises a translocation domain to enable entry of the non-cytotoxic protease into the cell cytosol. The translocation domain can be the natural translocation domain of the toxin or it can be a different translocation domain obtained from a microbial protein with translocation activity.
[0010] The above-mentioned TM replacement may be effected by conventional chemical conjugation techniques, which are well known to a skilled person. In this regard, reference is made to Hermanson, G. T. (1996), Bioconjugate techniques, Academic Press, and to Wong, S. S. (1991), Chemistry of protein conjugation and cross-linking, CRC Press.
[0011] Chemical conjugation is, however, often imprecise. For example, following conjugation, a TM may become joined to the remainder of the conjugate at more than one attachment site.
[0012] Chemical conjugation is also difficult to control. For example, a TM may become joined to the remainder of the modified toxin at an attachment site on the protease component and/or on the translocation component. This is problematic when attachment to only one of said components (preferably at a single site) is desired for therapeutic efficacy.
[0013] Thus, chemical conjugation results in a mixed population of modified toxin molecules, which is undesirable.
[0014] As an alternative to chemical conjugation, TM replacement may be effected by recombinant preparation of a single polypeptide fusion protein (see WO98/07864). This technique is based on the in vivo bacterial mechanism by which native clostridial neurotoxin (i.e. holotoxin) is prepared, and results in a fusion protein having the following structural arrangement:
NH2-[protease component]-[translocation component]-[TM]-COOH
[0015] According to WO98/07864, the TM is placed towards the C-terminal end of the fusion protein. The fusion protein is then activated by treatment with a protease, which cleaves at a site between the protease component and the translocation component. A di-chain protein is thus produced, comprising the protease component as a single polypeptide chain covalently attached (via a disulphide bridge) to another single polypeptide chain containing the translocation component plus TM. Whilst the WO98/07864 methodology follows (in terms of structural arrangement of the fusion protein) the natural expression system of clostridial holotoxin, the present inventors have found that this system may result in the production of certain fusion proteins that have a substantially-reduced binding ability for the intended target cell.
[0016] There is therefore a need for an alternative or improved system for constructing a non-cytotoxic fusion protein.
SUMMARY OF THE INVENTION
[0017] The present invention addresses one or more of the above-mentioned problems by providing a single chain, polypeptide fusion protein, comprising: [0018] a. a non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus in a nociceptive sensory afferent; [0019] b. a Targeting Moiety that is capable of binding to a Binding Site on the nociceptive sensory afferent, which Binding Site is capable of undergoing endocytosis to be incorporated into an endosome within the nociceptive sensory afferent; [0020] c. a protease cleavage site at which site the fusion protein is cleavable by a protease, wherein the protease cleavage site is located between the non-cytotoxic protease or fragment thereof and the Targeting Moiety; and [0021] d. a translocation domain that is capable of translocating the protease or protease fragment from within an endosome, across the endosomal membrane and into the cytosol of the nociceptive sensory afferent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1--Purification of a LC/A-nociceptin-HN/A fusion protein
[0023] Using the methodology outlined in Example 9, a LC/A-nociceptin-HN/A fusion protein was purified from E. coli BL21 cells. Briefly, the soluble products obtained following cell disruption were applied to a nickel-charged affinity capture column. Bound proteins were eluted with 100 mM imidazole, treated with Factor Xa to activate the fusion protein and remove the maltose-binding protein (MBP) tag, then re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE (Panel A) and Western blotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) were used as the primary antibody for Western blotting. The final purified material in the absence and presence of reducing agent is identified in the lanes marked [-] and [+] respectively.
[0024] FIG. 2--Purification of a nociceptin-LC/A-HN/A fusion protein
[0025] Using the methodology outlined in Example 9, a nociceptin-LC/A-HN/A fusion protein was purified from E. coli BL21 cells. Briefly, the soluble products obtained following cell disruption were applied to a nickel-charged affinity capture column. Bound proteins were eluted with 100 mM imidazole, treated with Factor Xa to activate the fusion protein and remove the maltose-binding protein (MBP) tag, then re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE (Panel A) and Western blotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) were used as the primary antibody for Western blotting. The final purified material in the absence and presence of reducing agent is identified in the lanes marked [-] and [+] respectively.
[0026] FIG. 3--Purification of a LC/C-nociceptin-HN/C fusion protein
[0027] Using the methodology outlined in Example 9, an LC/C-nociceptin-HN/C fusion protein was purified from E. coli BL21 cells. Briefly, the soluble products obtained following cell disruption were applied to a nickel-charged affinity capture column. Bound proteins were eluted with 100 mM imidazole, treated with Factor Xa to activate the fusion protein and remove the maltose-binding protein (MBP) tag, then re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE (Panel A) and Western blotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) were used as the primary antibody for Western blotting. The final purified material in the absence and presence of reducing agent is identified in the lanes marked [-] and [+] respectively.
[0028] FIG. 4--Purification of a LC/A-met enkephalin-HN/A fusion protein
[0029] Using the methodology outlined in Example 9, an LC/A-met enkephalin-HN/A fusion protein was purified from E. coli BL21 cells. Briefly, the soluble products obtained following cell disruption were applied to a nickel-charged affinity capture column. Bound proteins were eluted with 100 mM imidazole, treated with Factor Xa to activate the fusion protein and remove the maltose-binding protein (MBP) tag, then re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE. The final purified material in the absence and presence of reducing agent is identified in the lanes marked [-] and [+] respectively.
[0030] FIG. 5--Comparison of binding efficacy of a LC/A-nociceptin-HN/A fusion protein and a nociceptin-LC/A-HN/A fusion protein
[0031] The ability of nociceptin fusions to bind to the ORL1 receptor was assessed using a simple competition-based assay. Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of test material in the presence of 1 nM [3H]-nociceptin. The reduction in specific binding of the radiolabelled ligand was assessed by scintillation counting, and plotted in comparison to the efficacy of unlabelled ligand (Tocris nociceptin). It is clear that the LC/A-nociceptin-HN/A fusion is far superior to the nociceptin-LC/A-HN/A fusion at interacting with the ORL1 receptor.
[0032] FIG. 6--In vitro catalytic activity of a LC/A-nociceptin-HN/A fusion protein
[0033] The in vitro endopeptidase activity of the purified LC/A-nociceptin-HN/A fusion protein was determined essentially as described in Chaddock et al 2002, Prot. Express Purif. 25, 219-228. Briefly, SNAP-25 peptide immobilised to an ELISA plate was exposed to varying concentrations of fusion protein for 1 hour at 37° C. Following a series of washes, the amount of cleaved SNAP-25 peptide was quantified by reactivity with a specific antisera.
[0034] FIG. 7--Purification of a LC/A-nociceptin variant-HN/A fusion protein
[0035] Using the methodology outlined in Example 9, an LC/A-nociceptin variant-HN/A fusion protein was purified from E. coli BL21 cells. Briefly, the soluble products obtained following cell disruption were applied to a nickel-charged affinity capture column. Bound proteins were eluted with 100 mM imidazole, treated with Factor Xa to activate the fusion protein and remove the maltose-binding protein (MBP) tag, then re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE. The final purified material in the absence and presence of reducing agent is identified in the lanes marked [-] and [+] respectively.
[0036] FIG. 8--Comparison of binding efficacy of a LC/A-nociceptin-HN/A fusion protein and a LC/A-nociceptin variant-HN/A fusion protein
[0037] The ability of nociceptin fusions to bind to the ORL1 receptor was assessed using a simple competition-based assay. Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of test material in the presence of 1 nM [3H]-nociceptin. The reduction in specific binding of the radiolabelled ligand was assessed by scintillation counting, and plotted in comparison to the efficacy of unlabelled ligand (Tocris nociceptin). It is clear that the LC/A-nociceptin variant-HN/A fusion (CPNv-LHA) is superior to the LC/A-nociceptin variant-HN/A fusion (CPN-LHA) at interacting with the ORL1 receptor.
[0038] FIG. 9--Expressed/purified LC/A-nociceptin-HN/A fusion protein family with variable spacer length product(s)
[0039] Using the methodology outlined in Example 9, variants of the LC/A-CPN-HN/A fusion consisting of GS10, GS30 and HX27 are purified from E. coli cell paste. Samples from the purification of LC/A-CPN(GS10)-HN/A, LC/A-CPN(GS15)-HN/A, LC/A-CPN(GS25)-HN/A, LC/A-CPN(GS30)-HN/A and LC/A-CPN(HX27)-HN/A were assessed by SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis profile indicates purification of a disulphide-bonded di-chain species of the expected molecular mass of CPBE-A. Top panel: M=benchmark molecular mass markers; S=total E. coli protein soluble fraction; FT=proteins that did not bind to the Ni2+-charged Sepharose column; FUSION=fusion protein eluted by the addition of imidazole. Bottom panel: Lane 1=benchmark molecular mass markers; Lane 2=total E. coli protein soluble fraction; Lane 3=purified material following initial capture on Ni2+-charged Sepharose; Lane 4=Factor Xa treated material prior to final capture on Ni2+-charged Sepharose; Lane 5=purified final material post activation with Factor Xa (5 μl); Lane 6=purified final material post activation with Factor Xa (10 μl); Lane 7=purified final material post activation with Factor Xa (20 μl); Lane 8=purified final material post activation with Factor Xa+DTT (5 μl); Lane 9=purified final material post activation with Factor Xa+DTT (10 μl); Lane 10=purified final material post activation with Factor Xa+DTT (20 μl).
[0040] FIG. 10--Inhibition of SP release and cleavage of SNAP-25 by CPN-A
[0041] Briefly, primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of CPN-A for 24 hours. Cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis and plotted against fusion concentration (dashed line). Material was also recovered for an analysis of substance P content using a specific EIA kit. Inhibition of substance P release is illustrated by the solid line. The fusion concentration required to achieve 50% maximal SNAP-25 cleavage is estimated to be 6.30±2.48 nM.
[0042] FIG. 11--Inhibition of SP release and cleavage of SNAP-25 over extended time periods after exposure of DRG to CPN-A
[0043] Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of CPN-A for 24 hours. Botulinum neurotoxin (BoNT/A) was used as a control. After this initial exposure, extracellular material was removed by washing, and the cells incubated at 37° C. for varying periods of time. At specific time points, cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis and illustrated by the dotted lines. Material was also recovered for an analysis of substance P content using a specific EIA kit. Inhibition of substance P release is illustrated by the solid lines.
[0044] FIG. 12--Cleavage of SNAP-25 by CPNv-A Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of CPNv-A for 24 hours. Cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis. The fusion concentration required to achieve 50% maximal SNAP-25 cleavage is estimated to be 1.38±0.36 nM.
[0045] FIG. 13--Cleavage of SNAP-25 over extended time periods after exposure of DRG to CPNv-A Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of CPNv-A for 24 hours. CPN-A was used as a control. After this initial exposure, extracellular material was removed by washing, and the cells incubated at 37° C. for varying periods of time. At specific time points, cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis.
[0046] FIG. 14--CPNv-A fusion-mediated displacement of [3H]-nociceptin binding
[0047] The ability of nociceptin fusions to bind to the ORL1 receptor was assessed using a simple competition-based assay. Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of test material in the presence of 1 nM [3H]-nociceptin. The reduction in specific binding of the radiolabelled ligand was assessed by scintillation counting, and plotted in comparison to the efficacy of unlabelled ligand (Tocris nociceptin). It is clear that the LC/A-nociceptin variant-HN/A fusion (labelled as CPNv-LHnA) is superior to the LC/A-nociceptin-HN/A fusion (labelled as CPN-LHnA) at interacting with the ORL1 receptor.
[0048] FIG. 15--Expressed/purified CPNv(Ek)-A product
[0049] Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis profile indicates purification of a disulphide-bonded di-chain species of the expected molecular mass of CPNv(Ek)-A. Lane 1=benchmark molecular mass markers; Lane 2=total E. coli protein soluble fraction; Lane 3=purified material following initial capture on Ni2+-charged Sepharose; Lane 4=purified final material post activation with enterokinase (5 μl); Lane 5=purified final material post activation with enterokinase (10 μl); Lane 6=purified final material post activation with enterokinase (20 μl); Lane 7=purified final material post activation with enterokinase+DTT (5 μl); Lane 8=purified final material post activation with enterokinase+DTT (10 μl); Lane 9=purified final material post activation with enterokinase+DTT (20 μl).
[0050] FIG. 16--Cleavage of SNAP-25 by CPNv(Ek)-A
[0051] Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of CPNv(Ek)-A for 24 hours. Cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis. CPNv-A as prepared in Example 9 was used for comparison purposes. The percentage cleavage of SNAP-25 by CPNv(Ek)-A (labelled as En activated) and CPNv-A (labelled as Xa activated) are illustrated.
[0052] FIG. 17--Expressed/purified CPNv-C product
[0053] Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis profile indicates purification of a disulphide-bonded di-chain species of the expected molecular mass of CPNv-C. Lane 1=benchmark molecular mass markers; Lane 2=total E. coli protein soluble fraction; Lane 3=purified material following initial capture on Ni2+-charged Sepharose; Lane 4=Factor Xa treated material prior to final capture on Ni2+-charged Sepharose; Lane 5=purified material following second capture on Ni2+-charged Sepharose; Lane 6=final purified material; Lane 7=final purified material+DTT; Lane 8=benchmark molecular mass markers.
[0054] FIG. 18--Cleavage of syntaxin by CPNv-C
[0055] Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of CPNv-C for 24 hours. Cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-syntaxin to facilitate an assessment of syntaxin cleavage. The percentage of cleaved syntaxin was calculated by densitometric analysis. The fusion concentration required to achieve 50% maximal syntaxin cleavage is estimated to be 3.13±1.96 nM.
[0056] FIG. 19--CPN-A efficacy in the Acute Capsaicin-Induced Mechanical Allodynia model
[0057] The ability of an LC/A-nociceptin-HN/A fusion (CPN/A) to inhibit capsaicin-induced mechanical allodynia was evaluated following subcutaneous intraplantar injection in the rat hind paw. Test animals were evaluated for paw withdrawal frequency (PWF %) in response to a 10 g Von Frey filament stimulus series (10 stimuli×3 trials) prior to recruitment into the study (Pre-Treat); after subcutaneous intraplantar treatment with CPN/A but before capsaicin (Pre-CAP); and following capsaicin challenge post-injection of CPN/A (average of responses at 15' and 30'; CAP). Capsaicin challenge was achieved by injection of 10 μL of a 0.3% solution. Sample dilutions were prepared in 0.5% BSA/saline.
[0058] FIG. 20--CPN-A efficacy in the Streptozotocin (STZ)-Induced Peripheral Diabetic Neuropathy (Neuropathic Pain) model Male Sprague-Dawley rats (250-300 g) are treated with 65 mg/kg STZ in citrate buffer (I.V.) and blood glucose and lipid are measured weekly to define the readiness of the model. Paw Withdrawal Threshold (PWT) is measured in response to a Von Frey filament stimulus series over a period of time. Allodynia is said to be established when the PWT on two consecutive test dates (separated by 1 week) measures below 6 g on the scale. At this point, rats are randomized to either a saline group (negative efficacy control), gabapentin group (positive efficacy control) or a test group (CPN/A). Test materials (20-25 μl are injected subcutaneously as a single injection (except gabapentin) and the PWT is measured at 1 day post-treatment and periodically thereafter over a 2 week period. Gabapentin (30 mg/kg i.p. @ 3 ml/kg injection volume) is injected daily, 2 hours prior to the start of PWT testing.
[0059] FIG. 21--CPNv-A efficacy in the Acute Capsaicin-Induced Mechanical Allodynia model
[0060] The ability of an LC/A-nociceptin variant-HN/A fusion (CPNv/A) to inhibit capsaicin-induced mechanical allodynia was evaluated following subcutaneous intraplantar injection in the rat hind paw. Test animals were evaluated for paw withdrawal frequency (PWF %) in response to a 10 g Von Frey filament stimulus series (10 stimuli×3 trials) prior to recruitment into the study (Pre-Treat), after subcutaneous intraplantar treatment with CPNv/A but before capsaicin (Pre-CAP), and following capsaicin challenge post-injection of CPNv/A (average of responses at 15' and 30'; CAP). Capsaicin challenge was achieved by injection of 10 μL of a 0.3% solution. Sample dilutions were prepared in 0.5% BSA/saline. These data are expressed as a normalized paw withdrawal frequency differential, in which the difference between the peak response (post-capsaicin) and the baseline response (pre-capsaicin) is expressed as a percentage. With this analysis, it can be seen that CPNv/A is more potent than CPN/A since a lower dose of CPNv/A is required to achieve similar analgesic effect to that seen with CPN/A.
[0061] FIG. 22--Expressed/purified LC/A-CPLE-HN/A product
[0062] Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis profile indicates purification of a disulphide-bonded di-chain species of the expected molecular mass of CPLE-A. Lane 1=benchmark molecular mass markers; Lane 2=total E. coli protein soluble fraction; Lane 3=purified material following initial capture on Ni2+-charged Sepharose; Lane 4=Factor Xa treated material prior to final capture on Ni2+-charged Sepharose; Lane 5=purified material following second capture on Ni2+-charged Sepharose; Lane 6=final purified material; Lane 7=final purified material+DTT.
[0063] FIG. 23--Expressed/purified LC/A-CPBE-HN/A product
[0064] Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis profile indicates purification of a disulphide-bonded di-chain species of the expected molecular mass of CPBE-A. Lane 1=total E. coli protein soluble fraction; Lane 2=purified material following initial capture on Ni2+-charged Sepharose; Lane 3=Factor Xa treated material prior to final capture on Ni2+-charged Sepharose; Lane 4=purified final material post activation with Factor Xa (5 μl; Lane 5=purified final material post activation with Factor Xa (10 μl; Lane 6=purified final material post activation with Factor Xa (20 μl; Lane 7=purified final material post activation with Factor Xa+DTT (5 μl; Lane 8=purified final material post activation with Factor Xa+DTT (10 μl; Lane 9=purified final material post activation with Factor Xa+DTT (20 μl; Lane 10=benchmark molecular mass markers.
[0065] FIG. 24--Expressed/Purified CPOP-A Product
[0066] Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis profile indicates purification of a disulphide-bonded di-chain species of the expected molecular mass of CPOP-A. Lane 1=benchmark molecular mass markers; Lane 2=purified material following initial capture on Ni2+-charged Sepharose; Lane 3=Factor Xa treated material prior to final capture on Ni2+-charged Sepharose; Lane 4=purified material following second capture on Ni2+-charged Sepharose; Lane 5=purified final material post activation with Factor Xa (5 μl; Lane 6=purified final material post activation with Factor Xa (10 μl; Lane 7=purified final material post activation with Factor Xa (20 μl; Lane 8=purified final material post activation with Factor Xa+DTT (5 μl; Lane 9=purified final material post activation with Factor Xa+DTT (10 μl; Lane 10=purified final material post activation with Factor Xa+DTT (20 μl.
[0067] FIG. 25--Expressed/purified CPOPv-A product
[0068] Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis profile indicates purification of a disulphide-bonded di-chain species of the expected molecular mass of CPOPv-A. Lane 1=benchmark molecular mass markers; Lane 2=total E. coli protein soluble fraction; Lane 3=purified material following initial capture on Ni2+-charged Sepharose; Lane 4=Factor Xa treated material prior to final capture on Ni2+-charged Sepharose; Lane 5=purified final material post activation with Factor Xa (5 μl); Lane 6=purified final material post activation with Factor Xa (10 μl); Lane 7=purified final material post activation with Factor Xa (20 μl); Lane 8=purified final material post activation with Factor Xa+DTT (5 μl; Lane 9=purified final material post activation with Factor Xa+DTT (10 μl; Lane 10=purified final material post activation with Factor Xa+DTT (20 μl.
[0069] FIG. 26--In vitro SNAP-25 cleavage in a DRG cell model
[0070] Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of CPOPv-A for 24 hours. Cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis.
[0071] FIG. 27--Expressed/purified CPNv-A-FXa-HT (removable his-tag)
[0072] Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis profile indicates purification of a disulphide-bonded di-chain species of the expected molecular mass of CPNv-A-FXa-HT. Lane 1=benchmark molecular mass markers; Lane 2=total E. coli protein soluble fraction; Lane 3=Factor Xa treated material prior to final capture on Ni2+-charged Sepharose; Lane 4=purified final material post activation with Factor Xa; Lane 5=purified final material post activation with Factor Xa+DTT.
[0073] FIG. 28--In vitro efficacy of LC/A-nociceptin-HN/A fusion proteins with variable spacer length, as assessed by ligand competition assay
[0074] The ability of LC/A-nociceptin-HN/A fusions of variable spacer length to bind to the ORL1 receptor was assessed using a simple competition-based assay. Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of test material in the presence of 1 nM [3H]-nociceptin. The reduction in specific binding of the radiolabelled ligand was assessed by scintillation counting, and plotted in comparison to the efficacy of unlabelled ligand (Tocris nociceptin). The upper panel illustrates the displacement characteristics of the GS0, GS20, GS30 and Hx27 spacers, whilst the lower panel illustrates the displacement achieved by the GS10, GS15 and GS25 spaced fusion proteins. It is concluded that the GS0 and GS30 spacers are ineffective, and the GS10 is poorly effective, at displacing nociceptin from the ORL1 receptor.
[0075] FIG. 29--In vitro efficacy of LC/A-nociceptin-HN/A fusion proteins with variable spacer length, as assessed by in vitro SNAP-25 cleavage
[0076] Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of CPN-A (of variable spacer length) for 24 hours. Cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis. The poorly effective binding characteristics of the GS10 spaced fusion protein (see FIG. 28) are reflected in the higher concentrations of fusion required to achieve cleavage of intracellular SNAP-25. GS0 and GS30 spaced fusion proteins were completely ineffective (date not shown). GS15, 20 and 25 spaced fusion proteins were similarly effective.
[0077] FIG. 30--Cleavage of SNARE protein by dynorphin conjugates in embryonic spinal cord neurons (eSCNs)
[0078] Embryonic spinal cord neurons were exposed to varying concentrations of dynorphin conjugates of the present invention for 24 hours. Cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis. It is clear that LC/A-dynorphin-HN/A fusion is more potent than an unliganded LC/A-HN/A control molecule. The concentration of LC/A-dynorphin-HN/A fusion required to achieve 50% maximal SNAP-25 cleavage is estimated to be 35.3 nM and the concentration for the LC/A-HN/A control required to achieve 50% maximal SNAP-25 cleavage could not be determined due to it's low potency.
[0079] FIG. 31--Cleavage of SNARE protein by dynorphin conjugates in Chinese hamster ovary cells (CHO-K1 cells) transfected with OP2 receptor and SNAP-25
[0080] Chinese hamster ovary (CHO) cells were transfected so that they express the OP2 receptor. Said cells were further transfected to express a SNARE protein (SNAP-25). The transfected cells were exposed to varying concentrations of different dynorphin conjugates for 24 hours. Cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis. It is clear that LC/A-CPDY-HN/A conjugates are more potent than the unliganded LC/A-HN/A control molecule (labelled as LC/A-HN/A).
[0081] FIG. 32--Cleavage of SNARE protein by dynorphin conjugates in embryonic spinal cord neurons (eSCNs)
[0082] Embryonic spinal cord neurons were exposed to varying concentrations of dynorphin conjugates of the present invention for 24 hours. Cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis. It is clear that LC/A-CPDY-HN/A conjugates are more potent than the unliganded LC/A-HN/A control molecule (labelled as LC/A-HN/A).
[0083] FIG. 33--Kappa receptor activation studies with a range of dynorphin conjugates
[0084] Chinese hamster ovary (CHO) cells were transfected so that they express the OP2 receptor and SNAP-25. Said cells were used to measure cAMP deletion that occurs when the receptor is activated with a dynorphin ligand, using a FRET-based cAMP kit (LANCE kit from Perkin Elmer). The transfected cells were exposed to varying concentrations of dynorphin conjugates of the present invention for 2 hours. cAMP levels were then detected by addition of a detection mix containing a fluorescently labelled cAMP tracer (Europium-streptavadi/biotin-cAMP) and fluorescently (Alexa) labelled anti-cAMP antibody and incubating at room temperature for 24 hours. Then samples are excited at 320 nM and emitted light measured at 665 nM to determine cAMP levels. It is clear that LC/A-CPDY-HN/A conjugates are more potent than the unliganded LC/A-HN/A control molecule (labelled as LC/A-HN/A).
[0085] FIG. 34--Kappa receptor activation studies with a range of dynorphin conjugates
[0086] Chinese hamster ovary (CHO) cells were transfected so that they express the OP2 receptor (purchased from Perkin Elmer). Said cells were transfected so they express SNAP-25 and used to measure cAMP deletion that occurs when the receptor is activated with a dynorphin ligand, using a FRET-based cAMP kit (LANCE kit from Perkin Elmer). The transfected cells were exposed to varying concentrations of dynorphin conjugates of the present invention for 2 hours. cAMP levels were then detected by addition of a detection mix containing a fluorescently labelled cAMP tracer (Europium-streptavadi/biotin-cAMP) and fluorescently (Alexa) labelled anti-cAMP antibody and incubating at room temperature for 24 hours. Then samples are excited at 320 nM and emitted light measured at 665 nM to determine cAMP levels. It is clear from the figure by the reduction in maximum cAMP that the OP2 receptor is activated by LC/A-CPDY-HN/A (labelled as CPDY/A), LC/B-CPDY-HN/B (labelled as CPDY/B), LC/C-CPDY-HN/C (labelled as CPDY/C), and LC/D-CPDY-HN/D (labelled as CPDY/D). The concentration required to achieve 50% reduction in cAMP with LC/A-CPDY-HN/A, LC/B-CPDY-HN/B, LC/C-CPDY-HN/C (labelled as CPDY/, and LC/D-CPDY-HN/D is 10.47 nM, 14.79 nM, 14.79 nM and 23.99 nM, respectively. Dynorphin peptide containing amino acids 1-17 of dynorphin A (labelled as dynorphin (1-17) was more potent than the fusions; 0.15 nm concentration required to achieve 50% reduction of cAMP.
DETAILED DESCRIPTION OF THE INVENTION
[0087] The present inventors have found that the WO 98/07864 fusion protein system is not optimal for TMs requiring a N-terminal domain for interaction with a binding site on a nociceptive sensory afferent. This problem is particularly acute with TMs that require a specific N-terminus amino acid residue or a specific sequence of amino acid residues including the N-terminus amino acid residue for interaction with a binding site on a nociceptive sensory afferent.
[0088] In contrast to WO98/07864, the present invention provides a system for preparing non-cytotoxic conjugates, wherein the TM component of the fusion includes the relevant binding domain in an intra domain or an amino acid sequence located towards the middle (i.e. of the linear peptide sequence) of the TM, or preferably located towards the N-terminus of the TM, or more preferably at or near to the N-terminus. The N-terminal domain is capable of binding to a Binding Site on a nociceptive sensory afferent, and the TM preferably has a requirement for a specific and defined sequence of amino acid residue(s) to be free at its N-terminus.
[0089] The non-cytotoxic protease component of the present invention is a non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is capable of cleaving different but specific peptide bonds in one of three substrate proteins, namely synaptobrevin, syntaxin or SNAP-25, of the exocytic fusion apparatus in a nociceptive sensory afferent. These substrates are important components of the neurosecretory machinery. The non-cytotoxic protease component of the present invention is preferably a neisserial IgA protease or a fragment thereof or a clostridial neurotoxin L-chain or a fragment thereof. A particularly preferred non-cytotoxic protease component is a botulinum neurotoxin (BoNT) L-chain or a fragment thereof.
[0090] The translocation component of the present invention enables translocation of the non-cytotoxic protease (or fragment thereof) into the target cell such that functional expression of protease activity occurs within the cytosol of the target cell. The translocation component is preferably capable of forming ion-permeable pores in lipid membranes under conditions of low pH. Preferably it has been found to use only those portions of the protein molecule capable of pore-formation within the endosomal membrane. The translocation component may be obtained from a microbial protein source, in particular from a bacterial or viral protein source. Hence, in one embodiment, the translocation component is a translocating domain of an enzyme, such as a bacterial toxin or viral protein. The translocation component of the present invention is preferably a clostridial neurotoxin H-chain or a fragment thereof. Most preferably it is the HN domain (or a functional component thereof), wherein HN means a portion or fragment of the H-chain of a clostridial neurotoxin approximately equivalent to the amino-terminal half of the H-chain, or the domain corresponding to that fragment in the intact H-chain.
[0091] The TM component of the present invention is responsible for binding the fusion protein of the present invention to a Binding Site on a target cell. Thus, the TM component is simply a ligand through which a fusion protein of the present invention binds to a selected target cell.
[0092] In the context of the present invention, the target cell is a nociceptive sensory afferent, preferably a primary nociceptive afferent (e.g. an A-fibre such as an Aδ-fibre or a C-fibre). Thus, the fusion proteins of the present invention are capable of inhibiting neurotransmitter or neuromodulator [e.g. glutamate, substance P, calcitonin-gene related peptide (CGRP), and/or neuropeptide Y] release from discrete populations of nociceptive sensory afferent neurons. In use, the fusion proteins reduce or prevent the transmission of sensory afferent signals (e.g. neurotransmitters or neuromodulators) from peripheral to central pain fibres, and therefore have application as therapeutic molecules for the treatment of pain, in particular chronic pain.
[0093] It is routine to confirm that a TM binds to a nociceptive sensory afferent. For example, a simple radioactive displacement experiment may be employed in which tissue or cells representative of the nociceptive sensory afferent (for example DRGs) are exposed to labelled (e.g. tritiated) ligand in the presence of an excess of unlabelled ligand. In such an experiment, the relative proportions of non-specific and specific binding may be assessed, thereby allowing confirmation that the ligand binds to the nociceptive sensory afferent target cell. Optionally, the assay may include one or more binding antagonists, and the assay may further comprise observing a loss of ligand binding. Examples of this type of experiment can be found in Hulme, E. C. (1990), Receptor-binding studies, a brief outline, pp. 303-311, In Receptor biochemistry, A Practical Approach, Ed. E. C. Hulme, Oxford University Press.
[0094] The fusion proteins of the present invention generally demonstrate a reduced binding affinity (in the region of up to 100-fold) for nociceptive sensory afferent target cells when compared with the corresponding `free` TM. However, despite this observation, the fusion proteins of the present invention surprisingly demonstrate good efficacy. This can be attributed to two principal features. First, the non-cytotoxic protease component is catalytic--thus, the therapeutic effect of a few such molecules is rapidly amplified. Secondly, the receptors present on the nociceptive sensory afferents need only act as a gateway for entry of the therapeutic, and need not necessarily be stimulated to a level required in order to achieve a ligand-receptor mediated pharmacological response. Accordingly, the fusion proteins of the present invention may be administered at a dosage that is much lower that would be employed for other types of analgesic molecules such as NSAIDS, morphine, and gabapentin. The latter molecules are typically administered at high microgram to milligram (even up to hundreds of milligram) quantities, whereas the fusion proteins of the present invention may be administered at much lower dosages, typically at least 10-fold lower, and more typically at 100-fold lower.
[0095] The TM preferably comprises a maximum of 50 amino acid residues, more preferably a maximum of 40 amino acid residues, particularly preferably a maximum of 30 amino acid residues, and most preferably a maximum of 20 amino acid residues.
[0096] Opioids represent a preferred group of TMs of the present invention. Within this family of peptides is included enkephalins (met and leu), endomorphins 1 and 2, β-endorphin and dynorphin. Opioid peptides are frequently used in the clinic to modify the activity to nociceptors, and other cells involved in the pain response. As exemplified by the three-step World Health Organisation Analgesic Ladder, opioids have entry points into the pharmacological treatment of chronic cancer and non-cancer pain at all three stages, underlining their importance to the treatment of pain. Reference to opioids embraces fragments, variants and derivatives thereof, which retain the ability to bind to nociceptive sensory afferents.
[0097] The TM of the invention can also be a molecule that acts as an "agonist" at one or more of the receptors present on a nociceptive sensory afferent, more particularly on a primary nociceptive afferent. Conventionally, an agonist has been considered any molecule that can either increase or decrease activities within a cell, namely any molecule that simply causes an alteration of cell activity. For example, the conventional meaning of an agonist would include a chemical substance capable of combining with a receptor on a cell and initiating a reaction or activity, or a drug that induces an active response by activating receptors, whether the response is an increase or decrease in cellular activity.
[0098] However, for the purposes of this invention, an agonist is more specifically defined as a molecule that is capable of stimulating the process of exocytic fusion in a target cell, which process is susceptible to inhibition by a protease (or fragment thereof) capable of cleaving a protein of the exocytic fusion apparatus in said target cell.
[0099] Accordingly, the particular agonist definition of the present invention would exclude many molecules that would be conventionally considered as agonists. For example, nerve growth factor (NGF) is an agonist in respect of its ability to promote neuronal differentiation via binding to a TrkA receptor. However, NGF is not an agonist when assessed by the above criteria because it is not a principal inducer of exocytic fusion. In addition, the process that NGF stimulates (i.e. cell differentiation) is not susceptible to inhibition by the protease activity of a non-cytotoxic toxin molecule.
[0100] The agonist properties of a TM that binds to a receptor on a nociceptive afferent can be confirmed using the methods described in Example 10.
[0101] In a preferred embodiment of the invention, the target for the TM is the ORL1 receptor. This receptor is a member of the G-protein-coupled class of receptors, and has a seven transmembrane domain structure. The properties of the ORL1 receptor are discussed in detail in Mogil & Pasternak (2001), Pharmacological Reviews, Vol. 53, No. 3, pages 381-415.
[0102] In one embodiment, the TM is a molecule that binds (preferably that specifically binds) to the ORL1 receptor. More preferably, the TM is an "agonist" of the ORL1 receptor. The term "agonist" in this context is defined as above.
[0103] The agonist properties of a TM that binds to an ORL1 receptor can be confirmed using the methods described in Example 10. These methods are based on previous experiments [see Inoue et al. 1998 [Proc. Natl. Acad. Sci., 95, 10949-10953]), which confirm that the natural agonist of the ORL1 receptor, nociceptin, causes the induction of substance P release from nociceptive primary afferent neurons. This is supported by the fact that: [0104] the nociceptin-induced responses are abolished by specific NK1 receptor (the substance P receptor) antagonists; and [0105] pre-treatment of the cells with capsaicin (which depletes substance P from small diameter primary afferent neurons) attenuates the nociceptin-induced responses.
[0106] Similarly, Inoue et al. confirm that an intraplantar injection of botulinum neurotoxin type A abolishes the nociceptin-induced responses. Since it is known that BoNT inhibits the release of substance P from primary afferent neurons (Welch et al., 2000, Toxicon, 38, 245-258), this confirms the link between nociceptin-ORL1 interaction and subsequent release of substance P.
[0107] Thus, a TM can be said to have agonist activity at the ORL1 receptor if the TM causes an induction in the release of substance P from a nociceptive sensory afferent neuron (see Example 10).
[0108] In a particularly preferred embodiment of the invention, the TM is nociceptin--the natural ligand for the ORL1 receptor. Nociceptin targets the ORL1 receptor with high affinity. Examples of other preferred TMs include:
TABLE-US-00001 Code Sequence Ref. SEQ ID No. Nociceptin 1-17 FGGFTGARKSARKLANQ [1] 37, 38 Nociceptin 1-11 FGGFTGARKSA [1] 39, 40 Nociceptin [Y10]1-11 FGGFTGARKYA [1] 41, 42 Nociceptin [Y11]1-11 FGGFTGARKSY [1] 43, 44 Nociceptin [Y14]1-17 FGGFTGARKSARKYANQ [1] 45, 46 Nociceptin 1-13 FGGFTGARKSARK [2] 47, 48 Nociceptin [R14K15] 1- FGGFTGARKSARKRKNQ [3, 4] 49, 50 17 (also known in this specification as "variant" nociceptin) Peptide agonist Peptide agonists from [5] -- combinatorial library approach [1] Mogil & Pasternak, 2001, Pharmacol. Rev., 53, 381-415 [2] Maile et al., 2003, Neurosci. Lett., 350, 190-192 [3] Rizzi et al., 2002, J. Pharmacol. Exp. Therap., 300, 57-63 [4] Okada et al., 2000, Biochem. Biophys. Res. Commun., 278, 493-498 [5] Dooley et al., 1997, J Pharmacol Exp Ther. 283(2), 735-41.
[0109] The above-identified "variant" TM demonstrates particularly good binding affinity (when compared with natural nociceptin) for nociceptive sensory afferents. This is surprising as the amino acid modifications occur at a position away from the N-terminus of the TM. Moreover, the modifications are almost at the C-terminus of the TM, which in turn is attached to a large polypeptide sequence (i.e. the translocation domain). Generally speaking, a TM-containing fusion protein will demonstrate an approximate 100-fold reduction in binding ability vis-a-vis the TM per se. The above-mentioned "variant" TM per se demonstrates an approximate 3- to 10-fold increase in binding ability for a nociceptive sensory afferent (e.g. via the ORL1 receptor) vis-a-vis natural nociceptin. Thus, a "variant" TM-containing fusion might be expected to demonstrate an approximate 10-fold reduction in binding ability for a nociceptive sensory afferent (e.g. via the ORL1 receptor) vis-a-vis `free` nociceptin. However, the present inventors have demonstrated that such "variant" TM-containing fusion proteins demonstrate a binding ability that (most surprisingly) closely mirrors that of `free` nociceptin--see FIG. 14.
[0110] In the context of the present invention, the term opioid or an agonist of the ORL1 receptor (such as nociceptin, or any one of the peptides listed in the table above) embraces molecules having at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% homology with said opioid or agonist. The agonist homologues retain the agonist properties of nociceptin at the ORL1 receptor, which may be tested using the methods provided in Example 10. Similarly, an opioid homologue substantially retains the binding function of the opioid with which it shows high homology.
[0111] The invention also encompasses fragments, variants, and derivatives of any one of the TMs described above. These fragments, variants, and derivatives substantially retain the properties that are ascribed to said TMs.
[0112] In addition to the above-mentioned opioid and non-opioid classes of TMs, a variety of other polypeptides are suitable for targeting the fusion proteins of the present invention to nociceptive sensory afferents (e.g. to nociceptors). In this regard, particular reference is made to galanin and derivatives of galanin. Galanin receptors are found pre- and post-synaptically in DRGs (Liu & Hokfelt, (2002), Trends Pharm. Sci., 23(10), 468-74), and are enhanced in expression during neuropathic pain states. Proteinase-activated receptors (PARs) are also a preferred group of TMs of the present invention, most particularly PAR-2. It is known that agonists of PAR-2 induce/elicit acute inflammation, in part via a neurogenic mechanism. PAR2 is expressed by primary spinal afferent neurons, and PAR2 agonists stimulate release of substance P(SP) and calcitonin gene-related peptide (CGRP) in peripheral tissues
[0113] A particularly preferred set of TMs of the present invention includes:
TABLE-US-00002 Ligand Reference Nociceptin Guerrini, et al., (1997) J. Med. Chem., 40, pp. 1789-1793 β-endorphin Blanc, et al., (1983) J. Biol. Chem., 258(13), pp. 8277-8284 Endomorphin-1; Zadina, et al., (1997). Nature, 386, pp. Endomorphin-2 499-502 Dynorphin Fields & Basbaum (2002) Chapter 11, In The Textbook of Pain, Wall & Melzack eds. Met-enkephalin Fields & Basbaum (2002) Chapter 11, In The Textbook of Pain, Wall & Melzack eds. Leu-enkephalin Fields & Basbaum (2002) Chapter 11, In The Textbook of Pain, Wall & Melzack eds. Galanin Xu et al., (2000) Neuropeptides, 34 (3&4), 137-147 PAR-2 peptide Vergnolle et al., (2001) Nat. Med., 7(7), 821-826
[0114] The protease cleavage site of the present invention allows cleavage (preferably controlled cleavage) of the fusion protein at a position between the non-cytotoxic protease component and the TM component. It is this cleavage reaction that converts the fusion protein from a single chain polypeptide into a disulphide-linked, di-chain polypeptide.
[0115] According to a preferred embodiment of the present invention, the TM binds via a domain or amino acid sequence that is located away from the C-terminus of the TM. For example, the relevant binding domain may include an intra domain or an amino acid sequence located towards the middle (i.e. of the linear peptide sequence) of the TM. Preferably, the relevant binding domain is located towards the N-terminus of the TM, more preferably at or near to the N-terminus.
[0116] In one embodiment, the single chain polypeptide fusion may include more than one proteolytic cleavage site. However, where two or more such sites exist, they are different, thereby substantially preventing the occurrence of multiple cleavage events in the presence of a single protease. In another embodiment, it is preferred that the single chain polypeptide fusion has a single protease cleavage site.
[0117] The protease cleavage sequence(s) may be introduced (and/or any inherent cleavage sequence removed) at the DNA level by conventional means, such as by site-directed mutagenesis. Screening to confirm the presence of cleavage sequences may be performed manually or with the assistance of computer software (e.g. the MapDraw program by DNASTAR, Inc.).
[0118] Whilst any protease cleavage site may be employed, the following are preferred:
TABLE-US-00003 Enterokinase (DDDDK↓) Factor Xa (IEGR↓/IDGR↓) TEV(Tobacco Etch virus) (ENLYFQ↓G) Thrombin (LVPR↓GS) PreScission (LEVLFQ↓GP).
[0119] Also embraced by the term protease cleavage site is an intein, which is a self-cleaving sequence. The self-splicing reaction is controllable, for example by varying the concentration of reducing agent present.
[0120] In use, the protease cleavage site is cleaved and the N-terminal region (preferably the N-terminus) of the TM becomes exposed. The resulting polypeptide has a TM with an N-terminal domain or an intra domain that is substantially free from the remainder of the fusion protein. This arrangement ensures that the N-terminal component (or intra domain) of the TM may interact directly with a Binding Site on a target cell.
[0121] In a preferred embodiment, the TM and the protease cleavage site are distanced apart in the fusion protein by at most 10 amino acid residues, more preferably by at most 5 amino acid residues, and most preferably by zero amino acid residues. Thus, following cleavage of the protease cleavage site, a fusion is provided with a TM that has an N-terminal domain that is substantially free from the remainder of the fusion. This arrangement ensures that the N-terminal component of the Targeting Moiety may interact directly with a Binding Site on a target cell.
[0122] One advantage associated with the above-mentioned activation step is that the TM only becomes susceptible to N-terminal degradation once proteolytic cleavage of the fusion protein has occurred. In addition, the selection of a specific protease cleavage site permits selective activation of the polypeptide fusion into a di-chain conformation.
[0123] Construction of the single-chain polypeptide fusion of the present invention places the protease cleavage site between the TM and the non-cytotoxic protease component.
[0124] It is preferred that, in the single-chain fusion, the TM is located between the protease cleavage site and the translocation component. This ensures that the TM is attached to the translocation domain (i.e. as occurs with native clostridial holotoxin), though in the case of the present invention the order of the two components is reversed vis-a-vis native holotoxin. A further advantage with this arrangement is that the TM is located in an exposed loop region of the fusion protein, which has minimal structural effects on the conformation of the fusion protein. In this regard, said loop is variously referred to as the linker, the activation loop, the inter-domain linker, or just the surface exposed loop (Schiavo et al 2000, Phys. Rev., 80, 717-766; Turton et al., 2002, Trends Biochem. Sci., 27, 552-558).
[0125] In one embodiment, in the single chain polypeptide, the non-cytotoxic protease component and the translocation component are linked together by a disulphide bond. Thus, following cleavage of the protease cleavage site, the polypeptide assumes a di-chain conformation, wherein the protease and translocation components remain linked together by the disulphide bond. To this end, it is preferred that the protease and translocation components are distanced apart from one another in the single chain fusion protein by a maximum of 100 amino acid residues, more preferably a maximum of 80 amino acid residues, particularly preferably by a maximum of 60 amino acid residues, and most preferably by a maximum of 50 amino acid residues.
[0126] In one embodiment, the non-cytotoxic protease component forms a disulphide bond with the translocation component of the fusion protein. For example, the amino acid residue of the protease component that forms the disulphide bond is located within the last 20, preferably within the last 10 C-terminal amino acid residues of the protease component. Similarly, the amino acid residue within the translocation component that forms the second part of the disulphide bond may be located within the first 20, preferably within the first 10 N-terminal amino acid residues of the translocation component.
[0127] Alternatively, in the single chain polypeptide, the non-cytotoxic protease component and the TM may be linked together by a disulphide bond. In this regard, the amino acid residue of the TM that forms the disulphide bond is preferably located away from the N-terminus of the TM, more preferably towards to C-terminus of the TM.
[0128] In one embodiment, the non-cytotoxic protease component forms a disulphide bond with the TM component of the fusion protein. In this regard, the amino acid residue of the protease component that forms the disulphide bond is preferably located within the last 20, more preferably within the last 10 C-terminal amino acid residues of the protease component. Similarly, the amino acid residue within the TM component that forms the second part of the disulphide bond is preferably located within the last 20, more preferably within the last 10 C-terminal amino acid residues of the TM.
[0129] The above disulphide bond arrangements have the advantage that the protease and translocation components are arranged in a manner similar to that for native clostridial neurotoxin. By way of comparison, referring to the primary amino acid sequence for native clostridial neurotoxin, the respective cysteine amino acid residues are distanced apart by between 8 and 27 amino acid residues--taken from Popoff, M R & Marvaud, J-C, 1999, Structural & genomic features of clostridial neurotoxins, Chapter 9, in The Comprehensive Sourcebook of Bacterial Protein Toxins. Ed. Alouf & Freer:
TABLE-US-00004 `Native` length between Serotype1 Sequence C-C BoNT/A1 CVRGIITSKTKS----LDKGYNKALNDLC 23 BoNT/A2 CVRGIIPFKTKS----LDEGYNKALNDLC 23 BoNT/B CKSVKAPG-------------------IC 8 BoNT/C CHKAIDGRS----------LYNKTLDC 15 BoNT/D CLRLTK---------------NSRDDSTC 12 BoNT/E CKN-IVSVK----------GIRK---SIC 13 BoNT/F CKS-VIPRK----------GTKAPP-RLC 15 BoNT/G CKPVMYKNT----------GKSE----QC 13 TeNT CKKIIPPTNIRENLYNRTASLTDLGGELC 27 1Information from proteolytic strains only
[0130] The fusion protein may comprise one or more purification tags, which are located N-terminal to the protease component and/or C-terminal to the translocation component.
[0131] Whilst any purification tag may be employed, the following are preferred:
His-tag (e.g. 6× histidine), preferably as a C-terminal and/or N-terminal tag MBP-tag (maltose binding protein), preferably as an N-terminal tag GST-tag (glutathione-S-transferase), preferably as an N-terminal tag His-MBP-tag, preferably as an N-terminal tag GST-MBP-tag, preferably as an N-terminal tag Thioredoxin-tag, preferably as an N-terminal tag CBD-tag (Chitin Binding Domain), preferably as an N-terminal tag.
[0132] According to a further embodiment of the present invention, one or more peptide spacer molecules may be included in the fusion protein. For example, a peptide spacer may be employed between a purification tag and the rest of the fusion protein molecule (e.g. between an N-terminal purification tag and a protease component of the present invention; and/or between a C-terminal purification tag and a translocation component of the present invention). A peptide spacer may be also employed between the TM and translocation components of the present invention.
[0133] A variety of different spacer molecules may be employed in any of the fusion proteins of the present invention. Examples of such spacer molecules include those illustrated in FIGS. 28 and 29. Particular mention here is made to GS15, GS20, GS25, and Hx27--see FIGS. 28 and 29.
[0134] The present inventors have unexpectedly found that the fusion proteins (e.g. CPNv/A) of the present invention may demonstrate an improved binding activity for nociceptive sensory afferents when the size of the spacer is selected so that (in use) the C-terminus of the TM and the N-terminus of the translocation component are separated from one another by 40-105 angstroms, preferably by 50-100 angstroms, and more preferably by 50-90 angstroms. In another embodiment, the preferred spacers have an amino acid sequence of 11-29 amino acid residues, preferably 15-27 amino acid residues, and more preferably 20-27 amino acid residues. Suitable spacers may be routinely identified and obtained according to Crasto, C. J. and Feng, J. A. (2000) May, 13(5), pp. 309-312--see also http://www.fccc./edu/research/labs/feng/linker.html.
[0135] In accordance with a second aspect of the present invention, there is provided a DNA sequence that encodes the above-mentioned single chain polypeptide. In a preferred aspect of the present invention, the DNA sequence is prepared as part of a DNA vector, wherein the vector comprises a promoter and terminator.
[0136] In a preferred embodiment, the vector has a promoter selected from:
TABLE-US-00005 Promoter Induction Agent Typical Induction Condition Tac (hybrid) IPTG 0.2 mM (0.05-2.0 mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lac operator IPTG 0.2 mM (0.05-2.0 mM)
[0137] The DNA construct of the present invention is preferably designed in silico, and then synthesised by conventional DNA synthesis techniques.
[0138] The above-mentioned DNA sequence information is optionally modified for codon-biasing according to the ultimate host cell (e.g. E. coli) expression system that is to be employed.
[0139] The DNA backbone is preferably screened for any inherent nucleic acid sequence, which when transcribed and translated would produce an amino acid sequence corresponding to the protease cleave site encoded by the second peptide-coding sequence. This screening may be performed manually or with the assistance of computer software (e.g. the MapDraw program by DNASTAR, Inc.).
[0140] According to a further embodiment of the present invention, there is provided a method of preparing a non-cytotoxic agent, comprising: [0141] a. contacting a single-chain polypeptide fusion protein of the invention with a protease capable of cleaving the protease cleavage site; [0142] b. cleaving the protease cleavage site, and thereby forming a di-chain fusion protein.
[0143] This aspect provides a di-chain polypeptide, which generally mimics the structure of clostridial holotoxin. In more detail, the resulting di-chain polypeptide typically has a structure wherein: [0144] a. the first chain comprises the non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent; [0145] b. the second chain comprises the TM and the translocation domain that is capable of translocating the protease or protease fragment from within an endosome, across the endosomal membrane and into the cytosol of the nociceptive sensory afferent; and [0146] the first and second chains are disulphide linked together.
[0147] In use, the single chain or di-chain polypeptide of the invention treat, prevent or ameliorate pain.
[0148] In use, a therapeutically effective amount of a single chain or di-chain polypeptide of the invention is administered to a patient.
[0149] According to a further aspect of the present invention, there is provided use of a single chain or di-chain polypeptide of the invention, for the manufacture of a medicament for treating, preventing or ameliorating pain.
[0150] According to a related aspect, there is provided a method of treating, preventing or ameliorating pain in a subject, comprising administering to said patient a therapeutically effective amount of a single chain or di-chain polypeptide of the invention.
[0151] The compounds described here may be used to treat a patient suffering from one or more types of chronic pain including neuropathic pain, inflammatory pain, headache pain, somatic pain, visceral pain, and referred pain.
[0152] To "treat," as used here, means to deal with medically. It includes, for example, administering a compound of the invention to prevent pain or to lessen its severity.
[0153] The term "pain," as used here, means any unpleasant sensory experience, usually associated with a physical disorder. The physical disorder may or may not be apparent to a clinician. Pain is of two types: chronic and acute. An "acute pain" is a pain of short duration having a sudden onset. One type of acute pain, for example, is cutaneous pain felt on injury to the skin or other superficial tissues, such as caused by a cut or a burn. Cutaneous nociceptors terminate just below the skin, and due to the high concentration of nerve endings, produce a well-defined, localized pain of short duration. "Chronic pain" is a pain other than an acute pain. Chronic pain includes neuropathic pain, inflammatory pain, headache pain, somatic pain visceral pain and referred pain.
I. Neuropathic Pain
[0154] The compounds of the invention may be used to treat pain caused by or otherwise associated with any of the following neuropathic pain conditions. "Neuropathic pain" means abnormal sensory input, resulting in discomfort, from the peripheral nervous system, central nervous systems, or both.
A. Symptoms of Neuropathic Pain
[0155] Symptoms of neuropathic pain can involve persistent, spontaneous pain, as well as allodynia (a painful response to a stimulus that normally is not painful), hyperalgesia (an accentuated response to a painful stimulus that usually causes only a mild discomfort, such as a pin prick), or hyperpathia (where a short discomfort becomes a prolonged severe pain).
B. Causes of Neuropathic Pain
[0156] Neuropathic pain may be caused by any of the following.
1. A traumatic insult, such as, for example, a nerve compression injury (e.g., a nerve crush, a nerve stretch, a nerve entrapment or an incomplete nerve transsection); a spinal cord injury (e.g., a hemisection of the spinal cord); a limb amputation; a contusion; an inflammation (e.g., an inflammation of the spinal cord); or a surgical procedure. 2. An ischemic event, including, for example, a stroke and heart attack. 3. An infectious agent 4. Exposure to a toxic agent, including, for example, a drug, an alcohol, a heavy metal (e.g., lead, arsenic, mercury), an industrial agent (e.g., a solvent, fumes from a glue) or nitrous oxide. 5. A disease, including, for example, an inflammatory disorder, a neoplastic tumor, an acquired immune deficiency syndrome (AIDS), Lyme's disease, a leprosy, a metabolic disease, a peripheral nerve disorder, like neuroma, a mononeuropathy or a polyneuropathy.
C. Types of Neuropathic Pain
1. Neuralgia.
[0157] A neuralgia is a pain that radiates along the course of one or more specific nerves usually without any demonstrable pathological change in the nerve structure. The causes of neuralgia are varied. Chemical irritation, inflammation, trauma (including surgery), compression by nearby structures (for instance, tumors), and infections may all lead to neuralgia. In many cases, however, the cause is unknown or unidentifiable. Neuralgia is most common in elderly persons, but it may occur at any age. A neuralgia, includes, without limitation, a trigeminal neuralgia, a post-herpetic neuralgia, a postherpetic neuralgia, a glossopharyngeal neuralgia, a sciatica and an atypical facial pain.
[0158] Neuralgia is pain in the distribution of a nerve or nerves. Examples are trigeminal neuralgia, atypical facial pain, and postherpetic neuralgia (caused by shingles or herpes). The affected nerves are responsible for sensing touch, temperature and pressure in the facial area from the jaw to the forehead. The disorder generally causes short episodes of excruciating pain, usually for less than two minutes and on only one side of the face. The pain can be described in a variety of ways such as "stabbing," "sharp," "like lightning," "burning," and even "itchy". In the atypical form of TN, the pain can also present as severe or merely aching and last for extended periods. The pain associated with TN is recognized as one the most excruciating pains that can be experienced.
[0159] Simple stimuli such as eating, talking, washing the face, or any light touch or sensation can trigger an attack (even the sensation of a gentle breeze). The attacks can occur in clusters or as an isolated attack.
[0160] Symptoms include sharp, stabbing pain or constant, burning pain located anywhere, usually on or near the surface of the body, in the same location for each episode; pain along the path of a specific nerve; impaired function of affected body part due to pain, or muscle weakness due to concomitant motor nerve damage; increased sensitivity of the skin or numbness of the affected skin area (feeling similar to a local anesthetic such as a Novacaine shot); and any touch or pressure is interpreted as pain. Movement may also be painful.
[0161] Trigeminal neuralgia is the most common form of neuralgia. It affects the main sensory nerve of the face, the trigeminal nerve ("trigeminal" literally means "three origins", referring to the division of the nerve into 3 branches). This condition involves sudden and short attacks of severe pain on the side of the face, along the area supplied by the trigeminal nerve on that side. The pain attacks may be severe enough to cause a facial grimace, which is classically referred to as a painful tic (tic douloureux). Sometimes, the cause of trigeminal neuralgia is a blood vessel or small tumor pressing on the nerve. Disorders such as multiple sclerosis (an inflammatory disease affecting the brain and spinal cord), certain forms of arthritis, and diabetes (high blood sugar) may also cause trigeminal neuralgia, but a cause is not always identified. In this condition, certain movements such as chewing, talking, swallowing, or touching an area of the face may trigger a spasm of excruciating pain.
[0162] A related but rather uncommon neuralgia affects the glosso-pharyngeal nerve, which provides sensation to the throat. Symptoms of this neuralgia are short, shock-like episodes of pain located in the throat.
[0163] Neuralgia may occur after infections such as shingles, which is caused by the varicella-zoster virus, a type of herpesvirus. This neuralgia produces a constant burning pain after the shingles rash has healed. The pain is worsened by movement of or contact with the affected area. Not all of those diagnosed with shingles go on to experience postherpetic neuralgia, which can be more painful than shingles. The pain and sensitivity can last for months or even years. The pain is usually in the form of an intolerable sensitivity to any touch but especially light touch. Postherpetic neuralgia is not restricted to the face; it can occur anywhere on the body but usually occurs at the location of the shingles rash. Depression is not uncommon due to the pain and social isolation during the illness.
[0164] Postherpetic neuralgia may be debilitating long after signs of the original herpes infection have disappeared. Other infectious diseases that may cause neuralgia are syphilis and Lyme disease.
[0165] Diabetes is another common cause of neuralgia. This very common medical problem affects almost 1 out of every 20 Americans during adulthood. Diabetes damages the tiny arteries that supply circulation to the nerves, resulting in nerve fiber malfunction and sometimes nerve loss. Diabetes can produce almost any neuralgia, including trigeminal neuralgia, carpal tunnel syndrome (pain and numbness of the hand and wrist), and meralgia paresthetica (numbness and pain in the thigh due to damage to the lateral femoral cutaneous nerve). Strict control of blood sugar may prevent diabetic nerve damage and may accelerate recovery in patients who do develop neuralgia.
[0166] Other medical conditions that may be associated with neuralgias are chronic renal insufficiency and porphyria--a hereditary disease in which the body cannot rid itself of certain substances produced after the normal breakdown of blood in the body. Certain drugs may also cause this problem.
2. Deafferentation.
[0167] Deafferentation indicates a loss of the sensory input from a portion of the body, and can be caused by interruption of either peripheral sensory fibres or nerves from the central nervous system. A deafferentation pain syndrome, includes, without limitation, an injury to the brain or spinal cord, a post-stroke pain, a phantom pain, a paraplegia, a brachial plexus avulsion injuries, lumbar radiculopathies.
3. Complex Regional Pain Syndromes (CRPSs)
[0168] CRPS is a chronic pain syndrome resulting from sympathetically-maintained pain, and presents in two forms. CRPS 1 currently replaces the term "reflex sympathetic dystrophy syndrome". It is a chronic nerve disorder that occurs most often in the arms or legs after a minor or major injury. CRPS 1 is associated with severe pain; changes in the nails, bone, and skin; and an increased sensitivity to touch in the affected limb. CRPS 2 replaces the term causalgia, and results from an identified injury to the nerve. A CRPS, includes, without limitation, a CRPS Type I (reflex sympathetic dystrophy) and a CRPS Type II (causalgia).
4. Neuropathy.
[0169] A neuropathy is a functional or pathological change in a nerve and is characterized clinically by sensory or motor neuron abnormalities.
[0170] Central neuropathy is a functional or pathological change in the central nervous system.
[0171] Peripheral neuropathy is a functional or pathological change in one or more peripheral nerves. The peripheral nerves relay information from your central nervous system (brain and spinal cord) to muscles and other organs and from your skin, joints, and other organs back to your brain. Peripheral neuropathy occurs when these nerves fail to carry information to and from the brain and spinal cord, resulting in pain, loss of sensation, or inability to control muscles. In some cases, the failure of nerves that control blood vessels, intestines, and other organs results in abnormal blood pressure, digestion problems, and loss of other basic body processes. Risk factors for neuropathy include diabetes, heavy alcohol use, and exposure to certain chemicals and drugs. Some people have a hereditary predisposition for neuropathy. Prolonged pressure on a nerve is another risk for developing a nerve injury. Pressure injury may be caused by prolonged immobility (such as a long surgical procedure or lengthy illness) or compression of a nerve by casts, splints, braces, crutches, or other devices. Polyneuropathy implies a widespread process that usually affects both sides of the body equally. The symptoms depend on which type of nerve is affected. The three main types of nerves are sensory, motor, and autonomic. Neuropathy can affect any one or a combination of all three types of nerves. Symptoms also depend on whether the condition affects the whole body or just one nerve (as from an injury). The cause of chronic inflammatory polyneuropathy is an abnormal immune response. The specific antigens, immune processes, and triggering factors are variable and in many cases are unknown. It may occur in association with other conditions such as HIV, inflammatory bowel disease, lupus erythematosis, chronic active hepatitis, and blood cell abnormalities.
[0172] Peripheral neuropathy may involve a function or pathological change to a single nerve or nerve group (monneuropathy) or a function or pathological change affecting multiple nerves (polyneuropathy).
Peripheral Neuropathies
Hereditary Disorders
[0173] Charcot-Marie-Tooth disease [0174] Friedreich's ataxia
Systemic or Metabolic Disorders
[0174] [0175] Diabetes (diabetic neuropathy) [0176] Dietary deficiencies (especially vitamin B-12) [0177] Excessive alcohol use (alcoholic neuropathy) [0178] Uremia (from kidney failure) [0179] Cancer
Infectious or Inflammatory Conditions
[0179] [0180] AIDS [0181] Hepatitis [0182] Colorado tick fever [0183] diphtheria [0184] Guillain-Barre syndrome [0185] HIV infection without development of AIDS [0186] leprosy [0187] Lyme [0188] polyarteritis nodosa [0189] rheumatoid arthritis [0190] sarcoidosis [0191] Sjogren syndrome [0192] syphilis [0193] systemic lupus erythematosus [0194] amyloid
Exposure to Toxic Compounds
[0194] [0195] sniffing glue or other toxic compounds [0196] nitrous oxide [0197] industrial agents--especially solvents [0198] heavy metals (lead, arsenic, mercury, etc.) [0199] Neuropathy secondary to drugs like analgesic nephropathy
Miscellaneous Causes
[0199] [0200] ischemia (decreased oxygen/decreased blood flow) [0201] prolonged exposure to cold temperature
[0202] a. Polyneuropathy
[0203] Polyneuropathy is a peripheral neuropathy involving the loss of movement or sensation to an area caused by damage or destruction to multiple peripheral nerves. Polyneuropathic pain, includes, without limitation, post-polio syndrome, postmastectomy syndrome, diabetic neuropathy, alcohol neuropathy, amyloid, toxins, AIDS, hypothyroidism, uremia, vitamin deficiencies, chemotherapy-induced pain, 2',3'-didexoycytidine (ddC) treatment, Guillain-Barre syndrome or Fabry's disease.
[0204] b. Mononeuropathy
[0205] Mononeuropathy is a peripheral neuropathy involving loss of movement or sensation to an area caused by damage or destruction to a single peripheral nerve or nerve group. Mononeuropathy is most often caused by damage to a local area resulting from injury or trauma, although occasionally systemic disorders may cause isolated nerve damage (as with mononeuritis multiplex). The usual causes are direct trauma, prolonged pressure on the nerve, and compression of the nerve by swelling or injury to nearby body structures. The damage includes destruction of the myelin sheath (covering) of the nerve or of part of the nerve cell (the axon). This damage slows or prevents conduction of impulses through the nerve. Mononeuropathy may involve any part of the body. Mononeuropathic pain, includes, without limitation, a sciatic nerve dysfunction, a common peroneal nerve dysfunction. a radial nerve dysfunction, an ulnar nerve dysfunction, a cranial mononeuropathy VI, a cranial mononeuropathy VII, a cranial mononeuropathy III (compression type), a cranial mononeuropathy III (diabetic type), an axillary nerve dysfunction, a carpal tunnel syndrome, a femoral nerve dysfunction, a tibial nerve dysfunction, a Bell's palsy, a thoracic outlet syndrome, a carpal tunnel syndrome and a sixth (abducent) nerve palsy
[0206] c. Generalized Peripheral Neuropathies
[0207] Generalized peripheral neuropathis are symmetrical, and usually due to various systematic illnesses and disease processes that affect the peripheral nervous system in its entirety. They are further subdivided into several categories:
[0208] i. Distal axonopathies are the result of some metabolic or toxic derangement of neurons. They may be caused by metabolic diseases such as diabetes, renal failure, deficiency syndromes such as malnutrition and alcoholism, or the effects of toxins or drugs. Distal axonopathy (aka dying back neuropathy) is a type of peripheral neuropathy that results from some metabolic or toxic derangement of peripheral nervous system (PNS) neurons. It is the most common response of nerves to metabolic or toxic disturbances, and as such may be caused by metabolic diseases such as diabetes, renal failure, deficiency syndromes such as malnutrition and alcoholism, or the effects of toxins or drugs. The most common cause of distal axonopathy is diabetes, and the most common distal axonopathy is diabetic neuropathy.
[0209] ii. Myelinopathies are due to a primary attack on myelin causing an acute failure of impulse conduction. The most common cause is acute inflammatory demyelinating polyneuropathy (AIDP; aka Guillain-Barre syndrome), though other causes include chronic inflammatory demyelinating syndrome (CIDP), genetic metabolic disorders (e.g., leukodystrophy), or toxins. Myelinopathy is due to primary destruction of myelin or the myelinating Schwann cells, which leaves the axon intact, but causes an acute failure of impulse conduction. This demyelination slows down or completely blocks the conduction of electrical impulses through the nerve. The most common cause is acute inflammatory demyelinating polyneuropathy (AIDP, better known as Guillain-Barre syndrome), though other causes include chronic inflammatory demyelinating polyneuropathy (CIDP), genetic metabolic disorders (e.g., leukodystrophy or Charcot-Marie-Tooth disease), or toxins.
[0210] iii. Neuronopathies are the result of destruction of peripheral nervous system (PNS) neurons. They may be caused by motor neurone diseases, sensory neuronopathies (e.g., Herpes zoster), toxins or autonomic dysfunction. Neurotoxins may cause neuronopathies, such as the chemotherapy agent vincristine. Neuronopathy is dysfunction due to damage to neurons of the peripheral nervous system (PNS), resulting in a peripheral neuropathy. It may be caused by motor neurone diseases, sensory neuronopathies (e.g., Herpes zoster), toxic substances or autonomic dysfunction. A person with neuronopathy may present in different ways, depending on the cause, the way it affects the nerve cells, and the type of nerve cell that is most affected.
[0211] iv. Focal entrapment neuropathies (e.g., carpal tunnel syndrome).
II. Inflammatory Pain
[0212] The compounds of the invention may be used to treat pain caused by or otherwise associated with any of the following inflammatory conditions
A. Arthritic Disorder
[0213] Arthritic disorders include, for example, a rheumatoid arthritis; a juvenile rheumatoid arthritis; a systemic lupus erythematosus (SLE); a gouty arthritis; a scleroderma; an osteoarthritis; a psoriatic arthritis; an ankylosing spondylitis; a Reiter's syndrome (reactive arthritis); an adult Still's disease; an arthritis from a viral infection; an arthritis from a bacterial infection, such as, e.g., a gonococcal arthritis and a non-gonococcal bacterial arthritis (septic arthritis); a Tertiary Lyme disease; a tuberculous arthritis; and an arthritis from a fungal infection, such as, e.g. a blastomycosis
B. Autoimmune Diseases
[0214] Autoimmune diseases include, for example, a Guillain-Barre syndrome, a Hashimoto's thyroiditis, a pernicious anemia, an Addison's disease, a type I diabetes, a systemic lupus erythematosus, a dermatomyositis, a Sjogren's syndrome, a lupus erythematosus, a multiple sclerosis, a myasthenia gravis, a Reiter's syndrome and a Grave's disease.
C. Connective Tissue Disorder
[0215] Connective tissue disorders include, for example, a spondyloarthritis a dermatomyositis, and a fibromyalgia.
D. Injury
[0216] Inflammation caused by injury, including, for example, a crush, puncture, stretch of a tissue or joint, may cause chronic inflammatory pain.
E. Infection
[0217] Inflammation caused by infection, including, for example, a tuberculosis or an interstitial keratitis may cause chronic inflammatory pain.
F. Neuritis
[0218] Neuritis is an inflammatory process affecting a nerve or group of nerves. Symptoms depend on the nerves involved, but may include pain, paresthesias, paresis, or hypesthesia (numbness).
[0219] Examples include: [0220] a. Brachial neuritis
[0221] b. Retrobulbar neuropathy, an inflammatory process affecting the part of the optic nerve lying immediately behind the eyeball.
[0222] c. Optic neuropathy, an inflammatory process affecting the optic nerve causing sudden, reduced vision in the affected eye. The cause of optic neuritis is unknown. The sudden inflammation of the optic nerve (the nerve connecting the eye and the brain) leads to swelling and destruction of the myelin sheath. The inflammation may occasionally be the result of a viral infection, or it may be caused by autoimmune diseases such as multiple sclerosis. Risk factors are related to the possible causes.
[0223] d. Vestibular neuritis, a viral infection causing an inflammatory process affecting the vestibular nerve.
G. Joint Inflammation
[0224] Inflammation of the joint, such as that caused by bursitis or tendonitis, for example, may cause chronic inflammatory pain.
III. Headache Pain
[0225] The compounds of the invention may be used to treat pain caused by or otherwise associated with any of the following headache conditions. A headache (medically known as cephalgia) is a condition of mild to severe pain in the head; sometimes neck or upper back pain may also be interpreted as a headache. It may indicate an underlying local or systemic disease or be a disorder in itself.
A. Muscular/Myogenic Headache
[0226] Muscular/myogenic headaches appear to involve the tightening or tensing of facial and neck muscles; they may radiate to the forehead. Tension headache is the most common form of myogenic headache.
[0227] A tension headache is a condition involving pain or discomfort in the head, scalp, or neck, usually associated with muscle tightness in these areas. Tension headaches result from the contraction of neck and scalp muscles. One cause of this muscle contraction is a response to stress, depression or anxiety. Any activity that causes the head to be held in one position for a long time without moving can cause a headache. Such activities include typing or use of computers, fine work with the hands, and use of a microscope. Sleeping in a cold room or sleeping with the neck in an abnormal position may also trigger this type of headache. A tension-type headache, includes, without limitation, an episodic tension headache and a chronic tension headache.
B. Vascular Headache
[0228] The most common type of vascular headache is migraine. Other kinds of vascular headaches include cluster headaches, which cause repeated episodes of intense pain, and headaches resulting from high blood pressure
[0229] 1. Migraine
[0230] A migraine is a heterogeneous disorder that generally involves recurring headaches. Migraines are different from other headaches because they occur with other symptoms, such as, e.g., nausea, vomiting, or sensitivity to light. In most people, a throbbing pain is felt only on one side of the head. Clinical features such as type of aura symptoms, presence of prodromes, or associated symptoms such as vertigo, may be seen in subgroups of patients with different underlying pathophysiological and genetic mechanisms. A migraine headache, includes, without limitation, a migraine without aura (common migraine), a migraine with aura (classic migraine), a menstrual migraine, a migraine equivalent (acephalic headache), a complicated migraine, an abdominal migraine and a mixed tension migraine.
[0231] 2. Cluster Headache
[0232] Cluster headaches affect one side of the head (unilateral) and may be associated with tearing of the eyes and nasal congestion. They occurs in clusters, happening repeatedly every day at the same time for several weeks and then remitting.
D. High Blood Pressure Headache
E. Traction and Inflammatory Headache
[0233] Traction and inflammatory headaches are usually symptoms of other disorders, ranging from stroke to sinus infection.
F. Hormone Headache
G. Rebound Headache
[0234] Rebound headaches, also known as medication overuse headaches, occur when medication is taken too frequently to relieve headache. Rebound headaches frequently occur daily and can be very painful.
H. Chronic Sinusitis Headache
[0235] Sinusitis is inflammation, either bacterial, fungal, viral, allergic or autoimmune, of the paranasal sinuses. Chronic sinusitis is one of the most common complications of the common cold. Symptoms include: Nasal congestion; facial pain; headache; fever; general malaise; thick green or yellow discharge; feeling of facial `fullness` worsening on bending over. In a small number of cases, chronic maxillary sinusitis can also be brought on by the spreading of bacteria from a dental infection. Chronic hyperplastic eosinophilic sinusitis is a noninfective form of chronic sinusitis.
I. An Organic Headache
J. Ictal Headaches
[0236] Ital headaches are headaches associated with seizure activity.
IV. Somatic Pain
[0237] The compounds of the invention may be used to treat pain caused by or otherwise associated with any of the following somatic pain conditions. Somatic pain originates from ligaments, tendons, bones, blood vessels, and even nerves themselves. It is detected with somatic nociceptors. The scarcity of pain receptors in these areas produces a dull, poorly-localized pain of longer duration than cutaneous pain; examples include sprains and broken bones. Additional examples include the following.
A. Excessive Muscle Tension
[0238] Excessive muscle tension can be caused, for example, by a sprain or a strain.
B. Repetitive Motion Disorders
[0239] Repetitive motion disorders can result from overuse of the hands, wrists, elbows, shoulders, neck, back, hips, knees, feet, legs, or ankles.
C. Muscle Disorders
[0240] Muscle disorders causing somatic pain include, for example, a polymyositis, a dermatomyositis, a lupus, a fibromyalgia, a polymyalgia rheumatica, and a rhabdomyolysis.
D. Myalgia
[0241] Myalgia is muscle pain and is a symptom of many diseases and disorders. The most common cause for myalgia is either overuse or over-stretching of a muscle or group of muscles. Myalgia without a traumatic history is often due to viral infections. Longer-term myalgias may be indicative of a metabolic myopathy, some nutritional deficiencies or chronic fatigue syndrome.
E. Infection
[0242] Infection can cause somatic pain. Examples of such infection include, for example, an abscess in the muscle, a trichinosis, an influenza, a Lyme disease, a malaria, a Rocky Mountain spotted fever, Avian influenza, the common cold, community-acquired pneumonia, meningitis, monkeypox, Severe Acute Respiratory Syndrome, toxic shock syndrome, trichinosis, typhoid fever, and upper respiratory tract infection.
F. Drugs
[0243] Drugs can cause somatic pain. Such drugs include, for example, cocaine, a statin for lowering cholesterol (such as atorvastatin, simvastatin, and lovastatin), and an ACE inhibitor for lowering blood pressure (such as enalapril and captopril)
V. Visceral Pain
[0244] The compounds of the invention may be used to treat pain caused by or otherwise associated with any of the following visceral pain conditions. Visceral pain originates from body's viscera, or organs. Visceral nociceptors are located within body organs and internal cavities. The even greater scarcity of nociceptors in these areas produces pain that is usually more aching and of a longer duration than somatic pain. Visceral pain is extremely difficult to localise, and several injuries to visceral tissue exhibit "referred" pain, where the sensation is localised to an area completely unrelated to the site of injury. Examples of visceral pain include the following.
A. Functional Visceral Pain
[0245] Functional visceral pain includes, for example, an irritable bowel syndrome and a chronic functional abdominal pain (CFAP), a functional constipation and a functional dyspepsia, a non-cardiac chest pain (NCCP) and a chronic abdominal pain.
B. Chronic Gastrointestinal Inflammation
[0246] Chronic gastrointestinal inflammation includes, for example, a gastritis, an inflammatory bowel disease, like, e.g., a Crohn's disease, an ulcerative colitis, a microscopic colitis, a diverticulitis and a gastroenteritis; an interstitial cystitis; an intestinal ischemia; a cholecystitis; an appendicitis; a gastroesophageal reflux; an ulcer, a nephrolithiasis, an urinary tract infection, a pancreatitis and a hernia.
C. Autoimmune Pain
[0247] Autoimmune pain includes, for example, a sarcoidosis and a vasculitis.
D. Organic Visceral Pain
[0248] Organic visceral pain includes, for example, pain resulting from a traumatic, inflammatory or degenerative lesion of the gut or produced by a tumor impinging on sensory innervation.
E. Treatment-Induced Visceral Pain
[0249] Treatment-induced visceral pain includes, for example, a pain attendant to chemotherapy therapy or a pain attendant to radiation therapy.
VI. Referred Pain
[0250] The compounds of the invention may be used to treat pain caused by or otherwise associated with any of the following referred pain conditions.
[0251] Referred pain arises from pain localized to an area separate from the site of pain stimulation. Often, referred pain arises when a nerve is compressed or damaged at or near its origin. In this circumstance, the sensation of pain will generally be felt in the territory that the nerve serves, even though the damage originates elsewhere. A common example occurs in intervertebral disc herniation, in which a nerve root arising from the spinal cord is compressed by adjacent disc material. Although pain may arise from the damaged disc itself, pain will also be felt in the region served by the compressed nerve (for example, the thigh, knee, or foot). Relieving the pressure on the nerve root may ameliorate the referred pain, provided that permanent nerve damage has not occurred. Myocardial ischaemia (the loss of blood flow to a part of the heart muscle tissue) is possibly the best known example of referred pain; the sensation can occur in the upper chest as a restricted feeling, or as an ache in the left shoulder, arm or even hand.
[0252] The present invention addresses a wide range of pain conditions, in particular chronic pain conditions. Preferred conditions include cancerous and non-cancerous pain, inflammatory pain and neuropathic pain. The opioid-fusions of the present application are particularly suited to addressing inflammatory pain, though may be less suited to addressing neuropathic pain. The galanin-fusions are more suited to addressing neuropathic pain.
[0253] In use, the polypeptides of the present invention are typically employed in the form of a pharmaceutical composition in association with a pharmaceutical carrier, diluent and/or excipient, although the exact form of the composition may be tailored to the mode of administration. Administration is preferably to a mammal, more preferably to a human.
[0254] The polypeptides may, for example, be employed in the form of a sterile solution for intra-articular administration or intra-cranial administration. Spinal injection (e.g. epidural or intrathecal) is preferred.
[0255] The dosage ranges for administration of the polypeptides of the present invention are those to produce the desired therapeutic effect. It will be appreciated that the dosage range required depends on the precise nature of the components, the route of administration, the nature of the formulation, the age of the patient, the nature, extent or severity of the patient's condition, contraindications, if any, and the judgement of the attending physician.
[0256] Suitable daily dosages are in the range 0.0001-1 mg/kg, preferably 0.0001-0.5 mg/kg, more preferably 0.002-0.5 mg/kg, and particularly preferably 0.004-0.5 mg/kg. The unit dosage can vary from less that 1 microgram to 30 mg, but typically will be in the region of 0.01 to 1 mg per dose, which may be administered daily or preferably less frequently, such as weekly or six monthly.
[0257] A particularly preferred dosing regimen is based on 2.5 ng of fusion protein (e.g. CPNv/A) as the 1× dose. In this regard, preferred dosages are in the range 1×-100× (i.e. 2.5-250 ng). This dosage range is significantly lower (i.e. at least 10-fold, typically 100-fold lower) than would be employed with other types of analgesic molecules such as NSAIDS, morphine, and gabapentin. Moreover, the above-mentioned difference is considerably magnified when the same comparison is made on a molar basis--this is because the fusion proteins of the present invention have a considerably greater Mw than do conventional `small` molecule therapeutics.
[0258] Wide variations in the required dosage, however, are to be expected depending on the precise nature of the components, and the differing efficiencies of various routes of administration.
[0259] Variations in these dosage levels can be adjusted using standard empirical routines for optimisation, as is well understood in the art.
[0260] Compositions suitable for injection may be in the form of solutions, suspensions or emulsions, or dry powders which are dissolved or suspended in a suitable vehicle prior to use.
[0261] Fluid unit dosage forms are typically prepared utilising a pyrogen-free sterile vehicle. The active ingredients, depending on the vehicle and concentration used, can be either dissolved or suspended in the vehicle.
[0262] In preparing administrable solutions, the polypeptides can be dissolved in a vehicle, the solution being made isotonic if necessary by addition of sodium chloride and sterilised by filtration through a sterile filter using aseptic techniques before filling into suitable sterile vials or ampoules and sealing. Alternatively, if solution stability is adequate, the solution in its sealed containers may be sterilised by autoclaving.
[0263] Advantageously additives such as buffering, solubilising, stabilising, preservative or bactericidal, suspending or emulsifying agents may be dissolved in the vehicle.
[0264] Dry powders which are dissolved or suspended in a suitable vehicle prior to use may be prepared by filling pre-sterilised drug substance and other ingredients into a sterile container using aseptic technique in a sterile area.
[0265] Alternatively the polypeptides and other ingredients may be dissolved in an aqueous vehicle, the solution is sterilized by filtration and distributed into suitable containers using aseptic technique in a sterile area. The product is then freeze dried and the containers are sealed aseptically.
[0266] Parenteral suspensions, suitable for intramuscular, subcutaneous or intradermal injection, are prepared in substantially the same manner, except that the sterile components are suspended in the sterile vehicle, instead of being dissolved and sterilisation cannot be accomplished by filtration. The components may be isolated in a sterile state or alternatively it may be sterilised after isolation, e.g. by gamma irradiation.
[0267] Advantageously, a suspending agent for example polyvinylpyrrolidone is included in the composition/s to facilitate uniform distribution of the components.
Definitions Section
[0268] Targeting Moiety (TM) means any chemical structure associated with an agent that functionally interacts with a Binding Site to cause a physical association between the agent and the surface of a target cell. In the context of the present invention, the target cell is a nociceptive sensory afferent. The term TM embraces any molecule (i.e. a naturally occurring molecule, or a chemically/physically modified variant thereof) that is capable of binding to a Binding Site on the target cell, which Binding Site is capable of internalisation (e.g. endosome formation)--also referred to as receptor-mediated endocytosis. The TM may possess an endosomal membrane translocation function, in which case separate TM and Translocation Domain components need not be present in an agent of the present invention.
[0269] The TM of the present invention binds (preferably specifically binds) to a nociceptive sensory afferent (e.g. a primary nociceptive afferent). In this regard, specifically binds means that the TM binds to a nociceptive sensory afferent (e.g. a primary nociceptive afferent) with a greater affinity than it binds to other neurons such as non-nociceptive afferents, and/or to motor neurons (i.e. the natural target for clostridial neurotoxin holotoxin). The term "specifically binding" can also mean that a given TM binds to a given receptor, for example the ORL1 receptor, with a binding affinity (Ka) of 106 M-1 or greater, preferably 107 M-1 or greater, more preferably 108 M-1 or greater, and most preferably, 109 M-1 or greater.
[0270] For the purposes of this invention, an agonist is defined as a molecule that is capable of stimulating the process of exocytic fusion in a target cell, which process is susceptible to inhibition by a protease (or fragment thereof) capable of cleaving a protein of the exocytic fusion apparatus in said target cell.
[0271] Accordingly, the particular agonist definition of the present invention would exclude many molecules that would be conventionally considered as agonists.
[0272] For example, nerve growth factor (NGF) is an agonist in respect of its ability to promote neuronal differentiation via binding to a TrkA receptor. However, NGF is not an agonist when assessed by the above criteria because it is not a principal inducer of exocytic fusion. In addition, the process that NGF stimulates (i.e. cell differentiation) is not susceptible to inhibition by the protease activity of a non-cytotoxic toxin molecule.
[0273] The term "fragment", when used in relation to a protein, means a peptide having at least thirty-five, preferably at least twenty-five, more preferably at least twenty, and most preferably at least ten amino acid residues of the protein in question.
[0274] The term "variant", when used in relation to a protein, means a peptide or peptide fragment of the protein that contains one or more analogues of an amino acid (e.g. an unnatural amino acid), or a substituted linkage.
[0275] The term "derivative", when used in relation to a protein, means a protein that comprises the protein in question, and a further peptide sequence. The further peptide sequence should preferably not interfere with the basic folding and thus conformational structure of the original protein. Two or more peptides (or fragments, or variants) may be joined together to form a derivative. Alternatively, a peptide (or fragment, or variant) may be joined to an unrelated molecule (e.g. a second, unrelated peptide). Derivatives may be chemically synthesized, but will be typically prepared by recombinant nucleic acid methods. Additional components such as lipid, and/or polysaccharide, and/or polyketide components may be included.
[0276] Throughout this specification, reference to the "ORL1 receptor" embraces all members of the ORL1 receptor family. Members of the ORL1 receptor family typically have a seven transmembrane domain structure and are coupled to G-proteins of the Gi and G0 families. A method for determining the G-protein-stimulating activity of ligands of the ORL1 receptor is given in Example 12. A method for measuring reduction in cellular cAMP levels following ORL1 activation is given in Example 11. A further characteristic of members of the ORL1 receptor family is that they are typically able to bind nociceptin (the natural ligand of ORL1). As an example, all alternative splice variants of the ORL1 receptor, are members of the ORL1 receptor family.
[0277] The term non-cytotoxic means that the protease molecule in question does not kill the target cell to which it has been re-targeted.
[0278] The protease of the present invention embraces all naturally-occurring non-cytotoxic proteases that are capable of cleaving one or more proteins of the exocytic fusion apparatus in eukaryotic cells.
[0279] The protease of the present invention is preferably a bacterial protease (or fragment thereof). More preferably the bacterial protease is selected from the genera Clostridium or Neisseria (e.g. a clostridial L-chain, or a neisserial IgA protease preferably from N. gonorrhoeae).
[0280] The present invention also embraces modified non-cytotoxic proteases, which include amino acid sequences that do not occur in nature and/or synthetic amino acid residues, so long as the modified proteases still demonstrate the above-mentioned protease activity.
[0281] The protease of the present invention preferably demonstrates a serine or metalloprotease activity (e.g. endopeptidase activity). The protease is preferably specific for a SNARE protein (e.g. SNAP-25, synaptobrevin/VAMP, or syntaxin).
[0282] Particular mention is made to the protease domains of neurotoxins, for example the protease domains of bacterial neurotoxins. Thus, the present invention embraces the use of neurotoxin domains, which occur in nature, as well as recombinantly prepared versions of said naturally-occurring neurotoxins.
[0283] Exemplary neurotoxins are produced by clostridia, and the term clostridial neurotoxin embraces neurotoxins produced by C. tetani (TeNT), and by C. botulinum (BoNT) serotypes A-G, as well as the closely related BoNT-like neurotoxins produced by C. baratii and C. butyricum. The above-mentioned abbreviations are used throughout the present specification. For example, the nomenclature BoNT/A denotes the source of neurotoxin as BoNT (serotype A). Corresponding nomenclature applies to other BoNT serotypes.
[0284] The term L-chain fragment means a component of the L-chain of a neurotoxin, which fragment demonstrates a metalloprotease activity and is capable of proteolytically cleaving a vesicle and/or plasma membrane associated protein involved in cellular exocytosis.
[0285] A Translocation Domain is a molecule that enables translocation of a protease (or fragment thereof) into a target cell such that a functional expression of protease activity occurs within the cytosol of the target cell. Whether any molecule (e.g. a protein or peptide) possesses the requisite translocation function of the present invention may be confirmed by any one of a number of conventional assays.
[0286] For example, Shone C. (1987) describes an in vitro assay employing liposomes, which are challenged with a test molecule. Presence of the requisite translocation function is confirmed by release from the liposomes of K.sup.+ and/or labelled NAD, which may be readily monitored [see Shone C. (1987) Eur. J. Biochem; vol. 167(1): pp. 175-180].
[0287] A further example is provided by Blaustein R. (1987), which describes a simple in vitro assay employing planar phospholipid bilayer membranes. The membranes are challenged with a test molecule and the requisite translocation function is confirmed by an increase in conductance across said membranes [see Blaustein (1987) FEBS Letts; vol. 226, no. 1: pp. 115-120].
[0288] Additional methodology to enable assessment of membrane fusion and thus identification of Translocation Domains suitable for use in the present invention are provided by Methods in Enzymology Vol 220 and 221, Membrane Fusion Techniques, Parts A and B, Academic Press 1993.
[0289] The Translocation Domain is preferably capable of formation of ion-permeable pores in lipid membranes under conditions of low pH. Preferably it has been found to use only those portions of the protein molecule capable of pore-formation within the endosomal membrane.
[0290] The Translocation Domain may be obtained from a microbial protein source, in particular from a bacterial or viral protein source. Hence, in one embodiment, the Translocation Domain is a translocating domain of an enzyme, such as a bacterial toxin or viral protein.
[0291] It is well documented that certain domains of bacterial toxin molecules are capable of forming such pores. It is also known that certain translocation domains of virally expressed membrane fusion proteins are capable of forming such pores. Such domains may be employed in the present invention.
[0292] The Translocation Domain may be of a clostridial origin, namely the HN domain (or a functional component thereof). HN means a portion or fragment of the H-chain of a clostridial neurotoxin approximately equivalent to the amino-terminal half of the H-chain, or the domain corresponding to that fragment in the intact H-chain. It is preferred that the H-chain substantially lacks the natural binding function of the HC component of the H-chain. In this regard, the HC function may be removed by deletion of the HC amino acid sequence (either at the DNA synthesis level, or at the post-synthesis level by nuclease or protease treatment). Alternatively, the HC function may be inactivated by chemical or biological treatment. Thus, the H-chain is preferably incapable of binding to the Binding Site on a target cell to which native clostridial neurotoxin (i.e. holotoxin) binds.
[0293] In one embodiment, the translocation domain is a HN domain (or a fragment thereof) of a clostridial neurotoxin. Examples of suitable clostridial Translocation
[0294] Domains include: [0295] Botulinum type A neurotoxin--amino acid residues (449-871) [0296] Botulinum type B neurotoxin--amino acid residues (441-858) [0297] Botulinum type C neurotoxin--amino acid residues (442-866) [0298] Botulinum type D neurotoxin--amino acid residues (446-862) [0299] Botulinum type E neurotoxin--amino acid residues (423-845) [0300] Botulinum type F neurotoxin--amino acid residues (440-864) [0301] Botulinum type G neurotoxin--amino acid residues (442-863) [0302] Tetanus neurotoxin--amino acid residues (458-879)
[0303] For further details on the genetic basis of toxin production in Clostridium botulinum and C. tetani, we refer to Henderson et al (1997) in The Clostridia: Molecular Biology and Pathogenesis, Academic press.
[0304] The term HN embraces naturally-occurring neurotoxin HN portions, and modified HN portions having amino acid sequences that do not occur in nature and/or synthetic amino acid residues, so long as the modified HN portions still demonstrate the above-mentioned translocation function.
[0305] Alternatively, the Translocation Domain may be of a non-clostridial origin (see Table 4). Examples of non-clostridial Translocation Domain origins include, but not be restricted to, the translocation domain of diphtheria toxin [O=Keefe et al., Proc. Natl. Acad. Sci. USA (1992) 89, 6202-6206; Silverman et al., J. Biol. Chem. (1993) 269, 22524-22532; and London, E. (1992) Biochem. Biophys. Acta., 1112, pp. 25-51], the translocation domain of Pseudomonas exotoxin type A [Prior et al. Biochemistry (1992) 31, 3555-3559], the translocation domains of anthrax toxin [Blanke et al. Proc. Natl. Acad. Sci. USA (1996) 93, 8437-8442], a variety of fusogenic or hydrophobic peptides of translocating function [Plank et al. J. Biol. Chem. (1994) 269, 12918-12924; and Wagner et al (1992) PNAS, 89, pp. 7934-7938], and amphiphilic peptides [Murata et al (1992) Biochem., 31, pp. 1986-1992]. The Translocation Domain may mirror the Translocation Domain present in a naturally-occurring protein, or may include amino acid variations so long as the variations do not destroy the translocating ability of the Translocation Domain.
[0306] Particular examples of viral Translocation Domains suitable for use in the present invention include certain translocating domains of virally expressed membrane fusion proteins. For example, Wagner et al. (1992) and Murata et al. (1992) describe the translocation (i.e. membrane fusion and vesiculation) function of a number of fusogenic and amphiphilic peptides derived from the N-terminal region of influenza virus haemagglutinin. Other virally expressed membrane fusion proteins known to have the desired translocating activity are a translocating domain of a fusogenic peptide of Semliki Forest Virus (SFV), a translocating domain of vesicular stomatitis virus (VSV) glycoprotein G, a translocating domain of SER virus F protein and a translocating domain of Foamy virus envelope glycoprotein. Virally encoded Aspike proteins have particular application in the context of the present invention, for example, the E1 protein of SFV and the G protein of the G protein of VSV.
[0307] Use of the Translocation Domains listed in Table (below) includes use of sequence variants thereof. A variant may comprise one or more conservative nucleic acid substitutions and/or nucleic acid deletions or insertions, with the proviso that the variant possesses the requisite translocating function. A variant may also comprise one or more amino acid substitutions and/or amino acid deletions or insertions, so long as the variant possesses the requisite translocating function.
TABLE-US-00006 Translocation Amino acid domain source residues References Diphtheria 194-380 Silverman et al., 1994, J. Biol. toxin Chem. 269, 22524-22532 London E., 1992, Biochem. Biophys. Acta., 1113, 25-51 Domain II of 405-613 Prior et al., 1992, Biochemistry pseudomonas 31, 3555-3559 exotoxin Kihara & Pastan, 1994, Bioconj Chem. 5, 532-538 Influenza virus GLFGAIAGFIENGWE Plank et al., 1994, J. Biol. Chem. haemagglutinin GMIDGWYG, and 269, 12918-12924 Variants thereof Wagner et al., 1992, PNAS, 89, 7934-7938 Murata et al., 1992, Biochemistry 31, 1986-1992 Semliki Forest Translocation Kielian et al., 1996, J Cell Biol. virus fusogenic domain 134(4), 863-872 protein Vesicular 118-139 Yao et al., 2003, Virology 310(2), Stomatitis virus 319-332 glycoprotein G SER virus F Translocation Seth et al., 2003, J Virol 77(11) protein domain 6520-6527 Foamy virus Translocation Picard-Maureau et al., 2003, J envelope domain Virol. 77(8), 4722-4730 glycoprotein
SEQ ID NOs
[0308] Where an initial Met amino acid residue or a corresponding initial codon is indicated in any of the following SEQ ID NOs, said residue/codon is optional.
SEQ ID1 DNA sequence of the LC/A SEQ ID2 DNA sequence of the HN/A SEQ ID3 DNA sequence of the LC/B SEQ ID4 DNA sequence of the HN/B SEQ ID5 DNA sequence of the LC/C SEQ ID6 DNA sequence of the HN/C SEQ ID7 DNA sequence of the CPN-A linker SEQ ID8 DNA sequence of the A linker SEQ ID9 DNA sequence of the N-terminal presentation nociceptin insert SEQ ID10 DNA sequence of the CPN-C linker SEQ ID11 DNA sequence of the CPBE-A linker SEQ ID12 DNA sequence of the CPNvar-A linker SEQ ID13 DNA sequence of the LC/A-CPN-HN/A fusion SEQ ID14 Protein sequence of the LC/A-CPN-HN/A fusion SEQ ID15 DNA sequence of the N-LC/A-HN/A fusion SEQ ID16 Protein sequence of the N-LC/A-HN/A fusion SEQ ID17 DNA sequence of the LC/C-CPN-HN/C fusion SEQ ID18 Protein sequence of the LC/C-CPN-HN/C fusion SEQ ID19 DNA sequence of the LC/C-CPN-HN/C (A-linker) fusion SEQ ID20 Protein sequence of the LC/C-CPN-HN/C (A-linker) fusion SEQ ID21 DNA sequence of the LC/A-CPME-HN/A fusion SEQ ID22 Protein sequence of the LC/A-CPME-HN/A fusion SEQ ID23 DNA sequence of the LC/A-CPBE-HN/A fusion SEQ ID24 Protein sequence of the LC/A-CPBE-HN/A fusion SEQ ID25 DNA sequence of the LC/A-CPNv-HN/A fusion SEQ ID26 Protein sequence of the LC/A-CPNv-HN/A fusion SEQ ID27 DNA sequence of the LC/A-CPN[1-11]-HN/A fusion SEQ ID28 Protein sequence of the LC/A-CPN[1-11]-HN/A fusion SEQ ID29 DNA sequence of the LC/A-CPN[[Y10]1-11]-HN/A fusion SEQ ID30 Protein sequence of the LC/A-CPN[[Y10]1-11]-HN/A fusion SEQ ID31 DNA sequence of the LC/A-CPN[[Y11]1-11]-HN/A fusion SEQ ID32 Protein sequence of the LC/A-CPN[[Y11]1-11]-HN/A fusion SEQ ID33 DNA sequence of the LC/A-CPN[[Y14]1-17]-HN/A fusion SEQ ID34 Protein sequence of the LC/A-CPN[[Y14]1-17]-HN/A fusion SEQ ID35 DNA sequence of the LC/A-CPN[1-13]-HN/A fusion SEQ ID36 Protein sequence of the LC/A-CPN[1-13]-HN/A fusion SEQ ID37 DNA sequence of CPN[1-17]
SEQ ID38 Protein Sequence of CPN[1-17]
[0309] SEQ ID39 DNA sequence of CPN[1-11] SEQ ID40 Protein sequence of CPN[1-11] SEQ ID41 DNA sequence of CPN[[Y10]1-11] SEQ ID42 Protein sequence of CPN[[Y10]1-11] SEQ ID43 DNA sequence of CPN[[Y11]1-11] SEQ ID44 Protein sequence of CPN[[Y11]1-11] SEQ ID45 DNA sequence of CPN[[Y14]1-17] SEQ ID46 Protein sequence of CPN[[Y14]1-17] SEQ ID47 DNA sequence of CPN[1-13] SEQ ID48 Protein sequence of CPN[1-13] SEQ ID49 DNA sequence of CPNv (also known as N[[R14K15]1-17]) SEQ ID50 Protein sequence of CPNv (also known as N[[R14K15]1-17]) SEQ ID51 DNA sequence of the nociceptin-spacer-LC/A-HN/A fusion SEQ ID52 Protein sequence of the nociceptin-spacer-LC/A-HN/A fusion SEQ ID53 DNA sequence of the CPN-A GS10 linker SEQ ID54 DNA sequence of the CPN-A GS15 linker SEQ ID55 DNA sequence of the CPN-A GS25 linker SEQ ID56 DNA sequence of the CPN-A GS30 linker SEQ ID57 DNA sequence of the CPN-A HX27 linker SEQ ID58 DNA sequence of the LC/A-CPN(GS15)-HN/A fusion SEQ ID59 Protein sequence of the LC/A-CPN(GS15)-HN/A fusion SEQ ID60 DNA sequence of the LC/A-CPN(GS25)-HN/A fusion SEQ ID61 Protein sequence of the LC/A-CPN(GS25)-HN/A fusion SEQ ID62 DNA sequence of the CPNvar-A Enterokinase activatable linker SEQ ID63 DNA sequence of the LC/A-CPNv(Ek)-HN/A fusion SEQ ID64 Protein sequence of the LC/A-CPNv(Ek)-HN/A fusion SEQ ID65 DNA sequence of the CPNvar-A linker SEQ ID66 DNA sequence of the LC/C-CPNv-HN/C fusion (act. A) SEQ ID67 Protein sequence of the LC/C-CPNv-HN/C fusion (act. A) SEQ ID68 DNA sequence of the LC/A-CPLE-HN/A fusion SEQ ID69 Protein sequence of the LC/A-CPLE-HN/A fusion SEQ ID70 DNA sequence of the LC/A-CPOP-HN/A fusion SEQ ID71 Protein sequence of the LC/A-CPOP-HN/A fusion SEQ ID72 DNA sequence of the LC/A-CPOPv-HN/A fusion SEQ ID73 Protein sequence of the LC/A-CPOPv-HN/A fusion SEQ ID74 DNA sequence of the IgA protease SEQ ID75 DNA sequence of the IgA-CPNv-HN/A fusion SEQ ID76 Protein sequence of the IgA-CPNv-HN/A fusion SEQ ID77 DNA sequence of the FXa-HT SEQ ID78 DNA sequence of the CPNv-A-FXa-HT SEQ ID79 Protein sequence of the CPNv-A-FXa-HT fusion SEQ ID80 DNA sequence of the DT translocation domain SEQ ID81 DNA sequence of the CPLE-DT-A SEQ ID82 Protein sequence of the CPLE-DT-A fusion SEQ ID83 DNA sequence of the TeNT LC SEQ ID84 DNA sequence of the CPNv-TENT LC SEQ ID85 Protein sequence of the CPNV-TeNT LC fusion SEQ ID86 DNA sequence of the CPNvar-C linker SEQ ID87 DNA sequence of the LC/C-CPNv-HN/C fusion (act. C) SEQ ID88 Protein sequence of the LC/C-CPNv-HN/C fusion (act. C) SEQ ID89 Protein sequence of dynorphin SEQ ID90 DNA sequence of LC/A-CPDY-HN/A fusion SEQ ID91 Protein sequence of LC/A-CPDY-HN/A fusion SEQ ID92 Protein sequence of LC/A-CPDY(GS10)-HN/A fusion SEQ ID93 Protein sequence of LC/A-CPDY(GS15)-HN/A fusion SEQ ID94 Protein sequence of LC/A-CPDY(GS25)-HN/A fusion SEQ ID95 Protein sequence of LC/C-CPDY-HN/C fusion SEQ ID96 Protein sequence of IgA-CPDY-HN/A fusion SEQ ID97 Protein sequence of CPDY-TeNT LC fusion
EXAMPLES
Example 1
Preparation of a LC/A and HN/A Backbone Clones
[0310] The following procedure creates the LC and HN fragments for use as the component backbone for multidomain fusion expression. This example is based on preparation of a serotype A based clone (SEQ ID1 and SEQ ID2), though the procedures and methods are equally applicable to the other serotypes [illustrated by the sequence listing for serotype B (SEQ ID3 and SEQ ID4) and serotype C (SEQ ID5 and SEQ ID6)].
Preparation of Cloning and Expression Vectors
[0311] pCR 4 (Invitrogen) is the chosen standard cloning vector, selected due to the lack of restriction sequences within the vector and adjacent sequencing primer sites for easy construct confirmation. The expression vector is based on the pMAL (NEB) expression vector, which has the desired restriction sequences within the multiple cloning site in the correct orientation for construct insertion (BamHI-SalI-PstI-HindIII). A fragment of the expression vector has been removed to create a non-mobilisable plasmid and a variety of different fusion tags have been inserted to increase purification options.
Preparation of Protease (e.g. LC/A) Insert
[0312] The LC/A (SEQ ID1) is created by one of two ways:
[0313] The DNA sequence is designed by back translation of the LC/A amino acid sequence [obtained from freely available database sources such as GenBank (accession number P10845) or Swissprot (accession locus BXA1_CLOBO) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)]. BamHI/SalI recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence, maintaining the correct reading frame. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 Sep. 2004). This optimised DNA sequence containing the LC/A open reading frame (ORF) is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
[0314] The alternative method is to use PCR amplification from an existing DNA sequence with BamHI and SalI restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. Complementary oligonucleotide primers are chemically synthesised by a supplier (for example MWG or Sigma-Genosys), so that each pair has the ability to hybridize to the opposite strands (3' ends pointing "towards" each other) flanking the stretch of Clostridium target DNA, one oligonucleotide for each of the two DNA strands. To generate a PCR product the pair of short oligonucleotide primers specific for the Clostridium DNA sequence are mixed with the Clostridium DNA template and other reaction components and placed in a machine (the `PCR machine`) that can change the incubation temperature of the reaction tube automatically, cycling between approximately 94° C. (for denaturation), 55° C. (for oligonucleotide annealing), and 72° C. (for synthesis). Other reagents required for amplification of a PCR product include a DNA polymerase (such as Taq or Pfu polymerase), each of the four nucleotide dNTP building blocks of DNA in equimolar amounts (50-200 μM) and a buffer appropriate for the enzyme optimised for Mg2+ concentration (0.5-5 mM).
[0315] The amplification product is cloned into pCR 4 using either, TOPO TA cloning for Taq PCR products or Zero Blunt TOPO cloning for Pfu PCR products (both kits commercially available from Invitrogen). The resultant clone is checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis [for example, using Quickchange (Stratagene Inc.)].
Preparation of Translocation (e.g. HN) Insert
[0316] The HN/A (SEQ ID2) is created by one of two ways:
[0317] The DNA sequence is designed by back translation of the HN/A amino acid sequence [obtained from freely available database sources such as GenBank (accession number P10845) or Swissprot (accession locus BXA1_CLOBO)] using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)]. A PstI restriction sequence added to the N-terminus and XbaI-stop codon-HindIII to the C-terminus ensuring the correct reading frame is maintained. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 Sep. 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
[0318] The alternative method is to use PCR amplification from an existing DNA sequence with PstI and XbaI-stop codon-HindIII restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. The PCR amplification is performed as described above. The PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis [for example using Quickchange (Stratagene Inc.)].
Example 2
Preparation of a LC/A-nociceptin-HN/A Fusion Protein (Nociceptin is N-Terminal of the HN-Chain)
Preparation of Linker-nociceptin-Spacer Insert
[0319] The LC-HN linker can be designed from first principle, using the existing sequence information for the linker as the template. For example, the serotype A linker (in this case defined as the inter-domain polypeptide region that exists between the cysteines of the disulphide bridge between LC and HN) is 23 amino acids long and has the sequence VRGIITSKTKSLDKGYNKALNDL. Within this sequence, it is understood that proteolytic activation in nature leads to an HN domain that has an N-terminus of the sequence ALNDL. This sequence information is freely available from available database sources such as GenBank (accession number P10845) or Swissprot (accession locus BXA1_CLOBO). Into this linker a Factor Xa site, nociceptin and spacer are incorporated; and using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)], the DNA sequence encoding the linker-ligand-spacer region is determined. Restriction sites are then incorporated into the DNA sequence and can be arranged as BamHI-SalI-linker-protease site-nociceptin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID7). It is important to ensure the correct reading frame is maintained for the spacer, nociceptin and restriction sequences and that the XbaI sequence is not preceded by the bases, TC, which would result on DAM methylation. The DNA sequence is screened for restriction sequence incorporation, and any additional sequences are removed manually from the remaining sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example, GenBank Release 143, 13 Sep. 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
Preparation of the LC/A-nociceptin-HN/A Fusion
[0320] In order to create the LC-linker-nociceptin-spacer-HN construct (SEQ ID13), the pCR 4 vector encoding the linker (SEQ ID7) is cleaved with BamHI+SalI restriction enzymes. This cleaved vector then serves as the recipient vector for insertion and ligation of the LC/A DNA (SEQ ID1) cleaved with BamHI+SalI. The resulting plasmid DNA is then cleaved with PstI+XbaI restriction enzymes and serves as the recipient vector for the insertion and ligation of the HN/A DNA (SEQ ID2) cleaved with PstI+XbaI. The final construct contains the LC-linker-nociceptin-spacer-HN ORF (SEQ ID13) for transfer into expression vectors for expression to result in a fusion protein of the sequence illustrated in SEQ ID14.
Example 3
Preparation of a Nociceptin-LC/A-HN/A Fusion Protein (Nociceptin is N-Terminal of the LC-Chain)
[0321] The LC/A-HN/A backbone is constructed as described in Example 2 using the synthesised A serotype linker with the addition of a Factor Xa site for activation, arranged as BamHI-SalI-linker-protease site-linker-PstI-XbaI-stop codon-HindIII (SEQ ID8). The LC/A-HN/A backbone and the synthesised N-terminal presentation nociceptin insert (SEQ ID9) are cleaved with BamHI+HindIII restriction enzymes, gel purified and ligated together to create a nociceptin-spacer-LC-linker-HN. The ORF (SEQ ID15) is then cut out using restriction enzymes AvaI+XbaI for transfer into expression vectors for expression to result in a fusion protein of the sequence illustrated in SEQ ID16.
Example 4
Preparation of a LC/C-nociceptin-HN/C Fusion Protein
[0322] Following the methods used in Examples 1 and 2, the LC/C (SEQ ID5) and HN/C (SEQ ID6) are created and inserted into the C serotype linker arranged as BamHI-SalI-linker-protease site-nociceptin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID10). The final construct contains the LC-linker-nociceptin-spacer-HN ORF (SEQ ID17) for expression as a protein of the sequence illustrated in SEQ ID18.
Example 5
Preparation of a LC/C-nociceptin-HN/C Fusion Protein with a Serotype A Activation Sequence
[0323] Following the methods used in Examples 1 and 2, the LC/C (SEQ ID5) and HN/C (SEQ ID6) are created and inserted into the A serotype linker arranged as BamHI-SalI-linker-protease site-nociceptin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID7). The final construct contains the LC-linker-nociceptin-spacer-HN ORF (SEQ ID19) for expression as a protein of the sequence illustrated in SEQ ID20.
Example 6
Preparation of a LC/A-met Enkephalin-HN/A Fusion Protein
[0324] Due to the small, five-amino acid, size of the met-enkephalin ligand the LC/A-met enkephalin-HN/A fusion is created by site directed mutagenesis [for example using Quickchange (Stratagene Inc.)] using the LC/A-nociceptin-HN/A fusion (SEQ ID13) as a template. Oligonucleotides are designed encoding the YGGFM met-enkephalin peptide, ensuring standard E. coli codon usage is maintained and no additional restriction sites are incorporated, flanked by sequences complimentary to the linker region of the LC/A-nociceptin-HN/A fusion (SEQ ID13) either side on the nociceptin section. The SDM product is checked by sequencing and the final construct containing the LC-linker-met enkephalin-spacer-HN ORF (SEQ ID21) for expression as a protein of the sequence illustrated in SEQ ID22.
Example 7
Preparation of a LC/A-β Endorphin-HN/A Fusion Protein
[0325] Following the methods used in Examples 1 and 2, the LC/A (SEQ ID1) and HN/A (SEQ ID2) are created and inserted into the A serotype β endorphin linker arranged as BamHI-SalI-linker-protease site-β endorphin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID11). The final construct contains the LC-linker-β endorphin-spacer-HN ORF (SEQ ID23) for expression as a protein of the sequence illustrated in SEQ ID24.
Example 8
Preparation of a LC/A-nociceptin Variant-HN/A Fusion Protein
[0326] Following the methods used in Examples 1 and 2, the LC/A (SEQ ID1) and HN/A (SEQ ID2) are created and inserted into the A serotype nociceptin variant linker arranged as BamHI-SalI-linker-protease site-nociceptin variant-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID12). The final construct contains the LC-linker-nociceptin variant-spacer-HN ORF (SEQ ID25) for expression as a protein of the sequence illustrated in SEQ ID26.
Example 9
Purification Method for LC/A-nociceptin-HN/A Fusion Protein
[0327] Defrost falcon tube containing 25 ml 50 mM HEPES pH 7.2, 200 mM NaCl and approximately 10 g of E. coli BL21 cell paste. Make the thawed cell paste up to 80 ml with 50 mM HEPES pH 7.2, 200 mM NaCl and sonicate on ice 30 seconds on, 30 seconds off for 10 cycles at a power of 22 microns ensuring the sample remains cool. Spin the lysed cells at 18 000 rpm, 4° C. for 30 minutes. Load the supernatant onto a 0.1 M NiSO4 charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2, 200 mM NaCl. Using a step gradient of 10 and 40 mM imidazol, wash away the non-specific bound protein and elute the fusion protein with 100 mM imidazol. Dialyse the eluted fusion protein against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4° C. overnight and measure the OD of the dialysed fusion protein. Add 1 unit of factor Xa per 100 μg fusion protein and Incubate at 25° C. static overnight. Load onto a 0.1 M NiSO4 charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2, 200 mM NaCl. Wash column to baseline with 50 mM HEPES pH 7.2, 200 mM NaCl. Using a step gradient of 10 and 40 mM imidazol, wash away the non-specific bound protein and elute the fusion protein with 100 mM imidazol. Dialyse the eluted fusion protein against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4° C. overnight and concentrate the fusion to about 2 mg/ml, aliquot sample and freeze at -20° C. Test purified protein using OD, BCA, purity analysis and SNAP-25 assessments.
Example 10
Confirmation of TM Agonist Activity by Measuring Release of Substance P from Neuronal Cell Cultures
Materials
[0328] Substance P EIA is obtained from R&D Systems, UK.
Methods
[0329] Primary neuronal cultures of eDRG are established as described previously (Duggan et al., 2002). Substance P release from the cultures is assessed by EIA, essentially as described previously (Duggan et al., 2002). The TM of interest is added to the neuronal cultures (established for at least 2 weeks prior to treatment); control cultures are performed in parallel by addition of vehicle in place of TM. Stimulated (100 mM KCl) and basal release, together with total cell lysate content, of substance P are obtained for both control and TM treated cultures. Substance P immunoreactivity is measured using Substance P Enzyme Immunoassay Kits (Cayman Chemical Company, USA or R&D Systems, UK) according to manufacturers' instructions.
[0330] The amount of Substance P released by the neuronal cells in the presence of the TM of interest is compared to the release obtained in the presence and absence of 100 mM KCl. Stimulation of Substance P release by the TM of interest above the basal release, establishes that the TM of interest is an "agonist ligand" as defined in this specification. If desired the stimulation of Substance P release by the TM of interest can be compared to a standard Substance P release-curve produced using the natural ORL-1 receptor ligand, nociceptin (Tocris).
Example 11
Confirmation of ORL1 Receptor Activation by Measuring Forskolin-Stimulated cAMP Production
[0331] Confirmation that a given TM is acting via the ORL1 receptor is provided by the following test, in which the TMs ability to inhibit forskolin-stimulated cAMP production is assessed.
Materials
[0332] [3H]adenine and [14C]cAMP are obtained from GE Healthcare
Methods
[0333] The test is conducted essentially as described previously by Meunier et al. [Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature 377: 532-535, 1995] in intact transfected-CHO cells plated on 24-well plastic plates.
[0334] To the cells is added [3H]adenine (1.0 μCi) in 0.4 ml of culture medium. The cells remain at 37° C. for 2 h to allow the adenine to incorporate into the intracellular ATP pool. After 2 h, the cells are washed once with incubation buffer containing: 130 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.3 mM CaCl2, 1.2 mM MgSO4, 10 mM glucose, 1 mg/ml bovine serum albumin and 25 mM HEPES pH 7.4, and replaced with buffer containing forskolin (10 μM) and isobutylmethylxanthine (50 μM) with or without the TM of interest. After 10 min, the medium is aspirated and replaced with 0.5 ml, 0.2 M HCl. Approximately 1000 cpm of [14C]cAMP is added to each well and used as an internal standard. The contents of the wells are then transferred to columns of 0.65 g dry alumina powder. The columns are eluted with 4 ml of 5 mM HCl, 0.5 ml of 0.1 M ammonium acetate, then two additional millilitres of ammonium acetate. The final eluate is collected into scintillation vials and counted for 14C and tritium. Amounts collected are corrected for recovery of [14C]cAMP. TMs that are agonists at the ORL1 receptor cause a reduction in the level of cAMP produced in response to forskolin.
Example 12
Confirmation of ORL1 Receptor Activation Using a GTPγS Binding Functional Assay
[0335] Confirmation that a given TM is acting via the ORL1 receptor is also provided by the following test, a GTPγS binding functional assay.
Materials
[0336] [35S]GTPγS is obtained from GE Healthcare Wheatgerm agglutinin-coated (SPA) beads are obtained from GE Healthcare
Methods
[0337] This assay is carried out essentially as described by Traynor and Nahorski [Modulation by μ-opioid agonists of guanosine-5-O-(3-[35S]thio)triphosphate binding to membranes from human neuroblastoma SH-SY5Y cells. Mol. Pharmacol. 47: 848-854, 1995].
[0338] Cells are scraped from tissue culture dishes into 20 mM HEPES, 1 mM ethylenediaminetetraacetic acid, then centrifuged at 500×g for 10 min. Cells are resuspended in this buffer and homogenized with a Polytron Homogenizer.
[0339] The homogenate is centrifuged at 27,000×g for 15 min, and the pellet resuspended in buffer A, containing: 20 mM HEPES, 10 mM MgCl2, 100 mM NaCl, pH 7.4. The suspension is recentrifuged at 20,000×g and suspended once more in buffer A. For the binding assay, membranes (8-15 μg protein) are incubated with [35S]GTP S (50 μM), GDP (10 μM), with and without the TM of interest, in a total volume of 1.0 ml, for 60 min at 25° C. Samples are filtered over glass fibre filters and counted as described for the binding assays.
Example 13
Preparation of a LC/A-nociceptin-HN/A Fusion Protein (Nociceptin is N-Terminal of the HN-Chain)
[0340] The linker-nociceptin-spacer insert is prepared as described in Example 2.
Preparation of the LC/A-nociceptin-HN/A Fusion
[0341] In order to create the LC-linker-nociceptin-spacer-HN construct (SEQ ID13), the pCR 4 vector encoding the linker (SEQ ID7) is cleaved with BamHI+SalI restriction enzymes. This cleaved vector then serves as the recipient for insertion and ligation of the LC/A DNA (SEQ ID1) also cleaved with BamHI+SalI. The resulting plasmid DNA is then cleaved with BamHI+HindIII restriction enzymes and the LC/A-linker fragment inserted into a similarly cleaved vector containing a unique multiple cloning site for BamHI, SalI, PstI, and HindIII such as the pMAL vector (NEB). The HN/A DNA (SEQ ID2) is then cleaved with PstI+HindIII restriction enzymes and inserted into the similarly cleaved pMAL-LC/A-linker construct. The final construct contains the LC-linker-nociceptin-spacer-HN ORF (SEQ ID13) for expression as a protein of the sequence illustrated in SEQ ID14.
Example 14
Preparation of a Nociceptin-LC/A-HN/A Fusion Protein (Nociceptin is N-Terminal of the LC-Chain)
[0342] In order to create the nociceptin-spacer-LC/A-HN/A construct, an A serotype linker with the addition of a Factor Xa site for activation, arranged as BamHI-SalI-linker-protease site-linker-PstI-XbaI-stop codon-HindIII (SEQ ID8) is synthesised as described in Example 13. The pCR 4 vector encoding the linker is cleaved with BamHI+SalI restriction enzymes. This cleaved vector then serves as the recipient for insertion and ligation of the LC/A DNA (SEQ ID1) also cleaved with BamHI+SalI. The resulting plasmid DNA is then cleaved with BamHI+HindIII restriction enzymes and the LC/A-linker fragment inserted into a similarly cleaved vector containing the synthesised N-terminal presentation nociceptin insert (SEQ ID9). This construct is then cleaved with AvaI+HindIII and inserted into an expression vector such as the pMAL plasmid (NEB). The HN/A DNA (SEQ ID2) is then cleaved with PstI+HindIII restriction enzymes and inserted into the similarly cleaved pMAL-nociceptin-LC/A-linker construct. The final construct contains the nociceptin-spacer-LC/A-HN/A ORF (SEQ ID51) for expression as a protein of the sequence illustrated in SEQ ID52.
Example 15
Preparation and Purification of an LC/A-nociceptin-HN/A Fusion Protein Family with Variable Spacer Length
[0343] Using the same strategy as employed in Example 2, a range of DNA linkers were prepared that encoded nociceptin and variable spacer content. Using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)], the DNA sequence encoding the linker-ligand-spacer region is determined. Restriction sites are then incorporated into the DNA sequence and can be arranged as BamHI-SalI-linker-protease site-nociceptin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID53 to SEQ ID57). It is important to ensure the correct reading frame is maintained for the spacer, nociceptin and restriction sequences and that the XbaI sequence is not preceded by the bases, TC which would result on DAM methylation. The DNA sequence is screened for restriction sequence incorporation and any additional sequences are removed manually from the remaining sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 Sep. 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
[0344] The spacers that were created included:
TABLE-US-00007 TABLE 1 SEQ ID of the Code Protein sequence of the linker linker DNA GS10 ALAGGGGSALVLQ 53 GS15 ALAGGGGSGGGGSALVLQ 54 GS25 ALAGGGGSGGGGSGGGGSGGGGSALVLQ 55 GS30 ALAGGGGSGGGGSGGGGSGGGGSGGGGSALVLQ 56 HX27 ALAAEAAAKEAAAKEAAAKAGGGGSALVLQ 57
[0345] By way of example, in order to create the LC/A-CPN(GS15)-HN/A fusion construct (SEQ ID58), the pCR 4 vector encoding the linker (SEQ ID54) is cleaved with BamHI+SalI restriction enzymes. This cleaved vector then serves as the recipient vector for insertion and ligation of the LC/A DNA (SEQ ID1) also cleaved with BamHI+SalI. The resulting plasmid DNA is then cleaved with BamHI+HindIII restriction enzymes and the LC/A-linker fragment inserted into a similarly cleaved vector containing a unique multiple cloning site for BamHI, SalI, PstI, and HindIII such as the pMAL vector (NEB). The HN/A DNA (SEQ ID2) is then cleaved with PstI+HindIII restriction enzymes and inserted into the similarly cleaved pMAL-LC/A-linker construct. The final construct contains the LC/A-CPN(GS15)-HN/A ORF (SEQ ID58) for expression as a protein of the sequence illustrated in SEQ ID59.
[0346] As a further example, to create the LC/A-CPN(GS25)-HN/A fusion construct (SEQ ID60), the pCR 4 vector encoding the linker (SEQ ID55) is cleaved with BamHI+SalI restriction enzymes. This cleaved vector then serves as the recipient vector for insertion and ligation of the LC/A DNA (SEQ ID1) cleaved with BamHI+SalI. The resulting plasmid DNA is then cleaved with BamHI+HindIII restriction enzymes and the LC/A-linker fragment inserted into a similarly cleaved vector containing a unique multiple cloning site for BamHI, SalI, PstI, and HindIII such as the pMAL vector (NEB). The HN/A DNA (SEQ ID2) is then cleaved with PstI+HindIII restriction enzymes and inserted into the similarly cleaved pMAL-LC/A-linker construct. The final construct contains the LC/A-CPN(GS25)-HN/A ORF (SEQ ID60) for expression as a protein of the sequence illustrated in SEQ ID61.
[0347] Variants of the LC/A-CPN-HN/A fusion consisting of GS10, GS30 and HX27 are similarly created. Using the purification methodology described in Example 9, fusion protein is purified from E. coli cell paste. FIG. 9 illustrates the purified product obtained in the case of LC/A-CPN(GS10)-HN/A, LC/A-CPN(GS15)-HN/A, LC/A-CPN(GS25)-HN/A, LC/A-CPN(GS30)-HN/A and LC/A-CPN(HX27)-HN/A.
Example 16
Assessment of in Vitro Efficacy of an LC/A-nociceptin-HN/A Fusion
[0348] Fusion protein prepared according to Examples 2 and 9 was assessed in the eDRG neuronal cell model.
[0349] Assays for the inhibition of substance P release and cleavage of SNAP-25 have been previously reported (Duggan et al., 2002, J. Biol. Chem., 277, 34846-34852). Briefly, dorsal root ganglia neurons are harvested from 15-day-old fetal Sprague-Dawley rats and dissociated cells plated onto 24-well plates coated with Matrigel at a density of 1×106 cells/well. One day post-plating the cells are treated with 10 μM cytosine β-D-arabinofuranoside for 48 h. Cells are maintained in Dulbecco's minimal essential medium supplemented with 5% heat-inactivated fetal bovine serum, 5 mM L-glutamine, 0.6% D-glucose, 2% B27 supplement, and 100 ng/ml 2.5 S mouse nerve growth factor. Cultures are maintained for 2 weeks at 37° C. in 95% air/5% CO2 before addition of test materials.
[0350] Release of substance P from eDRG is assessed by enzyme-linked immunosorbent assay. Briefly, eDRG cells are washed twice with low potassium-balanced salt solution (BSS: 5 mM KCl, 137 mM NaCl, 1.2 mM MgCl2, 5 mM glucose, 0.44 mM KH2PO4, 20 mM HEPES, pH 7.4, 2 mM CaCl2). Basal samples are obtained by incubating each well for 5 min. with 1 ml of low potassium BSS. After removal of this buffer, the cells are stimulated to release by incubation with 1 ml of high potassium buffer (BSS as above with modification to include 100 mM KCl isotonically balanced with NaCl) for 5 min. All samples are removed to tubes on ice prior to assay of substance P. Total cell lysates are prepared by addition of 250 μl of 2 M acetic acid/0.1% trifluoroacetic acid to lyse the cells, centrifugal evaporation, and resuspension in 500 μl of assay buffer. Diluted samples are assessed for substance P content. Substance P immunoreactivity is measured using Substance P Enzyme Immunoassay Kits (Cayman Chemical Company or R&D Systems) according to manufacturers' instructions. Substance P is expressed in pg/ml relative to a standard substance P curve run in parallel.
[0351] SDS-PAGE and Western blot analysis were performed using standard protocols (Novex). SNAP-25 proteins were resolved on a 12% Tris/glycine polyacrylamide gel (Novex) and subsequently transferred to nitrocellulose membrane. The membranes were probed with a monoclonal antibody (SMI-81) that recognises cleaved and intact SNAP-25. Specific binding was visualised using peroxidase-conjugated secondary antibodies and a chemiluminescent detection system. Cleavage of SNAP-25 was quantified by scanning densitometry (Molecular Dynamics Personal SI, ImageQuant data analysis software). Percent SNAP-25 cleavage was calculated according to the formula: (Cleaved SNAP-25/(Cleaved+Intact SNAP-25))×100.
[0352] Following exposure of eDRG neurons to an LC/A-nociceptin-HN/A fusion (termed CPN-A), both inhibition of substance P release and cleavage of SNAP-25 are observed (FIG. 10). After 24 h exposure to the fusion, 50% of maximal SNAP-25 cleavage is achieved by a fusion concentration of 6.3±2.5 nM.
[0353] The effect of the fusion is also assessed at defined time points following a 16 h exposure of eDRG to CPN-A. FIG. 11 illustrates the prolonged duration of action of the CPN-A fusion protein, with measurable activity still being observed at 28 days post exposure.
Example 17
Assessment of in Vitro Efficacy of an LC/A-nociceptin Variant-HN/A Fusion
[0354] Fusion protein prepared according to Examples 8 and 9 was assessed in the eDRG neuronal cell mode using the method described in Example 16.
[0355] Following exposure of eDRG neurons to an LC/A-nociceptin variant-HN/A fusion (termed CPNv-A), both inhibition of substance P release and cleavage of SNAP-25 are observed. After 24 h exposure to the fusion, 50% of maximal SNAP-25 cleavage is achieved by a fusion concentration of 1.4±0.4 nM (FIG. 12).
[0356] The effect of the fusion is also assessed at defined time points following a 16 h exposure of eDRG to CPN-A. FIG. 13 illustrates the prolonged duration of action of the CPN-A fusion protein, with measurable activity still being observed at 24 days post exposure.
[0357] The binding capability of the CPNv-A fusion protein is also assessed in comparison to the CPN-A fusion. FIG. 14 illustrates the results of a competition experiment to determine binding efficacy at the ORL-1 receptor. CPNv-A is demonstrated to displace [3H]-nociceptin, thereby confirming that access to the receptor is possible with the ligand in the central presentation format.
Example 18
Preparation of an LC/A-nociceptin Variant-HN/A Fusion Protein that is Activated by Treatment with Enterokinase
[0358] Following the methods used in Examples 1 and 2, the LC/A (SEQ ID1) and HN/A (SEQ ID2) are created and inserted into the A serotype nociceptin variant linker arranged as BamHI-SalI-linker-enterokinase protease site-nociceptin variant-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID62). The final construct contains the LC-linker-nociceptin variant-spacer-HN ORF sequences (SEQ ID63) for expression as a protein of the sequence illustrated in SEQ ID64. The fusion protein is termed CPNv(Ek)-A. FIG. 15 illustrates the purification of CPNv(Ek)-A from E. coli following the methods used in Example 9 but using Enterokinase for activation at 0.00064 μg per 100 μg of fusion protein.
Example 19
Assessment of in Vitro Efficacy of a LC/A-nociceptin Variant-HN/A Fusion that has been Activated by Treatment with Enterokinase
[0359] The CPNv(Ek)-A prepared in Example 18 is obtained in a purified form and applied to the eDRG cell model to assess cleavage of SNAP-25 (using methodology from Example 16). FIG. 16 illustrates the cleavage of SNAP-25 following 24 h exposure of eDRG to CPNv(Ek)-A. The efficiency of cleavage is observed to be similar to that achieved with the Factor Xa-cleaved material, as recorded in Example 17.
Example 20
Preparation of an LC/C-nociceptin Variant-HN/C Fusion Protein with a Factor Xa Activation Linker Derived from Serotype A
[0360] Following the methods used in Example 4, the LC/C (SEQ ID5) and HN/C (SEQ ID6) are created and inserted into the A serotype nociceptin variant linker arranged as BamHI-SalI-linker-nociceptin variant-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID65). The final construct contains the LC-linker-nociceptin variant-spacer-HN ORF sequences (SEQ ID66) for expression as a protein of the sequence illustrated in SEQ ID67. The fusion protein is termed CPNv-C (act. A). FIG. 17 illustrates the purification of CPNv-C (act. A) from E. coli following the methods used in Example 9.
Example 21
Assessment of in Vitro Efficacy of an LC/C-nociceptin Variant-HN/C Fusion Protein
[0361] Following the methods used in Example 9, the CPNv-C (act. A) prepared in Example 20 is obtained in a purified form and applied to the eDRG cell model to assess cleavage of SNAP-25 (using methodology from Example 16). After 24 h exposure to the fusion, 50% of maximal syntaxin cleavage is achieved by a fusion concentration of 3.1±2.0 nM. FIG. 18 illustrates the cleavage of syntaxin following 24 h exposure of eDRG to CPNv-C (act. A).
Example 22
Assessment of in Vivo Efficacy of an LC/A-nociceptin-HN/A Fusion
[0362] The ability of an LC/A-nociceptin-HN/A fusion (CPN/A) to inhibit acute capsaicin-induced mechanical allodynia is evaluated following subcutaneous intraplantar injection in the rat hind paw. Test animals are evaluated for paw withdrawal frequency (PWF %) in response to a 10 g Von Frey filament stimulus series (10 stimuli×3 trials) prior to recruitment into the study, after subcutaneous treatment with CPN/A but before capsaicin, and following capsaicin challenge post-injection of CPN/A (average of responses at 15' and 30'). Capsaicin challenge is achieved by injection of 10 μL of a 0.3% solution. Sample dilutions are prepared in 0.5% BSA/saline. FIG. 19 illustrates the reversal of mechanical allodynia that is achieved by pre-treatment of the animals with a range of concentrations of LC/A-nociceptin-HN/A fusion.
[0363] The ability of an LC/A-nociceptin-HN/A fusion (CPN/A) to inhibit streptozotocin (STZ)-induced mechanical (tactile) allodynia in rats is evaluated. STZ-induced mechanical allodynia in rats is achieved by injection of streptozotocin (i.p. or i.v.) which yields destruction of pancreatic β-cells leading to loss of insulin production, with concomitant metabolic stress (hyperglycemia and hyperlipidemia). As such, STZ induces Type I diabetes. In addition, STZ treatment leads to progressive development of neuropathy, which serves as a model of chronic pain with hyperalgesia and allodynia that may reflect signs observed in diabetic humans (peripheral diabetic neuropathy).
[0364] Male Sprague-Dawley rats (250-300 g) are treated with 65 mg/kg STZ in citrate buffer (I.V.) and blood glucose and lipid are measured weekly to define the readiness of the model. Paw Withdrawal Threshold (PWT) is measured in response to a Von Frey filament stimulus series over a period of time. Allodynia is said to be established when the PWT on two consecutive test dates (separated by 1 week) measures below 6 g on the scale. At this point, rats are randomized to either a saline group (negative efficacy control), gabapentin group (positive efficacy control) or a test group (CPN/A). Test materials (20-25 μl are injected subcutaneously as a single injection (except gabapentin) and the PWT is measured at 1 day post-treatment and periodically thereafter over a 2-week period. Gabapentin (30 mg/kg i.p. @ 3 ml/kg injection volume) is injected daily, 2 hours prior to the start of PWT testing. FIG. 20 illustrates the reversal of allodynia achieved by pre-treatment of the animals with 750 ng of CPN/A. Data were obtained over a 2-week period after a single injection of CPN/A
Example 23
Assessment of in Vivo Efficacy of an LC/A-nociceptin Variant-HN/A Fusion
[0365] The ability of an LC/A-nociceptin variant-HN/A fusion (CPNv/A) to inhibit capsaicin-induced mechanical allodynia is evaluated following subcutaneous intraplantar injection in the rat hind paw. Test animals are evaluated for paw withdrawal frequency (PWF %) in response to a 10 g Von Frey filament stimulus series (10 stimuli×3 trials) prior to recruitment into the study (Pre-Treat); after subcutaneous intraplantar treatment with CPNv/A but before capsaicin (Pre-CAP); and following capsaicin challenge post-injection of CPNv/A (average of responses at 15' and 30'; CAP). Capsaicin challenge is achieved by injection of 10 μL of a 0.3% solution. Sample dilutions are prepared in 0.5% BSA/saline.
[0366] FIG. 21 illustrates the reversal of allodynia that is achieved by pre-treatment of the animals with a range of concentrations of LC/A-nociceptin variant-HN/A fusion in comparison to the reversal achieved with the addition of LC/A-nociceptin-HN/A fusion. These data are expressed as a normalized paw withdrawal frequency differential, in which the difference between the peak response (post-capsaicin) and the baseline response (pre-capsaicin) is expressed as a percentage. With this analysis, it can be seen that CPNv/A is more potent than CPN/A since a lower dose of CPNv/A is required to achieve similar analgesic effect to that seen with CPN/A.
Example 24
Preparation of an LC/A-leu Enkephalin-HN/A Fusion Protein
[0367] Due to the small, five-amino acid, size of the leu-enkephalin ligand the LC/A-leu enkephalin-HN/A fusion is created by site directed mutagenesis [for example using Quickchange (Stratagene Inc.)] using the LC/A-nociceptin-HN/A fusion (SEQ ID13) as a template. Oligonucleotides are designed encoding the YGGFL leu-enkephalin peptide, ensuring standard E. coli codon usage is maintained and no additional restriction sites are incorporated, flanked by sequences complimentary to the linker region of the LC/A-nociceptin-HN/A fusion (SEQ ID13) either side on the nociceptin section. The SDM product is checked by sequencing and the final construct containing the LC-linker-leu enkephalin-spacer-HN ORF (SEQ ID68) for expression as a protein of the sequence illustrated in SEQ ID69. The fusion protein is termed CPLE-A. FIG. 22 illustrates the purification of CPLE-A from E. coli following the methods used in Example 9.
Example 25
Expression and Purification of an LC/A-beta-endorphin-HN/A Fusion Protein
[0368] Following the methods used in Example 9, and with the LC/A-beta-endorphin-HN/A fusion protein (termed CPBE-A) created in Example 7, the CPBE-A is purified from E. coli. FIG. 23 illustrates the purified protein as analysed by SDS-PAGE.
Example 26
Preparation of an LC/A-nociceptin Mutant-HN/A Fusion Protein
[0369] Due to the single amino acid modification necessary to mutate the nociceptin sequence at position 1 from a Phe to a Tyr, the LC/A-nociceptin mutant-HN/A fusion is created by site directed mutagenesis [for example using Quickchange (Stratagene Inc.)] using the LC/A-nociceptin-HN/A fusion (SEQ ID13) as a template. Oligonucleotides are designed encoding tyrosine at position 1 of the nociceptin sequence, ensuring standard E. coli codon usage is maintained and no additional restriction sites are incorporated, flanked by sequences complimentary to the linker region of the LC/A-nociceptin-HN/A fusion (SEQ ID13) either side on the nociceptin section. The SDM product is checked by sequencing and the final construct containing the LC/A-nociceptin mutant-spacer-HN/A fusion ORF (SEQ ID70) for expression as a protein of the sequence illustrated in SEQ ID71. The fusion protein is termed CPOP-A. FIG. 24 illustrates the purification of CPOP-A from E. coli following the methods used in Example 9.
Example 27
Preparation and Assessment of an LC/A-nociceptin Variant Mutant-HN/A Fusion Protein
[0370] Due to the single amino acid modification necessary to mutate the nociceptin sequence at position 1 from a Phe to a Tyr, the LC/A-nociceptin variant mutant-HN/A fusion is created by site directed mutagenesis [for example using Quickchange (Stratagene Inc.)] using the LC/A-nociceptin variant-HN/A fusion (SEQ ID25) as a template. Oligonucleotides are designed encoding tyrosine at position 1 of the nociceptin sequence, ensuring standard E. coli codon usage is maintained and no additional restriction sites are incorporated, flanked by sequences complimentary to the linker region of the LC/A-nociceptin variant-HN/A fusion (SEQ ID25) either side on the nociceptin section. The SDM product is checked by sequencing and the final construct containing the LC/A-nociceptin mutant-spacer-HN/A fusion ORF (SEQ ID72) for expression as a protein of the sequence illustrated in SEQ ID73. The fusion protein is termed CPOPv-A. FIG. 25 illustrates the purification of CPOPv-A from E. coli following the methods used in Example 9.
[0371] Using methodology described in Example 16, CPOPv-A is assessed for its ability to cleave SNAP-25 in the eDRG cell model. FIG. 26 illustrates that CPOPv-A is able to cleave SNAP-25 in the eDRG model, achieving cleavage of 50% of the maximal SNAP-25 after exposure of the cells to approximately 5.9 nM fusion for 24 h.
Example 28
Preparation of an IgA Protease-nociceptin Variant-HN/A Fusion Protein
[0372] The IgA protease amino acid sequence was obtained from freely available database sources such as GenBank (accession number P09790). Information regarding the structure of the N. Gonorrhoeae IgA protease gene is available in the literature (Pohlner et al., Gene structure and extracellular secretion of Neisseria gonorrhoeae IgA protease, Nature, 1987, 325(6103), 458-62). Using Backtranslation tool v2.0 (Entelechon), the DNA sequence encoding the IgA protease modified for E. coli expression was determined. A BamHI recognition sequence was incorporated at the 5' end and a codon encoding a cysteine amino acid and SalI recognition sequence were incorporated at the 3' end of the IgA DNA. The DNA sequence was screened using MapDraw, (DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required for cloning were removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage was assessed Graphical Codon Usage Analyser (Geneart), and the % GC content and codon usage ratio assessed by reference to published codon usage tables. This optimised DNA sequence (SEQ ID74) containing the IgA open reading frame (ORF) is then commercially synthesized.
[0373] The IgA (SEQ ID74) is inserted into the LC-linker-nociceptin variant-spacer-HN ORF (SEQ ID25) using BamHI and SalI restriction enzymes to replace the LC with the IgA protease DNA. The final construct contains the IgA-linker-nociceptin variant-spacer-HN ORF (SEQ ID75) for expression as a protein of the sequence illustrated in SEQ ID76.
Example 29
Preparation and Assessment of a Nociceptin Targeted Endopeptidase Fusion Protein with a Removable Histidine Purification Tag
[0374] DNA was prepared that encoded a Factor Xa removable his-tag (his6), although it is clear that alternative proteases site such as Enterokinase and alternative purification tags such as longer histidine tags are also possible. Using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)], the DNA sequence encoding the Factor Xa removable his-tag region is determined. Restriction sites are then incorporated into the DNA sequence and can be arranged as NheI-linker-SpeI-PstI-HN/A-XbaI-LEIEGRSGHHHHHHStop codon-HindIII (SEQ ID77). The DNA sequence is screened for restriction sequence incorporated and any additional sequences are removed manually from the remaining sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 Sep. 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector. In order to create CPNv-A-FXa-HT (SEQ ID78, removable his-tag construct) the pCR 4 vector encoding the removable his-tag is cleaved with NheI and HindIII. The NheI-HindIII fragment is then inserted into the LC/A-CPNv-HN/A vector (SEQ ID25) that has also been cleaved by NheI and HindIII. The final construct contains the LC/A-linker-nociceptin variant-spacer-HN-FXa-Histag-HindIII ORF sequences (SEQ ID78) for expression as a protein of the sequence illustrated in SEQ ID79. FIG. 27 illustrates the purification of CPNv-A-FXa-HT from E. coli following the methods used in Example 9.
Example 30
Preparation of a Leu-Enkephalin Targeted Endopeptidase Fusion Protein Containing a Translocation Domain Derived from Diphtheria Toxin
[0375] The DNA sequence is designed by back translation of the amino acid sequence of the translocation domain of the diphtheria toxin (obtained from freely available database sources such as GenBank (accession number 1×DTT) using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)]. Restriction sites are then incorporated into the DNA sequence and can be arranged as NheI-Linker-SpeI-PstI-diphtheria translocation domain-XbaI-stop codon-HindIII (SEQ ID80). PstI/XbaI recognition sequences are incorporated at the 5' and 3' ends of the translocation domain respectively of the sequence maintaining the correct reading frame. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 Sep. 2004). This optimised DNA sequence containing the diphtheria translocation domain is then commercially synthesized as NheI-Linker-SpeI-PstI-diphtheria translocation domain-XbaI-stop codon-HindIII (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector (Invitrogen). The pCR 4 vector encoding the diphtheria translocation domain is cleaved with NheI and XbaI. The NheI-XbaI fragment is then inserted into the LC/A-CPLE-HN/A vector (SEQ ID68) that has also been cleaved by NheI and XbaI. The final construct contains the LC/A-leu-enkephalin-spacer-diphtheria translocation domain ORF sequences (SEQ ID81) for expression as a protein of the sequence illustrated in SEQ ID82.
Example 31
Preparation of a Nociceptin Variant Targeted Endopeptidase Fusion Protein Containing a LC Domain Derived from Tetanus Toxin
[0376] The DNA sequence is designed by back translation of the tetanus toxin LC amino acid sequence (obtained from freely available database sources such as GenBank (accession number X04436) using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)]. BamHI/SalI recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame (SEQ ID83). The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 Sep. 2004). This optimised DNA sequence containing the tetanus toxin LC open reading frame (ORF) is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector (invitrogen). The pCR 4 vector encoding the TeNT LC is cleaved with BamHI and SalI. The BamHI-SalI fragment is then inserted into the LC/A-CPNv-HN/A vector (SEQ ID25) that has also been cleaved by BamHI and SalI. The final construct contains the TeNT LC-linker-nociceptin variant-spacer-HN ORF sequences (SEQ ID84) for expression as a protein of the sequence illustrated in SEQ ID85.
Example 32
Preparation of an LC/C-nociceptin Variant-HN/C Fusion Protein with a Native Serotype C Linker that is Susceptible to Factor Xa Cleavage
[0377] Following the methods used in Example 4, the LC/C (SEQ ID5) and HN/C (SEQ ID6) are created and inserted into the C serotype nociceptin variant linker arranged as BamHI-SalI-linker-nociceptin variant-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID86). The final construct contains the LC-linker-nociceptin variant-spacer-HN ORF sequences (SEQ ID87) for expression as a protein of the sequence illustrated in SEQ ID88. The fusion protein is termed CPNv-C (act. C).
Example 33
Construction of CHO-K1 OP2 Receptor Activation Assay and SNAP-25 Cleavage Assay
Cell-Line Creation
[0378] CHO OP2 cell line was purchased from Perkin Elmer (ES-541-C, lot 451-719-A). Cells were transfected with SNAP-25 DNA using Lipofectamine® 2000 and incubated for 4 hours before media replacement. After 24 hours, cells were transferred to a T175 flask. 100 ug/ml Zeocin was added after a further 24 hours to begin selection of SNAP-25 expressing cells, and 5 ug/ml Blasticidin added to maintain selective pressure for the receptor. Cells were maintained in media containing selection agents for two weeks, passaging cells every two to three days to maintain 30-70% confluence. Cells were then diluted in selective media to achieve 0.5 cell per well in a 96 well microplate. After a few days, the plates were examined under a microscope, and those containing single colonies were marked. Media in these wells was changed weekly. As cells became confluent in the wells, they were transferred to T25 flasks. When they had expanded sufficiently each clone was seeded to 24 wells of a 96 well plate, plus a frozen stock vial created. LC/A-CPDY-HNA fusion and LC/A-HNA were applied to the cells for 24 hours, and then western blots performed to detect SNAP-25 cleavage. Clones from which SNAP-25 bands were strong and cleavage levels were high with fusion were maintained for further investigation. Full dose curves were run on these, and the clone (D30) with the highest differential between LC/A-CPDY-HNA fusion and LC/A-HNA cleavage levels was selected.
OP2 Receptor Activation Assay
[0379] The OP2 receptor activation measures the potency and intrinsic efficacy of ligands at OP2 receptor in transfected CHO-K1 cells by quantifying the reduction of forskolin-stimulated intracellular cAMP using a FRET-based cAMP (Perkin Elmer LANCE cAMP kit). After stimulation, a fluorescently labelled cAMP tracer (Europium-streptavadin/biotin-cAMP) and fluorescently (Alexa) labelled anti-cAMP antibody are added to the cells in a lysis buffer. cAMP from the cells competes with the cAMP tracer for antibody binding sites. When read, a light pulse at 320 nm excites the fluorescent portion (Europium) of the cAMP tracer. The energy emitted from the europium is transferred to the Alexa fluor-labelled antibodies bound to the tracer, generating a TR-FRET signal at 665 nm (Time-resolved fluorescence resonance energy transfer is based on the proximity of the donor label, europium, and the acceptor label, Alexa fluor, which have been brought together by a specific binding reaction). Residual energy from the europium produces light at 615 nm. In agonist treated cells there will be less cAMP to compete with the tracer so a dose dependant increase in signal at 665 nm will be observed compared with samples treated with forskolin alone. The signal at 665 nm signal is converted to cAMP concentration by interpolation to a cAMP standard curve which is included in each experiment.
Culture of Cells for Receptor Activation Assay:
[0380] Cells were seeded and cultured in T175 flasks containing Ham F12 with Glutamax, 10% Foetal bovine serum, 5 μg ml-1 Blasticidin and 100 μg ml-1 Zeocin. The flasks were incubated at 37° C. in a humidified environment containing 5% CO2 until 60-80% confluent. On the day of harvest the media was removed and the cells washed twice with 25 ml PBS. The cells were removed from the flask by addition of 10 ml of Tryple Express, and incubation at 37° C. for 10 min followed by gentle tapping of the flask. The dislodged cells were transferred to a 50 ml centrifuge tube and the flask washed twice with 10 ml media which was added to the cell suspension. The tube was centrifuged at 1300×g for 3 min and the supernatant removed. Cells were gently re-suspended in 10 ml media (if freezing cells) or assay buffer (if using `fresh` cells in assay), and a sample was removed for counting using a nucleocounter (ChemoMetec). Cells for use `fresh` in an assay were diluted further in assay buffer to the appropriate concentration. Cells harvested for freezing were re-centrifuged (1300×g; 3 min), the supernatant removed and cells re-suspended in Synth-a-freeze at 4° C. to 3×106 cells/ml. Cryovials containing 1 ml suspension each were placed in a chilled Nalgene Mr Frosty freezing container (-1° C./minute cooling rate), and left overnight in a -80° C. freezer. The following day vials were transferred to the vapour phase of a liquid nitrogen storage tank.
Dilution of Test Materials and Cell Assay
[0381] Using Gilson pipettes and Sigmacoted or lo-bind tips, test materials and standards were diluted to the appropriate concentrations in the wells of the first two columns of an eppendorf 500 μl deep-well lo-bind plate, in assay buffer containing 10 μM forskolin. The chosen concentrations in columns one and two were half a log unit apart. From these, serial 1:10 dilutions were made across the plate (using an electronic eight channel pipette with sigmacote or lo-bind tips) until eleven concentrations at half log intervals had been created. In the twelfth column, assay buffer only was added as a `basal`. Using a 12 channel digital pipette, 10 μM of sample from the lo-bind plate was transferred to the optiplate 96 well microplate.
[0382] To wells containing the standard curve, 10 ul of assay buffer was added using a multichannel digital pipette. To wells containing the test materials, 10 ul of cells in assay buffer at the appropriate concentration were added. Plates were sealed and incubated for 120 min at room temperature, for the first hour on an IKA MTS 2/4 orbital shaker set to maximum speed.
Detection
[0383] LANCE Eu-W8044 labelled streptavidin (Eu-SA) and Biotin-cAMP (b-cAMP) were diluted in cAMP Detection Buffer (both from Perkin Elmer LANCE cAMP kit) to create sub-stocks, at dilution ratios of 1:17 and 1:5, respectively. The final detection mix was prepared by diluting from the two sub stocks into detection buffer at a ratio of 1:125. The mixture was incubated for 15-30 min at room temperature before addition of 1:200 Alexa Fluor® 647-anti cAMP Antibody (Alexa-Fluor Ab). After briefly vortex mixing, 20 μl was immediately added to each well using a digital multichannel pipette. Microplate sealers were applied and plates incubated for 24 h at room temperature (for the first hour on an IKA MTS 2/4 orbital shaker set to maximum speed). Plate sealers were removed prior to reading on the Envision.
[0384] FIGS. 33 and 34 show that dynorphin conjugates with LC/A-HN/A, LC/B-HN/B, LC/C-HN/C and LC/D-HN/D backbones active the OP2 receptor.
CHO-K1 OP2 SNAP-25 Cleavage Assay
[0385] Cultures of cells were exposed to varying concentrations of fusion protein for 24 hours. Cellular proteins were separated by SDS-PAGE and western blotted with anti-SNAP-25 antibody to facilitate assessment of SNAP-25 cleavage. SNAP-25 cleavage calculated by densitometric analysis (Syngene).
Plating Cells
[0386] Prepare cells at 2×10e5 cells/ml and seed 125 μl per well of 96 well plate. Use the following media: 500 ml Gibco Ham F12 with Glutamax (product code 31765068), 50 ml FBS, 5 ug/ml Blasticidin (250 μl aliquot from box in freezer, G13) (Calbiochem #203351, 10 ml at 10 mg/ml), 100 ug/ml Zeocin (500 μl from box in freezer, G35). (Invitrogen from Fisher, 1 g in 8×1.25 ml tubes at 100 mg/ml product code VXR25001). Allow cells to grow for 24 hrs (37° C., 5 CO2, humidified atmosphere).
Cell Treatment
[0387] Prepare dilutions of test protein for a dose range of each test proteins (make up double (2×) the desired final concentrations because 125 μl will be applied directly onto 125 μl of media already in each well). Filter sterilize CHO KOR D30 feeding medium (20 ml syringe, 0.2 μm syringe filter) to make the dilutions. Add the filtered medium into 5 labelled bijoux's (7 ml tubes), 0.9 ml each using a Gilson pipette or multi-stepper. Dilute the stock test protein to 2000 nM (working stock solution 1) and 600 nM (working stock solution 2). Using a Gilson pipette prepare 10-fold serial dilutions of each working stock, by adding 100 μl to the next concentration in the series. Pipette up and down to mix thoroughly. Repeat to obtain 4 serial dilutions for solution 1, and 3 serial dilutions for solution 2. A 0 nM control (filtered feeding medium only) should also be prepared as a negative control for each plate. Repeat the above for each test protein. In each experiment a `standard` batch of material must be included as control/reference material, this is unliganded LC/A-HN/A.
Apply Diluted Sample to CHO KOR D30 Plates
[0388] Apply 125 μl of test sample (double concentration) per well. Each test sample should be applied to triplicate wells and each dose range should include a 0 nM control. Incubate for 24 hrs (37° C., 5% CO2, humidified atmosphere).
Cell Lysis
[0389] Prepare fresh lysis buffer (20 mls per plate) with 25% (4×) NuPAGE LDS sample buffer, 65% dH2O and 10% 1 M DTT. Remove medium from the CHO KOR D30 plate by inverting over a waste receptacle. Drain the remaining media from each well using a fine-tipped pipette. Lyse the cells by adding 125 μl of lysis buffer per well using a multi-stepper pipette. After a minimum of 20 mins, remove the buffer from each well to a 1.5 ml microcentrifuge tube. Tubes must be numbered to allowing tracking of the CHO KOR treatments throughout the blotting procedure. A1-A3 down to H1-H3 numbered 1-24, A4-A6 down to H4-H6 numbered 25-48, A7-A9 down to H7-H93 numbered 49-72, A10-A12 down to H10-H12 numbered 73-96. Vortex each sample and heat at 90° C. for 5-10 mins in a prewarmed heat block. Store at -20° C. or use on the same day on an SDS gel.
Gel Electrophoresis
[0390] If the sample has been stored o/n or longer, put in a heat block prewarmed to 90° C. for 5-10 mins. Set up SDS page gels, use 1 gel per 12 samples, prepare running buffer (1×, Invitrogen NuPAGE MOPS SDS Running Buffer (20×) (NP0001))≈800 ml/gel tank. Add 500 μl of NuPAGE antioxidant to the upper buffer chamber. Load 15 ul samples onto gel lanes from left to right as and load 2.5 ul of Invitrogen Magic Marker XP and 5 ul Invitrogen See Blue Plus 2 pre-stained standard and 15 ul of non-treated control. It is important to maximize the resolution of separation during SDS_PAGE. This can be achieved by running 12% bis-tris gels at 200 V for 1 hour and 25 minutes (until the pink (17 kDa) marker reaches the bottom of the tank).
Western Blotting
[0391] Complete a Semi-dry transfer: using an Invitrogen iBlot (use iBlot Programme 3 for 6 minutes). Put the nitrocellulose membranes in individual small trays. Incubate the membranes with blocking buffer solution (5 g Marvel milk powder per 100 ml 0.1% PBS/Tween) at room temperature, on a rocker, for 1 hour. Apply primary antibody (Anti-SNAP-25 1:1000 dilution) and incubate the membranes with primary antibody (diluted in blocking buffer) for 1 hour on a rocker at room temperature. Wash the membranes by rinsing 3 times with PBS/Tween (0.1%). Then apply the secondary (Anti-Rabbit-HRP conjugate diluted 1:1000) and incubate the membranes with secondary antibody (diluted in blocking buffer) at room temperature, on a rocker, for 1 hour. Wash the membranes by rinsing 3 times with PBS/Tween (0.1%), leave membrane a minimum of 20 mins for the last wash. Detect the bound antibody using Syngene: Drain blots of PBS/Tween, mix WestDura reagents 1:1 and add to blots for 5 minutes. Ensure enough solution is added to the membranes to completely cover them. Place membrane in Syngene tray, set up Syngene software for 5 min expose time.
[0392] FIG. 31 clearly shows that LC/A-CPDY-HN/A conjugates effectively cleave SNAP-25.
Example 34
Construction and Activation of Dynorphin Conjugates
Preparation of a LC/A and HN/A Backbone Clones
[0393] The following procedure creates the LC and HN fragments for use as the component backbone for multidomain fusion expression. This example is based on preparation of a serotype A based clone (SEQ ID1 and SEQ ID2), though the procedures and methods are equally applicable to the other serotypes [illustrated by the sequence listing for serotype B (SEQ ID3 and SEQ ID4) and serotype C (SEQ ID5 and SEQ ID6)].
Preparation of Cloning and Expression Vectors
[0394] pCR 4 (Invitrogen) is the chosen standard cloning vector, selected due to the lack of restriction sequences within the vector and adjacent sequencing primer sites for easy construct confirmation. The expression vector is based on the pMAL (NEB) expression vector, which has the desired restriction sequences within the multiple cloning site in the correct orientation for construct insertion (BamHI-SalI-PstI-HindIII). A fragment of the expression vector has been removed to create a non-mobilisable plasmid and a variety of different fusion tags have been inserted to increase purification options.
Preparation of Protease (e.g. LC/A) Insert
[0395] The LC/A (SEQ ID1) is created by one of two ways:
[0396] The DNA sequence is designed by back translation of the LC/A amino acid sequence [obtained from freely available database sources such as GenBank (accession number P10845) or Swissprot (accession locus BXA1_CLOBO) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)]. BamHI/SalI recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence, maintaining the correct reading frame. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 Sep. 2004). This optimised DNA sequence containing the LC/A open reading frame (ORF) is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
[0397] The alternative method is to use PCR amplification from an existing DNA sequence with BamHI and SalI restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. Complementary oligonucleotide primers are chemically synthesised by a supplier (for example MWG or Sigma-Genosys), so that each pair has the ability to hybridize to the opposite strands (3' ends pointing "towards" each other) flanking the stretch of Clostridium target DNA, one oligonucleotide for each of the two DNA strands. To generate a PCR product the pair of short oligonucleotide primers specific for the Clostridium DNA sequence are mixed with the Clostridium DNA template and other reaction components and placed in a machine (the `PCR machine`) that can change the incubation temperature of the reaction tube automatically, cycling between approximately 94° C. (for denaturation), 55° C. (for oligonucleotide annealing), and 72° C. (for synthesis). Other reagents required for amplification of a PCR product include a DNA polymerase (such as Taq or Pfu polymerase), each of the four nucleotide dNTP building blocks of DNA in equimolar amounts (50-200 μM) and a buffer appropriate for the enzyme optimised for Mg2+ concentration (0.5-5 mM).
[0398] The amplification product is cloned into pCR 4 using either, TOPO TA cloning for Taq PCR products or Zero Blunt TOPO cloning for Pfu PCR products (both kits commercially available from Invitrogen). The resultant clone is checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis [for example, using Quickchange (Stratagene Inc.)].
Preparation of Translocation (e.g. HN) Insert
[0399] The HN/A (SEQ ID2) is created by one of two ways:
[0400] The DNA sequence is designed by back translation of the HN/A amino acid sequence [obtained from freely available database sources such as GenBank (accession number P10845) or Swissprot (accession locus BXA1_CLOBO)] using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)]. A PstI restriction sequence added to the N-terminus and XbaI-stop codon-HindIII to the C-terminus ensuring the correct reading frame is maintained. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 Sep. 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
[0401] The alternative method is to use PCR amplification from an existing DNA sequence with PstI and XbaI-stop codon-HindIII restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. The PCR amplification is performed as described above. The PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis [for example using Quickchange (Stratagene Inc.)].
Preparation of Linker-Dynorphin-Spacer Insert
[0402] The LC-HN linker can be designed from first principle, using the existing sequence information for the linker as the template. For example, the serotype A linker (in this case defined as the inter-domain polypeptide region that exists between the cysteines of the disulphide bridge between LC and HN) is 23 amino acids long and has the sequence VRGIITSKTKSLDKGYNKALNDL. Within this sequence, it is understood that proteolytic activation in nature leads to an HN domain that has an N-terminus of the sequence ALNDL. This sequence information is freely available from available database sources such as GenBank (accession number P10845) or Swissprot (accession locus BXA1_CLOBO). Into this linker an enterokinase site, dynorphin and spacer are incorporated; and using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)], the DNA sequence encoding the linker-ligand-spacer region is determined. Restriction sites are then incorporated into the DNA sequence and can be arranged as BamH I-SalI-linker-protease site-dynorphin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII. It is important to ensure the correct reading frame is maintained for the spacer, dynorphin and restriction sequences and that the XbaI sequence is not preceded by the bases, TC, which would result on DAM methylation. The DNA sequence is screened for restriction sequence incorporation, and any additional sequences are removed manually from the remaining sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example, GenBank Release 143, 13 Sep. 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
Preparation of the LC/A-dynorphin-HN/A Fusion
[0403] In order to create the LC-linker-dynorphin-spacer-HN construct (SEQ ID90), the pCR 4 vector encoding the linker is cleaved with BamHI+SalI restriction enzymes. This cleaved vector then serves as the recipient vector for insertion and ligation of the LC/A DNA (SEQ ID1) cleaved with BamHI+SalI. The resulting plasmid DNA is then cleaved with PstI+XbaI restriction enzymes and serves as the recipient vector for the insertion and ligation of the HN/A DNA (SEQ ID2) cleaved with PstI+XbaI. The final construct contains the LC-linker-dynorphin-spacer-HN ORF (SEQ ID90) for transfer into expression vectors for expression to result in a fusion protein of the sequence illustrated in SEQ ID91.
Examples 35
Preparation and Purification of an LC/A-dynorphin-HN/A Fusion Protein Family with Variable Spacer Length
[0404] Using the same strategy as employed in Example 34, a range of DNA linkers were prepared that encoded dynorphin and variable spacer content. Using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)], the DNA sequence encoding the linker-ligand-spacer region is determined. Restriction sites are then incorporated into the DNA sequence and can be arranged as BamHI-SalI-linker-protease site-dynorphin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII. It is important to ensure the correct reading frame is maintained for the spacer, dynorphin and restriction sequences and that the XbaI sequence is not preceded by the bases, TC which would result on DAM methylation. The DNA sequence is screened for restriction sequence incorporation and any additional sequences are removed manually from the remaining sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 Sep. 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
[0405] The spacers that were created included:
TABLE-US-00008 SEQ ID of the Code Protein sequence of the linker linker DNA GS10 ALAGGGGSALVLQ 92 GS15 ALAGGGGSGGGGSALVLQ 93 GS25 ALAGGGGSGGGGSGGGGSGGGGSALVLQ 94
[0406] By way of example, in order to create the LC/A-CPDY(GS25)-HN/A fusion construct (SEQ ID94), the pCR 4 vector encoding the linker is cleaved with BamHI+SalI restriction enzymes. This cleaved vector then serves as the recipient vector for insertion and ligation of the LC/A DNA (SEQ ID1) also cleaved with BamHI+SalI. The resulting plasmid DNA is then cleaved with BamHI+HindIII restriction enzymes and the LC/A-linker fragment inserted into a similarly cleaved vector containing a unique multiple cloning site for BamHI, SalI, PstI, and HindIII such as the pMAL vector (NEB). The HN/A DNA (SEQ ID2) is then cleaved with PstI+HindIII restriction enzymes and inserted into the similarly cleaved pMAL-LC/A-linker construct. The final construct contains the LC/A-CPDY(GS25)-HN/A ORF for expression as a protein of the sequence illustrated in SEQ ID94.
Example 36
Purification Method for LC/A-Dynorphin-HN/A Fusion Protein
[0407] Defrost falcon tube containing 25 ml 50 mM HEPES pH 7.2, 200 mM NaCl and approximately 10 g of E. coli BL21 cell paste. Make the thawed cell paste up to 80 ml with 50 mM HEPES pH 7.2, 200 mM NaCl and sonicate on ice 30 seconds on, 30 seconds off for 10 cycles at a power of 22 microns ensuring the sample remains cool. Spin the lysed cells at 18 000 rpm, 4° C. for 30 minutes. Load the supernatant onto a 0.1 M NiSO4 charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2, 200 mM NaCl. Using a step gradient of 10 and 40 mM imidazol, wash away the non-specific bound protein and elute the fusion protein with 100 mM imidazol. Dialyse the eluted fusion protein against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4° C. overnight and measure the OD of the dialysed fusion protein. Add 3.2 μl of enterokinase (2 μg/ml) per 1 mg fusion protein and Incubate at 25° C. static overnight. Load onto a 0.1 M NiSO4 charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2, 200 mM NaCl. Wash column to baseline with 50 mM HEPES pH 7.2, 200 mM NaCl. Using a step gradient of 10 and 40 mM imidazol, wash away the non-specific bound protein and elute the fusion protein with 100 mM imidazol. Dialyse the eluted fusion protein against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4° C. overnight and concentrate the fusion to about 2 mg/ml, aliquot sample and freeze at -20° C. Test purified protein using OD, BCA, purity analysis and SNAP-25 assessments.
Example 37
Preparation of a LC/C-dynorphin-HN/C Fusion Protein with a Serotype A Activation Sequence
[0408] Following the methods used in Examples 1 and 2, the LC/C (SEQ ID5) and HN/C (SEQ ID6) are created and inserted into the A serotype linker arranged as BamHI-SalI-linker-protease site-dynorphin-NheI-spacer-SpeI-PstI-XbaI-stop codon-HindIII. The final construct contains the LC-linker-dynorphin-spacer-HN ORF for expression as a protein of the sequence illustrated in SEQ ID95.
Example 38
Preparation of an IgA Protease-Dynorphin Variant-HN/A Fusion Protein
[0409] The IgA protease amino acid sequence was obtained from freely available database sources such as GenBank (accession number P09790). Information regarding the structure of the N. Gonorrhoeae IgA protease gene is available in the literature (Pohlner et al., Gene structure and extracellular secretion of Neisseria gonorrhoeae IgA protease, Nature, 1987, 325(6103), 458-62). Using Backtranslation tool v2.0 (Entelechon), the DNA sequence encoding the IgA protease modified for E. coli expression was determined. A BamHI recognition sequence was incorporated at the 5' end and a codon encoding a cysteine amino acid and SalI recognition sequence were incorporated at the 3' end of the IgA DNA. The DNA sequence was screened using MapDraw, (DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required for cloning were removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage was assessed Graphical Codon Usage Analyser (Geneart), and the % GC content and codon usage ratio assessed by reference to published codon usage tables. This optimised DNA sequence (SEQ ID74) containing the IgA open reading frame (ORF) is then commercially synthesized.
[0410] The IgA (SEQ ID74) is inserted into the LC-linker-dynorphin-spacer-HN ORF (SEQ ID90) using BamHI and SalI restriction enzymes to replace the LC with the IgA protease DNA. The final construct contains the IgA-linker-dynorphin-spacer-HN ORF for expression as a protein of the sequence illustrated in SEQ ID96.
Example 39
Preparation of a Dynorphin Targeted Endopeptidase Fusion Protein Containing a LC Domain Derived from Tetanus Toxin
[0411] The DNA sequence is designed by back translation of the tetanus toxin LC amino acid sequence (obtained from freely available database sources such as GenBank (accession number X04436) using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)]. BamHI/SalI recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame (SEQ ID83). The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 Sep. 2004). This optimised DNA sequence containing the tetanus toxin LC open reading frame (ORF) is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector (invitrogen). The pCR 4 vector encoding the TeNT LC is cleaved with BamHI and SalI. The BamHI-SalI fragment is then inserted into the LC/A-dynorphin-HN/A vector (SEQ ID90) that has also been cleaved by BamHI and SalI. The final construct contains the TeNT LC-linker-dynorphin-spacer-HN ORF sequences for expression as a protein of the sequence illustrated in SEQ ID97.
Example 40
[0412] A method of treating, preventing or ameliorating pain in a subject, comprising administration to said patient a therapeutic effective amount of fusion protein, wherein said pain is selected from the group consisting of: chronic pain arising from malignant disease, chronic pain not caused by malignant disease (peripheral neuropathies).
Patient A
[0413] A 73 year old woman suffering from severe pain caused by posthepatic neuralgia is treated by a peripheral injection with fusion protein to reduce neurotransmitter release at the synapse of nerve terminals to reduce the pain. The patient experiences good analgesic effect within 2 hours of said injection.
Patient B
[0414] A 32 year old male suffering from phantom limb pain after having his left arm amputated following a car accident is treated by peripheral injection with fusion protein to reduce the pain. The patient experiences good analgesic effect within 1 hour of said injection.
Patient C
[0415] A 55 year male suffering from diabetic neuropathy is treated by a peripheral injection with fusion protein to reduce neurotransmitter release at the synapse of nerve terminals to reduce the pain. The patient experiences good analgesic effect within 4 hours of said injection.
Patient D
[0416] A 63 year old woman suffering from cancer pain is treated by a peripheral injection with fusion protein to reduce neurotransmitter release at the synapse of nerve terminals to reduce the pain. The patient experiences good analgesic effect within 4 hours of said injection.
[0417] All documents, books, manuals, papers, patents, published patent applications, guides, abstracts and other reference materials cited herein are incorporated by reference in their entirety. While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be appreciated by one skilled in the art from reading this disclosure that various changes in form and detail can be made without departing from the true scope of the invention.
Sequence CWU
1
9711302DNAArtificial SequenceSynthetic 1ggatccatgg agttcgttaa caaacagttc
aactataaag acccagttaa cggtgttgac 60attgcttaca tcaaaatccc gaacgctggc
cagatgcagc cggtaaaggc attcaaaatc 120cacaacaaaa tctgggttat cccggaacgt
gataccttta ctaacccgga agaaggtgac 180ctgaacccgc caccggaagc gaaacaggtg
ccggtatctt actatgactc cacctacctg 240tctaccgata acgaaaagga caactacctg
aaaggtgtta ctaaactgtt cgagcgtatt 300tactccaccg acctgggccg tatgctgctg
actagcatcg ttcgcggtat cccgttctgg 360ggcggttcta ccatcgatac cgaactgaaa
gtaatcgaca ctaactgcat caacgttatt 420cagccggacg gttcctatcg ttccgaagaa
ctgaacctgg tgatcatcgg cccgtctgct 480gatatcatcc agttcgagtg taagagcttt
ggtcacgaag ttctgaacct cacccgtaac 540ggctacggtt ccactcagta catccgtttc
tctccggact tcaccttcgg ttttgaagaa 600tccctggaag tagacacgaa cccactgctg
ggcgctggta aattcgcaac tgatcctgcg 660gttaccctgg ctcacgaact gattcatgca
ggccaccgcc tgtacggtat cgccatcaat 720ccgaaccgtg tcttcaaagt taacaccaac
gcgtattacg agatgtccgg tctggaagtt 780agcttcgaag aactgcgtac ttttggcggt
cacgacgcta aattcatcga ctctctgcaa 840gaaaacgagt tccgtctgta ctactataac
aagttcaaag atatcgcatc caccctgaac 900aaagcgaaat ccatcgtggg taccactgct
tctctccagt acatgaagaa cgtttttaaa 960gaaaaatacc tgctcagcga agacacctcc
ggcaaattct ctgtagacaa gttgaaattc 1020gataaacttt acaaaatgct gactgaaatt
tacaccgaag acaacttcgt taagttcttt 1080aaagttctga accgcaaaac ctatctgaac
ttcgacaagg cagtattcaa aatcaacatc 1140gtgccgaaag ttaactacac tatctacgat
ggtttcaacc tgcgtaacac caacctggct 1200gctaatttta acggccagaa cacggaaatc
aacaacatga acttcacaaa actgaaaaac 1260ttcactggtc tgttcgagtt ttacaagctg
ctgtgcgtcg ac 130221257DNAArtificial
SequenceSynthetic 2ctgcagtgta tcaaggttaa caactgggat ttattcttca gcccgagtga
agacaacttc 60accaacgacc tgaacaaagg tgaagaaatc acctcagata ctaacatcga
agcagccgaa 120gaaaacatct cgctggacct gatccagcag tactacctga cctttaattt
cgacaacgag 180ccggaaaaca tttctatcga aaacctgagc tctgatatca tcggccagct
ggaactgatg 240ccgaacatcg aacgtttccc aaacggtaaa aagtacgagc tggacaaata
taccatgttc 300cactacctgc gcgcgcagga atttgaacac ggcaaatccc gtatcgcact
gactaactcc 360gttaacgaag ctctgctcaa cccgtcccgt gtatacacct tcttctctag
cgactacgtg 420aaaaaggtca acaaagcgac tgaagctgca atgttcttgg gttgggttga
acagcttgtt 480tatgatttta ccgacgagac gtccgaagta tctactaccg acaaaattgc
ggatatcact 540atcatcatcc cgtacatcgg tccggctctg aacattggca acatgctgta
caaagacgac 600ttcgttggcg cactgatctt ctccggtgcg gtgatcctgc tggagttcat
cccggaaatc 660gccatcccgg tactgggcac ctttgctctg gtttcttaca ttgcaaacaa
ggttctgact 720gtacaaacca tcgacaacgc gctgagcaaa cgtaacgaaa aatgggatga
agtttacaaa 780tatatcgtga ccaactggct ggctaaggtt aatactcaga tcgacctcat
ccgcaaaaaa 840atgaaagaag cactggaaaa ccaggcggaa gctaccaagg caatcattaa
ctaccagtac 900aaccagtaca ccgaggaaga aaaaaacaac atcaacttca acatcgacga
tctgtcctct 960aaactgaacg aatccatcaa caaagctatg atcaacatca acaagttcct
gaaccagtgc 1020tctgtaagct atctgatgaa ctccatgatc ccgtacggtg ttaaacgtct
ggaggacttc 1080gatgcgtctc tgaaagacgc cctgctgaaa tacatttacg acaaccgtgg
cactctgatc 1140ggtcaggttg atcgtctgaa ggacaaagtg aacaatacct tatcgaccga
catccctttt 1200cagctcagta aatatgtcga taaccaacgc cttttgtcca ctctagacta
gaagctt 125731323DNAArtificial SequenceSynthetic 3ggatccatgc
cggttaccat caacaacttc aactacaacg acccgatcga caacaacaac 60atcattatga
tggaaccgcc gttcgcacgt ggtaccggac gttactacaa ggcttttaag 120atcaccgacc
gtatctggat catcccggaa cgttacacct tcggttacaa acctgaggac 180ttcaacaaga
gtagcgggat tttcaatcgt gacgtctgcg agtactatga tccagattat 240ctgaatacca
acgataagaa gaacatattc cttcagacta tgattaaact cttcaaccgt 300atcaaaagca
aaccgctcgg tgaaaaactc ctcgaaatga ttatcaacgg tatcccgtac 360ctcggtgacc
gtcgtgtccc gcttgaagag ttcaacacca acatcgcaag cgtcaccgtc 420aacaaactca
tcagcaaccc aggtgaagtc gaacgtaaaa aaggtatctt cgcaaacctc 480atcatcttcg
gtccgggtcc ggtcctcaac gaaaacgaaa ccatcgacat cggtatccag 540aaccacttcg
caagccgtga aggtttcggt ggtatcatgc agatgaaatt ctgcccggaa 600tacgtcagtg
tcttcaacaa cgtccaggaa aacaaaggtg caagcatctt caaccgtcgt 660ggttacttca
gcgacccggc actcatcctc atgcatgaac tcatccacgt cctccacggt 720ctctacggta
tcaaagttga cgacctcccg atcgtcccga acgagaagaa attcttcatg 780cagagcaccg
acgcaatcca ggctgaggaa ctctacacct tcggtggcca agacccaagt 840atcataaccc
cgtccaccga caaaagcatc tacgacaaag tcctccagaa cttcaggggt 900atcgtggaca
gactcaacaa agtcctcgtc tgcatcagcg acccgaacat caatatcaac 960atatacaaga
acaagttcaa agacaagtac aaattcgtcg aggacagcga aggcaaatac 1020agcatcgacg
tagaaagttt cgacaagctc tacaaaagcc tcatgttcgg tttcaccgaa 1080accaacatcg
ccgagaacta caagatcaag acaagggcaa gttacttcag cgacagcctc 1140ccgcctgtca
aaatcaagaa cctcttagac aacgagattt acacaattga agagggcttc 1200aacatcagtg
acaaagacat ggagaaggaa tacagaggtc agaacaaggc tatcaacaaa 1260caggcatacg
aggagatcag caaagaacac ctcgcagtct acaagatcca gatgtgcgtc 1320gac
132341260DNAArtificial SequenceSynthetic 4ctgcagtgca tcgacgttga
caacgaagac ctgttcttca tcgctgacaa aaacagcttc 60agtgacgacc tgagcaaaaa
cgaacgtatc gaatacaaca cccagagcaa ctacatcgaa 120aacgacttcc cgatcaacga
actgatcctg gacaccgacc tgataagtaa aatcgaactg 180ccgagcgaaa acaccgaaag
tctgaccgac ttcaacgttg acgttccggt ttacgaaaaa 240cagccggcta tcaagaaaat
cttcaccgac gaaaacacca tcttccagta cctgtacagc 300cagaccttcc cgctggacat
ccgtgacatc agtctgacca gcagtttcga cgacgctctg 360ctgttcagca acaaagttta
cagtttcttc agcatggact acatcaaaac cgctaacaaa 420gttgttgaag cagggctgtt
cgctggttgg gttaaacaga tcgttaacga cttcgttatc 480gaagctaaca aaagcaacac
tatggacaaa atcgctgaca tcagtctgat cgttccgtac 540atcggtctgg ctctgaacgt
tggtaacgaa accgctaaag gtaactttga aaacgctttc 600gagatcgctg gtgcaagcat
cctgctggag ttcatcccgg aactgctgat cccggttgtt 660ggtgctttcc tgctggaaag
ttacatcgac aacaaaaaca agatcatcaa aaccatcgac 720aacgctctga ccaaacgtaa
cgaaaaatgg agtgatatgt acggtctgat cgttgctcag 780tggctgagca ccgtcaacac
ccagttctac accatcaaag aaggtatgta caaagctctg 840aactaccagg ctcaggctct
ggaagagatc atcaaatacc gttacaacat ctacagtgag 900aaggaaaaga gtaacatcaa
catcgacttc aacgacatca acagcaaact gaacgaaggt 960atcaaccagg ctatcgacaa
catcaacaac ttcatcaacg gttgcagtgt tagctacctg 1020atgaagaaga tgatcccgct
ggctgttgaa aaactgctgg acttcgacaa caccctgaaa 1080aagaacctgc tgaactacat
cgacgaaaac aagctgtacc tgatcggtag tgctgaatac 1140gaaaaaagta aagtgaacaa
atacctgaag accatcatgc cgttcgacct gagtatctac 1200accaacgaca ccatcctgat
cgaaatgttc aacaaataca actctctaga ctagaagctt 126051329DNAArtificial
SequenceSynthetic 5ggatccgaat tcatgccgat caccatcaac aacttcaact acagcgatcc
ggtggataac 60aaaaacatcc tgtacctgga tacccatctg aataccctgg cgaacgaacc
ggaaaaagcg 120tttcgtatca ccggcaacat ttgggttatt ccggatcgtt ttagccgtaa
cagcaacccg 180aatctgaata aaccgccgcg tgttaccagc ccgaaaagcg gttattacga
tccgaactat 240ctgagcaccg atagcgataa agataccttc ctgaaagaaa tcatcaaact
gttcaaacgc 300atcaacagcc gtgaaattgg cgaagaactg atctatcgcc tgagcaccga
tattccgttt 360ccgggcaaca acaacacccc gatcaacacc tttgatttcg atgtggattt
caacagcgtt 420gatgttaaaa cccgccaggg taacaattgg gtgaaaaccg gcagcattaa
cccgagcgtg 480attattaccg gtccgcgcga aaacattatt gatccggaaa ccagcacctt
taaactgacc 540aacaacacct ttgcggcgca ggaaggtttt ggcgcgctga gcattattag
cattagcccg 600cgctttatgc tgacctatag caacgcgacc aacgatgttg gtgaaggccg
tttcagcaaa 660agcgaatttt gcatggaccc gatcctgatc ctgatgcatg aactgaacca
tgcgatgcat 720aacctgtatg gcatcgcgat tccgaacgat cagaccatta gcagcgtgac
cagcaacatc 780ttttacagcc agtacaacgt gaaactggaa tatgcggaaa tctatgcgtt
tggcggtccg 840accattgatc tgattccgaa aagcgcgcgc aaatacttcg aagaaaaagc
gctggattac 900tatcgcagca ttgcgaaacg tctgaacagc attaccaccg cgaatccgag
cagcttcaac 960aaatatatcg gcgaatataa acagaaactg atccgcaaat atcgctttgt
ggtggaaagc 1020agcggcgaag ttaccgttaa ccgcaataaa ttcgtggaac tgtacaacga
actgacccag 1080atcttcaccg aatttaacta tgcgaaaatc tataacgtgc agaaccgtaa
aatctacctg 1140agcaacgtgt ataccccggt gaccgcgaat attctggatg ataacgtgta
cgatatccag 1200aacggcttta acatcccgaa aagcaacctg aacgttctgt ttatgggcca
gaacctgagc 1260cgtaatccgg cgctgcgtaa agtgaacccg gaaaacatgc tgtacctgtt
caccaaattt 1320tgcgtcgac
132961263DNAArtificial SequenceSynthetic 6ctgcagtgtc
gtgaactgct ggtgaaaaac accgatctgc cgtttattgg cgatatcagc 60gatgtgaaaa
ccgatatctt cctgcgcaaa gatatcaacg aagaaaccga agtgatctac 120tacccggata
acgtgagcgt tgatcaggtg atcctgagca aaaacaccag cgaacatggt 180cagctggatc
tgctgtatcc gagcattgat agcgaaagcg aaattctgcc gggcgaaaac 240caggtgtttt
acgataaccg tacccagaac gtggattacc tgaacagcta ttactacctg 300gaaagccaga
aactgagcga taacgtggaa gattttacct ttacccgcag cattgaagaa 360gcgctggata
acagcgcgaa agtttacacc tattttccga ccctggcgaa caaagttaat 420gcgggtgttc
agggcggtct gtttctgatg tgggcgaacg atgtggtgga agatttcacc 480accaacatcc
tgcgtaaaga taccctggat aaaatcagcg atgttagcgc gattattccg 540tatattggtc
cggcgctgaa cattagcaat agcgtgcgtc gtggcaattt taccgaagcg 600tttgcggtta
ccggtgtgac cattctgctg gaagcgtttc cggaatttac cattccggcg 660ctgggtgcgt
ttgtgatcta tagcaaagtg caggaacgca acgaaatcat caaaaccatc 720gataactgcc
tggaacagcg tattaaacgc tggaaagata gctatgaatg gatgatgggc 780acctggctga
gccgtattat cacccagttc aacaacatca gctaccagat gtacgatagc 840ctgaactatc
aggcgggtgc gattaaagcg aaaatcgatc tggaatacaa aaaatacagc 900ggcagcgata
aagaaaacat caaaagccag gttgaaaacc tgaaaaacag cctggatgtg 960aaaattagcg
aagcgatgaa taacatcaac aaattcatcc gcgaatgcag cgtgacctac 1020ctgttcaaaa
acatgctgcc gaaagtgatc gatgaactga acgaatttga tcgcaacacc 1080aaagcgaaac
tgatcaacct gatcgatagc cacaacatta ttctggtggg cgaagtggat 1140aaactgaaag
cgaaagttaa caacagcttc cagaacacca tcccgtttaa catcttcagc 1200tataccaaca
acagcctgct gaaagatatc atcaacgaat acttcaatct agactagaag 1260ctt
12637207DNAArtificial SequenceSynthetic 7ggatccacgc acgtcgacgg catcattacc
tccaaaacta aatctctgat cgaaggtcgt 60tttggcggtt tcacgggcgc acgcaaatca
gcgcgtaaat tagctaacca ggcgctagcg 120ggcggtggcg gtagcggcgg tggcggtagc
ggcggtggcg gtagcgcact agtgctgcag 180acgcacggtc tagaatgata aaagctt
2078108DNAArtificial SequenceSynthetic
8ggatccacgc acgtcgacgg catcattacc tccaaaacta aatctctgat agaaggtaga
60aacaaagcgc tgaacctgca gacgcacggt ctagaatgat aaaagctt
1089186DNAArtificial SequenceSynthetic 9catatgaata acctcgggat tgagggtcgt
tttggcggtt tcacgggcgc acgcaaatca 60gcgcgtaaat tagctaacca gactagtggc
ggtgggggta gtggcggtgg cggttcgggc 120gggggtggga gccctagggg atccgtcgac
ctgcagggtc tagaagcgct agcgtgataa 180aagctt
18610180DNAArtificial SequenceSynthetic
10ggatccacgc acgtcgacgc gattgatggt cgttttggcg gtttcacggg cgcacgcaaa
60tcagcgcgta aattagctaa ccaggcgcta gcgggcggtg gcggtagcgg cggtggcggt
120agcggcggtg gcggtagcgc actagtgctg cagacgcacg gtctagaatg ataaaagctt
18011249DNAArtificial SequenceSynthetic 11ggatccacgc acgtcgacgg
catcattacc tccaaaacta aatctctgat cgaaggtcgt 60tacggtggtt tcatgacctc
tgaaaaatct cagaccccgc tggttaccct gttcaaaaac 120gctatcatca aaaacgctta
caaaaaaggt gaagcgctag cgggtggtgg tggttctggt 180ggtggtggtt ctggtggtgg
tggttctgca ctagtgctgc agacgcacgg tctagaatga 240taaaagctt
24912207DNAArtificial
SequenceSynthetic 12ggatccacgc acgtcgacgg catcattacc tccaaaacta
aatctctgat cgaaggtcgt 60tttggcggtt tcacgggcgc acgcaaatca gcgcgtaaac
gtaagaacca ggcgctagcg 120ggcggtggcg gtagcggcgg tggcggtagc ggcggtggcg
gtagcgcact agtgctgcag 180acgcacggtc tagaatgata aaagctt
207132709DNAArtificial SequenceSynthetic
13ggatccatgg agttcgttaa caaacagttc aactataaag acccagttaa cggtgttgac
60attgcttaca tcaaaatccc gaacgctggc cagatgcagc cggtaaaggc attcaaaatc
120cacaacaaaa tctgggttat cccggaacgt gataccttta ctaacccgga agaaggtgac
180ctgaacccgc caccggaagc gaaacaggtg ccggtatctt actatgactc cacctacctg
240tctaccgata acgaaaagga caactacctg aaaggtgtta ctaaactgtt cgagcgtatt
300tactccaccg acctgggccg tatgctgctg actagcatcg ttcgcggtat cccgttctgg
360ggcggttcta ccatcgatac cgaactgaaa gtaatcgaca ctaactgcat caacgttatt
420cagccggacg gttcctatcg ttccgaagaa ctgaacctgg tgatcatcgg cccgtctgct
480gatatcatcc agttcgagtg taagagcttt ggtcacgaag ttctgaacct cacccgtaac
540ggctacggtt ccactcagta catccgtttc tctccggact tcaccttcgg ttttgaagaa
600tccctggaag tagacacgaa cccactgctg ggcgctggta aattcgcaac tgatcctgcg
660gttaccctgg ctcacgaact gattcatgca ggccaccgcc tgtacggtat cgccatcaat
720ccgaaccgtg tcttcaaagt taacaccaac gcgtattacg agatgtccgg tctggaagtt
780agcttcgaag aactgcgtac ttttggcggt cacgacgcta aattcatcga ctctctgcaa
840gaaaacgagt tccgtctgta ctactataac aagttcaaag atatcgcatc caccctgaac
900aaagcgaaat ccatcgtggg taccactgct tctctccagt acatgaagaa cgtttttaaa
960gaaaaatacc tgctcagcga agacacctcc ggcaaattct ctgtagacaa gttgaaattc
1020gataaacttt acaaaatgct gactgaaatt tacaccgaag acaacttcgt taagttcttt
1080aaagttctga accgcaaaac ctatctgaac ttcgacaagg cagtattcaa aatcaacatc
1140gtgccgaaag ttaactacac tatctacgat ggtttcaacc tgcgtaacac caacctggct
1200gctaatttta acggccagaa cacggaaatc aacaacatga acttcacaaa actgaaaaac
1260ttcactggtc tgttcgagtt ttacaagctg ctgtgcgtcg acggcatcat tacctccaaa
1320actaaatctc tgatagaagg tagatttggc ggtttcacgg gcgcacgcaa atcagcgcgt
1380aaattagcta accaggcgct agcgggcggt ggcggtagcg gcggtggcgg tagcggcggt
1440ggcggtagcg cactagtgct gcagtgtatc aaggttaaca actgggattt attcttcagc
1500ccgagtgaag acaacttcac caacgacctg aacaaaggtg aagaaatcac ctcagatact
1560aacatcgaag cagccgaaga aaacatctcg ctggacctga tccagcagta ctacctgacc
1620tttaatttcg acaacgagcc ggaaaacatt tctatcgaaa acctgagctc tgatatcatc
1680ggccagctgg aactgatgcc gaacatcgaa cgtttcccaa acggtaaaaa gtacgagctg
1740gacaaatata ccatgttcca ctacctgcgc gcgcaggaat ttgaacacgg caaatcccgt
1800atcgcactga ctaactccgt taacgaagct ctgctcaacc cgtcccgtgt atacaccttc
1860ttctctagcg actacgtgaa aaaggtcaac aaagcgactg aagctgcaat gttcttgggt
1920tgggttgaac agcttgttta tgattttacc gacgagacgt ccgaagtatc tactaccgac
1980aaaattgcgg atatcactat catcatcccg tacatcggtc cggctctgaa cattggcaac
2040atgctgtaca aagacgactt cgttggcgca ctgatcttct ccggtgcggt gatcctgctg
2100gagttcatcc cggaaatcgc catcccggta ctgggcacct ttgctctggt ttcttacatt
2160gcaaacaagg ttctgactgt acaaaccatc gacaacgcgc tgagcaaacg taacgaaaaa
2220tgggatgaag tttacaaata tatcgtgacc aactggctgg ctaaggttaa tactcagatc
2280gacctcatcc gcaaaaaaat gaaagaagca ctggaaaacc aggcggaagc taccaaggca
2340atcattaact accagtacaa ccagtacacc gaggaagaaa aaaacaacat caacttcaac
2400atcgacgatc tgtcctctaa actgaacgaa tccatcaaca aagctatgat caacatcaac
2460aagttcctga accagtgctc tgtaagctat ctgatgaact ccatgatccc gtacggtgtt
2520aaacgtctgg aggacttcga tgcgtctctg aaagacgccc tgctgaaata catttacgac
2580aaccgtggca ctctgatcgg tcaggttgat cgtctgaagg acaaagtgaa caatacctta
2640tcgaccgaca tcccttttca gctcagtaaa tatgtcgata accaacgcct tttgtccact
2700ctagactag
270914902PRTArtificial SequenceSynthetic 14Gly Ser Met Glu Phe Val Asn
Lys Gln Phe Asn Tyr Lys Asp Pro Val1 5 10
15Asn Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala
Gly Gln Met 20 25 30Gln Pro
Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro 35
40 45Glu Arg Asp Thr Phe Thr Asn Pro Glu Glu
Gly Asp Leu Asn Pro Pro 50 55 60Pro
Glu Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu65
70 75 80Ser Thr Asp Asn Glu Lys
Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu 85
90 95Phe Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met
Leu Leu Thr Ser 100 105 110Ile
Val Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu 115
120 125Leu Lys Val Ile Asp Thr Asn Cys Ile
Asn Val Ile Gln Pro Asp Gly 130 135
140Ser Tyr Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala145
150 155 160Asp Ile Ile Gln
Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn 165
170 175Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln
Tyr Ile Arg Phe Ser Pro 180 185
190Asp Phe Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro
195 200 205Leu Leu Gly Ala Gly Lys Phe
Ala Thr Asp Pro Ala Val Thr Leu Ala 210 215
220His Glu Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile
Asn225 230 235 240Pro Asn
Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser
245 250 255Gly Leu Glu Val Ser Phe Glu
Glu Leu Arg Thr Phe Gly Gly His Asp 260 265
270Ala Lys Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu
Tyr Tyr 275 280 285Tyr Asn Lys Phe
Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser 290
295 300Ile Val Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys
Asn Val Phe Lys305 310 315
320Glu Lys Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp
325 330 335Lys Leu Lys Phe Asp
Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr 340
345 350Glu Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn
Arg Lys Thr Tyr 355 360 365Leu Asn
Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val 370
375 380Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg
Asn Thr Asn Leu Ala385 390 395
400Ala Asn Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr
405 410 415Lys Leu Lys Asn
Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys 420
425 430Val Asp Gly Ile Ile Thr Ser Lys Thr Lys Ser
Leu Ile Glu Gly Arg 435 440 445Phe
Gly Gly Phe Thr Gly Ala Arg Lys Ser Ala Arg Lys Leu Ala Asn 450
455 460Gln Ala Leu Ala Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly465 470 475
480Gly Gly Ser Ala Leu Val Leu Gln Cys Ile Lys Val Asn Asn Trp
Asp 485 490 495Leu Phe Phe
Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys 500
505 510Gly Glu Glu Ile Thr Ser Asp Thr Asn Ile
Glu Ala Ala Glu Glu Asn 515 520
525Ile Ser Leu Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp 530
535 540Asn Glu Pro Glu Asn Ile Ser Ile
Glu Asn Leu Ser Ser Asp Ile Ile545 550
555 560Gly Gln Leu Glu Leu Met Pro Asn Ile Glu Arg Phe
Pro Asn Gly Lys 565 570
575Lys Tyr Glu Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln
580 585 590Glu Phe Glu His Gly Lys
Ser Arg Ile Ala Leu Thr Asn Ser Val Asn 595 600
605Glu Ala Leu Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser
Ser Asp 610 615 620Tyr Val Lys Lys Val
Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly625 630
635 640Trp Val Glu Gln Leu Val Tyr Asp Phe Thr
Asp Glu Thr Ser Glu Val 645 650
655Ser Thr Thr Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile
660 665 670Gly Pro Ala Leu Asn
Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val 675
680 685Gly Ala Leu Ile Phe Ser Gly Ala Val Ile Leu Leu
Glu Phe Ile Pro 690 695 700Glu Ile Ala
Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile705
710 715 720Ala Asn Lys Val Leu Thr Val
Gln Thr Ile Asp Asn Ala Leu Ser Lys 725
730 735Arg Asn Glu Lys Trp Asp Glu Val Tyr Lys Tyr Ile
Val Thr Asn Trp 740 745 750Leu
Ala Lys Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys 755
760 765Glu Ala Leu Glu Asn Gln Ala Glu Ala
Thr Lys Ala Ile Ile Asn Tyr 770 775
780Gln Tyr Asn Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn785
790 795 800Ile Asp Asp Leu
Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met 805
810 815Ile Asn Ile Asn Lys Phe Leu Asn Gln Cys
Ser Val Ser Tyr Leu Met 820 825
830Asn Ser Met Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala
835 840 845Ser Leu Lys Asp Ala Leu Leu
Lys Tyr Ile Tyr Asp Asn Arg Gly Thr 850 855
860Leu Ile Gly Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr
Leu865 870 875 880Ser Thr
Asp Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg
885 890 895Leu Leu Ser Thr Leu Asp
900152736DNAArtificial SequenceSynthetic 15ctcgggattg agggtcgttt
tggcggtttc acgggcgcac gcaaatcagc gcgtaaatta 60gctaaccaga ctagtggcgg
tgggggtagt ggcggtggcg gttcgggcgg gggtgggagc 120cctaggggat ccatggagtt
cgttaacaaa cagttcaact ataaagaccc agttaacggt 180gttgacattg cttacatcaa
aatcccgaac gctggccaga tgcagccggt aaaggcattc 240aaaatccaca acaaaatctg
ggttatcccg gaacgtgata cctttactaa cccggaagaa 300ggtgacctga acccgccacc
ggaagcgaaa caggtgccgg tatcttacta tgactccacc 360tacctgtcta ccgataacga
aaaggacaac tacctgaaag gtgttactaa actgttcgag 420cgtatttact ccaccgacct
gggccgtatg ctgctgacta gcatcgttcg cggtatcccg 480ttctggggcg gttctaccat
cgataccgaa ctgaaagtaa tcgacactaa ctgcatcaac 540gttattcagc cggacggttc
ctatcgttcc gaagaactga acctggtgat catcggcccg 600tctgctgata tcatccagtt
cgagtgtaag agctttggtc acgaagttct gaacctcacc 660cgtaacggct acggttccac
tcagtacatc cgtttctctc cggacttcac cttcggtttt 720gaagaatccc tggaagtaga
cacgaaccca ctgctgggcg ctggtaaatt cgcaactgat 780cctgcggtta ccctggctca
cgaactgatt catgcaggcc accgcctgta cggtatcgcc 840atcaatccga accgtgtctt
caaagttaac accaacgcgt attacgagat gtccggtctg 900gaagttagct tcgaagaact
gcgtactttt ggcggtcacg acgctaaatt catcgactct 960ctgcaagaaa acgagttccg
tctgtactac tataacaagt tcaaagatat cgcatccacc 1020ctgaacaaag cgaaatccat
cgtgggtacc actgcttctc tccagtacat gaagaacgtt 1080tttaaagaaa aatacctgct
cagcgaagac acctccggca aattctctgt agacaagttg 1140aaattcgata aactttacaa
aatgctgact gaaatttaca ccgaagacaa cttcgttaag 1200ttctttaaag ttctgaaccg
caaaacctat ctgaacttcg acaaggcagt attcaaaatc 1260aacatcgtgc cgaaagttaa
ctacactatc tacgatggtt tcaacctgcg taacaccaac 1320ctggctgcta attttaacgg
ccagaacacg gaaatcaaca acatgaactt cacaaaactg 1380aaaaacttca ctggtctgtt
cgagttttac aagctgctgt gcgtcgacgg catcattacc 1440tccaaaacta aatctctgat
agaaggtaga aacaaagcgc tgaacgacct ctgtatcaag 1500gttaacaact gggatttatt
cttcagcccg agtgaagaca acttcaccaa cgacctgaac 1560aaaggtgaag aaatcacctc
agatactaac atcgaagcag ccgaagaaaa catctcgctg 1620gacctgatcc agcagtacta
cctgaccttt aatttcgaca acgagccgga aaacatttct 1680atcgaaaacc tgagctctga
tatcatcggc cagctggaac tgatgccgaa catcgaacgt 1740ttcccaaacg gtaaaaagta
cgagctggac aaatatacca tgttccacta cctgcgcgcg 1800caggaatttg aacacggcaa
atcccgtatc gcactgacta actccgttaa cgaagctctg 1860ctcaacccgt cccgtgtata
caccttcttc tctagcgact acgtgaaaaa ggtcaacaaa 1920gcgactgaag ctgcaatgtt
cttgggttgg gttgaacagc ttgtttatga ttttaccgac 1980gagacgtccg aagtatctac
taccgacaaa attgcggata tcactatcat catcccgtac 2040atcggtccgg ctctgaacat
tggcaacatg ctgtacaaag acgacttcgt tggcgcactg 2100atcttctccg gtgcggtgat
cctgctggag ttcatcccgg aaatcgccat cccggtactg 2160ggcacctttg ctctggtttc
ttacattgca aacaaggttc tgactgtaca aaccatcgac 2220aacgcgctga gcaaacgtaa
cgaaaaatgg gatgaagttt acaaatatat cgtgaccaac 2280tggctggcta aggttaatac
tcagatcgac ctcatccgca aaaaaatgaa agaagcactg 2340gaaaaccagg cggaagctac
caaggcaatc attaactacc agtacaacca gtacaccgag 2400gaagaaaaaa acaacatcaa
cttcaacatc gacgatctgt cctctaaact gaacgaatcc 2460atcaacaaag ctatgatcaa
catcaacaag ttcctgaacc agtgctctgt aagctatctg 2520atgaactcca tgatcccgta
cggtgttaaa cgtctggagg acttcgatgc gtctctgaaa 2580gacgccctgc tgaaatacat
ttacgacaac cgtggcactc tgatcggtca ggttgatcgt 2640ctgaaggaca aagtgaacaa
taccttatcg accgacatcc cttttcagct cagtaaatat 2700gtcgataacc aacgcctttt
gtccactcta gactag 273616911PRTArtificial
SequenceSynthetic 16Leu Gly Ile Glu Gly Arg Phe Gly Gly Phe Thr Gly Ala
Arg Lys Ser1 5 10 15Ala
Arg Lys Leu Ala Asn Gln Thr Ser Gly Gly Gly Gly Ser Gly Gly 20
25 30Gly Gly Ser Gly Gly Gly Gly Ser
Pro Arg Gly Ser Met Glu Phe Val 35 40
45Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly Val Asp Ile Ala
50 55 60Tyr Ile Lys Ile Pro Asn Ala Gly
Gln Met Gln Pro Val Lys Ala Phe65 70 75
80Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg Asp
Thr Phe Thr 85 90 95Asn
Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu Ala Lys Gln Val
100 105 110Pro Val Ser Tyr Tyr Asp Ser
Thr Tyr Leu Ser Thr Asp Asn Glu Lys 115 120
125Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu Arg Ile Tyr
Ser 130 135 140Thr Asp Leu Gly Arg Met
Leu Leu Thr Ser Ile Val Arg Gly Ile Pro145 150
155 160Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu
Lys Val Ile Asp Thr 165 170
175Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr Arg Ser Glu Glu
180 185 190Leu Asn Leu Val Ile Ile
Gly Pro Ser Ala Asp Ile Ile Gln Phe Glu 195 200
205Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr Arg Asn
Gly Tyr 210 215 220Gly Ser Thr Gln Tyr
Ile Arg Phe Ser Pro Asp Phe Thr Phe Gly Phe225 230
235 240Glu Glu Ser Leu Glu Val Asp Thr Asn Pro
Leu Leu Gly Ala Gly Lys 245 250
255Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu Leu Ile His Ala
260 265 270Gly His Arg Leu Tyr
Gly Ile Ala Ile Asn Pro Asn Arg Val Phe Lys 275
280 285Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu
Glu Val Ser Phe 290 295 300Glu Glu Leu
Arg Thr Phe Gly Gly His Asp Ala Lys Phe Ile Asp Ser305
310 315 320Leu Gln Glu Asn Glu Phe Arg
Leu Tyr Tyr Tyr Asn Lys Phe Lys Asp 325
330 335Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val
Gly Thr Thr Ala 340 345 350Ser
Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys Tyr Leu Leu Ser 355
360 365Glu Asp Thr Ser Gly Lys Phe Ser Val
Asp Lys Leu Lys Phe Asp Lys 370 375
380Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp Asn Phe Val Lys385
390 395 400Phe Phe Lys Val
Leu Asn Arg Lys Thr Tyr Leu Asn Phe Asp Lys Ala 405
410 415Val Phe Lys Ile Asn Ile Val Pro Lys Val
Asn Tyr Thr Ile Tyr Asp 420 425
430Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn Phe Asn Gly Gln
435 440 445Asn Thr Glu Ile Asn Asn Met
Asn Phe Thr Lys Leu Lys Asn Phe Thr 450 455
460Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Asp Gly Ile Ile
Thr465 470 475 480Ser Lys
Thr Lys Ser Leu Ile Glu Gly Arg Asn Lys Ala Leu Asn Asp
485 490 495Leu Cys Ile Lys Val Asn Asn
Trp Asp Leu Phe Phe Ser Pro Ser Glu 500 505
510Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu Ile Thr
Ser Asp 515 520 525Thr Asn Ile Glu
Ala Ala Glu Glu Asn Ile Ser Leu Asp Leu Ile Gln 530
535 540Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro
Glu Asn Ile Ser545 550 555
560Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu Glu Leu Met Pro
565 570 575Asn Ile Glu Arg Phe
Pro Asn Gly Lys Lys Tyr Glu Leu Asp Lys Tyr 580
585 590Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu
His Gly Lys Ser 595 600 605Arg Ile
Ala Leu Thr Asn Ser Val Asn Glu Ala Leu Leu Asn Pro Ser 610
615 620Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val
Lys Lys Val Asn Lys625 630 635
640Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu Gln Leu Val Tyr
645 650 655Asp Phe Thr Asp
Glu Thr Ser Glu Val Ser Thr Thr Asp Lys Ile Ala 660
665 670Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro
Ala Leu Asn Ile Gly 675 680 685Asn
Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu Ile Phe Ser Gly 690
695 700Ala Val Ile Leu Leu Glu Phe Ile Pro Glu
Ile Ala Ile Pro Val Leu705 710 715
720Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys Val Leu Thr
Val 725 730 735Gln Thr Ile
Asp Asn Ala Leu Ser Lys Arg Asn Glu Lys Trp Asp Glu 740
745 750Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu
Ala Lys Val Asn Thr Gln 755 760
765Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu Glu Asn Gln Ala 770
775 780Glu Ala Thr Lys Ala Ile Ile Asn
Tyr Gln Tyr Asn Gln Tyr Thr Glu785 790
795 800Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp
Leu Ser Ser Lys 805 810
815Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile Asn Lys Phe Leu
820 825 830Asn Gln Cys Ser Val Ser
Tyr Leu Met Asn Ser Met Ile Pro Tyr Gly 835 840
845Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys Asp Ala
Leu Leu 850 855 860Lys Tyr Ile Tyr Asp
Asn Arg Gly Thr Leu Ile Gly Gln Val Asp Arg865 870
875 880Leu Lys Asp Lys Val Asn Asn Thr Leu Ser
Thr Asp Ile Pro Phe Gln 885 890
895Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser Thr Leu Asp
900 905 910172715DNAArtificial
SequenceSynthetic 17ggatccgaat tcatgccgat caccatcaac aacttcaact
acagcgatcc ggtggataac 60aaaaacatcc tgtacctgga tacccatctg aataccctgg
cgaacgaacc ggaaaaagcg 120tttcgtatca ccggcaacat ttgggttatt ccggatcgtt
ttagccgtaa cagcaacccg 180aatctgaata aaccgccgcg tgttaccagc ccgaaaagcg
gttattacga tccgaactat 240ctgagcaccg atagcgataa agataccttc ctgaaagaaa
tcatcaaact gttcaaacgc 300atcaacagcc gtgaaattgg cgaagaactg atctatcgcc
tgagcaccga tattccgttt 360ccgggcaaca acaacacccc gatcaacacc tttgatttcg
atgtggattt caacagcgtt 420gatgttaaaa cccgccaggg taacaattgg gtgaaaaccg
gcagcattaa cccgagcgtg 480attattaccg gtccgcgcga aaacattatt gatccggaaa
ccagcacctt taaactgacc 540aacaacacct ttgcggcgca ggaaggtttt ggcgcgctga
gcattattag cattagcccg 600cgctttatgc tgacctatag caacgcgacc aacgatgttg
gtgaaggccg tttcagcaaa 660agcgaatttt gcatggaccc gatcctgatc ctgatgcatg
aactgaacca tgcgatgcat 720aacctgtatg gcatcgcgat tccgaacgat cagaccatta
gcagcgtgac cagcaacatc 780ttttacagcc agtacaacgt gaaactggaa tatgcggaaa
tctatgcgtt tggcggtccg 840accattgatc tgattccgaa aagcgcgcgc aaatacttcg
aagaaaaagc gctggattac 900tatcgcagca ttgcgaaacg tctgaacagc attaccaccg
cgaatccgag cagcttcaac 960aaatatatcg gcgaatataa acagaaactg atccgcaaat
atcgctttgt ggtggaaagc 1020agcggcgaag ttaccgttaa ccgcaataaa ttcgtggaac
tgtacaacga actgacccag 1080atcttcaccg aatttaacta tgcgaaaatc tataacgtgc
agaaccgtaa aatctacctg 1140agcaacgtgt ataccccggt gaccgcgaat attctggatg
ataacgtgta cgatatccag 1200aacggcttta acatcccgaa aagcaacctg aacgttctgt
ttatgggcca gaacctgagc 1260cgtaatccgg cgctgcgtaa agtgaacccg gaaaacatgc
tgtacctgtt caccaaattt 1320tgcgtcgacg cgatagatgg tagatttggc ggtttcacgg
gcgcacgcaa atcagcgcgt 1380aaattagcta accaggcgct agcgggcggt ggcggtagcg
gcggtggcgg tagcggcggt 1440ggcggtagcg cactagtgct gcagtgtcgt gaactgctgg
tgaaaaacac cgatctgccg 1500tttattggcg atatcagcga tgtgaaaacc gatatcttcc
tgcgcaaaga tatcaacgaa 1560gaaaccgaag tgatctacta cccggataac gtgagcgttg
atcaggtgat cctgagcaaa 1620aacaccagcg aacatggtca gctggatctg ctgtatccga
gcattgatag cgaaagcgaa 1680attctgccgg gcgaaaacca ggtgttttac gataaccgta
cccagaacgt ggattacctg 1740aacagctatt actacctgga aagccagaaa ctgagcgata
acgtggaaga ttttaccttt 1800acccgcagca ttgaagaagc gctggataac agcgcgaaag
tttacaccta ttttccgacc 1860ctggcgaaca aagttaatgc gggtgttcag ggcggtctgt
ttctgatgtg ggcgaacgat 1920gtggtggaag atttcaccac caacatcctg cgtaaagata
ccctggataa aatcagcgat 1980gttagcgcga ttattccgta tattggtccg gcgctgaaca
ttagcaatag cgtgcgtcgt 2040ggcaatttta ccgaagcgtt tgcggttacc ggtgtgacca
ttctgctgga agcgtttccg 2100gaatttacca ttccggcgct gggtgcgttt gtgatctata
gcaaagtgca ggaacgcaac 2160gaaatcatca aaaccatcga taactgcctg gaacagcgta
ttaaacgctg gaaagatagc 2220tatgaatgga tgatgggcac ctggctgagc cgtattatca
cccagttcaa caacatcagc 2280taccagatgt acgatagcct gaactatcag gcgggtgcga
ttaaagcgaa aatcgatctg 2340gaatacaaaa aatacagcgg cagcgataaa gaaaacatca
aaagccaggt tgaaaacctg 2400aaaaacagcc tggatgtgaa aattagcgaa gcgatgaata
acatcaacaa attcatccgc 2460gaatgcagcg tgacctacct gttcaaaaac atgctgccga
aagtgatcga tgaactgaac 2520gaatttgatc gcaacaccaa agcgaaactg atcaacctga
tcgatagcca caacattatt 2580ctggtgggcg aagtggataa actgaaagcg aaagttaaca
acagcttcca gaacaccatc 2640ccgtttaaca tcttcagcta taccaacaac agcctgctga
aagatatcat caacgaatac 2700ttcaatctag actag
271518904PRTArtificial SequenceSynthetic 18Gly Ser
Glu Phe Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp1 5
10 15Pro Val Asp Asn Lys Asn Ile Leu
Tyr Leu Asp Thr His Leu Asn Thr 20 25
30Leu Ala Asn Glu Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile
Trp 35 40 45Val Ile Pro Asp Arg
Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys 50 55
60Pro Pro Arg Val Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro
Asn Tyr65 70 75 80Leu
Ser Thr Asp Ser Asp Lys Asp Thr Phe Leu Lys Glu Ile Ile Lys
85 90 95Leu Phe Lys Arg Ile Asn Ser
Arg Glu Ile Gly Glu Glu Leu Ile Tyr 100 105
110Arg Leu Ser Thr Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr
Pro Ile 115 120 125Asn Thr Phe Asp
Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr 130
135 140Arg Gln Gly Asn Asn Trp Val Lys Thr Gly Ser Ile
Asn Pro Ser Val145 150 155
160Ile Ile Thr Gly Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr
165 170 175Phe Lys Leu Thr Asn
Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala 180
185 190Leu Ser Ile Ile Ser Ile Ser Pro Arg Phe Met Leu
Thr Tyr Ser Asn 195 200 205Ala Thr
Asn Asp Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys 210
215 220Met Asp Pro Ile Leu Ile Leu Met His Glu Leu
Asn His Ala Met His225 230 235
240Asn Leu Tyr Gly Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val
245 250 255Thr Ser Asn Ile
Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala 260
265 270Glu Ile Tyr Ala Phe Gly Gly Pro Thr Ile Asp
Leu Ile Pro Lys Ser 275 280 285Ala
Arg Lys Tyr Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile 290
295 300Ala Lys Arg Leu Asn Ser Ile Thr Thr Ala
Asn Pro Ser Ser Phe Asn305 310 315
320Lys Tyr Ile Gly Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg
Phe 325 330 335Val Val Glu
Ser Ser Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val 340
345 350Glu Leu Tyr Asn Glu Leu Thr Gln Ile Phe
Thr Glu Phe Asn Tyr Ala 355 360
365Lys Ile Tyr Asn Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr 370
375 380Thr Pro Val Thr Ala Asn Ile Leu
Asp Asp Asn Val Tyr Asp Ile Gln385 390
395 400Asn Gly Phe Asn Ile Pro Lys Ser Asn Leu Asn Val
Leu Phe Met Gly 405 410
415Gln Asn Leu Ser Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn
420 425 430Met Leu Tyr Leu Phe Thr
Lys Phe Cys Val Asp Ala Ile Asp Gly Arg 435 440
445Phe Gly Gly Phe Thr Gly Ala Arg Lys Ser Ala Arg Lys Leu
Ala Asn 450 455 460Gln Ala Leu Ala Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly465 470
475 480Gly Gly Ser Ala Leu Val Leu Gln Cys Arg
Glu Leu Leu Val Lys Asn 485 490
495Thr Asp Leu Pro Phe Ile Gly Asp Ile Ser Asp Val Lys Thr Asp Ile
500 505 510Phe Leu Arg Lys Asp
Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr Pro 515
520 525Asp Asn Val Ser Val Asp Gln Val Ile Leu Ser Lys
Asn Thr Ser Glu 530 535 540His Gly Gln
Leu Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu545
550 555 560Ile Leu Pro Gly Glu Asn Gln
Val Phe Tyr Asp Asn Arg Thr Gln Asn 565
570 575Val Asp Tyr Leu Asn Ser Tyr Tyr Tyr Leu Glu Ser
Gln Lys Leu Ser 580 585 590Asp
Asn Val Glu Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala Leu 595
600 605Asp Asn Ser Ala Lys Val Tyr Thr Tyr
Phe Pro Thr Leu Ala Asn Lys 610 615
620Val Asn Ala Gly Val Gln Gly Gly Leu Phe Leu Met Trp Ala Asn Asp625
630 635 640Val Val Glu Asp
Phe Thr Thr Asn Ile Leu Arg Lys Asp Thr Leu Asp 645
650 655Lys Ile Ser Asp Val Ser Ala Ile Ile Pro
Tyr Ile Gly Pro Ala Leu 660 665
670Asn Ile Ser Asn Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala
675 680 685Val Thr Gly Val Thr Ile Leu
Leu Glu Ala Phe Pro Glu Phe Thr Ile 690 695
700Pro Ala Leu Gly Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg
Asn705 710 715 720Glu Ile
Ile Lys Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg
725 730 735Trp Lys Asp Ser Tyr Glu Trp
Met Met Gly Thr Trp Leu Ser Arg Ile 740 745
750Ile Thr Gln Phe Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser
Leu Asn 755 760 765Tyr Gln Ala Gly
Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys 770
775 780Tyr Ser Gly Ser Asp Lys Glu Asn Ile Lys Ser Gln
Val Glu Asn Leu785 790 795
800Lys Asn Ser Leu Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile Asn
805 810 815Lys Phe Ile Arg Glu
Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu 820
825 830Pro Lys Val Ile Asp Glu Leu Asn Glu Phe Asp Arg
Asn Thr Lys Ala 835 840 845Lys Leu
Ile Asn Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly Glu 850
855 860Val Asp Lys Leu Lys Ala Lys Val Asn Asn Ser
Phe Gln Asn Thr Ile865 870 875
880Pro Phe Asn Ile Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp Ile
885 890 895Ile Asn Glu Tyr
Phe Asn Leu Asp 900192742DNAArtificial SequenceSynthetic
19ggatccgaat tcatgccgat caccatcaac aacttcaact acagcgatcc ggtggataac
60aaaaacatcc tgtacctgga tacccatctg aataccctgg cgaacgaacc ggaaaaagcg
120tttcgtatca ccggcaacat ttgggttatt ccggatcgtt ttagccgtaa cagcaacccg
180aatctgaata aaccgccgcg tgttaccagc ccgaaaagcg gttattacga tccgaactat
240ctgagcaccg atagcgataa agataccttc ctgaaagaaa tcatcaaact gttcaaacgc
300atcaacagcc gtgaaattgg cgaagaactg atctatcgcc tgagcaccga tattccgttt
360ccgggcaaca acaacacccc gatcaacacc tttgatttcg atgtggattt caacagcgtt
420gatgttaaaa cccgccaggg taacaattgg gtgaaaaccg gcagcattaa cccgagcgtg
480attattaccg gtccgcgcga aaacattatt gatccggaaa ccagcacctt taaactgacc
540aacaacacct ttgcggcgca ggaaggtttt ggcgcgctga gcattattag cattagcccg
600cgctttatgc tgacctatag caacgcgacc aacgatgttg gtgaaggccg tttcagcaaa
660agcgaatttt gcatggaccc gatcctgatc ctgatgcatg aactgaacca tgcgatgcat
720aacctgtatg gcatcgcgat tccgaacgat cagaccatta gcagcgtgac cagcaacatc
780ttttacagcc agtacaacgt gaaactggaa tatgcggaaa tctatgcgtt tggcggtccg
840accattgatc tgattccgaa aagcgcgcgc aaatacttcg aagaaaaagc gctggattac
900tatcgcagca ttgcgaaacg tctgaacagc attaccaccg cgaatccgag cagcttcaac
960aaatatatcg gcgaatataa acagaaactg atccgcaaat atcgctttgt ggtggaaagc
1020agcggcgaag ttaccgttaa ccgcaataaa ttcgtggaac tgtacaacga actgacccag
1080atcttcaccg aatttaacta tgcgaaaatc tataacgtgc agaaccgtaa aatctacctg
1140agcaacgtgt ataccccggt gaccgcgaat attctggatg ataacgtgta cgatatccag
1200aacggcttta acatcccgaa aagcaacctg aacgttctgt ttatgggcca gaacctgagc
1260cgtaatccgg cgctgcgtaa agtgaacccg gaaaacatgc tgtacctgtt caccaaattt
1320tgcgtcgacg gcatcattac ctccaaaact aaatctctga tagaaggtag atttggcggt
1380ttcacgggcg cacgcaaatc agcgcgtaaa ttagctaacc aggcgctagc gggcggtggc
1440ggtagcggcg gtggcggtag cggcggtggc ggtagcgcac tagtgctgca gtgtcgtgaa
1500ctgctggtga aaaacaccga tctgccgttt attggcgata tcagcgatgt gaaaaccgat
1560atcttcctgc gcaaagatat caacgaagaa accgaagtga tctactaccc ggataacgtg
1620agcgttgatc aggtgatcct gagcaaaaac accagcgaac atggtcagct ggatctgctg
1680tatccgagca ttgatagcga aagcgaaatt ctgccgggcg aaaaccaggt gttttacgat
1740aaccgtaccc agaacgtgga ttacctgaac agctattact acctggaaag ccagaaactg
1800agcgataacg tggaagattt tacctttacc cgcagcattg aagaagcgct ggataacagc
1860gcgaaagttt acacctattt tccgaccctg gcgaacaaag ttaatgcggg tgttcagggc
1920ggtctgtttc tgatgtgggc gaacgatgtg gtggaagatt tcaccaccaa catcctgcgt
1980aaagataccc tggataaaat cagcgatgtt agcgcgatta ttccgtatat tggtccggcg
2040ctgaacatta gcaatagcgt gcgtcgtggc aattttaccg aagcgtttgc ggttaccggt
2100gtgaccattc tgctggaagc gtttccggaa tttaccattc cggcgctggg tgcgtttgtg
2160atctatagca aagtgcagga acgcaacgaa atcatcaaaa ccatcgataa ctgcctggaa
2220cagcgtatta aacgctggaa agatagctat gaatggatga tgggcacctg gctgagccgt
2280attatcaccc agttcaacaa catcagctac cagatgtacg atagcctgaa ctatcaggcg
2340ggtgcgatta aagcgaaaat cgatctggaa tacaaaaaat acagcggcag cgataaagaa
2400aacatcaaaa gccaggttga aaacctgaaa aacagcctgg atgtgaaaat tagcgaagcg
2460atgaataaca tcaacaaatt catccgcgaa tgcagcgtga cctacctgtt caaaaacatg
2520ctgccgaaag tgatcgatga actgaacgaa tttgatcgca acaccaaagc gaaactgatc
2580aacctgatcg atagccacaa cattattctg gtgggcgaag tggataaact gaaagcgaaa
2640gttaacaaca gcttccagaa caccatcccg tttaacatct tcagctatac caacaacagc
2700ctgctgaaag atatcatcaa cgaatacttc aatctagact ag
274220913PRTArtificial SequenceSynthetic 20Gly Ser Glu Phe Met Pro Ile
Thr Ile Asn Asn Phe Asn Tyr Ser Asp1 5 10
15Pro Val Asp Asn Lys Asn Ile Leu Tyr Leu Asp Thr His
Leu Asn Thr 20 25 30Leu Ala
Asn Glu Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp 35
40 45Val Ile Pro Asp Arg Phe Ser Arg Asn Ser
Asn Pro Asn Leu Asn Lys 50 55 60Pro
Pro Arg Val Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr65
70 75 80Leu Ser Thr Asp Ser Asp
Lys Asp Thr Phe Leu Lys Glu Ile Ile Lys 85
90 95Leu Phe Lys Arg Ile Asn Ser Arg Glu Ile Gly Glu
Glu Leu Ile Tyr 100 105 110Arg
Leu Ser Thr Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile 115
120 125Asn Thr Phe Asp Phe Asp Val Asp Phe
Asn Ser Val Asp Val Lys Thr 130 135
140Arg Gln Gly Asn Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val145
150 155 160Ile Ile Thr Gly
Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr 165
170 175Phe Lys Leu Thr Asn Asn Thr Phe Ala Ala
Gln Glu Gly Phe Gly Ala 180 185
190Leu Ser Ile Ile Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn
195 200 205Ala Thr Asn Asp Val Gly Glu
Gly Arg Phe Ser Lys Ser Glu Phe Cys 210 215
220Met Asp Pro Ile Leu Ile Leu Met His Glu Leu Asn His Ala Met
His225 230 235 240Asn Leu
Tyr Gly Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val
245 250 255Thr Ser Asn Ile Phe Tyr Ser
Gln Tyr Asn Val Lys Leu Glu Tyr Ala 260 265
270Glu Ile Tyr Ala Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro
Lys Ser 275 280 285Ala Arg Lys Tyr
Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile 290
295 300Ala Lys Arg Leu Asn Ser Ile Thr Thr Ala Asn Pro
Ser Ser Phe Asn305 310 315
320Lys Tyr Ile Gly Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe
325 330 335Val Val Glu Ser Ser
Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val 340
345 350Glu Leu Tyr Asn Glu Leu Thr Gln Ile Phe Thr Glu
Phe Asn Tyr Ala 355 360 365Lys Ile
Tyr Asn Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr 370
375 380Thr Pro Val Thr Ala Asn Ile Leu Asp Asp Asn
Val Tyr Asp Ile Gln385 390 395
400Asn Gly Phe Asn Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly
405 410 415Gln Asn Leu Ser
Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn 420
425 430Met Leu Tyr Leu Phe Thr Lys Phe Cys Val Asp
Gly Ile Ile Thr Ser 435 440 445Lys
Thr Lys Ser Leu Ile Glu Gly Arg Phe Gly Gly Phe Thr Gly Ala 450
455 460Arg Lys Ser Ala Arg Lys Leu Ala Asn Gln
Ala Leu Ala Gly Gly Gly465 470 475
480Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Val
Leu 485 490 495Gln Cys Arg
Glu Leu Leu Val Lys Asn Thr Asp Leu Pro Phe Ile Gly 500
505 510Asp Ile Ser Asp Val Lys Thr Asp Ile Phe
Leu Arg Lys Asp Ile Asn 515 520
525Glu Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser Val Asp Gln 530
535 540Val Ile Leu Ser Lys Asn Thr Ser
Glu His Gly Gln Leu Asp Leu Leu545 550
555 560Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro
Gly Glu Asn Gln 565 570
575Val Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu Asn Ser Tyr
580 585 590Tyr Tyr Leu Glu Ser Gln
Lys Leu Ser Asp Asn Val Glu Asp Phe Thr 595 600
605Phe Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser Ala Lys
Val Tyr 610 615 620Thr Tyr Phe Pro Thr
Leu Ala Asn Lys Val Asn Ala Gly Val Gln Gly625 630
635 640Gly Leu Phe Leu Met Trp Ala Asn Asp Val
Val Glu Asp Phe Thr Thr 645 650
655Asn Ile Leu Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp Val Ser Ala
660 665 670Ile Ile Pro Tyr Ile
Gly Pro Ala Leu Asn Ile Ser Asn Ser Val Arg 675
680 685Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly
Val Thr Ile Leu 690 695 700Leu Glu Ala
Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly Ala Phe Val705
710 715 720Ile Tyr Ser Lys Val Gln Glu
Arg Asn Glu Ile Ile Lys Thr Ile Asp 725
730 735Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp
Ser Tyr Glu Trp 740 745 750Met
Met Gly Thr Trp Leu Ser Arg Ile Ile Thr Gln Phe Asn Asn Ile 755
760 765Ser Tyr Gln Met Tyr Asp Ser Leu Asn
Tyr Gln Ala Gly Ala Ile Lys 770 775
780Ala Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser Asp Lys Glu785
790 795 800Asn Ile Lys Ser
Gln Val Glu Asn Leu Lys Asn Ser Leu Asp Val Lys 805
810 815Ile Ser Glu Ala Met Asn Asn Ile Asn Lys
Phe Ile Arg Glu Cys Ser 820 825
830Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile Asp Glu Leu
835 840 845Asn Glu Phe Asp Arg Asn Thr
Lys Ala Lys Leu Ile Asn Leu Ile Asp 850 855
860Ser His Asn Ile Ile Leu Val Gly Glu Val Asp Lys Leu Lys Ala
Lys865 870 875 880Val Asn
Asn Ser Phe Gln Asn Thr Ile Pro Phe Asn Ile Phe Ser Tyr
885 890 895Thr Asn Asn Ser Leu Leu Lys
Asp Ile Ile Asn Glu Tyr Phe Asn Leu 900 905
910Asp212673DNAArtificial SequenceSynthetic 21ggatccatgg
agttcgttaa caaacagttc aactataaag acccagttaa cggtgttgac 60attgcttaca
tcaaaatccc gaacgctggc cagatgcagc cggtaaaggc attcaaaatc 120cacaacaaaa
tctgggttat cccggaacgt gataccttta ctaacccgga agaaggtgac 180ctgaacccgc
caccggaagc gaaacaggtg ccggtatctt actatgactc cacctacctg 240tctaccgata
acgaaaagga caactacctg aaaggtgtta ctaaactgtt cgagcgtatt 300tactccaccg
acctgggccg tatgctgctg actagcatcg ttcgcggtat cccgttctgg 360ggcggttcta
ccatcgatac cgaactgaaa gtaatcgaca ctaactgcat caacgttatt 420cagccggacg
gttcctatcg ttccgaagaa ctgaacctgg tgatcatcgg cccgtctgct 480gatatcatcc
agttcgagtg taagagcttt ggtcacgaag ttctgaacct cacccgtaac 540ggctacggtt
ccactcagta catccgtttc tctccggact tcaccttcgg ttttgaagaa 600tccctggaag
tagacacgaa cccactgctg ggcgctggta aattcgcaac tgatcctgcg 660gttaccctgg
ctcacgaact gattcatgca ggccaccgcc tgtacggtat cgccatcaat 720ccgaaccgtg
tcttcaaagt taacaccaac gcgtattacg agatgtccgg tctggaagtt 780agcttcgaag
aactgcgtac ttttggcggt cacgacgcta aattcatcga ctctctgcaa 840gaaaacgagt
tccgtctgta ctactataac aagttcaaag atatcgcatc caccctgaac 900aaagcgaaat
ccatcgtggg taccactgct tctctccagt acatgaagaa cgtttttaaa 960gaaaaatacc
tgctcagcga agacacctcc ggcaaattct ctgtagacaa gttgaaattc 1020gataaacttt
acaaaatgct gactgaaatt tacaccgaag acaacttcgt taagttcttt 1080aaagttctga
accgcaaaac ctatctgaac ttcgacaagg cagtattcaa aatcaacatc 1140gtgccgaaag
ttaactacac tatctacgat ggtttcaacc tgcgtaacac caacctggct 1200gctaatttta
acggccagaa cacggaaatc aacaacatga acttcacaaa actgaaaaac 1260ttcactggtc
tgttcgagtt ttacaagctg ctgtgcgtcg acggcatcat tacctccaaa 1320actaaatctc
tgatagaagg tagatacggt ggtttcatgg cgctagcggg cggtggcggt 1380agcggcggtg
gcggtagcgg cggtggcggt agcgcactag tgctgcagtg tatcaaggtt 1440aacaactggg
atttattctt cagcccgagt gaagacaact tcaccaacga cctgaacaaa 1500ggtgaagaaa
tcacctcaga tactaacatc gaagcagccg aagaaaacat ctcgctggac 1560ctgatccagc
agtactacct gacctttaat ttcgacaacg agccggaaaa catttctatc 1620gaaaacctga
gctctgatat catcggccag ctggaactga tgccgaacat cgaacgtttc 1680ccaaacggta
aaaagtacga gctggacaaa tataccatgt tccactacct gcgcgcgcag 1740gaatttgaac
acggcaaatc ccgtatcgca ctgactaact ccgttaacga agctctgctc 1800aacccgtccc
gtgtatacac cttcttctct agcgactacg tgaaaaaggt caacaaagcg 1860actgaagctg
caatgttctt gggttgggtt gaacagcttg tttatgattt taccgacgag 1920acgtccgaag
tatctactac cgacaaaatt gcggatatca ctatcatcat cccgtacatc 1980ggtccggctc
tgaacattgg caacatgctg tacaaagacg acttcgttgg cgcactgatc 2040ttctccggtg
cggtgatcct gctggagttc atcccggaaa tcgccatccc ggtactgggc 2100acctttgctc
tggtttctta cattgcaaac aaggttctga ctgtacaaac catcgacaac 2160gcgctgagca
aacgtaacga aaaatgggat gaagtttaca aatatatcgt gaccaactgg 2220ctggctaagg
ttaatactca gatcgacctc atccgcaaaa aaatgaaaga agcactggaa 2280aaccaggcgg
aagctaccaa ggcaatcatt aactaccagt acaaccagta caccgaggaa 2340gaaaaaaaca
acatcaactt caacatcgac gatctgtcct ctaaactgaa cgaatccatc 2400aacaaagcta
tgatcaacat caacaagttc ctgaaccagt gctctgtaag ctatctgatg 2460aactccatga
tcccgtacgg tgttaaacgt ctggaggact tcgatgcgtc tctgaaagac 2520gccctgctga
aatacattta cgacaaccgt ggcactctga tcggtcaggt tgatcgtctg 2580aaggacaaag
tgaacaatac cttatcgacc gacatccctt ttcagctcag taaatatgtc 2640gataaccaac
gccttttgtc cactctagac tag
267322890PRTArtificial SequenceSynthetic 22Gly Ser Met Glu Phe Val Asn
Lys Gln Phe Asn Tyr Lys Asp Pro Val1 5 10
15Asn Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala
Gly Gln Met 20 25 30Gln Pro
Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro 35
40 45Glu Arg Asp Thr Phe Thr Asn Pro Glu Glu
Gly Asp Leu Asn Pro Pro 50 55 60Pro
Glu Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu65
70 75 80Ser Thr Asp Asn Glu Lys
Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu 85
90 95Phe Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met
Leu Leu Thr Ser 100 105 110Ile
Val Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu 115
120 125Leu Lys Val Ile Asp Thr Asn Cys Ile
Asn Val Ile Gln Pro Asp Gly 130 135
140Ser Tyr Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala145
150 155 160Asp Ile Ile Gln
Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn 165
170 175Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln
Tyr Ile Arg Phe Ser Pro 180 185
190Asp Phe Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro
195 200 205Leu Leu Gly Ala Gly Lys Phe
Ala Thr Asp Pro Ala Val Thr Leu Ala 210 215
220His Glu Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile
Asn225 230 235 240Pro Asn
Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser
245 250 255Gly Leu Glu Val Ser Phe Glu
Glu Leu Arg Thr Phe Gly Gly His Asp 260 265
270Ala Lys Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu
Tyr Tyr 275 280 285Tyr Asn Lys Phe
Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser 290
295 300Ile Val Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys
Asn Val Phe Lys305 310 315
320Glu Lys Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp
325 330 335Lys Leu Lys Phe Asp
Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr 340
345 350Glu Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn
Arg Lys Thr Tyr 355 360 365Leu Asn
Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val 370
375 380Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg
Asn Thr Asn Leu Ala385 390 395
400Ala Asn Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr
405 410 415Lys Leu Lys Asn
Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys 420
425 430Val Asp Gly Ile Ile Thr Ser Lys Thr Lys Ser
Leu Ile Glu Gly Arg 435 440 445Tyr
Gly Gly Phe Met Ala Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly 450
455 460Gly Ser Gly Gly Gly Gly Ser Ala Leu Val
Leu Gln Cys Ile Lys Val465 470 475
480Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu Asp Asn Phe Thr
Asn 485 490 495Asp Leu Asn
Lys Gly Glu Glu Ile Thr Ser Asp Thr Asn Ile Glu Ala 500
505 510Ala Glu Glu Asn Ile Ser Leu Asp Leu Ile
Gln Gln Tyr Tyr Leu Thr 515 520
525Phe Asn Phe Asp Asn Glu Pro Glu Asn Ile Ser Ile Glu Asn Leu Ser 530
535 540Ser Asp Ile Ile Gly Gln Leu Glu
Leu Met Pro Asn Ile Glu Arg Phe545 550
555 560Pro Asn Gly Lys Lys Tyr Glu Leu Asp Lys Tyr Thr
Met Phe His Tyr 565 570
575Leu Arg Ala Gln Glu Phe Glu His Gly Lys Ser Arg Ile Ala Leu Thr
580 585 590Asn Ser Val Asn Glu Ala
Leu Leu Asn Pro Ser Arg Val Tyr Thr Phe 595 600
605Phe Ser Ser Asp Tyr Val Lys Lys Val Asn Lys Ala Thr Glu
Ala Ala 610 615 620Met Phe Leu Gly Trp
Val Glu Gln Leu Val Tyr Asp Phe Thr Asp Glu625 630
635 640Thr Ser Glu Val Ser Thr Thr Asp Lys Ile
Ala Asp Ile Thr Ile Ile 645 650
655Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly Asn Met Leu Tyr Lys
660 665 670Asp Asp Phe Val Gly
Ala Leu Ile Phe Ser Gly Ala Val Ile Leu Leu 675
680 685Glu Phe Ile Pro Glu Ile Ala Ile Pro Val Leu Gly
Thr Phe Ala Leu 690 695 700Val Ser Tyr
Ile Ala Asn Lys Val Leu Thr Val Gln Thr Ile Asp Asn705
710 715 720Ala Leu Ser Lys Arg Asn Glu
Lys Trp Asp Glu Val Tyr Lys Tyr Ile 725
730 735Val Thr Asn Trp Leu Ala Lys Val Asn Thr Gln Ile
Asp Leu Ile Arg 740 745 750Lys
Lys Met Lys Glu Ala Leu Glu Asn Gln Ala Glu Ala Thr Lys Ala 755
760 765Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr
Thr Glu Glu Glu Lys Asn Asn 770 775
780Ile Asn Phe Asn Ile Asp Asp Leu Ser Ser Lys Leu Asn Glu Ser Ile785
790 795 800Asn Lys Ala Met
Ile Asn Ile Asn Lys Phe Leu Asn Gln Cys Ser Val 805
810 815Ser Tyr Leu Met Asn Ser Met Ile Pro Tyr
Gly Val Lys Arg Leu Glu 820 825
830Asp Phe Asp Ala Ser Leu Lys Asp Ala Leu Leu Lys Tyr Ile Tyr Asp
835 840 845Asn Arg Gly Thr Leu Ile Gly
Gln Val Asp Arg Leu Lys Asp Lys Val 850 855
860Asn Asn Thr Leu Ser Thr Asp Ile Pro Phe Gln Leu Ser Lys Tyr
Val865 870 875 880Asp Asn
Gln Arg Leu Leu Ser Thr Leu Asp 885
890232751DNAArtificial SequenceSynthetic 23ggatccatgg agttcgttaa
caaacagttc aactataaag acccagttaa cggtgttgac 60attgcttaca tcaaaatccc
gaacgctggc cagatgcagc cggtaaaggc attcaaaatc 120cacaacaaaa tctgggttat
cccggaacgt gataccttta ctaacccgga agaaggtgac 180ctgaacccgc caccggaagc
gaaacaggtg ccggtatctt actatgactc cacctacctg 240tctaccgata acgaaaagga
caactacctg aaaggtgtta ctaaactgtt cgagcgtatt 300tactccaccg acctgggccg
tatgctgctg actagcatcg ttcgcggtat cccgttctgg 360ggcggttcta ccatcgatac
cgaactgaaa gtaatcgaca ctaactgcat caacgttatt 420cagccggacg gttcctatcg
ttccgaagaa ctgaacctgg tgatcatcgg cccgtctgct 480gatatcatcc agttcgagtg
taagagcttt ggtcacgaag ttctgaacct cacccgtaac 540ggctacggtt ccactcagta
catccgtttc tctccggact tcaccttcgg ttttgaagaa 600tccctggaag tagacacgaa
cccactgctg ggcgctggta aattcgcaac tgatcctgcg 660gttaccctgg ctcacgaact
gattcatgca ggccaccgcc tgtacggtat cgccatcaat 720ccgaaccgtg tcttcaaagt
taacaccaac gcgtattacg agatgtccgg tctggaagtt 780agcttcgaag aactgcgtac
ttttggcggt cacgacgcta aattcatcga ctctctgcaa 840gaaaacgagt tccgtctgta
ctactataac aagttcaaag atatcgcatc caccctgaac 900aaagcgaaat ccatcgtggg
taccactgct tctctccagt acatgaagaa cgtttttaaa 960gaaaaatacc tgctcagcga
agacacctcc ggcaaattct ctgtagacaa gttgaaattc 1020gataaacttt acaaaatgct
gactgaaatt tacaccgaag acaacttcgt taagttcttt 1080aaagttctga accgcaaaac
ctatctgaac ttcgacaagg cagtattcaa aatcaacatc 1140gtgccgaaag ttaactacac
tatctacgat ggtttcaacc tgcgtaacac caacctggct 1200gctaatttta acggccagaa
cacggaaatc aacaacatga acttcacaaa actgaaaaac 1260ttcactggtc tgttcgagtt
ttacaagctg ctgtgcgtcg acggcatcat tacctccaaa 1320actaaatctc tgatcgaagg
tcgttacggt ggtttcatga cctctgaaaa atctcagacc 1380ccgctggtta ccctgttcaa
aaacgctatc atcaaaaacg cttacaaaaa aggtgaagcg 1440ctagcgggtg gtggtggttc
tggtggtggt ggttctggtg gtggtggttc tgcactagtg 1500ctgcagtgta tcaaggttaa
caactgggat ttattcttca gcccgagtga agacaacttc 1560accaacgacc tgaacaaagg
tgaagaaatc acctcagata ctaacatcga agcagccgaa 1620gaaaacatct cgctggacct
gatccagcag tactacctga cctttaattt cgacaacgag 1680ccggaaaaca tttctatcga
aaacctgagc tctgatatca tcggccagct ggaactgatg 1740ccgaacatcg aacgtttccc
aaacggtaaa aagtacgagc tggacaaata taccatgttc 1800cactacctgc gcgcgcagga
atttgaacac ggcaaatccc gtatcgcact gactaactcc 1860gttaacgaag ctctgctcaa
cccgtcccgt gtatacacct tcttctctag cgactacgtg 1920aaaaaggtca acaaagcgac
tgaagctgca atgttcttgg gttgggttga acagcttgtt 1980tatgatttta ccgacgagac
gtccgaagta tctactaccg acaaaattgc ggatatcact 2040atcatcatcc cgtacatcgg
tccggctctg aacattggca acatgctgta caaagacgac 2100ttcgttggcg cactgatctt
ctccggtgcg gtgatcctgc tggagttcat cccggaaatc 2160gccatcccgg tactgggcac
ctttgctctg gtttcttaca ttgcaaacaa ggttctgact 2220gtacaaacca tcgacaacgc
gctgagcaaa cgtaacgaaa aatgggatga agtttacaaa 2280tatatcgtga ccaactggct
ggctaaggtt aatactcaga tcgacctcat ccgcaaaaaa 2340atgaaagaag cactggaaaa
ccaggcggaa gctaccaagg caatcattaa ctaccagtac 2400aaccagtaca ccgaggaaga
aaaaaacaac atcaacttca acatcgacga tctgtcctct 2460aaactgaacg aatccatcaa
caaagctatg atcaacatca acaagttcct gaaccagtgc 2520tctgtaagct atctgatgaa
ctccatgatc ccgtacggtg ttaaacgtct ggaggacttc 2580gatgcgtctc tgaaagacgc
cctgctgaaa tacatttacg acaaccgtgg cactctgatc 2640ggtcaggttg atcgtctgaa
ggacaaagtg aacaatacct tatcgaccga catccctttt 2700cagctcagta aatatgtcga
taaccaacgc cttttgtcca ctctagacta g 275124916PRTArtificial
SequenceSynthetic 24Gly Ser Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys
Asp Pro Val1 5 10 15Asn
Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met 20
25 30Gln Pro Val Lys Ala Phe Lys Ile
His Asn Lys Ile Trp Val Ile Pro 35 40
45Glu Arg Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro
50 55 60Pro Glu Ala Lys Gln Val Pro Val
Ser Tyr Tyr Asp Ser Thr Tyr Leu65 70 75
80Ser Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val
Thr Lys Leu 85 90 95Phe
Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser
100 105 110Ile Val Arg Gly Ile Pro Phe
Trp Gly Gly Ser Thr Ile Asp Thr Glu 115 120
125Leu Lys Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp
Gly 130 135 140Ser Tyr Arg Ser Glu Glu
Leu Asn Leu Val Ile Ile Gly Pro Ser Ala145 150
155 160Asp Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly
His Glu Val Leu Asn 165 170
175Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro
180 185 190Asp Phe Thr Phe Gly Phe
Glu Glu Ser Leu Glu Val Asp Thr Asn Pro 195 200
205Leu Leu Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr
Leu Ala 210 215 220His Glu Leu Ile His
Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn225 230
235 240Pro Asn Arg Val Phe Lys Val Asn Thr Asn
Ala Tyr Tyr Glu Met Ser 245 250
255Gly Leu Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp
260 265 270Ala Lys Phe Ile Asp
Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr 275
280 285Tyr Asn Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn
Lys Ala Lys Ser 290 295 300Ile Val Gly
Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys305
310 315 320Glu Lys Tyr Leu Leu Ser Glu
Asp Thr Ser Gly Lys Phe Ser Val Asp 325
330 335Lys Leu Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr
Glu Ile Tyr Thr 340 345 350Glu
Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr 355
360 365Leu Asn Phe Asp Lys Ala Val Phe Lys
Ile Asn Ile Val Pro Lys Val 370 375
380Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala385
390 395 400Ala Asn Phe Asn
Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr 405
410 415Lys Leu Lys Asn Phe Thr Gly Leu Phe Glu
Phe Tyr Lys Leu Leu Cys 420 425
430Val Asp Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Ile Glu Gly Arg
435 440 445Tyr Gly Gly Phe Met Thr Ser
Glu Lys Ser Gln Thr Pro Leu Val Thr 450 455
460Leu Phe Lys Asn Ala Ile Ile Lys Asn Ala Tyr Lys Lys Gly Glu
Ala465 470 475 480Leu Ala
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
485 490 495Ser Ala Leu Val Leu Gln Cys
Ile Lys Val Asn Asn Trp Asp Leu Phe 500 505
510Phe Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys
Gly Glu 515 520 525Glu Ile Thr Ser
Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser 530
535 540Leu Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn
Phe Asp Asn Glu545 550 555
560Pro Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln
565 570 575Leu Glu Leu Met Pro
Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr 580
585 590Glu Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg
Ala Gln Glu Phe 595 600 605Glu His
Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala 610
615 620Leu Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe
Ser Ser Asp Tyr Val625 630 635
640Lys Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val
645 650 655Glu Gln Leu Val
Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr 660
665 670Thr Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile
Pro Tyr Ile Gly Pro 675 680 685Ala
Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala 690
695 700Leu Ile Phe Ser Gly Ala Val Ile Leu Leu
Glu Phe Ile Pro Glu Ile705 710 715
720Ala Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala
Asn 725 730 735Lys Val Leu
Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn 740
745 750Glu Lys Trp Asp Glu Val Tyr Lys Tyr Ile
Val Thr Asn Trp Leu Ala 755 760
765Lys Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala 770
775 780Leu Glu Asn Gln Ala Glu Ala Thr
Lys Ala Ile Ile Asn Tyr Gln Tyr785 790
795 800Asn Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn
Phe Asn Ile Asp 805 810
815Asp Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn
820 825 830Ile Asn Lys Phe Leu Asn
Gln Cys Ser Val Ser Tyr Leu Met Asn Ser 835 840
845Met Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala
Ser Leu 850 855 860Lys Asp Ala Leu Leu
Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile865 870
875 880Gly Gln Val Asp Arg Leu Lys Asp Lys Val
Asn Asn Thr Leu Ser Thr 885 890
895Asp Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu
900 905 910Ser Thr Leu Asp
915252709DNAArtificial SequenceSynthetic 25ggatccatgg agttcgttaa
caaacagttc aactataaag acccagttaa cggtgttgac 60attgcttaca tcaaaatccc
gaacgctggc cagatgcagc cggtaaaggc attcaaaatc 120cacaacaaaa tctgggttat
cccggaacgt gataccttta ctaacccgga agaaggtgac 180ctgaacccgc caccggaagc
gaaacaggtg ccggtatctt actatgactc cacctacctg 240tctaccgata acgaaaagga
caactacctg aaaggtgtta ctaaactgtt cgagcgtatt 300tactccaccg acctgggccg
tatgctgctg actagcatcg ttcgcggtat cccgttctgg 360ggcggttcta ccatcgatac
cgaactgaaa gtaatcgaca ctaactgcat caacgttatt 420cagccggacg gttcctatcg
ttccgaagaa ctgaacctgg tgatcatcgg cccgtctgct 480gatatcatcc agttcgagtg
taagagcttt ggtcacgaag ttctgaacct cacccgtaac 540ggctacggtt ccactcagta
catccgtttc tctccggact tcaccttcgg ttttgaagaa 600tccctggaag tagacacgaa
cccactgctg ggcgctggta aattcgcaac tgatcctgcg 660gttaccctgg ctcacgaact
gattcatgca ggccaccgcc tgtacggtat cgccatcaat 720ccgaaccgtg tcttcaaagt
taacaccaac gcgtattacg agatgtccgg tctggaagtt 780agcttcgaag aactgcgtac
ttttggcggt cacgacgcta aattcatcga ctctctgcaa 840gaaaacgagt tccgtctgta
ctactataac aagttcaaag atatcgcatc caccctgaac 900aaagcgaaat ccatcgtggg
taccactgct tctctccagt acatgaagaa cgtttttaaa 960gaaaaatacc tgctcagcga
agacacctcc ggcaaattct ctgtagacaa gttgaaattc 1020gataaacttt acaaaatgct
gactgaaatt tacaccgaag acaacttcgt taagttcttt 1080aaagttctga accgcaaaac
ctatctgaac ttcgacaagg cagtattcaa aatcaacatc 1140gtgccgaaag ttaactacac
tatctacgat ggtttcaacc tgcgtaacac caacctggct 1200gctaatttta acggccagaa
cacggaaatc aacaacatga acttcacaaa actgaaaaac 1260ttcactggtc tgttcgagtt
ttacaagctg ctgtgcgtcg acggcatcat tacctccaaa 1320actaaatctc tgatagaagg
tagatttggc ggtttcacgg gcgcacgcaa atcagcgcgt 1380aaacgtaaga accaggcgct
agcgggcggt ggcggtagcg gcggtggcgg tagcggcggt 1440ggcggtagcg cactagtgct
gcagtgtatc aaggttaaca actgggattt attcttcagc 1500ccgagtgaag acaacttcac
caacgacctg aacaaaggtg aagaaatcac ctcagatact 1560aacatcgaag cagccgaaga
aaacatctcg ctggacctga tccagcagta ctacctgacc 1620tttaatttcg acaacgagcc
ggaaaacatt tctatcgaaa acctgagctc tgatatcatc 1680ggccagctgg aactgatgcc
gaacatcgaa cgtttcccaa acggtaaaaa gtacgagctg 1740gacaaatata ccatgttcca
ctacctgcgc gcgcaggaat ttgaacacgg caaatcccgt 1800atcgcactga ctaactccgt
taacgaagct ctgctcaacc cgtcccgtgt atacaccttc 1860ttctctagcg actacgtgaa
aaaggtcaac aaagcgactg aagctgcaat gttcttgggt 1920tgggttgaac agcttgttta
tgattttacc gacgagacgt ccgaagtatc tactaccgac 1980aaaattgcgg atatcactat
catcatcccg tacatcggtc cggctctgaa cattggcaac 2040atgctgtaca aagacgactt
cgttggcgca ctgatcttct ccggtgcggt gatcctgctg 2100gagttcatcc cggaaatcgc
catcccggta ctgggcacct ttgctctggt ttcttacatt 2160gcaaacaagg ttctgactgt
acaaaccatc gacaacgcgc tgagcaaacg taacgaaaaa 2220tgggatgaag tttacaaata
tatcgtgacc aactggctgg ctaaggttaa tactcagatc 2280gacctcatcc gcaaaaaaat
gaaagaagca ctggaaaacc aggcggaagc taccaaggca 2340atcattaact accagtacaa
ccagtacacc gaggaagaaa aaaacaacat caacttcaac 2400atcgacgatc tgtcctctaa
actgaacgaa tccatcaaca aagctatgat caacatcaac 2460aagttcctga accagtgctc
tgtaagctat ctgatgaact ccatgatccc gtacggtgtt 2520aaacgtctgg aggacttcga
tgcgtctctg aaagacgccc tgctgaaata catttacgac 2580aaccgtggca ctctgatcgg
tcaggttgat cgtctgaagg acaaagtgaa caatacctta 2640tcgaccgaca tcccttttca
gctcagtaaa tatgtcgata accaacgcct tttgtccact 2700ctagactag
270926902PRTArtificial
SequenceSynthetic 26Gly Ser Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys
Asp Pro Val1 5 10 15Asn
Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met 20
25 30Gln Pro Val Lys Ala Phe Lys Ile
His Asn Lys Ile Trp Val Ile Pro 35 40
45Glu Arg Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro
50 55 60Pro Glu Ala Lys Gln Val Pro Val
Ser Tyr Tyr Asp Ser Thr Tyr Leu65 70 75
80Ser Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val
Thr Lys Leu 85 90 95Phe
Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser
100 105 110Ile Val Arg Gly Ile Pro Phe
Trp Gly Gly Ser Thr Ile Asp Thr Glu 115 120
125Leu Lys Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp
Gly 130 135 140Ser Tyr Arg Ser Glu Glu
Leu Asn Leu Val Ile Ile Gly Pro Ser Ala145 150
155 160Asp Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly
His Glu Val Leu Asn 165 170
175Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro
180 185 190Asp Phe Thr Phe Gly Phe
Glu Glu Ser Leu Glu Val Asp Thr Asn Pro 195 200
205Leu Leu Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr
Leu Ala 210 215 220His Glu Leu Ile His
Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn225 230
235 240Pro Asn Arg Val Phe Lys Val Asn Thr Asn
Ala Tyr Tyr Glu Met Ser 245 250
255Gly Leu Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp
260 265 270Ala Lys Phe Ile Asp
Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr 275
280 285Tyr Asn Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn
Lys Ala Lys Ser 290 295 300Ile Val Gly
Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys305
310 315 320Glu Lys Tyr Leu Leu Ser Glu
Asp Thr Ser Gly Lys Phe Ser Val Asp 325
330 335Lys Leu Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr
Glu Ile Tyr Thr 340 345 350Glu
Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr 355
360 365Leu Asn Phe Asp Lys Ala Val Phe Lys
Ile Asn Ile Val Pro Lys Val 370 375
380Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala385
390 395 400Ala Asn Phe Asn
Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr 405
410 415Lys Leu Lys Asn Phe Thr Gly Leu Phe Glu
Phe Tyr Lys Leu Leu Cys 420 425
430Val Asp Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Ile Glu Gly Arg
435 440 445Phe Gly Gly Phe Thr Gly Ala
Arg Lys Ser Ala Arg Lys Arg Lys Asn 450 455
460Gln Ala Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly465 470 475 480Gly Gly
Ser Ala Leu Val Leu Gln Cys Ile Lys Val Asn Asn Trp Asp
485 490 495Leu Phe Phe Ser Pro Ser Glu
Asp Asn Phe Thr Asn Asp Leu Asn Lys 500 505
510Gly Glu Glu Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu
Glu Asn 515 520 525Ile Ser Leu Asp
Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp 530
535 540Asn Glu Pro Glu Asn Ile Ser Ile Glu Asn Leu Ser
Ser Asp Ile Ile545 550 555
560Gly Gln Leu Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys
565 570 575Lys Tyr Glu Leu Asp
Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln 580
585 590Glu Phe Glu His Gly Lys Ser Arg Ile Ala Leu Thr
Asn Ser Val Asn 595 600 605Glu Ala
Leu Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp 610
615 620Tyr Val Lys Lys Val Asn Lys Ala Thr Glu Ala
Ala Met Phe Leu Gly625 630 635
640Trp Val Glu Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val
645 650 655Ser Thr Thr Asp
Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile 660
665 670Gly Pro Ala Leu Asn Ile Gly Asn Met Leu Tyr
Lys Asp Asp Phe Val 675 680 685Gly
Ala Leu Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro 690
695 700Glu Ile Ala Ile Pro Val Leu Gly Thr Phe
Ala Leu Val Ser Tyr Ile705 710 715
720Ala Asn Lys Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser
Lys 725 730 735Arg Asn Glu
Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp 740
745 750Leu Ala Lys Val Asn Thr Gln Ile Asp Leu
Ile Arg Lys Lys Met Lys 755 760
765Glu Ala Leu Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr 770
775 780Gln Tyr Asn Gln Tyr Thr Glu Glu
Glu Lys Asn Asn Ile Asn Phe Asn785 790
795 800Ile Asp Asp Leu Ser Ser Lys Leu Asn Glu Ser Ile
Asn Lys Ala Met 805 810
815Ile Asn Ile Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met
820 825 830Asn Ser Met Ile Pro Tyr
Gly Val Lys Arg Leu Glu Asp Phe Asp Ala 835 840
845Ser Leu Lys Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg
Gly Thr 850 855 860Leu Ile Gly Gln Val
Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu865 870
875 880Ser Thr Asp Ile Pro Phe Gln Leu Ser Lys
Tyr Val Asp Asn Gln Arg 885 890
895Leu Leu Ser Thr Leu Asp 900272691DNAArtificial
SequenceSynthetic 27ggatccatgg agttcgttaa caaacagttc aactataaag
acccagttaa cggtgttgac 60attgcttaca tcaaaatccc gaacgctggc cagatgcagc
cggtaaaggc attcaaaatc 120cacaacaaaa tctgggttat cccggaacgt gataccttta
ctaacccgga agaaggtgac 180ctgaacccgc caccggaagc gaaacaggtg ccggtatctt
actatgactc cacctacctg 240tctaccgata acgaaaagga caactacctg aaaggtgtta
ctaaactgtt cgagcgtatt 300tactccaccg acctgggccg tatgctgctg actagcatcg
ttcgcggtat cccgttctgg 360ggcggttcta ccatcgatac cgaactgaaa gtaatcgaca
ctaactgcat caacgttatt 420cagccggacg gttcctatcg ttccgaagaa ctgaacctgg
tgatcatcgg cccgtctgct 480gatatcatcc agttcgagtg taagagcttt ggtcacgaag
ttctgaacct cacccgtaac 540ggctacggtt ccactcagta catccgtttc tctccggact
tcaccttcgg ttttgaagaa 600tccctggaag tagacacgaa cccactgctg ggcgctggta
aattcgcaac tgatcctgcg 660gttaccctgg ctcacgaact gattcatgca ggccaccgcc
tgtacggtat cgccatcaat 720ccgaaccgtg tcttcaaagt taacaccaac gcgtattacg
agatgtccgg tctggaagtt 780agcttcgaag aactgcgtac ttttggcggt cacgacgcta
aattcatcga ctctctgcaa 840gaaaacgagt tccgtctgta ctactataac aagttcaaag
atatcgcatc caccctgaac 900aaagcgaaat ccatcgtggg taccactgct tctctccagt
acatgaagaa cgtttttaaa 960gaaaaatacc tgctcagcga agacacctcc ggcaaattct
ctgtagacaa gttgaaattc 1020gataaacttt acaaaatgct gactgaaatt tacaccgaag
acaacttcgt taagttcttt 1080aaagttctga accgcaaaac ctatctgaac ttcgacaagg
cagtattcaa aatcaacatc 1140gtgccgaaag ttaactacac tatctacgat ggtttcaacc
tgcgtaacac caacctggct 1200gctaatttta acggccagaa cacggaaatc aacaacatga
acttcacaaa actgaaaaac 1260ttcactggtc tgttcgagtt ttacaagctg ctgtgcgtcg
acggcatcat tacctccaaa 1320actaaatctc tgatagaagg tagatttggc ggtttcacgg
gcgcacgcaa atcagcggcg 1380ctagcgggcg gtggcggtag cggcggtggc ggtagcggcg
gtggcggtag cgcactagtg 1440ctgcagtgta tcaaggttaa caactgggat ttattcttca
gcccgagtga agacaacttc 1500accaacgacc tgaacaaagg tgaagaaatc acctcagata
ctaacatcga agcagccgaa 1560gaaaacatct cgctggacct gatccagcag tactacctga
cctttaattt cgacaacgag 1620ccggaaaaca tttctatcga aaacctgagc tctgatatca
tcggccagct ggaactgatg 1680ccgaacatcg aacgtttccc aaacggtaaa aagtacgagc
tggacaaata taccatgttc 1740cactacctgc gcgcgcagga atttgaacac ggcaaatccc
gtatcgcact gactaactcc 1800gttaacgaag ctctgctcaa cccgtcccgt gtatacacct
tcttctctag cgactacgtg 1860aaaaaggtca acaaagcgac tgaagctgca atgttcttgg
gttgggttga acagcttgtt 1920tatgatttta ccgacgagac gtccgaagta tctactaccg
acaaaattgc ggatatcact 1980atcatcatcc cgtacatcgg tccggctctg aacattggca
acatgctgta caaagacgac 2040ttcgttggcg cactgatctt ctccggtgcg gtgatcctgc
tggagttcat cccggaaatc 2100gccatcccgg tactgggcac ctttgctctg gtttcttaca
ttgcaaacaa ggttctgact 2160gtacaaacca tcgacaacgc gctgagcaaa cgtaacgaaa
aatgggatga agtttacaaa 2220tatatcgtga ccaactggct ggctaaggtt aatactcaga
tcgacctcat ccgcaaaaaa 2280atgaaagaag cactggaaaa ccaggcggaa gctaccaagg
caatcattaa ctaccagtac 2340aaccagtaca ccgaggaaga aaaaaacaac atcaacttca
acatcgacga tctgtcctct 2400aaactgaacg aatccatcaa caaagctatg atcaacatca
acaagttcct gaaccagtgc 2460tctgtaagct atctgatgaa ctccatgatc ccgtacggtg
ttaaacgtct ggaggacttc 2520gatgcgtctc tgaaagacgc cctgctgaaa tacatttacg
acaaccgtgg cactctgatc 2580ggtcaggttg atcgtctgaa ggacaaagtg aacaatacct
tatcgaccga catccctttt 2640cagctcagta aatatgtcga taaccaacgc cttttgtcca
ctctagacta g 269128896PRTArtificial SequenceSynthetic 28Gly
Ser Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val1
5 10 15Asn Gly Val Asp Ile Ala Tyr
Ile Lys Ile Pro Asn Ala Gly Gln Met 20 25
30Gln Pro Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val
Ile Pro 35 40 45Glu Arg Asp Thr
Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro 50 55
60Pro Glu Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser
Thr Tyr Leu65 70 75
80Ser Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu
85 90 95Phe Glu Arg Ile Tyr Ser
Thr Asp Leu Gly Arg Met Leu Leu Thr Ser 100
105 110Ile Val Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr
Ile Asp Thr Glu 115 120 125Leu Lys
Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly 130
135 140Ser Tyr Arg Ser Glu Glu Leu Asn Leu Val Ile
Ile Gly Pro Ser Ala145 150 155
160Asp Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn
165 170 175Leu Thr Arg Asn
Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro 180
185 190Asp Phe Thr Phe Gly Phe Glu Glu Ser Leu Glu
Val Asp Thr Asn Pro 195 200 205Leu
Leu Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala 210
215 220His Glu Leu Ile His Ala Gly His Arg Leu
Tyr Gly Ile Ala Ile Asn225 230 235
240Pro Asn Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met
Ser 245 250 255Gly Leu Glu
Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp 260
265 270Ala Lys Phe Ile Asp Ser Leu Gln Glu Asn
Glu Phe Arg Leu Tyr Tyr 275 280
285Tyr Asn Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser 290
295 300Ile Val Gly Thr Thr Ala Ser Leu
Gln Tyr Met Lys Asn Val Phe Lys305 310
315 320Glu Lys Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys
Phe Ser Val Asp 325 330
335Lys Leu Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr
340 345 350Glu Asp Asn Phe Val Lys
Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr 355 360
365Leu Asn Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro
Lys Val 370 375 380Asn Tyr Thr Ile Tyr
Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala385 390
395 400Ala Asn Phe Asn Gly Gln Asn Thr Glu Ile
Asn Asn Met Asn Phe Thr 405 410
415Lys Leu Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys
420 425 430Val Asp Gly Ile Ile
Thr Ser Lys Thr Lys Ser Leu Ile Glu Gly Arg 435
440 445Phe Gly Gly Phe Thr Gly Ala Arg Lys Ser Ala Ala
Leu Ala Gly Gly 450 455 460Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Val465
470 475 480Leu Gln Cys Ile Lys Val Asn
Asn Trp Asp Leu Phe Phe Ser Pro Ser 485
490 495Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu
Glu Ile Thr Ser 500 505 510Asp
Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu Asp Leu Ile 515
520 525Gln Gln Tyr Tyr Leu Thr Phe Asn Phe
Asp Asn Glu Pro Glu Asn Ile 530 535
540Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu Glu Leu Met545
550 555 560Pro Asn Ile Glu
Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu Asp Lys 565
570 575Tyr Thr Met Phe His Tyr Leu Arg Ala Gln
Glu Phe Glu His Gly Lys 580 585
590Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu Leu Asn Pro
595 600 605Ser Arg Val Tyr Thr Phe Phe
Ser Ser Asp Tyr Val Lys Lys Val Asn 610 615
620Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu Gln Leu
Val625 630 635 640Tyr Asp
Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr Asp Lys Ile
645 650 655Ala Asp Ile Thr Ile Ile Ile
Pro Tyr Ile Gly Pro Ala Leu Asn Ile 660 665
670Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu Ile
Phe Ser 675 680 685Gly Ala Val Ile
Leu Leu Glu Phe Ile Pro Glu Ile Ala Ile Pro Val 690
695 700Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn
Lys Val Leu Thr705 710 715
720Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu Lys Trp Asp
725 730 735Glu Val Tyr Lys Tyr
Ile Val Thr Asn Trp Leu Ala Lys Val Asn Thr 740
745 750Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala
Leu Glu Asn Gln 755 760 765Ala Glu
Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr Thr 770
775 780Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile
Asp Asp Leu Ser Ser785 790 795
800Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile Asn Lys Phe
805 810 815Leu Asn Gln Cys
Ser Val Ser Tyr Leu Met Asn Ser Met Ile Pro Tyr 820
825 830Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser
Leu Lys Asp Ala Leu 835 840 845Leu
Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly Gln Val Asp 850
855 860Arg Leu Lys Asp Lys Val Asn Asn Thr Leu
Ser Thr Asp Ile Pro Phe865 870 875
880Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser Thr Leu
Asp 885 890
895292691DNAArtificial SequenceSynthetic 29ggatccatgg agttcgttaa
caaacagttc aactataaag acccagttaa cggtgttgac 60attgcttaca tcaaaatccc
gaacgctggc cagatgcagc cggtaaaggc attcaaaatc 120cacaacaaaa tctgggttat
cccggaacgt gataccttta ctaacccgga agaaggtgac 180ctgaacccgc caccggaagc
gaaacaggtg ccggtatctt actatgactc cacctacctg 240tctaccgata acgaaaagga
caactacctg aaaggtgtta ctaaactgtt cgagcgtatt 300tactccaccg acctgggccg
tatgctgctg actagcatcg ttcgcggtat cccgttctgg 360ggcggttcta ccatcgatac
cgaactgaaa gtaatcgaca ctaactgcat caacgttatt 420cagccggacg gttcctatcg
ttccgaagaa ctgaacctgg tgatcatcgg cccgtctgct 480gatatcatcc agttcgagtg
taagagcttt ggtcacgaag ttctgaacct cacccgtaac 540ggctacggtt ccactcagta
catccgtttc tctccggact tcaccttcgg ttttgaagaa 600tccctggaag tagacacgaa
cccactgctg ggcgctggta aattcgcaac tgatcctgcg 660gttaccctgg ctcacgaact
gattcatgca ggccaccgcc tgtacggtat cgccatcaat 720ccgaaccgtg tcttcaaagt
taacaccaac gcgtattacg agatgtccgg tctggaagtt 780agcttcgaag aactgcgtac
ttttggcggt cacgacgcta aattcatcga ctctctgcaa 840gaaaacgagt tccgtctgta
ctactataac aagttcaaag atatcgcatc caccctgaac 900aaagcgaaat ccatcgtggg
taccactgct tctctccagt acatgaagaa cgtttttaaa 960gaaaaatacc tgctcagcga
agacacctcc ggcaaattct ctgtagacaa gttgaaattc 1020gataaacttt acaaaatgct
gactgaaatt tacaccgaag acaacttcgt taagttcttt 1080aaagttctga accgcaaaac
ctatctgaac ttcgacaagg cagtattcaa aatcaacatc 1140gtgccgaaag ttaactacac
tatctacgat ggtttcaacc tgcgtaacac caacctggct 1200gctaatttta acggccagaa
cacggaaatc aacaacatga acttcacaaa actgaaaaac 1260ttcactggtc tgttcgagtt
ttacaagctg ctgtgcgtcg acggcatcat tacctccaaa 1320actaaatctc tgatagaagg
tagatttggc ggtttcacgg gcgcacgcaa atatgcggcg 1380ctagcgggcg gtggcggtag
cggcggtggc ggtagcggcg gtggcggtag cgcactagtg 1440ctgcagtgta tcaaggttaa
caactgggat ttattcttca gcccgagtga agacaacttc 1500accaacgacc tgaacaaagg
tgaagaaatc acctcagata ctaacatcga agcagccgaa 1560gaaaacatct cgctggacct
gatccagcag tactacctga cctttaattt cgacaacgag 1620ccggaaaaca tttctatcga
aaacctgagc tctgatatca tcggccagct ggaactgatg 1680ccgaacatcg aacgtttccc
aaacggtaaa aagtacgagc tggacaaata taccatgttc 1740cactacctgc gcgcgcagga
atttgaacac ggcaaatccc gtatcgcact gactaactcc 1800gttaacgaag ctctgctcaa
cccgtcccgt gtatacacct tcttctctag cgactacgtg 1860aaaaaggtca acaaagcgac
tgaagctgca atgttcttgg gttgggttga acagcttgtt 1920tatgatttta ccgacgagac
gtccgaagta tctactaccg acaaaattgc ggatatcact 1980atcatcatcc cgtacatcgg
tccggctctg aacattggca acatgctgta caaagacgac 2040ttcgttggcg cactgatctt
ctccggtgcg gtgatcctgc tggagttcat cccggaaatc 2100gccatcccgg tactgggcac
ctttgctctg gtttcttaca ttgcaaacaa ggttctgact 2160gtacaaacca tcgacaacgc
gctgagcaaa cgtaacgaaa aatgggatga agtttacaaa 2220tatatcgtga ccaactggct
ggctaaggtt aatactcaga tcgacctcat ccgcaaaaaa 2280atgaaagaag cactggaaaa
ccaggcggaa gctaccaagg caatcattaa ctaccagtac 2340aaccagtaca ccgaggaaga
aaaaaacaac atcaacttca acatcgacga tctgtcctct 2400aaactgaacg aatccatcaa
caaagctatg atcaacatca acaagttcct gaaccagtgc 2460tctgtaagct atctgatgaa
ctccatgatc ccgtacggtg ttaaacgtct ggaggacttc 2520gatgcgtctc tgaaagacgc
cctgctgaaa tacatttacg acaaccgtgg cactctgatc 2580ggtcaggttg atcgtctgaa
ggacaaagtg aacaatacct tatcgaccga catccctttt 2640cagctcagta aatatgtcga
taaccaacgc cttttgtcca ctctagacta g 269130896PRTArtificial
SequenceSynthetic 30Gly Ser Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys
Asp Pro Val1 5 10 15Asn
Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met 20
25 30Gln Pro Val Lys Ala Phe Lys Ile
His Asn Lys Ile Trp Val Ile Pro 35 40
45Glu Arg Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro
50 55 60Pro Glu Ala Lys Gln Val Pro Val
Ser Tyr Tyr Asp Ser Thr Tyr Leu65 70 75
80Ser Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val
Thr Lys Leu 85 90 95Phe
Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser
100 105 110Ile Val Arg Gly Ile Pro Phe
Trp Gly Gly Ser Thr Ile Asp Thr Glu 115 120
125Leu Lys Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp
Gly 130 135 140Ser Tyr Arg Ser Glu Glu
Leu Asn Leu Val Ile Ile Gly Pro Ser Ala145 150
155 160Asp Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly
His Glu Val Leu Asn 165 170
175Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro
180 185 190Asp Phe Thr Phe Gly Phe
Glu Glu Ser Leu Glu Val Asp Thr Asn Pro 195 200
205Leu Leu Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr
Leu Ala 210 215 220His Glu Leu Ile His
Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn225 230
235 240Pro Asn Arg Val Phe Lys Val Asn Thr Asn
Ala Tyr Tyr Glu Met Ser 245 250
255Gly Leu Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp
260 265 270Ala Lys Phe Ile Asp
Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr 275
280 285Tyr Asn Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn
Lys Ala Lys Ser 290 295 300Ile Val Gly
Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys305
310 315 320Glu Lys Tyr Leu Leu Ser Glu
Asp Thr Ser Gly Lys Phe Ser Val Asp 325
330 335Lys Leu Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr
Glu Ile Tyr Thr 340 345 350Glu
Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr 355
360 365Leu Asn Phe Asp Lys Ala Val Phe Lys
Ile Asn Ile Val Pro Lys Val 370 375
380Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala385
390 395 400Ala Asn Phe Asn
Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr 405
410 415Lys Leu Lys Asn Phe Thr Gly Leu Phe Glu
Phe Tyr Lys Leu Leu Cys 420 425
430Val Asp Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Ile Glu Gly Arg
435 440 445Phe Gly Gly Phe Thr Gly Ala
Arg Lys Tyr Ala Ala Leu Ala Gly Gly 450 455
460Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu
Val465 470 475 480Leu Gln
Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser
485 490 495Glu Asp Asn Phe Thr Asn Asp
Leu Asn Lys Gly Glu Glu Ile Thr Ser 500 505
510Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu Asp
Leu Ile 515 520 525Gln Gln Tyr Tyr
Leu Thr Phe Asn Phe Asp Asn Glu Pro Glu Asn Ile 530
535 540Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln
Leu Glu Leu Met545 550 555
560Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu Asp Lys
565 570 575Tyr Thr Met Phe His
Tyr Leu Arg Ala Gln Glu Phe Glu His Gly Lys 580
585 590Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala
Leu Leu Asn Pro 595 600 605Ser Arg
Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys Lys Val Asn 610
615 620Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp
Val Glu Gln Leu Val625 630 635
640Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr Asp Lys Ile
645 650 655Ala Asp Ile Thr
Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile 660
665 670Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly
Ala Leu Ile Phe Ser 675 680 685Gly
Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala Ile Pro Val 690
695 700Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile
Ala Asn Lys Val Leu Thr705 710 715
720Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu Lys Trp
Asp 725 730 735Glu Val Tyr
Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys Val Asn Thr 740
745 750Gln Ile Asp Leu Ile Arg Lys Lys Met Lys
Glu Ala Leu Glu Asn Gln 755 760
765Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr Thr 770
775 780Glu Glu Glu Lys Asn Asn Ile Asn
Phe Asn Ile Asp Asp Leu Ser Ser785 790
795 800Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn
Ile Asn Lys Phe 805 810
815Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met Ile Pro Tyr
820 825 830Gly Val Lys Arg Leu Glu
Asp Phe Asp Ala Ser Leu Lys Asp Ala Leu 835 840
845Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly Gln
Val Asp 850 855 860Arg Leu Lys Asp Lys
Val Asn Asn Thr Leu Ser Thr Asp Ile Pro Phe865 870
875 880Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg
Leu Leu Ser Thr Leu Asp 885 890
895312691DNAArtificial SequenceSynthetic 31ggatccatgg agttcgttaa
caaacagttc aactataaag acccagttaa cggtgttgac 60attgcttaca tcaaaatccc
gaacgctggc cagatgcagc cggtaaaggc attcaaaatc 120cacaacaaaa tctgggttat
cccggaacgt gataccttta ctaacccgga agaaggtgac 180ctgaacccgc caccggaagc
gaaacaggtg ccggtatctt actatgactc cacctacctg 240tctaccgata acgaaaagga
caactacctg aaaggtgtta ctaaactgtt cgagcgtatt 300tactccaccg acctgggccg
tatgctgctg actagcatcg ttcgcggtat cccgttctgg 360ggcggttcta ccatcgatac
cgaactgaaa gtaatcgaca ctaactgcat caacgttatt 420cagccggacg gttcctatcg
ttccgaagaa ctgaacctgg tgatcatcgg cccgtctgct 480gatatcatcc agttcgagtg
taagagcttt ggtcacgaag ttctgaacct cacccgtaac 540ggctacggtt ccactcagta
catccgtttc tctccggact tcaccttcgg ttttgaagaa 600tccctggaag tagacacgaa
cccactgctg ggcgctggta aattcgcaac tgatcctgcg 660gttaccctgg ctcacgaact
gattcatgca ggccaccgcc tgtacggtat cgccatcaat 720ccgaaccgtg tcttcaaagt
taacaccaac gcgtattacg agatgtccgg tctggaagtt 780agcttcgaag aactgcgtac
ttttggcggt cacgacgcta aattcatcga ctctctgcaa 840gaaaacgagt tccgtctgta
ctactataac aagttcaaag atatcgcatc caccctgaac 900aaagcgaaat ccatcgtggg
taccactgct tctctccagt acatgaagaa cgtttttaaa 960gaaaaatacc tgctcagcga
agacacctcc ggcaaattct ctgtagacaa gttgaaattc 1020gataaacttt acaaaatgct
gactgaaatt tacaccgaag acaacttcgt taagttcttt 1080aaagttctga accgcaaaac
ctatctgaac ttcgacaagg cagtattcaa aatcaacatc 1140gtgccgaaag ttaactacac
tatctacgat ggtttcaacc tgcgtaacac caacctggct 1200gctaatttta acggccagaa
cacggaaatc aacaacatga acttcacaaa actgaaaaac 1260ttcactggtc tgttcgagtt
ttacaagctg ctgtgcgtcg acggcatcat tacctccaaa 1320actaaatctc tgatagaagg
tagatttggc ggtttcacgg gcgcacgcaa atcatatgcg 1380ctagcgggcg gtggcggtag
cggcggtggc ggtagcggcg gtggcggtag cgcactagtg 1440ctgcagtgta tcaaggttaa
caactgggat ttattcttca gcccgagtga agacaacttc 1500accaacgacc tgaacaaagg
tgaagaaatc acctcagata ctaacatcga agcagccgaa 1560gaaaacatct cgctggacct
gatccagcag tactacctga cctttaattt cgacaacgag 1620ccggaaaaca tttctatcga
aaacctgagc tctgatatca tcggccagct ggaactgatg 1680ccgaacatcg aacgtttccc
aaacggtaaa aagtacgagc tggacaaata taccatgttc 1740cactacctgc gcgcgcagga
atttgaacac ggcaaatccc gtatcgcact gactaactcc 1800gttaacgaag ctctgctcaa
cccgtcccgt gtatacacct tcttctctag cgactacgtg 1860aaaaaggtca acaaagcgac
tgaagctgca atgttcttgg gttgggttga acagcttgtt 1920tatgatttta ccgacgagac
gtccgaagta tctactaccg acaaaattgc ggatatcact 1980atcatcatcc cgtacatcgg
tccggctctg aacattggca acatgctgta caaagacgac 2040ttcgttggcg cactgatctt
ctccggtgcg gtgatcctgc tggagttcat cccggaaatc 2100gccatcccgg tactgggcac
ctttgctctg gtttcttaca ttgcaaacaa ggttctgact 2160gtacaaacca tcgacaacgc
gctgagcaaa cgtaacgaaa aatgggatga agtttacaaa 2220tatatcgtga ccaactggct
ggctaaggtt aatactcaga tcgacctcat ccgcaaaaaa 2280atgaaagaag cactggaaaa
ccaggcggaa gctaccaagg caatcattaa ctaccagtac 2340aaccagtaca ccgaggaaga
aaaaaacaac atcaacttca acatcgacga tctgtcctct 2400aaactgaacg aatccatcaa
caaagctatg atcaacatca acaagttcct gaaccagtgc 2460tctgtaagct atctgatgaa
ctccatgatc ccgtacggtg ttaaacgtct ggaggacttc 2520gatgcgtctc tgaaagacgc
cctgctgaaa tacatttacg acaaccgtgg cactctgatc 2580ggtcaggttg atcgtctgaa
ggacaaagtg aacaatacct tatcgaccga catccctttt 2640cagctcagta aatatgtcga
taaccaacgc cttttgtcca ctctagacta g 269132896PRTArtificial
SequenceSynthetic 32Gly Ser Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys
Asp Pro Val1 5 10 15Asn
Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met 20
25 30Gln Pro Val Lys Ala Phe Lys Ile
His Asn Lys Ile Trp Val Ile Pro 35 40
45Glu Arg Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro
50 55 60Pro Glu Ala Lys Gln Val Pro Val
Ser Tyr Tyr Asp Ser Thr Tyr Leu65 70 75
80Ser Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val
Thr Lys Leu 85 90 95Phe
Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser
100 105 110Ile Val Arg Gly Ile Pro Phe
Trp Gly Gly Ser Thr Ile Asp Thr Glu 115 120
125Leu Lys Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp
Gly 130 135 140Ser Tyr Arg Ser Glu Glu
Leu Asn Leu Val Ile Ile Gly Pro Ser Ala145 150
155 160Asp Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly
His Glu Val Leu Asn 165 170
175Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro
180 185 190Asp Phe Thr Phe Gly Phe
Glu Glu Ser Leu Glu Val Asp Thr Asn Pro 195 200
205Leu Leu Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr
Leu Ala 210 215 220His Glu Leu Ile His
Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn225 230
235 240Pro Asn Arg Val Phe Lys Val Asn Thr Asn
Ala Tyr Tyr Glu Met Ser 245 250
255Gly Leu Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp
260 265 270Ala Lys Phe Ile Asp
Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr 275
280 285Tyr Asn Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn
Lys Ala Lys Ser 290 295 300Ile Val Gly
Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys305
310 315 320Glu Lys Tyr Leu Leu Ser Glu
Asp Thr Ser Gly Lys Phe Ser Val Asp 325
330 335Lys Leu Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr
Glu Ile Tyr Thr 340 345 350Glu
Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr 355
360 365Leu Asn Phe Asp Lys Ala Val Phe Lys
Ile Asn Ile Val Pro Lys Val 370 375
380Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala385
390 395 400Ala Asn Phe Asn
Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr 405
410 415Lys Leu Lys Asn Phe Thr Gly Leu Phe Glu
Phe Tyr Lys Leu Leu Cys 420 425
430Val Asp Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Ile Glu Gly Arg
435 440 445Phe Gly Gly Phe Thr Gly Ala
Arg Lys Ser Tyr Ala Leu Ala Gly Gly 450 455
460Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu
Val465 470 475 480Leu Gln
Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser
485 490 495Glu Asp Asn Phe Thr Asn Asp
Leu Asn Lys Gly Glu Glu Ile Thr Ser 500 505
510Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu Asp
Leu Ile 515 520 525Gln Gln Tyr Tyr
Leu Thr Phe Asn Phe Asp Asn Glu Pro Glu Asn Ile 530
535 540Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln
Leu Glu Leu Met545 550 555
560Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu Asp Lys
565 570 575Tyr Thr Met Phe His
Tyr Leu Arg Ala Gln Glu Phe Glu His Gly Lys 580
585 590Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala
Leu Leu Asn Pro 595 600 605Ser Arg
Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys Lys Val Asn 610
615 620Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp
Val Glu Gln Leu Val625 630 635
640Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr Asp Lys Ile
645 650 655Ala Asp Ile Thr
Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile 660
665 670Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly
Ala Leu Ile Phe Ser 675 680 685Gly
Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala Ile Pro Val 690
695 700Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile
Ala Asn Lys Val Leu Thr705 710 715
720Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu Lys Trp
Asp 725 730 735Glu Val Tyr
Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys Val Asn Thr 740
745 750Gln Ile Asp Leu Ile Arg Lys Lys Met Lys
Glu Ala Leu Glu Asn Gln 755 760
765Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr Thr 770
775 780Glu Glu Glu Lys Asn Asn Ile Asn
Phe Asn Ile Asp Asp Leu Ser Ser785 790
795 800Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn
Ile Asn Lys Phe 805 810
815Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met Ile Pro Tyr
820 825 830Gly Val Lys Arg Leu Glu
Asp Phe Asp Ala Ser Leu Lys Asp Ala Leu 835 840
845Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly Gln
Val Asp 850 855 860Arg Leu Lys Asp Lys
Val Asn Asn Thr Leu Ser Thr Asp Ile Pro Phe865 870
875 880Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg
Leu Leu Ser Thr Leu Asp 885 890
895332709DNAArtificial SequenceSynthetic 33ggatccatgg agttcgttaa
caaacagttc aactataaag acccagttaa cggtgttgac 60attgcttaca tcaaaatccc
gaacgctggc cagatgcagc cggtaaaggc attcaaaatc 120cacaacaaaa tctgggttat
cccggaacgt gataccttta ctaacccgga agaaggtgac 180ctgaacccgc caccggaagc
gaaacaggtg ccggtatctt actatgactc cacctacctg 240tctaccgata acgaaaagga
caactacctg aaaggtgtta ctaaactgtt cgagcgtatt 300tactccaccg acctgggccg
tatgctgctg actagcatcg ttcgcggtat cccgttctgg 360ggcggttcta ccatcgatac
cgaactgaaa gtaatcgaca ctaactgcat caacgttatt 420cagccggacg gttcctatcg
ttccgaagaa ctgaacctgg tgatcatcgg cccgtctgct 480gatatcatcc agttcgagtg
taagagcttt ggtcacgaag ttctgaacct cacccgtaac 540ggctacggtt ccactcagta
catccgtttc tctccggact tcaccttcgg ttttgaagaa 600tccctggaag tagacacgaa
cccactgctg ggcgctggta aattcgcaac tgatcctgcg 660gttaccctgg ctcacgaact
gattcatgca ggccaccgcc tgtacggtat cgccatcaat 720ccgaaccgtg tcttcaaagt
taacaccaac gcgtattacg agatgtccgg tctggaagtt 780agcttcgaag aactgcgtac
ttttggcggt cacgacgcta aattcatcga ctctctgcaa 840gaaaacgagt tccgtctgta
ctactataac aagttcaaag atatcgcatc caccctgaac 900aaagcgaaat ccatcgtggg
taccactgct tctctccagt acatgaagaa cgtttttaaa 960gaaaaatacc tgctcagcga
agacacctcc ggcaaattct ctgtagacaa gttgaaattc 1020gataaacttt acaaaatgct
gactgaaatt tacaccgaag acaacttcgt taagttcttt 1080aaagttctga accgcaaaac
ctatctgaac ttcgacaagg cagtattcaa aatcaacatc 1140gtgccgaaag ttaactacac
tatctacgat ggtttcaacc tgcgtaacac caacctggct 1200gctaatttta acggccagaa
cacggaaatc aacaacatga acttcacaaa actgaaaaac 1260ttcactggtc tgttcgagtt
ttacaagctg ctgtgcgtcg acggcatcat tacctccaaa 1320actaaatctc tgatagaagg
tagatttggc ggtttcacgg gcgcacgcaa atcagcgcgt 1380aaatatgcta accaggcgct
agcgggcggt ggcggtagcg gcggtggcgg tagcggcggt 1440ggcggtagcg cactagtgct
gcagtgtatc aaggttaaca actgggattt attcttcagc 1500ccgagtgaag acaacttcac
caacgacctg aacaaaggtg aagaaatcac ctcagatact 1560aacatcgaag cagccgaaga
aaacatctcg ctggacctga tccagcagta ctacctgacc 1620tttaatttcg acaacgagcc
ggaaaacatt tctatcgaaa acctgagctc tgatatcatc 1680ggccagctgg aactgatgcc
gaacatcgaa cgtttcccaa acggtaaaaa gtacgagctg 1740gacaaatata ccatgttcca
ctacctgcgc gcgcaggaat ttgaacacgg caaatcccgt 1800atcgcactga ctaactccgt
taacgaagct ctgctcaacc cgtcccgtgt atacaccttc 1860ttctctagcg actacgtgaa
aaaggtcaac aaagcgactg aagctgcaat gttcttgggt 1920tgggttgaac agcttgttta
tgattttacc gacgagacgt ccgaagtatc tactaccgac 1980aaaattgcgg atatcactat
catcatcccg tacatcggtc cggctctgaa cattggcaac 2040atgctgtaca aagacgactt
cgttggcgca ctgatcttct ccggtgcggt gatcctgctg 2100gagttcatcc cggaaatcgc
catcccggta ctgggcacct ttgctctggt ttcttacatt 2160gcaaacaagg ttctgactgt
acaaaccatc gacaacgcgc tgagcaaacg taacgaaaaa 2220tgggatgaag tttacaaata
tatcgtgacc aactggctgg ctaaggttaa tactcagatc 2280gacctcatcc gcaaaaaaat
gaaagaagca ctggaaaacc aggcggaagc taccaaggca 2340atcattaact accagtacaa
ccagtacacc gaggaagaaa aaaacaacat caacttcaac 2400atcgacgatc tgtcctctaa
actgaacgaa tccatcaaca aagctatgat caacatcaac 2460aagttcctga accagtgctc
tgtaagctat ctgatgaact ccatgatccc gtacggtgtt 2520aaacgtctgg aggacttcga
tgcgtctctg aaagacgccc tgctgaaata catttacgac 2580aaccgtggca ctctgatcgg
tcaggttgat cgtctgaagg acaaagtgaa caatacctta 2640tcgaccgaca tcccttttca
gctcagtaaa tatgtcgata accaacgcct tttgtccact 2700ctagactag
270934902PRTArtificial
SequenceSynthetic 34Gly Ser Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys
Asp Pro Val1 5 10 15Asn
Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met 20
25 30Gln Pro Val Lys Ala Phe Lys Ile
His Asn Lys Ile Trp Val Ile Pro 35 40
45Glu Arg Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro
50 55 60Pro Glu Ala Lys Gln Val Pro Val
Ser Tyr Tyr Asp Ser Thr Tyr Leu65 70 75
80Ser Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val
Thr Lys Leu 85 90 95Phe
Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser
100 105 110Ile Val Arg Gly Ile Pro Phe
Trp Gly Gly Ser Thr Ile Asp Thr Glu 115 120
125Leu Lys Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp
Gly 130 135 140Ser Tyr Arg Ser Glu Glu
Leu Asn Leu Val Ile Ile Gly Pro Ser Ala145 150
155 160Asp Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly
His Glu Val Leu Asn 165 170
175Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro
180 185 190Asp Phe Thr Phe Gly Phe
Glu Glu Ser Leu Glu Val Asp Thr Asn Pro 195 200
205Leu Leu Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr
Leu Ala 210 215 220His Glu Leu Ile His
Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn225 230
235 240Pro Asn Arg Val Phe Lys Val Asn Thr Asn
Ala Tyr Tyr Glu Met Ser 245 250
255Gly Leu Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp
260 265 270Ala Lys Phe Ile Asp
Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr 275
280 285Tyr Asn Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn
Lys Ala Lys Ser 290 295 300Ile Val Gly
Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys305
310 315 320Glu Lys Tyr Leu Leu Ser Glu
Asp Thr Ser Gly Lys Phe Ser Val Asp 325
330 335Lys Leu Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr
Glu Ile Tyr Thr 340 345 350Glu
Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr 355
360 365Leu Asn Phe Asp Lys Ala Val Phe Lys
Ile Asn Ile Val Pro Lys Val 370 375
380Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala385
390 395 400Ala Asn Phe Asn
Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr 405
410 415Lys Leu Lys Asn Phe Thr Gly Leu Phe Glu
Phe Tyr Lys Leu Leu Cys 420 425
430Val Asp Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Ile Glu Gly Arg
435 440 445Phe Gly Gly Phe Thr Gly Ala
Arg Lys Ser Ala Arg Lys Tyr Ala Asn 450 455
460Gln Ala Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly465 470 475 480Gly Gly
Ser Ala Leu Val Leu Gln Cys Ile Lys Val Asn Asn Trp Asp
485 490 495Leu Phe Phe Ser Pro Ser Glu
Asp Asn Phe Thr Asn Asp Leu Asn Lys 500 505
510Gly Glu Glu Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu
Glu Asn 515 520 525Ile Ser Leu Asp
Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp 530
535 540Asn Glu Pro Glu Asn Ile Ser Ile Glu Asn Leu Ser
Ser Asp Ile Ile545 550 555
560Gly Gln Leu Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys
565 570 575Lys Tyr Glu Leu Asp
Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln 580
585 590Glu Phe Glu His Gly Lys Ser Arg Ile Ala Leu Thr
Asn Ser Val Asn 595 600 605Glu Ala
Leu Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp 610
615 620Tyr Val Lys Lys Val Asn Lys Ala Thr Glu Ala
Ala Met Phe Leu Gly625 630 635
640Trp Val Glu Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val
645 650 655Ser Thr Thr Asp
Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile 660
665 670Gly Pro Ala Leu Asn Ile Gly Asn Met Leu Tyr
Lys Asp Asp Phe Val 675 680 685Gly
Ala Leu Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro 690
695 700Glu Ile Ala Ile Pro Val Leu Gly Thr Phe
Ala Leu Val Ser Tyr Ile705 710 715
720Ala Asn Lys Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser
Lys 725 730 735Arg Asn Glu
Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp 740
745 750Leu Ala Lys Val Asn Thr Gln Ile Asp Leu
Ile Arg Lys Lys Met Lys 755 760
765Glu Ala Leu Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr 770
775 780Gln Tyr Asn Gln Tyr Thr Glu Glu
Glu Lys Asn Asn Ile Asn Phe Asn785 790
795 800Ile Asp Asp Leu Ser Ser Lys Leu Asn Glu Ser Ile
Asn Lys Ala Met 805 810
815Ile Asn Ile Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met
820 825 830Asn Ser Met Ile Pro Tyr
Gly Val Lys Arg Leu Glu Asp Phe Asp Ala 835 840
845Ser Leu Lys Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg
Gly Thr 850 855 860Leu Ile Gly Gln Val
Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu865 870
875 880Ser Thr Asp Ile Pro Phe Gln Leu Ser Lys
Tyr Val Asp Asn Gln Arg 885 890
895Leu Leu Ser Thr Leu Asp 900352697DNAArtificial
SequenceSynthetic 35ggatccatgg agttcgttaa caaacagttc aactataaag
acccagttaa cggtgttgac 60attgcttaca tcaaaatccc gaacgctggc cagatgcagc
cggtaaaggc attcaaaatc 120cacaacaaaa tctgggttat cccggaacgt gataccttta
ctaacccgga agaaggtgac 180ctgaacccgc caccggaagc gaaacaggtg ccggtatctt
actatgactc cacctacctg 240tctaccgata acgaaaagga caactacctg aaaggtgtta
ctaaactgtt cgagcgtatt 300tactccaccg acctgggccg tatgctgctg actagcatcg
ttcgcggtat cccgttctgg 360ggcggttcta ccatcgatac cgaactgaaa gtaatcgaca
ctaactgcat caacgttatt 420cagccggacg gttcctatcg ttccgaagaa ctgaacctgg
tgatcatcgg cccgtctgct 480gatatcatcc agttcgagtg taagagcttt ggtcacgaag
ttctgaacct cacccgtaac 540ggctacggtt ccactcagta catccgtttc tctccggact
tcaccttcgg ttttgaagaa 600tccctggaag tagacacgaa cccactgctg ggcgctggta
aattcgcaac tgatcctgcg 660gttaccctgg ctcacgaact gattcatgca ggccaccgcc
tgtacggtat cgccatcaat 720ccgaaccgtg tcttcaaagt taacaccaac gcgtattacg
agatgtccgg tctggaagtt 780agcttcgaag aactgcgtac ttttggcggt cacgacgcta
aattcatcga ctctctgcaa 840gaaaacgagt tccgtctgta ctactataac aagttcaaag
atatcgcatc caccctgaac 900aaagcgaaat ccatcgtggg taccactgct tctctccagt
acatgaagaa cgtttttaaa 960gaaaaatacc tgctcagcga agacacctcc ggcaaattct
ctgtagacaa gttgaaattc 1020gataaacttt acaaaatgct gactgaaatt tacaccgaag
acaacttcgt taagttcttt 1080aaagttctga accgcaaaac ctatctgaac ttcgacaagg
cagtattcaa aatcaacatc 1140gtgccgaaag ttaactacac tatctacgat ggtttcaacc
tgcgtaacac caacctggct 1200gctaatttta acggccagaa cacggaaatc aacaacatga
acttcacaaa actgaaaaac 1260ttcactggtc tgttcgagtt ttacaagctg ctgtgcgtcg
acggcatcat tacctccaaa 1320actaaatctc tgatagaagg tagatttggc ggtttcacgg
gcgcacgcaa atcagcgcgt 1380aaagcgctag cgggcggtgg cggtagcggc ggtggcggta
gcggcggtgg cggtagcgca 1440ctagtgctgc agtgtatcaa ggttaacaac tgggatttat
tcttcagccc gagtgaagac 1500aacttcacca acgacctgaa caaaggtgaa gaaatcacct
cagatactaa catcgaagca 1560gccgaagaaa acatctcgct ggacctgatc cagcagtact
acctgacctt taatttcgac 1620aacgagccgg aaaacatttc tatcgaaaac ctgagctctg
atatcatcgg ccagctggaa 1680ctgatgccga acatcgaacg tttcccaaac ggtaaaaagt
acgagctgga caaatatacc 1740atgttccact acctgcgcgc gcaggaattt gaacacggca
aatcccgtat cgcactgact 1800aactccgtta acgaagctct gctcaacccg tcccgtgtat
acaccttctt ctctagcgac 1860tacgtgaaaa aggtcaacaa agcgactgaa gctgcaatgt
tcttgggttg ggttgaacag 1920cttgtttatg attttaccga cgagacgtcc gaagtatcta
ctaccgacaa aattgcggat 1980atcactatca tcatcccgta catcggtccg gctctgaaca
ttggcaacat gctgtacaaa 2040gacgacttcg ttggcgcact gatcttctcc ggtgcggtga
tcctgctgga gttcatcccg 2100gaaatcgcca tcccggtact gggcaccttt gctctggttt
cttacattgc aaacaaggtt 2160ctgactgtac aaaccatcga caacgcgctg agcaaacgta
acgaaaaatg ggatgaagtt 2220tacaaatata tcgtgaccaa ctggctggct aaggttaata
ctcagatcga cctcatccgc 2280aaaaaaatga aagaagcact ggaaaaccag gcggaagcta
ccaaggcaat cattaactac 2340cagtacaacc agtacaccga ggaagaaaaa aacaacatca
acttcaacat cgacgatctg 2400tcctctaaac tgaacgaatc catcaacaaa gctatgatca
acatcaacaa gttcctgaac 2460cagtgctctg taagctatct gatgaactcc atgatcccgt
acggtgttaa acgtctggag 2520gacttcgatg cgtctctgaa agacgccctg ctgaaataca
tttacgacaa ccgtggcact 2580ctgatcggtc aggttgatcg tctgaaggac aaagtgaaca
ataccttatc gaccgacatc 2640ccttttcagc tcagtaaata tgtcgataac caacgccttt
tgtccactct agactag 269736898PRTArtificial SequenceSynthetic 36Gly
Ser Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val1
5 10 15Asn Gly Val Asp Ile Ala Tyr
Ile Lys Ile Pro Asn Ala Gly Gln Met 20 25
30Gln Pro Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val
Ile Pro 35 40 45Glu Arg Asp Thr
Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro 50 55
60Pro Glu Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser
Thr Tyr Leu65 70 75
80Ser Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu
85 90 95Phe Glu Arg Ile Tyr Ser
Thr Asp Leu Gly Arg Met Leu Leu Thr Ser 100
105 110Ile Val Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr
Ile Asp Thr Glu 115 120 125Leu Lys
Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly 130
135 140Ser Tyr Arg Ser Glu Glu Leu Asn Leu Val Ile
Ile Gly Pro Ser Ala145 150 155
160Asp Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn
165 170 175Leu Thr Arg Asn
Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro 180
185 190Asp Phe Thr Phe Gly Phe Glu Glu Ser Leu Glu
Val Asp Thr Asn Pro 195 200 205Leu
Leu Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala 210
215 220His Glu Leu Ile His Ala Gly His Arg Leu
Tyr Gly Ile Ala Ile Asn225 230 235
240Pro Asn Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met
Ser 245 250 255Gly Leu Glu
Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp 260
265 270Ala Lys Phe Ile Asp Ser Leu Gln Glu Asn
Glu Phe Arg Leu Tyr Tyr 275 280
285Tyr Asn Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser 290
295 300Ile Val Gly Thr Thr Ala Ser Leu
Gln Tyr Met Lys Asn Val Phe Lys305 310
315 320Glu Lys Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys
Phe Ser Val Asp 325 330
335Lys Leu Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr
340 345 350Glu Asp Asn Phe Val Lys
Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr 355 360
365Leu Asn Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro
Lys Val 370 375 380Asn Tyr Thr Ile Tyr
Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala385 390
395 400Ala Asn Phe Asn Gly Gln Asn Thr Glu Ile
Asn Asn Met Asn Phe Thr 405 410
415Lys Leu Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys
420 425 430Val Asp Gly Ile Ile
Thr Ser Lys Thr Lys Ser Leu Ile Glu Gly Arg 435
440 445Phe Gly Gly Phe Thr Gly Ala Arg Lys Ser Ala Arg
Lys Ala Leu Ala 450 455 460Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala465
470 475 480Leu Val Leu Gln Cys Ile Lys
Val Asn Asn Trp Asp Leu Phe Phe Ser 485
490 495Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys
Gly Glu Glu Ile 500 505 510Thr
Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu Asp 515
520 525Leu Ile Gln Gln Tyr Tyr Leu Thr Phe
Asn Phe Asp Asn Glu Pro Glu 530 535
540Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu Glu545
550 555 560Leu Met Pro Asn
Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu 565
570 575Asp Lys Tyr Thr Met Phe His Tyr Leu Arg
Ala Gln Glu Phe Glu His 580 585
590Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu Leu
595 600 605Asn Pro Ser Arg Val Tyr Thr
Phe Phe Ser Ser Asp Tyr Val Lys Lys 610 615
620Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu
Gln625 630 635 640Leu Val
Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr Asp
645 650 655Lys Ile Ala Asp Ile Thr Ile
Ile Ile Pro Tyr Ile Gly Pro Ala Leu 660 665
670Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala
Leu Ile 675 680 685Phe Ser Gly Ala
Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala Ile 690
695 700Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile
Ala Asn Lys Val705 710 715
720Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu Lys
725 730 735Trp Asp Glu Val Tyr
Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys Val 740
745 750Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys
Glu Ala Leu Glu 755 760 765Asn Gln
Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn Gln 770
775 780Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe
Asn Ile Asp Asp Leu785 790 795
800Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile Asn
805 810 815Lys Phe Leu Asn
Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met Ile 820
825 830Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp
Ala Ser Leu Lys Asp 835 840 845Ala
Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly Gln 850
855 860Val Asp Arg Leu Lys Asp Lys Val Asn Asn
Thr Leu Ser Thr Asp Ile865 870 875
880Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser
Thr 885 890 895Leu
Asp3751DNAArtificial SequenceSynthetic 37tttggcggtt tcacgggcgc acgcaaatca
gcgcgtaaat tagctaacca g 513817PRTArtificial
SequenceSynthetic 38Phe Gly Gly Phe Thr Gly Ala Arg Lys Ser Ala Arg Lys
Leu Ala Asn1 5 10
15Gln3933DNAArtificial SequenceSynthetic 39tttggcggtt tcacgggcgc
acgcaaatca gcg 334011PRTArtificial
SequenceSynthetic 40Phe Gly Gly Phe Thr Gly Ala Arg Lys Ser Ala1
5 104133DNAArtificial SequenceSynthetic
41tttggcggtt tcacgggcgc acgcaaatat gcg
334211PRTArtificial SequenceSynthetic 42Phe Gly Gly Phe Thr Gly Ala Arg
Lys Tyr Ala1 5 104333DNAArtificial
SequenceSynthetic 43tttggcggtt tcacgggcgc acgcaaatca tat
334411PRTArtificial SequenceSynthetic 44Phe Gly Gly Phe
Thr Gly Ala Arg Lys Ser Tyr1 5
104551DNAArtificial SequenceSynthetic 45tttggcggtt tcacgggcgc acgcaaatca
gcgcgtaaat atgctaacca g 514617PRTArtificial
SequenceSynthetic 46Phe Gly Gly Phe Thr Gly Ala Arg Lys Ser Ala Arg Lys
Tyr Ala Asn1 5 10
15Gln4739DNAArtificial SequenceSynthetic 47tttggcggtt tcacgggcgc
acgcaaatca gcgcgtaaa 394813PRTArtificial
SequenceSynthetic 48Phe Gly Gly Phe Thr Gly Ala Arg Lys Ser Ala Arg Lys1
5 104951DNAArtificial SequenceSynthetic
49tttggcggtt tcacgggcgc acgcaaatca gcgcgtaaac gcaaaaacca g
515017PRTArtificial SequenceSynthetic 50Phe Gly Gly Phe Thr Gly Ala Arg
Lys Ser Ala Arg Lys Arg Lys Asn1 5 10
15Gln512736DNAArtificial SequenceSynthetic 51ctcgggattg
agggtcgttt tggcggtttc acgggcgcac gcaaatcagc gcgtaaatta 60gctaaccaga
ctagtggcgg tgggggtagt ggcggtggcg gttcgggcgg gggtgggagc 120cctaggggat
ccatggagtt cgttaacaaa cagttcaact ataaagaccc agttaacggt 180gttgacattg
cttacatcaa aatcccgaac gctggccaga tgcagccggt aaaggcattc 240aaaatccaca
acaaaatctg ggttatcccg gaacgtgata cctttactaa cccggaagaa 300ggtgacctga
acccgccacc ggaagcgaaa caggtgccgg tatcttacta tgactccacc 360tacctgtcta
ccgataacga aaaggacaac tacctgaaag gtgttactaa actgttcgag 420cgtatttact
ccaccgacct gggccgtatg ctgctgacta gcatcgttcg cggtatcccg 480ttctggggcg
gttctaccat cgataccgaa ctgaaagtaa tcgacactaa ctgcatcaac 540gttattcagc
cggacggttc ctatcgttcc gaagaactga acctggtgat catcggcccg 600tctgctgata
tcatccagtt cgagtgtaag agctttggtc acgaagttct gaacctcacc 660cgtaacggct
acggttccac tcagtacatc cgtttctctc cggacttcac cttcggtttt 720gaagaatccc
tggaagtaga cacgaaccca ctgctgggcg ctggtaaatt cgcaactgat 780cctgcggtta
ccctggctca cgaactgatt catgcaggcc accgcctgta cggtatcgcc 840atcaatccga
accgtgtctt caaagttaac accaacgcgt attacgagat gtccggtctg 900gaagttagct
tcgaagaact gcgtactttt ggcggtcacg acgctaaatt catcgactct 960ctgcaagaaa
acgagttccg tctgtactac tataacaagt tcaaagatat cgcatccacc 1020ctgaacaaag
cgaaatccat cgtgggtacc actgcttctc tccagtacat gaagaacgtt 1080tttaaagaaa
aatacctgct cagcgaagac acctccggca aattctctgt agacaagttg 1140aaattcgata
aactttacaa aatgctgact gaaatttaca ccgaagacaa cttcgttaag 1200ttctttaaag
ttctgaaccg caaaacctat ctgaacttcg acaaggcagt attcaaaatc 1260aacatcgtgc
cgaaagttaa ctacactatc tacgatggtt tcaacctgcg taacaccaac 1320ctggctgcta
attttaacgg ccagaacacg gaaatcaaca acatgaactt cacaaaactg 1380aaaaacttca
ctggtctgtt cgagttttac aagctgctgt gcgtcgacgg catcattacc 1440tccaaaacta
aatctctgat agaaggtaga aacaaagcgc tgaacctgca gtgtatcaag 1500gttaacaact
gggatttatt cttcagcccg agtgaagaca acttcaccaa cgacctgaac 1560aaaggtgaag
aaatcacctc agatactaac atcgaagcag ccgaagaaaa catctcgctg 1620gacctgatcc
agcagtacta cctgaccttt aatttcgaca acgagccgga aaacatttct 1680atcgaaaacc
tgagctctga tatcatcggc cagctggaac tgatgccgaa catcgaacgt 1740ttcccaaacg
gtaaaaagta cgagctggac aaatatacca tgttccacta cctgcgcgcg 1800caggaatttg
aacacggcaa atcccgtatc gcactgacta actccgttaa cgaagctctg 1860ctcaacccgt
cccgtgtata caccttcttc tctagcgact acgtgaaaaa ggtcaacaaa 1920gcgactgaag
ctgcaatgtt cttgggttgg gttgaacagc ttgtttatga ttttaccgac 1980gagacgtccg
aagtatctac taccgacaaa attgcggata tcactatcat catcccgtac 2040atcggtccgg
ctctgaacat tggcaacatg ctgtacaaag acgacttcgt tggcgcactg 2100atcttctccg
gtgcggtgat cctgctggag ttcatcccgg aaatcgccat cccggtactg 2160ggcacctttg
ctctggtttc ttacattgca aacaaggttc tgactgtaca aaccatcgac 2220aacgcgctga
gcaaacgtaa cgaaaaatgg gatgaagttt acaaatatat cgtgaccaac 2280tggctggcta
aggttaatac tcagatcgac ctcatccgca aaaaaatgaa agaagcactg 2340gaaaaccagg
cggaagctac caaggcaatc attaactacc agtacaacca gtacaccgag 2400gaagaaaaaa
acaacatcaa cttcaacatc gacgatctgt cctctaaact gaacgaatcc 2460atcaacaaag
ctatgatcaa catcaacaag ttcctgaacc agtgctctgt aagctatctg 2520atgaactcca
tgatcccgta cggtgttaaa cgtctggagg acttcgatgc gtctctgaaa 2580gacgccctgc
tgaaatacat ttacgacaac cgtggcactc tgatcggtca ggttgatcgt 2640ctgaaggaca
aagtgaacaa taccttatcg accgacatcc cttttcagct cagtaaatat 2700gtcgataacc
aacgcctttt gtccactcta gactag
273652911PRTArtificial SequenceSynthetic 52Leu Gly Ile Glu Gly Arg Phe
Gly Gly Phe Thr Gly Ala Arg Lys Ser1 5 10
15Ala Arg Lys Leu Ala Asn Gln Thr Ser Gly Gly Gly Gly
Ser Gly Gly 20 25 30Gly Gly
Ser Gly Gly Gly Gly Ser Pro Arg Gly Ser Met Glu Phe Val 35
40 45Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val
Asn Gly Val Asp Ile Ala 50 55 60Tyr
Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro Val Lys Ala Phe65
70 75 80Lys Ile His Asn Lys Ile
Trp Val Ile Pro Glu Arg Asp Thr Phe Thr 85
90 95Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu
Ala Lys Gln Val 100 105 110Pro
Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr Asp Asn Glu Lys 115
120 125Asp Asn Tyr Leu Lys Gly Val Thr Lys
Leu Phe Glu Arg Ile Tyr Ser 130 135
140Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val Arg Gly Ile Pro145
150 155 160Phe Trp Gly Gly
Ser Thr Ile Asp Thr Glu Leu Lys Val Ile Asp Thr 165
170 175Asn Cys Ile Asn Val Ile Gln Pro Asp Gly
Ser Tyr Arg Ser Glu Glu 180 185
190Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile Ile Gln Phe Glu
195 200 205Cys Lys Ser Phe Gly His Glu
Val Leu Asn Leu Thr Arg Asn Gly Tyr 210 215
220Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe Thr Phe Gly
Phe225 230 235 240Glu Glu
Ser Leu Glu Val Asp Thr Asn Pro Leu Leu Gly Ala Gly Lys
245 250 255Phe Ala Thr Asp Pro Ala Val
Thr Leu Ala His Glu Leu Ile His Ala 260 265
270Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn Arg Val
Phe Lys 275 280 285Val Asn Thr Asn
Ala Tyr Tyr Glu Met Ser Gly Leu Glu Val Ser Phe 290
295 300Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys
Phe Ile Asp Ser305 310 315
320Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn Lys Phe Lys Asp
325 330 335Ile Ala Ser Thr Leu
Asn Lys Ala Lys Ser Ile Val Gly Thr Thr Ala 340
345 350Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys
Tyr Leu Leu Ser 355 360 365Glu Asp
Thr Ser Gly Lys Phe Ser Val Asp Lys Leu Lys Phe Asp Lys 370
375 380Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu
Asp Asn Phe Val Lys385 390 395
400Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn Phe Asp Lys Ala
405 410 415Val Phe Lys Ile
Asn Ile Val Pro Lys Val Asn Tyr Thr Ile Tyr Asp 420
425 430Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala
Asn Phe Asn Gly Gln 435 440 445Asn
Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu Lys Asn Phe Thr 450
455 460Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys
Val Asp Gly Ile Ile Thr465 470 475
480Ser Lys Thr Lys Ser Leu Ile Glu Gly Arg Asn Lys Ala Leu Asn
Leu 485 490 495Gln Cys Ile
Lys Val Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu 500
505 510Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly
Glu Glu Ile Thr Ser Asp 515 520
525Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu Asp Leu Ile Gln 530
535 540Gln Tyr Tyr Leu Thr Phe Asn Phe
Asp Asn Glu Pro Glu Asn Ile Ser545 550
555 560Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu
Glu Leu Met Pro 565 570
575Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu Asp Lys Tyr
580 585 590Thr Met Phe His Tyr Leu
Arg Ala Gln Glu Phe Glu His Gly Lys Ser 595 600
605Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu Leu Asn
Pro Ser 610 615 620Arg Val Tyr Thr Phe
Phe Ser Ser Asp Tyr Val Lys Lys Val Asn Lys625 630
635 640Ala Thr Glu Ala Ala Met Phe Leu Gly Trp
Val Glu Gln Leu Val Tyr 645 650
655Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr Asp Lys Ile Ala
660 665 670Asp Ile Thr Ile Ile
Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly 675
680 685Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu
Ile Phe Ser Gly 690 695 700Ala Val Ile
Leu Leu Glu Phe Ile Pro Glu Ile Ala Ile Pro Val Leu705
710 715 720Gly Thr Phe Ala Leu Val Ser
Tyr Ile Ala Asn Lys Val Leu Thr Val 725
730 735Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu
Lys Trp Asp Glu 740 745 750Val
Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys Val Asn Thr Gln 755
760 765Ile Asp Leu Ile Arg Lys Lys Met Lys
Glu Ala Leu Glu Asn Gln Ala 770 775
780Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr Thr Glu785
790 795 800Glu Glu Lys Asn
Asn Ile Asn Phe Asn Ile Asp Asp Leu Ser Ser Lys 805
810 815Leu Asn Glu Ser Ile Asn Lys Ala Met Ile
Asn Ile Asn Lys Phe Leu 820 825
830Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met Ile Pro Tyr Gly
835 840 845Val Lys Arg Leu Glu Asp Phe
Asp Ala Ser Leu Lys Asp Ala Leu Leu 850 855
860Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly Gln Val Asp
Arg865 870 875 880Leu Lys
Asp Lys Val Asn Asn Thr Leu Ser Thr Asp Ile Pro Phe Gln
885 890 895Leu Ser Lys Tyr Val Asp Asn
Gln Arg Leu Leu Ser Thr Leu Asp 900 905
91053177DNAArtificial SequenceSynthetic 53ggatccacgc acgtcgacgg
catcattacc tccaaaacta aatctctgat agaaggtaga 60tttggcggtt tcacgggcgc
acgcaaatca gcgcgtaaat tagctaacca ggcgctagcg 120ggtggtggtg gttctgcact
agtgctgcag acgcacggtc tagaatgata aaagctt 17754192DNAArtificial
SequenceSynthetic 54ggatccacgc acgtcgacgg catcattacc tccaaaacta
aatctctgat agaaggtaga 60tttggcggtt tcacgggcgc acgcaaatca gcgcgtaaat
tagctaacca ggcgctagcg 120ggtggtggtg gttctggtgg tggtggttct gcactagtgc
tgcagacgca cggtctagaa 180tgataaaagc tt
19255222DNAArtificial SequenceSynthetic
55ggatccacgc acgtcgacgg catcattacc tccaaaacta aatctctgat agaaggtaga
60tttggcggtt tcacgggcgc acgcaaatca gcgcgtaaat tagctaacca ggcgctagcg
120ggtggtggtg gttctggtgg tggtggttct ggtggtggtg gttctggtgg tggtggttct
180gcactagtgc tgcagacgca cggtctagaa tgataaaagc tt
22256237DNAArtificial SequenceSynthetic 56ggatccacgc acgtcgacgg
catcattacc tccaaaacta aatctctgat agaaggtaga 60tttggcggtt tcacgggcgc
acgcaaatca gcgcgtaaat tagctaacca ggcgctagcg 120ggtggtggtg gttctggtgg
tggtggttct ggtggtggtg gttctggtgg tggtggttct 180ggtggtggtg gttctgcact
agtgctgcag acgcacggtc tagaatgata aaagctt 23757228DNAArtificial
SequenceSynthetic 57ggatccacgc acgtcgacgg catcattacc tccaaaacta
aatctctgat agaaggtaga 60tttggcggtt tcacgggcgc acgcaaatca gcgcgtaaat
tagctaacca ggcgctagcg 120gctgaagctg ctgctaaaga agctgctgct aaagaagctg
ctgctaaagc tggtggcggt 180ggttccgcac tagtgctgca gacgcacggt ctagaatgat
aaaagctt 228582694DNAArtificial SequenceSynthetic
58ggatccatgg agttcgttaa caaacagttc aactataaag acccagttaa cggtgttgac
60attgcttaca tcaaaatccc gaacgctggc cagatgcagc cggtaaaggc attcaaaatc
120cacaacaaaa tctgggttat cccggaacgt gataccttta ctaacccgga agaaggtgac
180ctgaacccgc caccggaagc gaaacaggtg ccggtatctt actatgactc cacctacctg
240tctaccgata acgaaaagga caactacctg aaaggtgtta ctaaactgtt cgagcgtatt
300tactccaccg acctgggccg tatgctgctg actagcatcg ttcgcggtat cccgttctgg
360ggcggttcta ccatcgatac cgaactgaaa gtaatcgaca ctaactgcat caacgttatt
420cagccggacg gttcctatcg ttccgaagaa ctgaacctgg tgatcatcgg cccgtctgct
480gatatcatcc agttcgagtg taagagcttt ggtcacgaag ttctgaacct cacccgtaac
540ggctacggtt ccactcagta catccgtttc tctccggact tcaccttcgg ttttgaagaa
600tccctggaag tagacacgaa cccactgctg ggcgctggta aattcgcaac tgatcctgcg
660gttaccctgg ctcacgaact gattcatgca ggccaccgcc tgtacggtat cgccatcaat
720ccgaaccgtg tcttcaaagt taacaccaac gcgtattacg agatgtccgg tctggaagtt
780agcttcgaag aactgcgtac ttttggcggt cacgacgcta aattcatcga ctctctgcaa
840gaaaacgagt tccgtctgta ctactataac aagttcaaag atatcgcatc caccctgaac
900aaagcgaaat ccatcgtggg taccactgct tctctccagt acatgaagaa cgtttttaaa
960gaaaaatacc tgctcagcga agacacctcc ggcaaattct ctgtagacaa gttgaaattc
1020gataaacttt acaaaatgct gactgaaatt tacaccgaag acaacttcgt taagttcttt
1080aaagttctga accgcaaaac ctatctgaac ttcgacaagg cagtattcaa aatcaacatc
1140gtgccgaaag ttaactacac tatctacgat ggtttcaacc tgcgtaacac caacctggct
1200gctaatttta acggccagaa cacggaaatc aacaacatga acttcacaaa actgaaaaac
1260ttcactggtc tgttcgagtt ttacaagctg ctgtgcgtcg acggcatcat tacctccaaa
1320actaaatctc tgatagaagg tagatttggc ggtttcacgg gcgcacgcaa atcagcgcgt
1380aaattagcta accaggcgct agcgggtggt ggtggttctg gtggtggtgg ttctgcacta
1440gtgctgcagt gtatcaaggt taacaactgg gatttattct tcagcccgag tgaagacaac
1500ttcaccaacg acctgaacaa aggtgaagaa atcacctcag atactaacat cgaagcagcc
1560gaagaaaaca tctcgctgga cctgatccag cagtactacc tgacctttaa tttcgacaac
1620gagccggaaa acatttctat cgaaaacctg agctctgata tcatcggcca gctggaactg
1680atgccgaaca tcgaacgttt cccaaacggt aaaaagtacg agctggacaa atataccatg
1740ttccactacc tgcgcgcgca ggaatttgaa cacggcaaat cccgtatcgc actgactaac
1800tccgttaacg aagctctgct caacccgtcc cgtgtataca ccttcttctc tagcgactac
1860gtgaaaaagg tcaacaaagc gactgaagct gcaatgttct tgggttgggt tgaacagctt
1920gtttatgatt ttaccgacga gacgtccgaa gtatctacta ccgacaaaat tgcggatatc
1980actatcatca tcccgtacat cggtccggct ctgaacattg gcaacatgct gtacaaagac
2040gacttcgttg gcgcactgat cttctccggt gcggtgatcc tgctggagtt catcccggaa
2100atcgccatcc cggtactggg cacctttgct ctggtttctt acattgcaaa caaggttctg
2160actgtacaaa ccatcgacaa cgcgctgagc aaacgtaacg aaaaatggga tgaagtttac
2220aaatatatcg tgaccaactg gctggctaag gttaatactc agatcgacct catccgcaaa
2280aaaatgaaag aagcactgga aaaccaggcg gaagctacca aggcaatcat taactaccag
2340tacaaccagt acaccgagga agaaaaaaac aacatcaact tcaacatcga cgatctgtcc
2400tctaaactga acgaatccat caacaaagct atgatcaaca tcaacaagtt cctgaaccag
2460tgctctgtaa gctatctgat gaactccatg atcccgtacg gtgttaaacg tctggaggac
2520ttcgatgcgt ctctgaaaga cgccctgctg aaatacattt acgacaaccg tggcactctg
2580atcggtcagg ttgatcgtct gaaggacaaa gtgaacaata ccttatcgac cgacatccct
2640tttcagctca gtaaatatgt cgataaccaa cgccttttgt ccactctaga ctag
269459897PRTArtificial SequenceSynthetic 59Gly Ser Met Glu Phe Val Asn
Lys Gln Phe Asn Tyr Lys Asp Pro Val1 5 10
15Asn Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala
Gly Gln Met 20 25 30Gln Pro
Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro 35
40 45Glu Arg Asp Thr Phe Thr Asn Pro Glu Glu
Gly Asp Leu Asn Pro Pro 50 55 60Pro
Glu Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu65
70 75 80Ser Thr Asp Asn Glu Lys
Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu 85
90 95Phe Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met
Leu Leu Thr Ser 100 105 110Ile
Val Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu 115
120 125Leu Lys Val Ile Asp Thr Asn Cys Ile
Asn Val Ile Gln Pro Asp Gly 130 135
140Ser Tyr Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala145
150 155 160Asp Ile Ile Gln
Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn 165
170 175Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln
Tyr Ile Arg Phe Ser Pro 180 185
190Asp Phe Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro
195 200 205Leu Leu Gly Ala Gly Lys Phe
Ala Thr Asp Pro Ala Val Thr Leu Ala 210 215
220His Glu Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile
Asn225 230 235 240Pro Asn
Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser
245 250 255Gly Leu Glu Val Ser Phe Glu
Glu Leu Arg Thr Phe Gly Gly His Asp 260 265
270Ala Lys Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu
Tyr Tyr 275 280 285Tyr Asn Lys Phe
Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser 290
295 300Ile Val Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys
Asn Val Phe Lys305 310 315
320Glu Lys Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp
325 330 335Lys Leu Lys Phe Asp
Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr 340
345 350Glu Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn
Arg Lys Thr Tyr 355 360 365Leu Asn
Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val 370
375 380Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg
Asn Thr Asn Leu Ala385 390 395
400Ala Asn Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr
405 410 415Lys Leu Lys Asn
Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys 420
425 430Val Asp Gly Ile Ile Thr Ser Lys Thr Lys Ser
Leu Ile Glu Gly Arg 435 440 445Phe
Gly Gly Phe Thr Gly Ala Arg Lys Ser Ala Arg Lys Leu Ala Asn 450
455 460Gln Ala Leu Ala Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Ala Leu465 470 475
480Val Leu Gln Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe Ser
Pro 485 490 495Ser Glu Asp
Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu Ile Thr 500
505 510Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu
Asn Ile Ser Leu Asp Leu 515 520
525Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro Glu Asn 530
535 540Ile Ser Ile Glu Asn Leu Ser Ser
Asp Ile Ile Gly Gln Leu Glu Leu545 550
555 560Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys
Tyr Glu Leu Asp 565 570
575Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu His Gly
580 585 590Lys Ser Arg Ile Ala Leu
Thr Asn Ser Val Asn Glu Ala Leu Leu Asn 595 600
605Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys
Lys Val 610 615 620Asn Lys Ala Thr Glu
Ala Ala Met Phe Leu Gly Trp Val Glu Gln Leu625 630
635 640Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu
Val Ser Thr Thr Asp Lys 645 650
655Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn
660 665 670Ile Gly Asn Met Leu
Tyr Lys Asp Asp Phe Val Gly Ala Leu Ile Phe 675
680 685Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu
Ile Ala Ile Pro 690 695 700Val Leu Gly
Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys Val Leu705
710 715 720Thr Val Gln Thr Ile Asp Asn
Ala Leu Ser Lys Arg Asn Glu Lys Trp 725
730 735Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu
Ala Lys Val Asn 740 745 750Thr
Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu Glu Asn 755
760 765Gln Ala Glu Ala Thr Lys Ala Ile Ile
Asn Tyr Gln Tyr Asn Gln Tyr 770 775
780Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu Ser785
790 795 800Ser Lys Leu Asn
Glu Ser Ile Asn Lys Ala Met Ile Asn Ile Asn Lys 805
810 815Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu
Met Asn Ser Met Ile Pro 820 825
830Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys Asp Ala
835 840 845Leu Leu Lys Tyr Ile Tyr Asp
Asn Arg Gly Thr Leu Ile Gly Gln Val 850 855
860Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp Ile
Pro865 870 875 880Phe Gln
Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser Thr Leu
885 890 895Asp602724DNAArtificial
SequenceSynthetic 60ggatccatgg agttcgttaa caaacagttc aactataaag
acccagttaa cggtgttgac 60attgcttaca tcaaaatccc gaacgctggc cagatgcagc
cggtaaaggc attcaaaatc 120cacaacaaaa tctgggttat cccggaacgt gataccttta
ctaacccgga agaaggtgac 180ctgaacccgc caccggaagc gaaacaggtg ccggtatctt
actatgactc cacctacctg 240tctaccgata acgaaaagga caactacctg aaaggtgtta
ctaaactgtt cgagcgtatt 300tactccaccg acctgggccg tatgctgctg actagcatcg
ttcgcggtat cccgttctgg 360ggcggttcta ccatcgatac cgaactgaaa gtaatcgaca
ctaactgcat caacgttatt 420cagccggacg gttcctatcg ttccgaagaa ctgaacctgg
tgatcatcgg cccgtctgct 480gatatcatcc agttcgagtg taagagcttt ggtcacgaag
ttctgaacct cacccgtaac 540ggctacggtt ccactcagta catccgtttc tctccggact
tcaccttcgg ttttgaagaa 600tccctggaag tagacacgaa cccactgctg ggcgctggta
aattcgcaac tgatcctgcg 660gttaccctgg ctcacgaact gattcatgca ggccaccgcc
tgtacggtat cgccatcaat 720ccgaaccgtg tcttcaaagt taacaccaac gcgtattacg
agatgtccgg tctggaagtt 780agcttcgaag aactgcgtac ttttggcggt cacgacgcta
aattcatcga ctctctgcaa 840gaaaacgagt tccgtctgta ctactataac aagttcaaag
atatcgcatc caccctgaac 900aaagcgaaat ccatcgtggg taccactgct tctctccagt
acatgaagaa cgtttttaaa 960gaaaaatacc tgctcagcga agacacctcc ggcaaattct
ctgtagacaa gttgaaattc 1020gataaacttt acaaaatgct gactgaaatt tacaccgaag
acaacttcgt taagttcttt 1080aaagttctga accgcaaaac ctatctgaac ttcgacaagg
cagtattcaa aatcaacatc 1140gtgccgaaag ttaactacac tatctacgat ggtttcaacc
tgcgtaacac caacctggct 1200gctaatttta acggccagaa cacggaaatc aacaacatga
acttcacaaa actgaaaaac 1260ttcactggtc tgttcgagtt ttacaagctg ctgtgcgtcg
acggcatcat tacctccaaa 1320actaaatctc tgatagaagg tagatttggc ggtttcacgg
gcgcacgcaa atcagcgcgt 1380aaattagcta accaggcgct agcgggtggt ggtggttctg
gtggtggtgg ttctggtggt 1440ggtggttctg gtggtggtgg ttctgcacta gtgctgcagt
gtatcaaggt taacaactgg 1500gatttattct tcagcccgag tgaagacaac ttcaccaacg
acctgaacaa aggtgaagaa 1560atcacctcag atactaacat cgaagcagcc gaagaaaaca
tctcgctgga cctgatccag 1620cagtactacc tgacctttaa tttcgacaac gagccggaaa
acatttctat cgaaaacctg 1680agctctgata tcatcggcca gctggaactg atgccgaaca
tcgaacgttt cccaaacggt 1740aaaaagtacg agctggacaa atataccatg ttccactacc
tgcgcgcgca ggaatttgaa 1800cacggcaaat cccgtatcgc actgactaac tccgttaacg
aagctctgct caacccgtcc 1860cgtgtataca ccttcttctc tagcgactac gtgaaaaagg
tcaacaaagc gactgaagct 1920gcaatgttct tgggttgggt tgaacagctt gtttatgatt
ttaccgacga gacgtccgaa 1980gtatctacta ccgacaaaat tgcggatatc actatcatca
tcccgtacat cggtccggct 2040ctgaacattg gcaacatgct gtacaaagac gacttcgttg
gcgcactgat cttctccggt 2100gcggtgatcc tgctggagtt catcccggaa atcgccatcc
cggtactggg cacctttgct 2160ctggtttctt acattgcaaa caaggttctg actgtacaaa
ccatcgacaa cgcgctgagc 2220aaacgtaacg aaaaatggga tgaagtttac aaatatatcg
tgaccaactg gctggctaag 2280gttaatactc agatcgacct catccgcaaa aaaatgaaag
aagcactgga aaaccaggcg 2340gaagctacca aggcaatcat taactaccag tacaaccagt
acaccgagga agaaaaaaac 2400aacatcaact tcaacatcga cgatctgtcc tctaaactga
acgaatccat caacaaagct 2460atgatcaaca tcaacaagtt cctgaaccag tgctctgtaa
gctatctgat gaactccatg 2520atcccgtacg gtgttaaacg tctggaggac ttcgatgcgt
ctctgaaaga cgccctgctg 2580aaatacattt acgacaaccg tggcactctg atcggtcagg
ttgatcgtct gaaggacaaa 2640gtgaacaata ccttatcgac cgacatccct tttcagctca
gtaaatatgt cgataaccaa 2700cgccttttgt ccactctaga ctag
272461907PRTArtificial SequenceSynthetic 61Gly Ser
Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val1 5
10 15Asn Gly Val Asp Ile Ala Tyr Ile
Lys Ile Pro Asn Ala Gly Gln Met 20 25
30Gln Pro Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile
Pro 35 40 45Glu Arg Asp Thr Phe
Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro 50 55
60Pro Glu Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr
Tyr Leu65 70 75 80Ser
Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu
85 90 95Phe Glu Arg Ile Tyr Ser Thr
Asp Leu Gly Arg Met Leu Leu Thr Ser 100 105
110Ile Val Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp
Thr Glu 115 120 125Leu Lys Val Ile
Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly 130
135 140Ser Tyr Arg Ser Glu Glu Leu Asn Leu Val Ile Ile
Gly Pro Ser Ala145 150 155
160Asp Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn
165 170 175Leu Thr Arg Asn Gly
Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro 180
185 190Asp Phe Thr Phe Gly Phe Glu Glu Ser Leu Glu Val
Asp Thr Asn Pro 195 200 205Leu Leu
Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala 210
215 220His Glu Leu Ile His Ala Gly His Arg Leu Tyr
Gly Ile Ala Ile Asn225 230 235
240Pro Asn Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser
245 250 255Gly Leu Glu Val
Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp 260
265 270Ala Lys Phe Ile Asp Ser Leu Gln Glu Asn Glu
Phe Arg Leu Tyr Tyr 275 280 285Tyr
Asn Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser 290
295 300Ile Val Gly Thr Thr Ala Ser Leu Gln Tyr
Met Lys Asn Val Phe Lys305 310 315
320Glu Lys Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val
Asp 325 330 335Lys Leu Lys
Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr 340
345 350Glu Asp Asn Phe Val Lys Phe Phe Lys Val
Leu Asn Arg Lys Thr Tyr 355 360
365Leu Asn Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val 370
375 380Asn Tyr Thr Ile Tyr Asp Gly Phe
Asn Leu Arg Asn Thr Asn Leu Ala385 390
395 400Ala Asn Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn
Met Asn Phe Thr 405 410
415Lys Leu Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys
420 425 430Val Asp Gly Ile Ile Thr
Ser Lys Thr Lys Ser Leu Ile Glu Gly Arg 435 440
445Phe Gly Gly Phe Thr Gly Ala Arg Lys Ser Ala Arg Lys Leu
Ala Asn 450 455 460Gln Ala Leu Ala Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly465 470
475 480Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu
Val Leu Gln Cys Ile Lys 485 490
495Val Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu Asp Asn Phe Thr
500 505 510Asn Asp Leu Asn Lys
Gly Glu Glu Ile Thr Ser Asp Thr Asn Ile Glu 515
520 525Ala Ala Glu Glu Asn Ile Ser Leu Asp Leu Ile Gln
Gln Tyr Tyr Leu 530 535 540Thr Phe Asn
Phe Asp Asn Glu Pro Glu Asn Ile Ser Ile Glu Asn Leu545
550 555 560Ser Ser Asp Ile Ile Gly Gln
Leu Glu Leu Met Pro Asn Ile Glu Arg 565
570 575Phe Pro Asn Gly Lys Lys Tyr Glu Leu Asp Lys Tyr
Thr Met Phe His 580 585 590Tyr
Leu Arg Ala Gln Glu Phe Glu His Gly Lys Ser Arg Ile Ala Leu 595
600 605Thr Asn Ser Val Asn Glu Ala Leu Leu
Asn Pro Ser Arg Val Tyr Thr 610 615
620Phe Phe Ser Ser Asp Tyr Val Lys Lys Val Asn Lys Ala Thr Glu Ala625
630 635 640Ala Met Phe Leu
Gly Trp Val Glu Gln Leu Val Tyr Asp Phe Thr Asp 645
650 655Glu Thr Ser Glu Val Ser Thr Thr Asp Lys
Ile Ala Asp Ile Thr Ile 660 665
670Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly Asn Met Leu Tyr
675 680 685Lys Asp Asp Phe Val Gly Ala
Leu Ile Phe Ser Gly Ala Val Ile Leu 690 695
700Leu Glu Phe Ile Pro Glu Ile Ala Ile Pro Val Leu Gly Thr Phe
Ala705 710 715 720Leu Val
Ser Tyr Ile Ala Asn Lys Val Leu Thr Val Gln Thr Ile Asp
725 730 735Asn Ala Leu Ser Lys Arg Asn
Glu Lys Trp Asp Glu Val Tyr Lys Tyr 740 745
750Ile Val Thr Asn Trp Leu Ala Lys Val Asn Thr Gln Ile Asp
Leu Ile 755 760 765Arg Lys Lys Met
Lys Glu Ala Leu Glu Asn Gln Ala Glu Ala Thr Lys 770
775 780Ala Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr Thr Glu
Glu Glu Lys Asn785 790 795
800Asn Ile Asn Phe Asn Ile Asp Asp Leu Ser Ser Lys Leu Asn Glu Ser
805 810 815Ile Asn Lys Ala Met
Ile Asn Ile Asn Lys Phe Leu Asn Gln Cys Ser 820
825 830Val Ser Tyr Leu Met Asn Ser Met Ile Pro Tyr Gly
Val Lys Arg Leu 835 840 845Glu Asp
Phe Asp Ala Ser Leu Lys Asp Ala Leu Leu Lys Tyr Ile Tyr 850
855 860Asp Asn Arg Gly Thr Leu Ile Gly Gln Val Asp
Arg Leu Lys Asp Lys865 870 875
880Val Asn Asn Thr Leu Ser Thr Asp Ile Pro Phe Gln Leu Ser Lys Tyr
885 890 895Val Asp Asn Gln
Arg Leu Leu Ser Thr Leu Asp 900
90562207DNAArtificial SequenceSynthetic 62ggatccacgc acgtcgacgg
catcattacc tccaaaacta aatctgacga tgacgataaa 60tttggcggtt tcacgggcgc
acgcaaatca gcgcgtaaac gtaagaacca ggcgctagcg 120ggcggtggcg gtagcggcgg
tggcggtagc ggcggtggcg gtagcgcact agtgctgcag 180acgcacggtc tagaatgata
aaagctt 207632709DNAArtificial
SequenceSynthetic 63ggatccatgg agttcgttaa caaacagttc aactataaag
acccagttaa cggtgttgac 60attgcttaca tcaaaatccc gaacgctggc cagatgcagc
cggtaaaggc attcaaaatc 120cacaacaaaa tctgggttat cccggaacgt gataccttta
ctaacccgga agaaggtgac 180ctgaacccgc caccggaagc gaaacaggtg ccggtatctt
actatgactc cacctacctg 240tctaccgata acgaaaagga caactacctg aaaggtgtta
ctaaactgtt cgagcgtatt 300tactccaccg acctgggccg tatgctgctg actagcatcg
ttcgcggtat cccgttctgg 360ggcggttcta ccatcgatac cgaactgaaa gtaatcgaca
ctaactgcat caacgttatt 420cagccggacg gttcctatcg ttccgaagaa ctgaacctgg
tgatcatcgg cccgtctgct 480gatatcatcc agttcgagtg taagagcttt ggtcacgaag
ttctgaacct cacccgtaac 540ggctacggtt ccactcagta catccgtttc tctccggact
tcaccttcgg ttttgaagaa 600tccctggaag tagacacgaa cccactgctg ggcgctggta
aattcgcaac tgatcctgcg 660gttaccctgg ctcacgaact gattcatgca ggccaccgcc
tgtacggtat cgccatcaat 720ccgaaccgtg tcttcaaagt taacaccaac gcgtattacg
agatgtccgg tctggaagtt 780agcttcgaag aactgcgtac ttttggcggt cacgacgcta
aattcatcga ctctctgcaa 840gaaaacgagt tccgtctgta ctactataac aagttcaaag
atatcgcatc caccctgaac 900aaagcgaaat ccatcgtggg taccactgct tctctccagt
acatgaagaa cgtttttaaa 960gaaaaatacc tgctcagcga agacacctcc ggcaaattct
ctgtagacaa gttgaaattc 1020gataaacttt acaaaatgct gactgaaatt tacaccgaag
acaacttcgt taagttcttt 1080aaagttctga accgcaaaac ctatctgaac ttcgacaagg
cagtattcaa aatcaacatc 1140gtgccgaaag ttaactacac tatctacgat ggtttcaacc
tgcgtaacac caacctggct 1200gctaatttta acggccagaa cacggaaatc aacaacatga
acttcacaaa actgaaaaac 1260ttcactggtc tgttcgagtt ttacaagctg ctgtgcgtcg
acggcatcat tacctccaaa 1320actaaatctg acgatgacga taaatttggc ggtttcacgg
gcgcacgcaa atcagcgcgt 1380aaacgtaaga accaggcgct agcgggcggt ggcggtagcg
gcggtggcgg tagcggcggt 1440ggcggtagcg cactagtgct gcagtgtatc aaggttaaca
actgggattt attcttcagc 1500ccgagtgaag acaacttcac caacgacctg aacaaaggtg
aagaaatcac ctcagatact 1560aacatcgaag cagccgaaga aaacatctcg ctggacctga
tccagcagta ctacctgacc 1620tttaatttcg acaacgagcc ggaaaacatt tctatcgaaa
acctgagctc tgatatcatc 1680ggccagctgg aactgatgcc gaacatcgaa cgtttcccaa
acggtaaaaa gtacgagctg 1740gacaaatata ccatgttcca ctacctgcgc gcgcaggaat
ttgaacacgg caaatcccgt 1800atcgcactga ctaactccgt taacgaagct ctgctcaacc
cgtcccgtgt atacaccttc 1860ttctctagcg actacgtgaa aaaggtcaac aaagcgactg
aagctgcaat gttcttgggt 1920tgggttgaac agcttgttta tgattttacc gacgagacgt
ccgaagtatc tactaccgac 1980aaaattgcgg atatcactat catcatcccg tacatcggtc
cggctctgaa cattggcaac 2040atgctgtaca aagacgactt cgttggcgca ctgatcttct
ccggtgcggt gatcctgctg 2100gagttcatcc cggaaatcgc catcccggta ctgggcacct
ttgctctggt ttcttacatt 2160gcaaacaagg ttctgactgt acaaaccatc gacaacgcgc
tgagcaaacg taacgaaaaa 2220tgggatgaag tttacaaata tatcgtgacc aactggctgg
ctaaggttaa tactcagatc 2280gacctcatcc gcaaaaaaat gaaagaagca ctggaaaacc
aggcggaagc taccaaggca 2340atcattaact accagtacaa ccagtacacc gaggaagaaa
aaaacaacat caacttcaac 2400atcgacgatc tgtcctctaa actgaacgaa tccatcaaca
aagctatgat caacatcaac 2460aagttcctga accagtgctc tgtaagctat ctgatgaact
ccatgatccc gtacggtgtt 2520aaacgtctgg aggacttcga tgcgtctctg aaagacgccc
tgctgaaata catttacgac 2580aaccgtggca ctctgatcgg tcaggttgat cgtctgaagg
acaaagtgaa caatacctta 2640tcgaccgaca tcccttttca gctcagtaaa tatgtcgata
accaacgcct tttgtccact 2700ctagactag
270964902PRTArtificial SequenceSynthetic 64Gly Ser
Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val1 5
10 15Asn Gly Val Asp Ile Ala Tyr Ile
Lys Ile Pro Asn Ala Gly Gln Met 20 25
30Gln Pro Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile
Pro 35 40 45Glu Arg Asp Thr Phe
Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro 50 55
60Pro Glu Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr
Tyr Leu65 70 75 80Ser
Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu
85 90 95Phe Glu Arg Ile Tyr Ser Thr
Asp Leu Gly Arg Met Leu Leu Thr Ser 100 105
110Ile Val Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp
Thr Glu 115 120 125Leu Lys Val Ile
Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly 130
135 140Ser Tyr Arg Ser Glu Glu Leu Asn Leu Val Ile Ile
Gly Pro Ser Ala145 150 155
160Asp Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn
165 170 175Leu Thr Arg Asn Gly
Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro 180
185 190Asp Phe Thr Phe Gly Phe Glu Glu Ser Leu Glu Val
Asp Thr Asn Pro 195 200 205Leu Leu
Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala 210
215 220His Glu Leu Ile His Ala Gly His Arg Leu Tyr
Gly Ile Ala Ile Asn225 230 235
240Pro Asn Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser
245 250 255Gly Leu Glu Val
Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp 260
265 270Ala Lys Phe Ile Asp Ser Leu Gln Glu Asn Glu
Phe Arg Leu Tyr Tyr 275 280 285Tyr
Asn Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser 290
295 300Ile Val Gly Thr Thr Ala Ser Leu Gln Tyr
Met Lys Asn Val Phe Lys305 310 315
320Glu Lys Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val
Asp 325 330 335Lys Leu Lys
Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr 340
345 350Glu Asp Asn Phe Val Lys Phe Phe Lys Val
Leu Asn Arg Lys Thr Tyr 355 360
365Leu Asn Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val 370
375 380Asn Tyr Thr Ile Tyr Asp Gly Phe
Asn Leu Arg Asn Thr Asn Leu Ala385 390
395 400Ala Asn Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn
Met Asn Phe Thr 405 410
415Lys Leu Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys
420 425 430Val Asp Gly Ile Ile Thr
Ser Lys Thr Lys Ser Asp Asp Asp Asp Lys 435 440
445Phe Gly Gly Phe Thr Gly Ala Arg Lys Ser Ala Arg Lys Arg
Lys Asn 450 455 460Gln Ala Leu Ala Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly465 470
475 480Gly Gly Ser Ala Leu Val Leu Gln Cys Ile
Lys Val Asn Asn Trp Asp 485 490
495Leu Phe Phe Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys
500 505 510Gly Glu Glu Ile Thr
Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn 515
520 525Ile Ser Leu Asp Leu Ile Gln Gln Tyr Tyr Leu Thr
Phe Asn Phe Asp 530 535 540Asn Glu Pro
Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile545
550 555 560Gly Gln Leu Glu Leu Met Pro
Asn Ile Glu Arg Phe Pro Asn Gly Lys 565
570 575Lys Tyr Glu Leu Asp Lys Tyr Thr Met Phe His Tyr
Leu Arg Ala Gln 580 585 590Glu
Phe Glu His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn 595
600 605Glu Ala Leu Leu Asn Pro Ser Arg Val
Tyr Thr Phe Phe Ser Ser Asp 610 615
620Tyr Val Lys Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly625
630 635 640Trp Val Glu Gln
Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val 645
650 655Ser Thr Thr Asp Lys Ile Ala Asp Ile Thr
Ile Ile Ile Pro Tyr Ile 660 665
670Gly Pro Ala Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val
675 680 685Gly Ala Leu Ile Phe Ser Gly
Ala Val Ile Leu Leu Glu Phe Ile Pro 690 695
700Glu Ile Ala Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr
Ile705 710 715 720Ala Asn
Lys Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys
725 730 735Arg Asn Glu Lys Trp Asp Glu
Val Tyr Lys Tyr Ile Val Thr Asn Trp 740 745
750Leu Ala Lys Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys
Met Lys 755 760 765Glu Ala Leu Glu
Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr 770
775 780Gln Tyr Asn Gln Tyr Thr Glu Glu Glu Lys Asn Asn
Ile Asn Phe Asn785 790 795
800Ile Asp Asp Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met
805 810 815Ile Asn Ile Asn Lys
Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met 820
825 830Asn Ser Met Ile Pro Tyr Gly Val Lys Arg Leu Glu
Asp Phe Asp Ala 835 840 845Ser Leu
Lys Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr 850
855 860Leu Ile Gly Gln Val Asp Arg Leu Lys Asp Lys
Val Asn Asn Thr Leu865 870 875
880Ser Thr Asp Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg
885 890 895Leu Leu Ser Thr
Leu Asp 90065207DNAArtificial SequenceSynthetic 65ggatccacgc
acgtcgacgg catcattacc tccaaaacta aatctctgat agaaggtaga 60tttggcggtt
tcacgggcgc acgcaaatca gcgcgtaaac gtaagaacca ggcgctagcg 120ggcggtggcg
gtagcggcgg tggcggtagc ggcggtggcg gtagcgcact agtgctgcag 180acgcacggtc
tagaatgata aaagctt
207662742DNAArtificial SequenceSynthetic 66ggatccgaat tcatgccgat
caccatcaac aacttcaact acagcgatcc ggtggataac 60aaaaacatcc tgtacctgga
tacccatctg aataccctgg cgaacgaacc ggaaaaagcg 120tttcgtatca ccggcaacat
ttgggttatt ccggatcgtt ttagccgtaa cagcaacccg 180aatctgaata aaccgccgcg
tgttaccagc ccgaaaagcg gttattacga tccgaactat 240ctgagcaccg atagcgataa
agataccttc ctgaaagaaa tcatcaaact gttcaaacgc 300atcaacagcc gtgaaattgg
cgaagaactg atctatcgcc tgagcaccga tattccgttt 360ccgggcaaca acaacacccc
gatcaacacc tttgatttcg atgtggattt caacagcgtt 420gatgttaaaa cccgccaggg
taacaattgg gtgaaaaccg gcagcattaa cccgagcgtg 480attattaccg gtccgcgcga
aaacattatt gatccggaaa ccagcacctt taaactgacc 540aacaacacct ttgcggcgca
ggaaggtttt ggcgcgctga gcattattag cattagcccg 600cgctttatgc tgacctatag
caacgcgacc aacgatgttg gtgaaggccg tttcagcaaa 660agcgaatttt gcatggaccc
gatcctgatc ctgatgcatg aactgaacca tgcgatgcat 720aacctgtatg gcatcgcgat
tccgaacgat cagaccatta gcagcgtgac cagcaacatc 780ttttacagcc agtacaacgt
gaaactggaa tatgcggaaa tctatgcgtt tggcggtccg 840accattgatc tgattccgaa
aagcgcgcgc aaatacttcg aagaaaaagc gctggattac 900tatcgcagca ttgcgaaacg
tctgaacagc attaccaccg cgaatccgag cagcttcaac 960aaatatatcg gcgaatataa
acagaaactg atccgcaaat atcgctttgt ggtggaaagc 1020agcggcgaag ttaccgttaa
ccgcaataaa ttcgtggaac tgtacaacga actgacccag 1080atcttcaccg aatttaacta
tgcgaaaatc tataacgtgc agaaccgtaa aatctacctg 1140agcaacgtgt ataccccggt
gaccgcgaat attctggatg ataacgtgta cgatatccag 1200aacggcttta acatcccgaa
aagcaacctg aacgttctgt ttatgggcca gaacctgagc 1260cgtaatccgg cgctgcgtaa
agtgaacccg gaaaacatgc tgtacctgtt caccaaattt 1320tgcgtcgacg gcatcattac
ctccaaaact aaatctctga tagaaggtag atttggcggt 1380ttcacgggcg cacgcaaatc
agcgcgtaaa cgtaagaacc aggcgctagc gggcggtggc 1440ggtagcggcg gtggcggtag
cggcggtggc ggtagcgcac tagtgctgca gtgtcgtgaa 1500ctgctggtga aaaacaccga
tctgccgttt attggcgata tcagcgatgt gaaaaccgat 1560atcttcctgc gcaaagatat
caacgaagaa accgaagtga tctactaccc ggataacgtg 1620agcgttgatc aggtgatcct
gagcaaaaac accagcgaac atggtcagct ggatctgctg 1680tatccgagca ttgatagcga
aagcgaaatt ctgccgggcg aaaaccaggt gttttacgat 1740aaccgtaccc agaacgtgga
ttacctgaac agctattact acctggaaag ccagaaactg 1800agcgataacg tggaagattt
tacctttacc cgcagcattg aagaagcgct ggataacagc 1860gcgaaagttt acacctattt
tccgaccctg gcgaacaaag ttaatgcggg tgttcagggc 1920ggtctgtttc tgatgtgggc
gaacgatgtg gtggaagatt tcaccaccaa catcctgcgt 1980aaagataccc tggataaaat
cagcgatgtt agcgcgatta ttccgtatat tggtccggcg 2040ctgaacatta gcaatagcgt
gcgtcgtggc aattttaccg aagcgtttgc ggttaccggt 2100gtgaccattc tgctggaagc
gtttccggaa tttaccattc cggcgctggg tgcgtttgtg 2160atctatagca aagtgcagga
acgcaacgaa atcatcaaaa ccatcgataa ctgcctggaa 2220cagcgtatta aacgctggaa
agatagctat gaatggatga tgggcacctg gctgagccgt 2280attatcaccc agttcaacaa
catcagctac cagatgtacg atagcctgaa ctatcaggcg 2340ggtgcgatta aagcgaaaat
cgatctggaa tacaaaaaat acagcggcag cgataaagaa 2400aacatcaaaa gccaggttga
aaacctgaaa aacagcctgg atgtgaaaat tagcgaagcg 2460atgaataaca tcaacaaatt
catccgcgaa tgcagcgtga cctacctgtt caaaaacatg 2520ctgccgaaag tgatcgatga
actgaacgaa tttgatcgca acaccaaagc gaaactgatc 2580aacctgatcg atagccacaa
cattattctg gtgggcgaag tggataaact gaaagcgaaa 2640gttaacaaca gcttccagaa
caccatcccg tttaacatct tcagctatac caacaacagc 2700ctgctgaaag atatcatcaa
cgaatacttc aatctagact ag 274267913PRTArtificial
SequenceSynthetic 67Gly Ser Glu Phe Met Pro Ile Thr Ile Asn Asn Phe Asn
Tyr Ser Asp1 5 10 15Pro
Val Asp Asn Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr 20
25 30Leu Ala Asn Glu Pro Glu Lys Ala
Phe Arg Ile Thr Gly Asn Ile Trp 35 40
45Val Ile Pro Asp Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys
50 55 60Pro Pro Arg Val Thr Ser Pro Lys
Ser Gly Tyr Tyr Asp Pro Asn Tyr65 70 75
80Leu Ser Thr Asp Ser Asp Lys Asp Thr Phe Leu Lys Glu
Ile Ile Lys 85 90 95Leu
Phe Lys Arg Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr
100 105 110Arg Leu Ser Thr Asp Ile Pro
Phe Pro Gly Asn Asn Asn Thr Pro Ile 115 120
125Asn Thr Phe Asp Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys
Thr 130 135 140Arg Gln Gly Asn Asn Trp
Val Lys Thr Gly Ser Ile Asn Pro Ser Val145 150
155 160Ile Ile Thr Gly Pro Arg Glu Asn Ile Ile Asp
Pro Glu Thr Ser Thr 165 170
175Phe Lys Leu Thr Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala
180 185 190Leu Ser Ile Ile Ser Ile
Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn 195 200
205Ala Thr Asn Asp Val Gly Glu Gly Arg Phe Ser Lys Ser Glu
Phe Cys 210 215 220Met Asp Pro Ile Leu
Ile Leu Met His Glu Leu Asn His Ala Met His225 230
235 240Asn Leu Tyr Gly Ile Ala Ile Pro Asn Asp
Gln Thr Ile Ser Ser Val 245 250
255Thr Ser Asn Ile Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala
260 265 270Glu Ile Tyr Ala Phe
Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser 275
280 285Ala Arg Lys Tyr Phe Glu Glu Lys Ala Leu Asp Tyr
Tyr Arg Ser Ile 290 295 300Ala Lys Arg
Leu Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn305
310 315 320Lys Tyr Ile Gly Glu Tyr Lys
Gln Lys Leu Ile Arg Lys Tyr Arg Phe 325
330 335Val Val Glu Ser Ser Gly Glu Val Thr Val Asn Arg
Asn Lys Phe Val 340 345 350Glu
Leu Tyr Asn Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn Tyr Ala 355
360 365Lys Ile Tyr Asn Val Gln Asn Arg Lys
Ile Tyr Leu Ser Asn Val Tyr 370 375
380Thr Pro Val Thr Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln385
390 395 400Asn Gly Phe Asn
Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly 405
410 415Gln Asn Leu Ser Arg Asn Pro Ala Leu Arg
Lys Val Asn Pro Glu Asn 420 425
430Met Leu Tyr Leu Phe Thr Lys Phe Cys Val Asp Gly Ile Ile Thr Ser
435 440 445Lys Thr Lys Ser Leu Ile Glu
Gly Arg Phe Gly Gly Phe Thr Gly Ala 450 455
460Arg Lys Ser Ala Arg Lys Arg Lys Asn Gln Ala Leu Ala Gly Gly
Gly465 470 475 480Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Val Leu
485 490 495Gln Cys Arg Glu Leu Leu Val
Lys Asn Thr Asp Leu Pro Phe Ile Gly 500 505
510Asp Ile Ser Asp Val Lys Thr Asp Ile Phe Leu Arg Lys Asp
Ile Asn 515 520 525Glu Glu Thr Glu
Val Ile Tyr Tyr Pro Asp Asn Val Ser Val Asp Gln 530
535 540Val Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gln
Leu Asp Leu Leu545 550 555
560Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly Glu Asn Gln
565 570 575Val Phe Tyr Asp Asn
Arg Thr Gln Asn Val Asp Tyr Leu Asn Ser Tyr 580
585 590Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn Val
Glu Asp Phe Thr 595 600 605Phe Thr
Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser Ala Lys Val Tyr 610
615 620Thr Tyr Phe Pro Thr Leu Ala Asn Lys Val Asn
Ala Gly Val Gln Gly625 630 635
640Gly Leu Phe Leu Met Trp Ala Asn Asp Val Val Glu Asp Phe Thr Thr
645 650 655Asn Ile Leu Arg
Lys Asp Thr Leu Asp Lys Ile Ser Asp Val Ser Ala 660
665 670Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile
Ser Asn Ser Val Arg 675 680 685Arg
Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val Thr Ile Leu 690
695 700Leu Glu Ala Phe Pro Glu Phe Thr Ile Pro
Ala Leu Gly Ala Phe Val705 710 715
720Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys Thr Ile
Asp 725 730 735Asn Cys Leu
Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser Tyr Glu Trp 740
745 750Met Met Gly Thr Trp Leu Ser Arg Ile Ile
Thr Gln Phe Asn Asn Ile 755 760
765Ser Tyr Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala Gly Ala Ile Lys 770
775 780Ala Lys Ile Asp Leu Glu Tyr Lys
Lys Tyr Ser Gly Ser Asp Lys Glu785 790
795 800Asn Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser
Leu Asp Val Lys 805 810
815Ile Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser
820 825 830Val Thr Tyr Leu Phe Lys
Asn Met Leu Pro Lys Val Ile Asp Glu Leu 835 840
845Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn Leu
Ile Asp 850 855 860Ser His Asn Ile Ile
Leu Val Gly Glu Val Asp Lys Leu Lys Ala Lys865 870
875 880Val Asn Asn Ser Phe Gln Asn Thr Ile Pro
Phe Asn Ile Phe Ser Tyr 885 890
895Thr Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr Phe Asn Leu
900 905
910Asp682673DNAArtificial SequenceSynthetic 68ggatccatgg agttcgttaa
caaacagttc aactataaag acccagttaa cggtgttgac 60attgcttaca tcaaaatccc
gaacgctggc cagatgcagc cggtaaaggc attcaaaatc 120cacaacaaaa tctgggttat
cccggaacgt gataccttta ctaacccgga agaaggtgac 180ctgaacccgc caccggaagc
gaaacaggtg ccggtatctt actatgactc cacctacctg 240tctaccgata acgaaaagga
caactacctg aaaggtgtta ctaaactgtt cgagcgtatt 300tactccaccg acctgggccg
tatgctgctg actagcatcg ttcgcggtat cccgttctgg 360ggcggttcta ccatcgatac
cgaactgaaa gtaatcgaca ctaactgcat caacgttatt 420cagccggacg gttcctatcg
ttccgaagaa ctgaacctgg tgatcatcgg cccgtctgct 480gatatcatcc agttcgagtg
taagagcttt ggtcacgaag ttctgaacct cacccgtaac 540ggctacggtt ccactcagta
catccgtttc tctccggact tcaccttcgg ttttgaagaa 600tccctggaag tagacacgaa
cccactgctg ggcgctggta aattcgcaac tgatcctgcg 660gttaccctgg ctcacgaact
gattcatgca ggccaccgcc tgtacggtat cgccatcaat 720ccgaaccgtg tcttcaaagt
taacaccaac gcgtattacg agatgtccgg tctggaagtt 780agcttcgaag aactgcgtac
ttttggcggt cacgacgcta aattcatcga ctctctgcaa 840gaaaacgagt tccgtctgta
ctactataac aagttcaaag atatcgcatc caccctgaac 900aaagcgaaat ccatcgtggg
taccactgct tctctccagt acatgaagaa cgtttttaaa 960gaaaaatacc tgctcagcga
agacacctcc ggcaaattct ctgtagacaa gttgaaattc 1020gataaacttt acaaaatgct
gactgaaatt tacaccgaag acaacttcgt taagttcttt 1080aaagttctga accgcaaaac
ctatctgaac ttcgacaagg cagtattcaa aatcaacatc 1140gtgccgaaag ttaactacac
tatctacgat ggtttcaacc tgcgtaacac caacctggct 1200gctaatttta acggccagaa
cacggaaatc aacaacatga acttcacaaa actgaaaaac 1260ttcactggtc tgttcgagtt
ttacaagctg ctgtgcgtcg acggcatcat tacctccaaa 1320actaaatctc tgatagaagg
tagatacggt ggtttcctgg cgctagcggg cggtggcggt 1380agcggcggtg gcggtagcgg
cggtggcggt agcgcactag tgctgcagtg tatcaaggtt 1440aacaactggg atttattctt
cagcccgagt gaagacaact tcaccaacga cctgaacaaa 1500ggtgaagaaa tcacctcaga
tactaacatc gaagcagccg aagaaaacat ctcgctggac 1560ctgatccagc agtactacct
gacctttaat ttcgacaacg agccggaaaa catttctatc 1620gaaaacctga gctctgatat
catcggccag ctggaactga tgccgaacat cgaacgtttc 1680ccaaacggta aaaagtacga
gctggacaaa tataccatgt tccactacct gcgcgcgcag 1740gaatttgaac acggcaaatc
ccgtatcgca ctgactaact ccgttaacga agctctgctc 1800aacccgtccc gtgtatacac
cttcttctct agcgactacg tgaaaaaggt caacaaagcg 1860actgaagctg caatgttctt
gggttgggtt gaacagcttg tttatgattt taccgacgag 1920acgtccgaag tatctactac
cgacaaaatt gcggatatca ctatcatcat cccgtacatc 1980ggtccggctc tgaacattgg
caacatgctg tacaaagacg acttcgttgg cgcactgatc 2040ttctccggtg cggtgatcct
gctggagttc atcccggaaa tcgccatccc ggtactgggc 2100acctttgctc tggtttctta
cattgcaaac aaggttctga ctgtacaaac catcgacaac 2160gcgctgagca aacgtaacga
aaaatgggat gaagtttaca aatatatcgt gaccaactgg 2220ctggctaagg ttaatactca
gatcgacctc atccgcaaaa aaatgaaaga agcactggaa 2280aaccaggcgg aagctaccaa
ggcaatcatt aactaccagt acaaccagta caccgaggaa 2340gaaaaaaaca acatcaactt
caacatcgac gatctgtcct ctaaactgaa cgaatccatc 2400aacaaagcta tgatcaacat
caacaagttc ctgaaccagt gctctgtaag ctatctgatg 2460aactccatga tcccgtacgg
tgttaaacgt ctggaggact tcgatgcgtc tctgaaagac 2520gccctgctga aatacattta
cgacaaccgt ggcactctga tcggtcaggt tgatcgtctg 2580aaggacaaag tgaacaatac
cttatcgacc gacatccctt ttcagctcag taaatatgtc 2640gataaccaac gccttttgtc
cactctagac tag 267369890PRTArtificial
SequenceSynthetic 69Gly Ser Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys
Asp Pro Val1 5 10 15Asn
Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met 20
25 30Gln Pro Val Lys Ala Phe Lys Ile
His Asn Lys Ile Trp Val Ile Pro 35 40
45Glu Arg Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro
50 55 60Pro Glu Ala Lys Gln Val Pro Val
Ser Tyr Tyr Asp Ser Thr Tyr Leu65 70 75
80Ser Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val
Thr Lys Leu 85 90 95Phe
Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser
100 105 110Ile Val Arg Gly Ile Pro Phe
Trp Gly Gly Ser Thr Ile Asp Thr Glu 115 120
125Leu Lys Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp
Gly 130 135 140Ser Tyr Arg Ser Glu Glu
Leu Asn Leu Val Ile Ile Gly Pro Ser Ala145 150
155 160Asp Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly
His Glu Val Leu Asn 165 170
175Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro
180 185 190Asp Phe Thr Phe Gly Phe
Glu Glu Ser Leu Glu Val Asp Thr Asn Pro 195 200
205Leu Leu Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr
Leu Ala 210 215 220His Glu Leu Ile His
Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn225 230
235 240Pro Asn Arg Val Phe Lys Val Asn Thr Asn
Ala Tyr Tyr Glu Met Ser 245 250
255Gly Leu Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp
260 265 270Ala Lys Phe Ile Asp
Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr 275
280 285Tyr Asn Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn
Lys Ala Lys Ser 290 295 300Ile Val Gly
Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys305
310 315 320Glu Lys Tyr Leu Leu Ser Glu
Asp Thr Ser Gly Lys Phe Ser Val Asp 325
330 335Lys Leu Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr
Glu Ile Tyr Thr 340 345 350Glu
Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr 355
360 365Leu Asn Phe Asp Lys Ala Val Phe Lys
Ile Asn Ile Val Pro Lys Val 370 375
380Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala385
390 395 400Ala Asn Phe Asn
Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr 405
410 415Lys Leu Lys Asn Phe Thr Gly Leu Phe Glu
Phe Tyr Lys Leu Leu Cys 420 425
430Val Asp Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Ile Glu Gly Arg
435 440 445Tyr Gly Gly Phe Leu Ala Leu
Ala Gly Gly Gly Gly Ser Gly Gly Gly 450 455
460Gly Ser Gly Gly Gly Gly Ser Ala Leu Val Leu Gln Cys Ile Lys
Val465 470 475 480Asn Asn
Trp Asp Leu Phe Phe Ser Pro Ser Glu Asp Asn Phe Thr Asn
485 490 495Asp Leu Asn Lys Gly Glu Glu
Ile Thr Ser Asp Thr Asn Ile Glu Ala 500 505
510Ala Glu Glu Asn Ile Ser Leu Asp Leu Ile Gln Gln Tyr Tyr
Leu Thr 515 520 525Phe Asn Phe Asp
Asn Glu Pro Glu Asn Ile Ser Ile Glu Asn Leu Ser 530
535 540Ser Asp Ile Ile Gly Gln Leu Glu Leu Met Pro Asn
Ile Glu Arg Phe545 550 555
560Pro Asn Gly Lys Lys Tyr Glu Leu Asp Lys Tyr Thr Met Phe His Tyr
565 570 575Leu Arg Ala Gln Glu
Phe Glu His Gly Lys Ser Arg Ile Ala Leu Thr 580
585 590Asn Ser Val Asn Glu Ala Leu Leu Asn Pro Ser Arg
Val Tyr Thr Phe 595 600 605Phe Ser
Ser Asp Tyr Val Lys Lys Val Asn Lys Ala Thr Glu Ala Ala 610
615 620Met Phe Leu Gly Trp Val Glu Gln Leu Val Tyr
Asp Phe Thr Asp Glu625 630 635
640Thr Ser Glu Val Ser Thr Thr Asp Lys Ile Ala Asp Ile Thr Ile Ile
645 650 655Ile Pro Tyr Ile
Gly Pro Ala Leu Asn Ile Gly Asn Met Leu Tyr Lys 660
665 670Asp Asp Phe Val Gly Ala Leu Ile Phe Ser Gly
Ala Val Ile Leu Leu 675 680 685Glu
Phe Ile Pro Glu Ile Ala Ile Pro Val Leu Gly Thr Phe Ala Leu 690
695 700Val Ser Tyr Ile Ala Asn Lys Val Leu Thr
Val Gln Thr Ile Asp Asn705 710 715
720Ala Leu Ser Lys Arg Asn Glu Lys Trp Asp Glu Val Tyr Lys Tyr
Ile 725 730 735Val Thr Asn
Trp Leu Ala Lys Val Asn Thr Gln Ile Asp Leu Ile Arg 740
745 750Lys Lys Met Lys Glu Ala Leu Glu Asn Gln
Ala Glu Ala Thr Lys Ala 755 760
765Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr Thr Glu Glu Glu Lys Asn Asn 770
775 780Ile Asn Phe Asn Ile Asp Asp Leu
Ser Ser Lys Leu Asn Glu Ser Ile785 790
795 800Asn Lys Ala Met Ile Asn Ile Asn Lys Phe Leu Asn
Gln Cys Ser Val 805 810
815Ser Tyr Leu Met Asn Ser Met Ile Pro Tyr Gly Val Lys Arg Leu Glu
820 825 830Asp Phe Asp Ala Ser Leu
Lys Asp Ala Leu Leu Lys Tyr Ile Tyr Asp 835 840
845Asn Arg Gly Thr Leu Ile Gly Gln Val Asp Arg Leu Lys Asp
Lys Val 850 855 860Asn Asn Thr Leu Ser
Thr Asp Ile Pro Phe Gln Leu Ser Lys Tyr Val865 870
875 880Asp Asn Gln Arg Leu Leu Ser Thr Leu Asp
885 890702709DNAArtificial SequenceSynthetic
70ggatccatgg agttcgttaa caaacagttc aactataaag acccagttaa cggtgttgac
60attgcttaca tcaaaatccc gaacgctggc cagatgcagc cggtaaaggc attcaaaatc
120cacaacaaaa tctgggttat cccggaacgt gataccttta ctaacccgga agaaggtgac
180ctgaacccgc caccggaagc gaaacaggtg ccggtatctt actatgactc cacctacctg
240tctaccgata acgaaaagga caactacctg aaaggtgtta ctaaactgtt cgagcgtatt
300tactccaccg acctgggccg tatgctgctg actagcatcg ttcgcggtat cccgttctgg
360ggcggttcta ccatcgatac cgaactgaaa gtaatcgaca ctaactgcat caacgttatt
420cagccggacg gttcctatcg ttccgaagaa ctgaacctgg tgatcatcgg cccgtctgct
480gatatcatcc agttcgagtg taagagcttt ggtcacgaag ttctgaacct cacccgtaac
540ggctacggtt ccactcagta catccgtttc tctccggact tcaccttcgg ttttgaagaa
600tccctggaag tagacacgaa cccactgctg ggcgctggta aattcgcaac tgatcctgcg
660gttaccctgg ctcacgaact gattcatgca ggccaccgcc tgtacggtat cgccatcaat
720ccgaaccgtg tcttcaaagt taacaccaac gcgtattacg agatgtccgg tctggaagtt
780agcttcgaag aactgcgtac ttttggcggt cacgacgcta aattcatcga ctctctgcaa
840gaaaacgagt tccgtctgta ctactataac aagttcaaag atatcgcatc caccctgaac
900aaagcgaaat ccatcgtggg taccactgct tctctccagt acatgaagaa cgtttttaaa
960gaaaaatacc tgctcagcga agacacctcc ggcaaattct ctgtagacaa gttgaaattc
1020gataaacttt acaaaatgct gactgaaatt tacaccgaag acaacttcgt taagttcttt
1080aaagttctga accgcaaaac ctatctgaac ttcgacaagg cagtattcaa aatcaacatc
1140gtgccgaaag ttaactacac tatctacgat ggtttcaacc tgcgtaacac caacctggct
1200gctaatttta acggccagaa cacggaaatc aacaacatga acttcacaaa actgaaaaac
1260ttcactggtc tgttcgagtt ttacaagctg ctgtgcgtcg acggcatcat tacctccaaa
1320actaaatctc tgatagaagg tagatatggc ggtttcacgg gcgcacgcaa atcagcgcgt
1380aaattagcta accaggcgct agcgggcggt ggcggtagcg gcggtggcgg tagcggcggt
1440ggcggtagcg cactagtgct gcagtgtatc aaggttaaca actgggattt attcttcagc
1500ccgagtgaag acaacttcac caacgacctg aacaaaggtg aagaaatcac ctcagatact
1560aacatcgaag cagccgaaga aaacatctcg ctggacctga tccagcagta ctacctgacc
1620tttaatttcg acaacgagcc ggaaaacatt tctatcgaaa acctgagctc tgatatcatc
1680ggccagctgg aactgatgcc gaacatcgaa cgtttcccaa acggtaaaaa gtacgagctg
1740gacaaatata ccatgttcca ctacctgcgc gcgcaggaat ttgaacacgg caaatcccgt
1800atcgcactga ctaactccgt taacgaagct ctgctcaacc cgtcccgtgt atacaccttc
1860ttctctagcg actacgtgaa aaaggtcaac aaagcgactg aagctgcaat gttcttgggt
1920tgggttgaac agcttgttta tgattttacc gacgagacgt ccgaagtatc tactaccgac
1980aaaattgcgg atatcactat catcatcccg tacatcggtc cggctctgaa cattggcaac
2040atgctgtaca aagacgactt cgttggcgca ctgatcttct ccggtgcggt gatcctgctg
2100gagttcatcc cggaaatcgc catcccggta ctgggcacct ttgctctggt ttcttacatt
2160gcaaacaagg ttctgactgt acaaaccatc gacaacgcgc tgagcaaacg taacgaaaaa
2220tgggatgaag tttacaaata tatcgtgacc aactggctgg ctaaggttaa tactcagatc
2280gacctcatcc gcaaaaaaat gaaagaagca ctggaaaacc aggcggaagc taccaaggca
2340atcattaact accagtacaa ccagtacacc gaggaagaaa aaaacaacat caacttcaac
2400atcgacgatc tgtcctctaa actgaacgaa tccatcaaca aagctatgat caacatcaac
2460aagttcctga accagtgctc tgtaagctat ctgatgaact ccatgatccc gtacggtgtt
2520aaacgtctgg aggacttcga tgcgtctctg aaagacgccc tgctgaaata catttacgac
2580aaccgtggca ctctgatcgg tcaggttgat cgtctgaagg acaaagtgaa caatacctta
2640tcgaccgaca tcccttttca gctcagtaaa tatgtcgata accaacgcct tttgtccact
2700ctagactag
270971902PRTArtificial SequenceSynthetic 71Gly Ser Met Glu Phe Val Asn
Lys Gln Phe Asn Tyr Lys Asp Pro Val1 5 10
15Asn Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala
Gly Gln Met 20 25 30Gln Pro
Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro 35
40 45Glu Arg Asp Thr Phe Thr Asn Pro Glu Glu
Gly Asp Leu Asn Pro Pro 50 55 60Pro
Glu Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu65
70 75 80Ser Thr Asp Asn Glu Lys
Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu 85
90 95Phe Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met
Leu Leu Thr Ser 100 105 110Ile
Val Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu 115
120 125Leu Lys Val Ile Asp Thr Asn Cys Ile
Asn Val Ile Gln Pro Asp Gly 130 135
140Ser Tyr Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala145
150 155 160Asp Ile Ile Gln
Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn 165
170 175Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln
Tyr Ile Arg Phe Ser Pro 180 185
190Asp Phe Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro
195 200 205Leu Leu Gly Ala Gly Lys Phe
Ala Thr Asp Pro Ala Val Thr Leu Ala 210 215
220His Glu Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile
Asn225 230 235 240Pro Asn
Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser
245 250 255Gly Leu Glu Val Ser Phe Glu
Glu Leu Arg Thr Phe Gly Gly His Asp 260 265
270Ala Lys Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu
Tyr Tyr 275 280 285Tyr Asn Lys Phe
Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser 290
295 300Ile Val Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys
Asn Val Phe Lys305 310 315
320Glu Lys Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp
325 330 335Lys Leu Lys Phe Asp
Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr 340
345 350Glu Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn
Arg Lys Thr Tyr 355 360 365Leu Asn
Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val 370
375 380Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg
Asn Thr Asn Leu Ala385 390 395
400Ala Asn Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr
405 410 415Lys Leu Lys Asn
Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys 420
425 430Val Asp Gly Ile Ile Thr Ser Lys Thr Lys Ser
Leu Ile Glu Gly Arg 435 440 445Tyr
Gly Gly Phe Thr Gly Ala Arg Lys Ser Ala Arg Lys Leu Ala Asn 450
455 460Gln Ala Leu Ala Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly465 470 475
480Gly Gly Ser Ala Leu Val Leu Gln Cys Ile Lys Val Asn Asn Trp
Asp 485 490 495Leu Phe Phe
Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys 500
505 510Gly Glu Glu Ile Thr Ser Asp Thr Asn Ile
Glu Ala Ala Glu Glu Asn 515 520
525Ile Ser Leu Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp 530
535 540Asn Glu Pro Glu Asn Ile Ser Ile
Glu Asn Leu Ser Ser Asp Ile Ile545 550
555 560Gly Gln Leu Glu Leu Met Pro Asn Ile Glu Arg Phe
Pro Asn Gly Lys 565 570
575Lys Tyr Glu Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln
580 585 590Glu Phe Glu His Gly Lys
Ser Arg Ile Ala Leu Thr Asn Ser Val Asn 595 600
605Glu Ala Leu Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser
Ser Asp 610 615 620Tyr Val Lys Lys Val
Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly625 630
635 640Trp Val Glu Gln Leu Val Tyr Asp Phe Thr
Asp Glu Thr Ser Glu Val 645 650
655Ser Thr Thr Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile
660 665 670Gly Pro Ala Leu Asn
Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val 675
680 685Gly Ala Leu Ile Phe Ser Gly Ala Val Ile Leu Leu
Glu Phe Ile Pro 690 695 700Glu Ile Ala
Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile705
710 715 720Ala Asn Lys Val Leu Thr Val
Gln Thr Ile Asp Asn Ala Leu Ser Lys 725
730 735Arg Asn Glu Lys Trp Asp Glu Val Tyr Lys Tyr Ile
Val Thr Asn Trp 740 745 750Leu
Ala Lys Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys 755
760 765Glu Ala Leu Glu Asn Gln Ala Glu Ala
Thr Lys Ala Ile Ile Asn Tyr 770 775
780Gln Tyr Asn Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn785
790 795 800Ile Asp Asp Leu
Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met 805
810 815Ile Asn Ile Asn Lys Phe Leu Asn Gln Cys
Ser Val Ser Tyr Leu Met 820 825
830Asn Ser Met Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala
835 840 845Ser Leu Lys Asp Ala Leu Leu
Lys Tyr Ile Tyr Asp Asn Arg Gly Thr 850 855
860Leu Ile Gly Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr
Leu865 870 875 880Ser Thr
Asp Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg
885 890 895Leu Leu Ser Thr Leu Asp
900722709DNAArtificial SequenceSynthetic 72ggatccatgg agttcgttaa
caaacagttc aactataaag acccagttaa cggtgttgac 60attgcttaca tcaaaatccc
gaacgctggc cagatgcagc cggtaaaggc attcaaaatc 120cacaacaaaa tctgggttat
cccggaacgt gataccttta ctaacccgga agaaggtgac 180ctgaacccgc caccggaagc
gaaacaggtg ccggtatctt actatgactc cacctacctg 240tctaccgata acgaaaagga
caactacctg aaaggtgtta ctaaactgtt cgagcgtatt 300tactccaccg acctgggccg
tatgctgctg actagcatcg ttcgcggtat cccgttctgg 360ggcggttcta ccatcgatac
cgaactgaaa gtaatcgaca ctaactgcat caacgttatt 420cagccggacg gttcctatcg
ttccgaagaa ctgaacctgg tgatcatcgg cccgtctgct 480gatatcatcc agttcgagtg
taagagcttt ggtcacgaag ttctgaacct cacccgtaac 540ggctacggtt ccactcagta
catccgtttc tctccggact tcaccttcgg ttttgaagaa 600tccctggaag tagacacgaa
cccactgctg ggcgctggta aattcgcaac tgatcctgcg 660gttaccctgg ctcacgaact
gattcatgca ggccaccgcc tgtacggtat cgccatcaat 720ccgaaccgtg tcttcaaagt
taacaccaac gcgtattacg agatgtccgg tctggaagtt 780agcttcgaag aactgcgtac
ttttggcggt cacgacgcta aattcatcga ctctctgcaa 840gaaaacgagt tccgtctgta
ctactataac aagttcaaag atatcgcatc caccctgaac 900aaagcgaaat ccatcgtggg
taccactgct tctctccagt acatgaagaa cgtttttaaa 960gaaaaatacc tgctcagcga
agacacctcc ggcaaattct ctgtagacaa gttgaaattc 1020gataaacttt acaaaatgct
gactgaaatt tacaccgaag acaacttcgt taagttcttt 1080aaagttctga accgcaaaac
ctatctgaac ttcgacaagg cagtattcaa aatcaacatc 1140gtgccgaaag ttaactacac
tatctacgat ggtttcaacc tgcgtaacac caacctggct 1200gctaatttta acggccagaa
cacggaaatc aacaacatga acttcacaaa actgaaaaac 1260ttcactggtc tgttcgagtt
ttacaagctg ctgtgcgtcg acggcatcat tacctccaaa 1320actaaatctc tgatagaagg
tagatatggc ggtttcacgg gcgcacgcaa atcagcgcgt 1380aaacgtaaga accaggcgct
agcgggcggt ggcggtagcg gcggtggcgg tagcggcggt 1440ggcggtagcg cactagtgct
gcagtgtatc aaggttaaca actgggattt attcttcagc 1500ccgagtgaag acaacttcac
caacgacctg aacaaaggtg aagaaatcac ctcagatact 1560aacatcgaag cagccgaaga
aaacatctcg ctggacctga tccagcagta ctacctgacc 1620tttaatttcg acaacgagcc
ggaaaacatt tctatcgaaa acctgagctc tgatatcatc 1680ggccagctgg aactgatgcc
gaacatcgaa cgtttcccaa acggtaaaaa gtacgagctg 1740gacaaatata ccatgttcca
ctacctgcgc gcgcaggaat ttgaacacgg caaatcccgt 1800atcgcactga ctaactccgt
taacgaagct ctgctcaacc cgtcccgtgt atacaccttc 1860ttctctagcg actacgtgaa
aaaggtcaac aaagcgactg aagctgcaat gttcttgggt 1920tgggttgaac agcttgttta
tgattttacc gacgagacgt ccgaagtatc tactaccgac 1980aaaattgcgg atatcactat
catcatcccg tacatcggtc cggctctgaa cattggcaac 2040atgctgtaca aagacgactt
cgttggcgca ctgatcttct ccggtgcggt gatcctgctg 2100gagttcatcc cggaaatcgc
catcccggta ctgggcacct ttgctctggt ttcttacatt 2160gcaaacaagg ttctgactgt
acaaaccatc gacaacgcgc tgagcaaacg taacgaaaaa 2220tgggatgaag tttacaaata
tatcgtgacc aactggctgg ctaaggttaa tactcagatc 2280gacctcatcc gcaaaaaaat
gaaagaagca ctggaaaacc aggcggaagc taccaaggca 2340atcattaact accagtacaa
ccagtacacc gaggaagaaa aaaacaacat caacttcaac 2400atcgacgatc tgtcctctaa
actgaacgaa tccatcaaca aagctatgat caacatcaac 2460aagttcctga accagtgctc
tgtaagctat ctgatgaact ccatgatccc gtacggtgtt 2520aaacgtctgg aggacttcga
tgcgtctctg aaagacgccc tgctgaaata catttacgac 2580aaccgtggca ctctgatcgg
tcaggttgat cgtctgaagg acaaagtgaa caatacctta 2640tcgaccgaca tcccttttca
gctcagtaaa tatgtcgata accaacgcct tttgtccact 2700ctagactag
270973902PRTArtificial
SequenceSynthetic 73Gly Ser Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys
Asp Pro Val1 5 10 15Asn
Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met 20
25 30Gln Pro Val Lys Ala Phe Lys Ile
His Asn Lys Ile Trp Val Ile Pro 35 40
45Glu Arg Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro
50 55 60Pro Glu Ala Lys Gln Val Pro Val
Ser Tyr Tyr Asp Ser Thr Tyr Leu65 70 75
80Ser Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val
Thr Lys Leu 85 90 95Phe
Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser
100 105 110Ile Val Arg Gly Ile Pro Phe
Trp Gly Gly Ser Thr Ile Asp Thr Glu 115 120
125Leu Lys Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp
Gly 130 135 140Ser Tyr Arg Ser Glu Glu
Leu Asn Leu Val Ile Ile Gly Pro Ser Ala145 150
155 160Asp Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly
His Glu Val Leu Asn 165 170
175Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro
180 185 190Asp Phe Thr Phe Gly Phe
Glu Glu Ser Leu Glu Val Asp Thr Asn Pro 195 200
205Leu Leu Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr
Leu Ala 210 215 220His Glu Leu Ile His
Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn225 230
235 240Pro Asn Arg Val Phe Lys Val Asn Thr Asn
Ala Tyr Tyr Glu Met Ser 245 250
255Gly Leu Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp
260 265 270Ala Lys Phe Ile Asp
Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr 275
280 285Tyr Asn Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn
Lys Ala Lys Ser 290 295 300Ile Val Gly
Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys305
310 315 320Glu Lys Tyr Leu Leu Ser Glu
Asp Thr Ser Gly Lys Phe Ser Val Asp 325
330 335Lys Leu Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr
Glu Ile Tyr Thr 340 345 350Glu
Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr 355
360 365Leu Asn Phe Asp Lys Ala Val Phe Lys
Ile Asn Ile Val Pro Lys Val 370 375
380Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala385
390 395 400Ala Asn Phe Asn
Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr 405
410 415Lys Leu Lys Asn Phe Thr Gly Leu Phe Glu
Phe Tyr Lys Leu Leu Cys 420 425
430Val Asp Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Ile Glu Gly Arg
435 440 445Tyr Gly Gly Phe Thr Gly Ala
Arg Lys Ser Ala Arg Lys Arg Lys Asn 450 455
460Gln Ala Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly465 470 475 480Gly Gly
Ser Ala Leu Val Leu Gln Cys Ile Lys Val Asn Asn Trp Asp
485 490 495Leu Phe Phe Ser Pro Ser Glu
Asp Asn Phe Thr Asn Asp Leu Asn Lys 500 505
510Gly Glu Glu Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu
Glu Asn 515 520 525Ile Ser Leu Asp
Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp 530
535 540Asn Glu Pro Glu Asn Ile Ser Ile Glu Asn Leu Ser
Ser Asp Ile Ile545 550 555
560Gly Gln Leu Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys
565 570 575Lys Tyr Glu Leu Asp
Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln 580
585 590Glu Phe Glu His Gly Lys Ser Arg Ile Ala Leu Thr
Asn Ser Val Asn 595 600 605Glu Ala
Leu Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp 610
615 620Tyr Val Lys Lys Val Asn Lys Ala Thr Glu Ala
Ala Met Phe Leu Gly625 630 635
640Trp Val Glu Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val
645 650 655Ser Thr Thr Asp
Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile 660
665 670Gly Pro Ala Leu Asn Ile Gly Asn Met Leu Tyr
Lys Asp Asp Phe Val 675 680 685Gly
Ala Leu Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro 690
695 700Glu Ile Ala Ile Pro Val Leu Gly Thr Phe
Ala Leu Val Ser Tyr Ile705 710 715
720Ala Asn Lys Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser
Lys 725 730 735Arg Asn Glu
Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp 740
745 750Leu Ala Lys Val Asn Thr Gln Ile Asp Leu
Ile Arg Lys Lys Met Lys 755 760
765Glu Ala Leu Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr 770
775 780Gln Tyr Asn Gln Tyr Thr Glu Glu
Glu Lys Asn Asn Ile Asn Phe Asn785 790
795 800Ile Asp Asp Leu Ser Ser Lys Leu Asn Glu Ser Ile
Asn Lys Ala Met 805 810
815Ile Asn Ile Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met
820 825 830Asn Ser Met Ile Pro Tyr
Gly Val Lys Arg Leu Glu Asp Phe Asp Ala 835 840
845Ser Leu Lys Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg
Gly Thr 850 855 860Leu Ile Gly Gln Val
Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu865 870
875 880Ser Thr Asp Ile Pro Phe Gln Leu Ser Lys
Tyr Val Asp Asn Gln Arg 885 890
895Leu Leu Ser Thr Leu Asp 900742889DNAArtificial
SequenceSynthetic 74ggatccttgg tacgagatga cgttgactat caaattttcc
gcgactttgc ggaaaataaa 60ggtaagtttt tcgtcggcgc cacagacctg tccgtcaaaa
ataagagagg ccagaacatc 120ggtaacgcac tgagcaacgt ccctatgatt gattttagtg
tagcggacgt taataaacgg 180attgcaaccg tcgttgatcc gcagtatgct gtcagcgtca
aacatgctaa agcggaagtt 240catacgttct attacgggca atataacggc cataacgatg
tggctgataa agaaaatgaa 300tatcgcgtgg tcgagcagaa caattacgaa ccgcacaaag
cgtggggcgc gagtaattta 360ggccgcctgg aggactataa catggcccgt ttcaataaat
tcgtgaccga ggtagcaccg 420atcgccccca cagatgctgg tgggggcctg gatacctaca
aagataaaaa ccgcttctct 480agcttcgtgc gcattggcgc cggtcgtcag ctcgtgtacg
agaagggtgt ctatcaccag 540gaaggtaatg aaaaggggta cgacctccgt gatttgtccc
aggcgtatcg ctacgctatt 600gccggaaccc cgtataaaga tattaatatc gatcaaacca
tgaataccga aggcctaatt 660ggtttcggga atcataataa gcaatatagc gcagaagagc
taaagcaggc cctcagccaa 720gatgcgttaa ccaattacgg agtgttaggc gatagcggca
gtccgctgtt tgccttcgat 780aaacagaaaa atcaatgggt gtttctgggc acttatgatt
attgggccgg atatggtaaa 840aagagctggc aggaatggaa tatttataaa aaggaattcg
cagacaaaat caagcagcat 900gacaacgcag gtacggtgaa ggggaacggc gaacatcact
ggaagacgac cggcacgaat 960agtcatatcg gatcgacggc cgttcgcctg gcgaacaatg
agggcgatgc aaacaatggg 1020caaaacgtga cctttgagga caacggtacc ctggtcctta
accagaacat aaatcagggc 1080gcgggaggct tgttctttaa aggcgactat actgttaagg
gagcaaacaa tgacatcacc 1140tggttagggg ccggtattga cgttgcggat ggaaaaaagg
tggtttggca ggttaaaaac 1200cctaacgggg accggctggc aaaaatcggc aaagggacat
tggaaattaa tggtaccggt 1260gtgaatcagg gtcagctgaa agtgggagat gggaccgtga
ttctgaacca gaaagcagac 1320gctgacaaaa aggtgcaagc ctttagccaa gtaggaattg
ttagtggtcg tggcacactc 1380gtcttgaact caagcaacca aataaatccg gataacctgt
actttggatt tcgtggcgga 1440cgcctggatg ctaacgggaa tgatctgacc tttgaacata
tccgtaacgt tgacgagggt 1500gcgcgcatag ttaatcataa tactgaccat gcatcaacta
tcaccttgac cgggaaaagt 1560ctgattacaa acccaaactc tctgtcagta cattccatcc
agaatgatta tgatgaagac 1620gattactcat actattaccg gccgcgtaga ccaattccac
aaggtaaaga tctttattac 1680aaaaattacc gttattacgc attaaaatcc ggagggcggc
tgaatgcacc tatgccggaa 1740aatggcgtgg ccgaaaacaa tgactggatt tttatgggtt
atactcaaga agaggctcgc 1800aaaaatgcaa tgaaccataa aaataaccga aggatcggtg
atttcggcgg atttttcgat 1860gaggaaaatg gtaaaggtca caatggtgcg ctgaatctaa
attttaacgg caaaagtgcc 1920cagaaacgtt tccttctgac tggtggcgct aatctgaatg
gtaaaatcag tgtgacgcag 1980ggtaacgtgc tgctttctgg ccggccaact ccgcatgcac
gtgattttgt aaataaatcg 2040agcgctcgta aagatgcgca tttttctaaa aataacgagg
tcgtgtttga agatgactgg 2100ataaatcgca cctttaaagc ggcagaaatc gcggttaatc
agagtgcgag cttttcatcg 2160ggtaggaatg tatctgatat tacagcaaac attacagcca
ctgataatgc gaaggtcaac 2220ctgggttata aaaacggtga tgaagtttgt gttcgatcgg
attacacggg ctatgttacc 2280tgcaacactg gcaatctgtc tgataaagcg cttaactctt
ttgacgccac gcgcattaac 2340gggaatgtga acctgaacca aaacgctgcc ttggtacttg
gtaaggccgc gttgtggggt 2400aaaattcagg gccagggcaa ctcccgtgtg tctctgaacc
agcactcgaa gtggcacctg 2460acgggggact cgcaggtgca caacttgtcc ctggccgata
gccatattca ccttaacaat 2520gcgtccgatg cccagtcagc taataaatat catacgatca
aaatcaatca cctctctggc 2580aacggtcact ttcactactt aacggattta gcaaaaaact
taggggataa agtcctggta 2640aaagaatcag cgagcggaca ttatcagtta catgtacaga
acaaaacagg cgagccaaat 2700caggaaggcc ttgacttatt tgatgcttca tcggtacaag
atcgttccag actgttcgtt 2760tcactcgcga atcactacgt tgatctgggt gcgctgcgct
atactataaa gacggaaaat 2820ggcataacac gcctctataa tccctatgcc ggtaacggcc
gtccggtgaa acctgctccc 2880tgcgtcgac
2889754296DNAArtificial SequenceSynthetic
75ggatccttgg tacgagatga cgttgactat caaattttcc gcgactttgc ggaaaataaa
60ggtaagtttt tcgtcggcgc cacagacctg tccgtcaaaa ataagagagg ccagaacatc
120ggtaacgcac tgagcaacgt ccctatgatt gattttagtg tagcggacgt taataaacgg
180attgcaaccg tcgttgatcc gcagtatgct gtcagcgtca aacatgctaa agcggaagtt
240catacgttct attacgggca atataacggc cataacgatg tggctgataa agaaaatgaa
300tatcgcgtgg tcgagcagaa caattacgaa ccgcacaaag cgtggggcgc gagtaattta
360ggccgcctgg aggactataa catggcccgt ttcaataaat tcgtgaccga ggtagcaccg
420atcgccccca cagatgctgg tgggggcctg gatacctaca aagataaaaa ccgcttctct
480agcttcgtgc gcattggcgc cggtcgtcag ctcgtgtacg agaagggtgt ctatcaccag
540gaaggtaatg aaaaggggta cgacctccgt gatttgtccc aggcgtatcg ctacgctatt
600gccggaaccc cgtataaaga tattaatatc gatcaaacca tgaataccga aggcctaatt
660ggtttcggga atcataataa gcaatatagc gcagaagagc taaagcaggc cctcagccaa
720gatgcgttaa ccaattacgg agtgttaggc gatagcggca gtccgctgtt tgccttcgat
780aaacagaaaa atcaatgggt gtttctgggc acttatgatt attgggccgg atatggtaaa
840aagagctggc aggaatggaa tatttataaa aaggaattcg cagacaaaat caagcagcat
900gacaacgcag gtacggtgaa ggggaacggc gaacatcact ggaagacgac cggcacgaat
960agtcatatcg gatcgacggc cgttcgcctg gcgaacaatg agggcgatgc aaacaatggg
1020caaaacgtga cctttgagga caacggtacc ctggtcctta accagaacat aaatcagggc
1080gcgggaggct tgttctttaa aggcgactat actgttaagg gagcaaacaa tgacatcacc
1140tggttagggg ccggtattga cgttgcggat ggaaaaaagg tggtttggca ggttaaaaac
1200cctaacgggg accggctggc aaaaatcggc aaagggacat tggaaattaa tggtaccggt
1260gtgaatcagg gtcagctgaa agtgggagat gggaccgtga ttctgaacca gaaagcagac
1320gctgacaaaa aggtgcaagc ctttagccaa gtaggaattg ttagtggtcg tggcacactc
1380gtcttgaact caagcaacca aataaatccg gataacctgt actttggatt tcgtggcgga
1440cgcctggatg ctaacgggaa tgatctgacc tttgaacata tccgtaacgt tgacgagggt
1500gcgcgcatag ttaatcataa tactgaccat gcatcaacta tcaccttgac cgggaaaagt
1560ctgattacaa acccaaactc tctgtcagta cattccatcc agaatgatta tgatgaagac
1620gattactcat actattaccg gccgcgtaga ccaattccac aaggtaaaga tctttattac
1680aaaaattacc gttattacgc attaaaatcc ggagggcggc tgaatgcacc tatgccggaa
1740aatggcgtgg ccgaaaacaa tgactggatt tttatgggtt atactcaaga agaggctcgc
1800aaaaatgcaa tgaaccataa aaataaccga aggatcggtg atttcggcgg atttttcgat
1860gaggaaaatg gtaaaggtca caatggtgcg ctgaatctaa attttaacgg caaaagtgcc
1920cagaaacgtt tccttctgac tggtggcgct aatctgaatg gtaaaatcag tgtgacgcag
1980ggtaacgtgc tgctttctgg ccggccaact ccgcatgcac gtgattttgt aaataaatcg
2040agcgctcgta aagatgcgca tttttctaaa aataacgagg tcgtgtttga agatgactgg
2100ataaatcgca cctttaaagc ggcagaaatc gcggttaatc agagtgcgag cttttcatcg
2160ggtaggaatg tatctgatat tacagcaaac attacagcca ctgataatgc gaaggtcaac
2220ctgggttata aaaacggtga tgaagtttgt gttcgatcgg attacacggg ctatgttacc
2280tgcaacactg gcaatctgtc tgataaagcg cttaactctt ttgacgccac gcgcattaac
2340gggaatgtga acctgaacca aaacgctgcc ttggtacttg gtaaggccgc gttgtggggt
2400aaaattcagg gccagggcaa ctcccgtgtg tctctgaacc agcactcgaa gtggcacctg
2460acgggggact cgcaggtgca caacttgtcc ctggccgata gccatattca ccttaacaat
2520gcgtccgatg cccagtcagc taataaatat catacgatca aaatcaatca cctctctggc
2580aacggtcact ttcactactt aacggattta gcaaaaaact taggggataa agtcctggta
2640aaagaatcag cgagcggaca ttatcagtta catgtacaga acaaaacagg cgagccaaat
2700caggaaggcc ttgacttatt tgatgcttca tcggtacaag atcgttccag actgttcgtt
2760tcactcgcga atcactacgt tgatctgggt gcgctgcgct atactataaa gacggaaaat
2820ggcataacac gcctctataa tccctatgcc ggtaacggcc gtccggtgaa acctgctccc
2880tgcgtcgacg gcatcattac ctccaaaact aaatctctga tagaaggtag atttggcggt
2940ttcacgggcg cacgcaaatc agcgcgtaaa cgtaagaacc aggcgctagc gggcggtggc
3000ggtagcggcg gtggcggtag cggcggtggc ggtagcgcac tagtgctgca gtgtatcaag
3060gttaacaact gggatttatt cttcagcccg agtgaagaca acttcaccaa cgacctgaac
3120aaaggtgaag aaatcacctc agatactaac atcgaagcag ccgaagaaaa catctcgctg
3180gacctgatcc agcagtacta cctgaccttt aatttcgaca acgagccgga aaacatttct
3240atcgaaaacc tgagctctga tatcatcggc cagctggaac tgatgccgaa catcgaacgt
3300ttcccaaacg gtaaaaagta cgagctggac aaatatacca tgttccacta cctgcgcgcg
3360caggaatttg aacacggcaa atcccgtatc gcactgacta actccgttaa cgaagctctg
3420ctcaacccgt cccgtgtata caccttcttc tctagcgact acgtgaaaaa ggtcaacaaa
3480gcgactgaag ctgcaatgtt cttgggttgg gttgaacagc ttgtttatga ttttaccgac
3540gagacgtccg aagtatctac taccgacaaa attgcggata tcactatcat catcccgtac
3600atcggtccgg ctctgaacat tggcaacatg ctgtacaaag acgacttcgt tggcgcactg
3660atcttctccg gtgcggtgat cctgctggag ttcatcccgg aaatcgccat cccggtactg
3720ggcacctttg ctctggtttc ttacattgca aacaaggttc tgactgtaca aaccatcgac
3780aacgcgctga gcaaacgtaa cgaaaaatgg gatgaagttt acaaatatat cgtgaccaac
3840tggctggcta aggttaatac tcagatcgac ctcatccgca aaaaaatgaa agaagcactg
3900gaaaaccagg cggaagctac caaggcaatc attaactacc agtacaacca gtacaccgag
3960gaagaaaaaa acaacatcaa cttcaacatc gacgatctgt cctctaaact gaacgaatcc
4020atcaacaaag ctatgatcaa catcaacaag ttcctgaacc agtgctctgt aagctatctg
4080atgaactcca tgatcccgta cggtgttaaa cgtctggagg acttcgatgc gtctctgaaa
4140gacgccctgc tgaaatacat ttacgacaac cgtggcactc tgatcggtca ggttgatcgt
4200ctgaaggaca aagtgaacaa taccttatcg accgacatcc cttttcagct cagtaaatat
4260gtcgataacc aacgcctttt gtccactcta gactag
4296761431PRTArtificial SequenceSynthetic 76Gly Ser Leu Val Arg Asp Asp
Val Asp Tyr Gln Ile Phe Arg Asp Phe1 5 10
15Ala Glu Asn Lys Gly Lys Phe Phe Val Gly Ala Thr Asp
Leu Ser Val 20 25 30Lys Asn
Lys Arg Gly Gln Asn Ile Gly Asn Ala Leu Ser Asn Val Pro 35
40 45Met Ile Asp Phe Ser Val Ala Asp Val Asn
Lys Arg Ile Ala Thr Val 50 55 60Val
Asp Pro Gln Tyr Ala Val Ser Val Lys His Ala Lys Ala Glu Val65
70 75 80His Thr Phe Tyr Tyr Gly
Gln Tyr Asn Gly His Asn Asp Val Ala Asp 85
90 95Lys Glu Asn Glu Tyr Arg Val Val Glu Gln Asn Asn
Tyr Glu Pro His 100 105 110Lys
Ala Trp Gly Ala Ser Asn Leu Gly Arg Leu Glu Asp Tyr Asn Met 115
120 125Ala Arg Phe Asn Lys Phe Val Thr Glu
Val Ala Pro Ile Ala Pro Thr 130 135
140Asp Ala Gly Gly Gly Leu Asp Thr Tyr Lys Asp Lys Asn Arg Phe Ser145
150 155 160Ser Phe Val Arg
Ile Gly Ala Gly Arg Gln Leu Val Tyr Glu Lys Gly 165
170 175Val Tyr His Gln Glu Gly Asn Glu Lys Gly
Tyr Asp Leu Arg Asp Leu 180 185
190Ser Gln Ala Tyr Arg Tyr Ala Ile Ala Gly Thr Pro Tyr Lys Asp Ile
195 200 205Asn Ile Asp Gln Thr Met Asn
Thr Glu Gly Leu Ile Gly Phe Gly Asn 210 215
220His Asn Lys Gln Tyr Ser Ala Glu Glu Leu Lys Gln Ala Leu Ser
Gln225 230 235 240Asp Ala
Leu Thr Asn Tyr Gly Val Leu Gly Asp Ser Gly Ser Pro Leu
245 250 255Phe Ala Phe Asp Lys Gln Lys
Asn Gln Trp Val Phe Leu Gly Thr Tyr 260 265
270Asp Tyr Trp Ala Gly Tyr Gly Lys Lys Ser Trp Gln Glu Trp
Asn Ile 275 280 285Tyr Lys Lys Glu
Phe Ala Asp Lys Ile Lys Gln His Asp Asn Ala Gly 290
295 300Thr Val Lys Gly Asn Gly Glu His His Trp Lys Thr
Thr Gly Thr Asn305 310 315
320Ser His Ile Gly Ser Thr Ala Val Arg Leu Ala Asn Asn Glu Gly Asp
325 330 335Ala Asn Asn Gly Gln
Asn Val Thr Phe Glu Asp Asn Gly Thr Leu Val 340
345 350Leu Asn Gln Asn Ile Asn Gln Gly Ala Gly Gly Leu
Phe Phe Lys Gly 355 360 365Asp Tyr
Thr Val Lys Gly Ala Asn Asn Asp Ile Thr Trp Leu Gly Ala 370
375 380Gly Ile Asp Val Ala Asp Gly Lys Lys Val Val
Trp Gln Val Lys Asn385 390 395
400Pro Asn Gly Asp Arg Leu Ala Lys Ile Gly Lys Gly Thr Leu Glu Ile
405 410 415Asn Gly Thr Gly
Val Asn Gln Gly Gln Leu Lys Val Gly Asp Gly Thr 420
425 430Val Ile Leu Asn Gln Lys Ala Asp Ala Asp Lys
Lys Val Gln Ala Phe 435 440 445Ser
Gln Val Gly Ile Val Ser Gly Arg Gly Thr Leu Val Leu Asn Ser 450
455 460Ser Asn Gln Ile Asn Pro Asp Asn Leu Tyr
Phe Gly Phe Arg Gly Gly465 470 475
480Arg Leu Asp Ala Asn Gly Asn Asp Leu Thr Phe Glu His Ile Arg
Asn 485 490 495Val Asp Glu
Gly Ala Arg Ile Val Asn His Asn Thr Asp His Ala Ser 500
505 510Thr Ile Thr Leu Thr Gly Lys Ser Leu Ile
Thr Asn Pro Asn Ser Leu 515 520
525Ser Val His Ser Ile Gln Asn Asp Tyr Asp Glu Asp Asp Tyr Ser Tyr 530
535 540Tyr Tyr Arg Pro Arg Arg Pro Ile
Pro Gln Gly Lys Asp Leu Tyr Tyr545 550
555 560Lys Asn Tyr Arg Tyr Tyr Ala Leu Lys Ser Gly Gly
Arg Leu Asn Ala 565 570
575Pro Met Pro Glu Asn Gly Val Ala Glu Asn Asn Asp Trp Ile Phe Met
580 585 590Gly Tyr Thr Gln Glu Glu
Ala Arg Lys Asn Ala Met Asn His Lys Asn 595 600
605Asn Arg Arg Ile Gly Asp Phe Gly Gly Phe Phe Asp Glu Glu
Asn Gly 610 615 620Lys Gly His Asn Gly
Ala Leu Asn Leu Asn Phe Asn Gly Lys Ser Ala625 630
635 640Gln Lys Arg Phe Leu Leu Thr Gly Gly Ala
Asn Leu Asn Gly Lys Ile 645 650
655Ser Val Thr Gln Gly Asn Val Leu Leu Ser Gly Arg Pro Thr Pro His
660 665 670Ala Arg Asp Phe Val
Asn Lys Ser Ser Ala Arg Lys Asp Ala His Phe 675
680 685Ser Lys Asn Asn Glu Val Val Phe Glu Asp Asp Trp
Ile Asn Arg Thr 690 695 700Phe Lys Ala
Ala Glu Ile Ala Val Asn Gln Ser Ala Ser Phe Ser Ser705
710 715 720Gly Arg Asn Val Ser Asp Ile
Thr Ala Asn Ile Thr Ala Thr Asp Asn 725
730 735Ala Lys Val Asn Leu Gly Tyr Lys Asn Gly Asp Glu
Val Cys Val Arg 740 745 750Ser
Asp Tyr Thr Gly Tyr Val Thr Cys Asn Thr Gly Asn Leu Ser Asp 755
760 765Lys Ala Leu Asn Ser Phe Asp Ala Thr
Arg Ile Asn Gly Asn Val Asn 770 775
780Leu Asn Gln Asn Ala Ala Leu Val Leu Gly Lys Ala Ala Leu Trp Gly785
790 795 800Lys Ile Gln Gly
Gln Gly Asn Ser Arg Val Ser Leu Asn Gln His Ser 805
810 815Lys Trp His Leu Thr Gly Asp Ser Gln Val
His Asn Leu Ser Leu Ala 820 825
830Asp Ser His Ile His Leu Asn Asn Ala Ser Asp Ala Gln Ser Ala Asn
835 840 845Lys Tyr His Thr Ile Lys Ile
Asn His Leu Ser Gly Asn Gly His Phe 850 855
860His Tyr Leu Thr Asp Leu Ala Lys Asn Leu Gly Asp Lys Val Leu
Val865 870 875 880Lys Glu
Ser Ala Ser Gly His Tyr Gln Leu His Val Gln Asn Lys Thr
885 890 895Gly Glu Pro Asn Gln Glu Gly
Leu Asp Leu Phe Asp Ala Ser Ser Val 900 905
910Gln Asp Arg Ser Arg Leu Phe Val Ser Leu Ala Asn His Tyr
Val Asp 915 920 925Leu Gly Ala Leu
Arg Tyr Thr Ile Lys Thr Glu Asn Gly Ile Thr Arg 930
935 940Leu Tyr Asn Pro Tyr Ala Gly Asn Gly Arg Pro Val
Lys Pro Ala Pro945 950 955
960Cys Val Asp Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Ile Glu Gly
965 970 975Arg Phe Gly Gly Phe
Thr Gly Ala Arg Lys Ser Ala Arg Lys Arg Lys 980
985 990Asn Gln Ala Leu Ala Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly 995 1000 1005Gly
Gly Gly Ser Ala Leu Val Leu Gln Cys Ile Lys Val Asn Asn 1010
1015 1020Trp Asp Leu Phe Phe Ser Pro Ser Glu
Asp Asn Phe Thr Asn Asp 1025 1030
1035Leu Asn Lys Gly Glu Glu Ile Thr Ser Asp Thr Asn Ile Glu Ala
1040 1045 1050Ala Glu Glu Asn Ile Ser
Leu Asp Leu Ile Gln Gln Tyr Tyr Leu 1055 1060
1065Thr Phe Asn Phe Asp Asn Glu Pro Glu Asn Ile Ser Ile Glu
Asn 1070 1075 1080Leu Ser Ser Asp Ile
Ile Gly Gln Leu Glu Leu Met Pro Asn Ile 1085 1090
1095Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu Asp Lys
Tyr Thr 1100 1105 1110Met Phe His Tyr
Leu Arg Ala Gln Glu Phe Glu His Gly Lys Ser 1115
1120 1125Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala
Leu Leu Asn Pro 1130 1135 1140Ser Arg
Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys Lys Val 1145
1150 1155Asn Lys Ala Thr Glu Ala Ala Met Phe Leu
Gly Trp Val Glu Gln 1160 1165 1170Leu
Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr 1175
1180 1185Asp Lys Ile Ala Asp Ile Thr Ile Ile
Ile Pro Tyr Ile Gly Pro 1190 1195
1200Ala Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly
1205 1210 1215Ala Leu Ile Phe Ser Gly
Ala Val Ile Leu Leu Glu Phe Ile Pro 1220 1225
1230Glu Ile Ala Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser
Tyr 1235 1240 1245Ile Ala Asn Lys Val
Leu Thr Val Gln Thr Ile Asp Asn Ala Leu 1250 1255
1260Ser Lys Arg Asn Glu Lys Trp Asp Glu Val Tyr Lys Tyr
Ile Val 1265 1270 1275Thr Asn Trp Leu
Ala Lys Val Asn Thr Gln Ile Asp Leu Ile Arg 1280
1285 1290Lys Lys Met Lys Glu Ala Leu Glu Asn Gln Ala
Glu Ala Thr Lys 1295 1300 1305Ala Ile
Ile Asn Tyr Gln Tyr Asn Gln Tyr Thr Glu Glu Glu Lys 1310
1315 1320Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu
Ser Ser Lys Leu Asn 1325 1330 1335Glu
Ser Ile Asn Lys Ala Met Ile Asn Ile Asn Lys Phe Leu Asn 1340
1345 1350Gln Cys Ser Val Ser Tyr Leu Met Asn
Ser Met Ile Pro Tyr Gly 1355 1360
1365Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys Asp Ala Leu
1370 1375 1380Leu Lys Tyr Ile Tyr Asp
Asn Arg Gly Thr Leu Ile Gly Gln Val 1385 1390
1395Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp
Ile 1400 1405 1410Pro Phe Gln Leu Ser
Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser 1415 1420
1425Thr Leu Asp 1430771357DNAArtificial
SequenceSynthetic 77gctagcgggc ggtggcggta gcggcggtgg cggtagcggc
ggtggcggta gcgcactagt 60gctgcagtgt atcaaggtta acaactggga tttattcttc
agcccgagtg aagacaactt 120caccaacgac ctgaacaaag gtgaagaaat cacctcagat
actaacatcg aagcagccga 180agaaaacatc tcgctggacc tgatccagca gtactacctg
acctttaatt tcgacaacga 240gccggaaaac atttctatcg aaaacctgag ctctgatatc
atcggccagc tggaactgat 300gccgaacatc gaacgtttcc caaacggtaa aaagtacgag
ctggacaaat ataccatgtt 360ccactacctg cgcgcgcagg aatttgaaca cggcaaatcc
cgtatcgcac tgactaactc 420cgttaacgaa gctctgctca acccgtcccg tgtatacacc
ttcttctcta gcgactacgt 480gaaaaaggtc aacaaagcga ctgaagctgc aatgttcttg
ggttgggttg aacagcttgt 540ttatgatttt accgacgaga cgtccgaagt atctactacc
gacaaaattg cggatatcac 600tatcatcatc ccgtacatcg gtccggctct gaacattggc
aacatgctgt acaaagacga 660cttcgttggc gcactgatct tctccggtgc ggtgatcctg
ctggagttca tcccggaaat 720cgccatcccg gtactgggca cctttgctct ggtttcttac
attgcaaaca aggttctgac 780tgtacaaacc atcgacaacg cgctgagcaa acgtaacgaa
aaatgggatg aagtttacaa 840atatatcgtg accaactggc tggctaaggt taatactcag
atcgacctca tccgcaaaaa 900aatgaaagaa gcactggaaa accaggcgga agctaccaag
gcaatcatta actaccagta 960caaccagtac accgaggaag aaaaaaacaa catcaacttc
aacatcgacg atctgtcctc 1020taaactgaac gaatccatca acaaagctat gatcaacatc
aacaagttcc tgaaccagtg 1080ctctgtaagc tatctgatga actccatgat cccgtacggt
gttaaacgtc tggaggactt 1140cgatgcgtct ctgaaagacg ccctgctgaa atacatttac
gacaaccgtg gcactctgat 1200cggtcaggtt gatcgtctga aggacaaagt gaacaatacc
ttatcgaccg acatcccttt 1260tcagctcagt aaatatgtcg ataaccaacg ccttttgtcc
actctagaaa tagaaggtag 1320aagtgggcac catcaccatc accattaatg aaagctt
1357782745DNAArtificial SequenceSynthetic
78ggatccatgg agttcgttaa caaacagttc aactataaag acccagttaa cggtgttgac
60attgcttaca tcaaaatccc gaacgctggc cagatgcagc cggtaaaggc attcaaaatc
120cacaacaaaa tctgggttat cccggaacgt gataccttta ctaacccgga agaaggtgac
180ctgaacccgc caccggaagc gaaacaggtg ccggtatctt actatgactc cacctacctg
240tctaccgata acgaaaagga caactacctg aaaggtgtta ctaaactgtt cgagcgtatt
300tactccaccg acctgggccg tatgctgctg actagcatcg ttcgcggtat cccgttctgg
360ggcggttcta ccatcgatac cgaactgaaa gtaatcgaca ctaactgcat caacgttatt
420cagccggacg gttcctatcg ttccgaagaa ctgaacctgg tgatcatcgg cccgtctgct
480gatatcatcc agttcgagtg taagagcttt ggtcacgaag ttctgaacct cacccgtaac
540ggctacggtt ccactcagta catccgtttc tctccggact tcaccttcgg ttttgaagaa
600tccctggaag tagacacgaa cccactgctg ggcgctggta aattcgcaac tgatcctgcg
660gttaccctgg ctcacgaact gattcatgca ggccaccgcc tgtacggtat cgccatcaat
720ccgaaccgtg tcttcaaagt taacaccaac gcgtattacg agatgtccgg tctggaagtt
780agcttcgaag aactgcgtac ttttggcggt cacgacgcta aattcatcga ctctctgcaa
840gaaaacgagt tccgtctgta ctactataac aagttcaaag atatcgcatc caccctgaac
900aaagcgaaat ccatcgtggg taccactgct tctctccagt acatgaagaa cgtttttaaa
960gaaaaatacc tgctcagcga agacacctcc ggcaaattct ctgtagacaa gttgaaattc
1020gataaacttt acaaaatgct gactgaaatt tacaccgaag acaacttcgt taagttcttt
1080aaagttctga accgcaaaac ctatctgaac ttcgacaagg cagtattcaa aatcaacatc
1140gtgccgaaag ttaactacac tatctacgat ggtttcaacc tgcgtaacac caacctggct
1200gctaatttta acggccagaa cacggaaatc aacaacatga acttcacaaa actgaaaaac
1260ttcactggtc tgttcgagtt ttacaagctg ctgtgcgtcg acggcatcat tacctccaaa
1320actaaatctc tgatagaagg tagatttggc ggtttcacgg gcgcacgcaa atcagcgcgt
1380aaacgtaaga accaggcgct agcgggcggt ggcggtagcg gcggtggcgg tagcggcggt
1440ggcggtagcg cactagtgct gcagtgtatc aaggttaaca actgggattt attcttcagc
1500ccgagtgaag acaacttcac caacgacctg aacaaaggtg aagaaatcac ctcagatact
1560aacatcgaag cagccgaaga aaacatctcg ctggacctga tccagcagta ctacctgacc
1620tttaatttcg acaacgagcc ggaaaacatt tctatcgaaa acctgagctc tgatatcatc
1680ggccagctgg aactgatgcc gaacatcgaa cgtttcccaa acggtaaaaa gtacgagctg
1740gacaaatata ccatgttcca ctacctgcgc gcgcaggaat ttgaacacgg caaatcccgt
1800atcgcactga ctaactccgt taacgaagct ctgctcaacc cgtcccgtgt atacaccttc
1860ttctctagcg actacgtgaa aaaggtcaac aaagcgactg aagctgcaat gttcttgggt
1920tgggttgaac agcttgttta tgattttacc gacgagacgt ccgaagtatc tactaccgac
1980aaaattgcgg atatcactat catcatcccg tacatcggtc cggctctgaa cattggcaac
2040atgctgtaca aagacgactt cgttggcgca ctgatcttct ccggtgcggt gatcctgctg
2100gagttcatcc cggaaatcgc catcccggta ctgggcacct ttgctctggt ttcttacatt
2160gcaaacaagg ttctgactgt acaaaccatc gacaacgcgc tgagcaaacg taacgaaaaa
2220tgggatgaag tttacaaata tatcgtgacc aactggctgg ctaaggttaa tactcagatc
2280gacctcatcc gcaaaaaaat gaaagaagca ctggaaaacc aggcggaagc taccaaggca
2340atcattaact accagtacaa ccagtacacc gaggaagaaa aaaacaacat caacttcaac
2400atcgacgatc tgtcctctaa actgaacgaa tccatcaaca aagctatgat caacatcaac
2460aagttcctga accagtgctc tgtaagctat ctgatgaact ccatgatccc gtacggtgtt
2520aaacgtctgg aggacttcga tgcgtctctg aaagacgccc tgctgaaata catttacgac
2580aaccgtggca ctctgatcgg tcaggttgat cgtctgaagg acaaagtgaa caatacctta
2640tcgaccgaca tcccttttca gctcagtaaa tatgtcgata accaacgcct tttgtccact
2700ctagaaatag aaggtagaag tgggcaccat caccatcacc attaa
274579914PRTArtificial SequenceSynthetic 79Gly Ser Met Glu Phe Val Asn
Lys Gln Phe Asn Tyr Lys Asp Pro Val1 5 10
15Asn Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala
Gly Gln Met 20 25 30Gln Pro
Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro 35
40 45Glu Arg Asp Thr Phe Thr Asn Pro Glu Glu
Gly Asp Leu Asn Pro Pro 50 55 60Pro
Glu Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu65
70 75 80Ser Thr Asp Asn Glu Lys
Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu 85
90 95Phe Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met
Leu Leu Thr Ser 100 105 110Ile
Val Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu 115
120 125Leu Lys Val Ile Asp Thr Asn Cys Ile
Asn Val Ile Gln Pro Asp Gly 130 135
140Ser Tyr Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala145
150 155 160Asp Ile Ile Gln
Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn 165
170 175Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln
Tyr Ile Arg Phe Ser Pro 180 185
190Asp Phe Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro
195 200 205Leu Leu Gly Ala Gly Lys Phe
Ala Thr Asp Pro Ala Val Thr Leu Ala 210 215
220His Glu Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile
Asn225 230 235 240Pro Asn
Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser
245 250 255Gly Leu Glu Val Ser Phe Glu
Glu Leu Arg Thr Phe Gly Gly His Asp 260 265
270Ala Lys Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu
Tyr Tyr 275 280 285Tyr Asn Lys Phe
Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser 290
295 300Ile Val Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys
Asn Val Phe Lys305 310 315
320Glu Lys Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp
325 330 335Lys Leu Lys Phe Asp
Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr 340
345 350Glu Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn
Arg Lys Thr Tyr 355 360 365Leu Asn
Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val 370
375 380Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg
Asn Thr Asn Leu Ala385 390 395
400Ala Asn Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr
405 410 415Lys Leu Lys Asn
Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys 420
425 430Val Asp Gly Ile Ile Thr Ser Lys Thr Lys Ser
Leu Ile Glu Gly Arg 435 440 445Phe
Gly Gly Phe Thr Gly Ala Arg Lys Ser Ala Arg Lys Arg Lys Asn 450
455 460Gln Ala Leu Ala Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly465 470 475
480Gly Gly Ser Ala Leu Val Leu Gln Cys Ile Lys Val Asn Asn Trp
Asp 485 490 495Leu Phe Phe
Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys 500
505 510Gly Glu Glu Ile Thr Ser Asp Thr Asn Ile
Glu Ala Ala Glu Glu Asn 515 520
525Ile Ser Leu Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp 530
535 540Asn Glu Pro Glu Asn Ile Ser Ile
Glu Asn Leu Ser Ser Asp Ile Ile545 550
555 560Gly Gln Leu Glu Leu Met Pro Asn Ile Glu Arg Phe
Pro Asn Gly Lys 565 570
575Lys Tyr Glu Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln
580 585 590Glu Phe Glu His Gly Lys
Ser Arg Ile Ala Leu Thr Asn Ser Val Asn 595 600
605Glu Ala Leu Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser
Ser Asp 610 615 620Tyr Val Lys Lys Val
Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly625 630
635 640Trp Val Glu Gln Leu Val Tyr Asp Phe Thr
Asp Glu Thr Ser Glu Val 645 650
655Ser Thr Thr Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile
660 665 670Gly Pro Ala Leu Asn
Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val 675
680 685Gly Ala Leu Ile Phe Ser Gly Ala Val Ile Leu Leu
Glu Phe Ile Pro 690 695 700Glu Ile Ala
Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile705
710 715 720Ala Asn Lys Val Leu Thr Val
Gln Thr Ile Asp Asn Ala Leu Ser Lys 725
730 735Arg Asn Glu Lys Trp Asp Glu Val Tyr Lys Tyr Ile
Val Thr Asn Trp 740 745 750Leu
Ala Lys Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys 755
760 765Glu Ala Leu Glu Asn Gln Ala Glu Ala
Thr Lys Ala Ile Ile Asn Tyr 770 775
780Gln Tyr Asn Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn785
790 795 800Ile Asp Asp Leu
Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met 805
810 815Ile Asn Ile Asn Lys Phe Leu Asn Gln Cys
Ser Val Ser Tyr Leu Met 820 825
830Asn Ser Met Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala
835 840 845Ser Leu Lys Asp Ala Leu Leu
Lys Tyr Ile Tyr Asp Asn Arg Gly Thr 850 855
860Leu Ile Gly Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr
Leu865 870 875 880Ser Thr
Asp Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg
885 890 895Leu Leu Ser Thr Leu Glu Ile
Glu Gly Arg Ser Gly His His His His 900 905
910His His80619DNAArtificial SequenceSynthetic 80gctagcgggc
ggtggcggta gcggcggtgg cggtagcggc ggtggcggta gcgcactagt 60gctgcagtgt
atcaatctgg attgggacgt aatccgtgat aagaccaaaa caaaaatcga 120gtctttgaaa
gaacacggcc cgatcaaaaa taagatgtct gaatcaccca ataaaactgt 180ttcggaggaa
aaagcgaaac agtatttgga agagtttcat caaaccgcgc ttgaacatcc 240ggagctcagt
gaactgaaaa cagtgacggg aacgaatcct gtttttgcag gcgcaaacta 300tgcggcttgg
gccgtgaatg ttgcccaagt aattgatagt gagaccgcag acaacctgga 360aaagacgacc
gcagcgttaa gcattttacc ggggattggt tccgtgatgg gtatagcgga 420tggagcggtc
caccataaca ctgaggaaat tgtcgcccag tcaatcgctc tgagttccct 480gatggttgca
caggctatcc cactcgtggg ggaactggtt gacataggtt tcgccgccta 540caacttcgta
gaaagcatta ttaatctttt tcaggtggtg cataacagct acaaccgccc 600tctagaatga
taaaagctt
619811971DNAArtificial SequenceSynthetic 81ggatccatgg agttcgttaa
caaacagttc aactataaag acccagttaa cggtgttgac 60attgcttaca tcaaaatccc
gaacgctggc cagatgcagc cggtaaaggc attcaaaatc 120cacaacaaaa tctgggttat
cccggaacgt gataccttta ctaacccgga agaaggtgac 180ctgaacccgc caccggaagc
gaaacaggtg ccggtatctt actatgactc cacctacctg 240tctaccgata acgaaaagga
caactacctg aaaggtgtta ctaaactgtt cgagcgtatt 300tactccaccg acctgggccg
tatgctgctg actagcatcg ttcgcggtat cccgttctgg 360ggcggttcta ccatcgatac
cgaactgaaa gtaatcgaca ctaactgcat caacgttatt 420cagccggacg gttcctatcg
ttccgaagaa ctgaacctgg tgatcatcgg cccgtctgct 480gatatcatcc agttcgagtg
taagagcttt ggtcacgaag ttctgaacct cacccgtaac 540ggctacggtt ccactcagta
catccgtttc tctccggact tcaccttcgg ttttgaagaa 600tccctggaag tagacacgaa
cccactgctg ggcgctggta aattcgcaac tgatcctgcg 660gttaccctgg ctcacgaact
gattcatgca ggccaccgcc tgtacggtat cgccatcaat 720ccgaaccgtg tcttcaaagt
taacaccaac gcgtattacg agatgtccgg tctggaagtt 780agcttcgaag aactgcgtac
ttttggcggt cacgacgcta aattcatcga ctctctgcaa 840gaaaacgagt tccgtctgta
ctactataac aagttcaaag atatcgcatc caccctgaac 900aaagcgaaat ccatcgtggg
taccactgct tctctccagt acatgaagaa cgtttttaaa 960gaaaaatacc tgctcagcga
agacacctcc ggcaaattct ctgtagacaa gttgaaattc 1020gataaacttt acaaaatgct
gactgaaatt tacaccgaag acaacttcgt taagttcttt 1080aaagttctga accgcaaaac
ctatctgaac ttcgacaagg cagtattcaa aatcaacatc 1140gtgccgaaag ttaactacac
tatctacgat ggtttcaacc tgcgtaacac caacctggct 1200gctaatttta acggccagaa
cacggaaatc aacaacatga acttcacaaa actgaaaaac 1260ttcactggtc tgttcgagtt
ttacaagctg ctgtgcgtcg acggcatcat tacctccaaa 1320actaaatctc tgatagaagg
tagatacggt ggtttcctgg cgctagcggg cggtggcggt 1380agcggcggtg gcggtagcgg
cggtggcggt agcgcactag tgctgcagtg tatcaatctg 1440gattgggacg taatccgtga
taagaccaaa acaaaaatcg agtctttgaa agaacacggc 1500ccgatcaaaa ataagatgtc
tgaatcaccc aataaaactg tttcggagga aaaagcgaaa 1560cagtatttgg aagagtttca
tcaaaccgcg cttgaacatc cggagctcag tgaactgaaa 1620acagtgacgg gaacgaatcc
tgtttttgca ggcgcaaact atgcggcttg ggccgtgaat 1680gttgcccaag taattgatag
tgagaccgca gacaacctgg aaaagacgac cgcagcgtta 1740agcattttac cggggattgg
ttccgtgatg ggtatagcgg atggagcggt ccaccataac 1800actgaggaaa ttgtcgccca
gtcaatcgct ctgagttccc tgatggttgc acaggctatc 1860ccactcgtgg gggaactggt
tgacataggt ttcgccgcct acaacttcgt agaaagcatt 1920attaatcttt ttcaggtggt
gcataacagc tacaaccgcc ctctagaatg a 197182656PRTArtificial
SequenceSynthetic 82Gly Ser Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys
Asp Pro Val1 5 10 15Asn
Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met 20
25 30Gln Pro Val Lys Ala Phe Lys Ile
His Asn Lys Ile Trp Val Ile Pro 35 40
45Glu Arg Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro
50 55 60Pro Glu Ala Lys Gln Val Pro Val
Ser Tyr Tyr Asp Ser Thr Tyr Leu65 70 75
80Ser Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val
Thr Lys Leu 85 90 95Phe
Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser
100 105 110Ile Val Arg Gly Ile Pro Phe
Trp Gly Gly Ser Thr Ile Asp Thr Glu 115 120
125Leu Lys Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp
Gly 130 135 140Ser Tyr Arg Ser Glu Glu
Leu Asn Leu Val Ile Ile Gly Pro Ser Ala145 150
155 160Asp Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly
His Glu Val Leu Asn 165 170
175Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro
180 185 190Asp Phe Thr Phe Gly Phe
Glu Glu Ser Leu Glu Val Asp Thr Asn Pro 195 200
205Leu Leu Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr
Leu Ala 210 215 220His Glu Leu Ile His
Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn225 230
235 240Pro Asn Arg Val Phe Lys Val Asn Thr Asn
Ala Tyr Tyr Glu Met Ser 245 250
255Gly Leu Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp
260 265 270Ala Lys Phe Ile Asp
Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr 275
280 285Tyr Asn Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn
Lys Ala Lys Ser 290 295 300Ile Val Gly
Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys305
310 315 320Glu Lys Tyr Leu Leu Ser Glu
Asp Thr Ser Gly Lys Phe Ser Val Asp 325
330 335Lys Leu Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr
Glu Ile Tyr Thr 340 345 350Glu
Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr 355
360 365Leu Asn Phe Asp Lys Ala Val Phe Lys
Ile Asn Ile Val Pro Lys Val 370 375
380Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala385
390 395 400Ala Asn Phe Asn
Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr 405
410 415Lys Leu Lys Asn Phe Thr Gly Leu Phe Glu
Phe Tyr Lys Leu Leu Cys 420 425
430Val Asp Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Ile Glu Gly Arg
435 440 445Tyr Gly Gly Phe Leu Ala Leu
Ala Gly Gly Gly Gly Ser Gly Gly Gly 450 455
460Gly Ser Gly Gly Gly Gly Ser Ala Leu Val Leu Gln Cys Ile Asn
Leu465 470 475 480Asp Trp
Asp Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu
485 490 495Lys Glu His Gly Pro Ile Lys
Asn Lys Met Ser Glu Ser Pro Asn Lys 500 505
510Thr Val Ser Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe
His Gln 515 520 525Thr Ala Leu Glu
His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly 530
535 540Thr Asn Pro Val Phe Ala Gly Ala Asn Tyr Ala Ala
Trp Ala Val Asn545 550 555
560Val Ala Gln Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr
565 570 575Thr Ala Ala Leu Ser
Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile 580
585 590Ala Asp Gly Ala Val His His Asn Thr Glu Glu Ile
Val Ala Gln Ser 595 600 605Ile Ala
Leu Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly 610
615 620Glu Leu Val Asp Ile Gly Phe Ala Ala Tyr Asn
Phe Val Glu Ser Ile625 630 635
640Ile Asn Leu Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Leu Glu
645 650
655831329DNAArtificial SequenceSynthetic 83ggatccatgc ctattactat
taacaatttt cgttatagcg atcccgtcaa caatgacacc 60attatcatga tggaaccgcc
atattgcaaa ggactggaca tttactataa agccttcaag 120attactgacc gcatttggat
tgttccagag cgttacgagt tcgggacgaa accagaagat 180tttaacccgc cttcatcgct
gatcgaagga gcatcagagt attacgatcc gaactatctg 240cgtacggaca gcgataaaga
ccgcttctta cagaccatgg tcaaactttt taaccgtatt 300aagaacaatg tggccggaga
agcactcttg gataagatta tcaacgcgat tccatacctg 360ggcaattctt acagcctgct
ggataaattt gacacaaata gtaattcagt cagctttaac 420ctgttagaac aagatccgag
tggcgcaacc acgaagtctg ccatgctgac aaatctgatc 480atttttggtc caggtcctgt
actgaataaa aatgaagtac gcggcatcgt tctccgcgtg 540gacaataaga actacttccc
atgccgtgac ggcttcggtt cgatcatgca gatggctttc 600tgtccggagt acgttccgac
gtttgataat gttattgaga atatcacgag tttaacaatc 660ggtaagtcaa aatattttca
agatccggcc cttctcctta tgcatgaact gattcacgtg 720ctgcacggct tatatggtat
gcaagtgtcc tcgcatgaaa tcattccgtc caaacaggaa 780atttatatgc agcataccta
cccgatttca gctgaagagt tgtttacgtt tggtggccag 840gacgcgaatt tgatctccat
cgacatcaaa aacgatctgt atgagaaaac attaaatgac 900tataaagcga ttgcgaacaa
actgtctcag gtgactagct gcaacgatcc taacattgat 960attgattcct acaaacaaat
ttatcaacag aaataccagt tcgataaaga cagcaatggt 1020cagtatatcg taaacgaaga
taaatttcag atcctgtata acagcattat gtatggcttt 1080accgaaattg agttggggaa
gaaatttaac attaaaaccc gtctgtctta ttttagtatg 1140aaccatgatc cggtgaaaat
ccccaatctg cttgatgata ccatttataa tgataccgaa 1200gggttcaaca ttgaatctaa
ggatctgaaa tccgaataca aaggccaaaa tatgcgtgtt 1260aatactaacg ctttccgtaa
tgttgatggt agtggactcg tctcgaaact gattgggttg 1320tgtgtcgac
1329842736DNAArtificial
SequenceSynthetic 84ggatccatgc ctattactat taacaatttt cgttatagcg
atcccgtcaa caatgacacc 60attatcatga tggaaccgcc atattgcaaa ggactggaca
tttactataa agccttcaag 120attactgacc gcatttggat tgttccagag cgttacgagt
tcgggacgaa accagaagat 180tttaacccgc cttcatcgct gatcgaagga gcatcagagt
attacgatcc gaactatctg 240cgtacggaca gcgataaaga ccgcttctta cagaccatgg
tcaaactttt taaccgtatt 300aagaacaatg tggccggaga agcactcttg gataagatta
tcaacgcgat tccatacctg 360ggcaattctt acagcctgct ggataaattt gacacaaata
gtaattcagt cagctttaac 420ctgttagaac aagatccgag tggcgcaacc acgaagtctg
ccatgctgac aaatctgatc 480atttttggtc caggtcctgt actgaataaa aatgaagtac
gcggcatcgt tctccgcgtg 540gacaataaga actacttccc atgccgtgac ggcttcggtt
cgatcatgca gatggctttc 600tgtccggagt acgttccgac gtttgataat gttattgaga
atatcacgag tttaacaatc 660ggtaagtcaa aatattttca agatccggcc cttctcctta
tgcatgaact gattcacgtg 720ctgcacggct tatatggtat gcaagtgtcc tcgcatgaaa
tcattccgtc caaacaggaa 780atttatatgc agcataccta cccgatttca gctgaagagt
tgtttacgtt tggtggccag 840gacgcgaatt tgatctccat cgacatcaaa aacgatctgt
atgagaaaac attaaatgac 900tataaagcga ttgcgaacaa actgtctcag gtgactagct
gcaacgatcc taacattgat 960attgattcct acaaacaaat ttatcaacag aaataccagt
tcgataaaga cagcaatggt 1020cagtatatcg taaacgaaga taaatttcag atcctgtata
acagcattat gtatggcttt 1080accgaaattg agttggggaa gaaatttaac attaaaaccc
gtctgtctta ttttagtatg 1140aaccatgatc cggtgaaaat ccccaatctg cttgatgata
ccatttataa tgataccgaa 1200gggttcaaca ttgaatctaa ggatctgaaa tccgaataca
aaggccaaaa tatgcgtgtt 1260aatactaacg ctttccgtaa tgttgatggt agtggactcg
tctcgaaact gattgggttg 1320tgtgtcgacg gcatcattac ctccaaaact aaatctctga
tagaaggtag atttggcggt 1380ttcacgggcg cacgcaaatc agcgcgtaaa cgtaagaacc
aggcgctagc gggcggtggc 1440ggtagcggcg gtggcggtag cggcggtggc ggtagcgcac
tagtgctgca gtgtatcaag 1500gttaacaact gggatttatt cttcagcccg agtgaagaca
acttcaccaa cgacctgaac 1560aaaggtgaag aaatcacctc agatactaac atcgaagcag
ccgaagaaaa catctcgctg 1620gacctgatcc agcagtacta cctgaccttt aatttcgaca
acgagccgga aaacatttct 1680atcgaaaacc tgagctctga tatcatcggc cagctggaac
tgatgccgaa catcgaacgt 1740ttcccaaacg gtaaaaagta cgagctggac aaatatacca
tgttccacta cctgcgcgcg 1800caggaatttg aacacggcaa atcccgtatc gcactgacta
actccgttaa cgaagctctg 1860ctcaacccgt cccgtgtata caccttcttc tctagcgact
acgtgaaaaa ggtcaacaaa 1920gcgactgaag ctgcaatgtt cttgggttgg gttgaacagc
ttgtttatga ttttaccgac 1980gagacgtccg aagtatctac taccgacaaa attgcggata
tcactatcat catcccgtac 2040atcggtccgg ctctgaacat tggcaacatg ctgtacaaag
acgacttcgt tggcgcactg 2100atcttctccg gtgcggtgat cctgctggag ttcatcccgg
aaatcgccat cccggtactg 2160ggcacctttg ctctggtttc ttacattgca aacaaggttc
tgactgtaca aaccatcgac 2220aacgcgctga gcaaacgtaa cgaaaaatgg gatgaagttt
acaaatatat cgtgaccaac 2280tggctggcta aggttaatac tcagatcgac ctcatccgca
aaaaaatgaa agaagcactg 2340gaaaaccagg cggaagctac caaggcaatc attaactacc
agtacaacca gtacaccgag 2400gaagaaaaaa acaacatcaa cttcaacatc gacgatctgt
cctctaaact gaacgaatcc 2460atcaacaaag ctatgatcaa catcaacaag ttcctgaacc
agtgctctgt aagctatctg 2520atgaactcca tgatcccgta cggtgttaaa cgtctggagg
acttcgatgc gtctctgaaa 2580gacgccctgc tgaaatacat ttacgacaac cgtggcactc
tgatcggtca ggttgatcgt 2640ctgaaggaca aagtgaacaa taccttatcg accgacatcc
cttttcagct cagtaaatat 2700gtcgataacc aacgcctttt gtccactcta gactag
273685911PRTArtificial SequenceSynthetic 85Gly Ser
Met Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val1 5
10 15Asn Asn Asp Thr Ile Ile Met Met
Glu Pro Pro Tyr Cys Lys Gly Leu 20 25
30Asp Ile Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile
Val 35 40 45Pro Glu Arg Tyr Glu
Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro 50 55
60Ser Ser Leu Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn
Tyr Leu65 70 75 80Arg
Thr Asp Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu
85 90 95Phe Asn Arg Ile Lys Asn Asn
Val Ala Gly Glu Ala Leu Leu Asp Lys 100 105
110Ile Ile Asn Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu
Leu Asp 115 120 125Lys Phe Asp Thr
Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln 130
135 140Asp Pro Ser Gly Ala Thr Thr Lys Ser Ala Met Leu
Thr Asn Leu Ile145 150 155
160Ile Phe Gly Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile
165 170 175Val Leu Arg Val Asp
Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe 180
185 190Gly Ser Ile Met Gln Met Ala Phe Cys Pro Glu Tyr
Val Pro Thr Phe 195 200 205Asp Asn
Val Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys 210
215 220Tyr Phe Gln Asp Pro Ala Leu Leu Leu Met His
Glu Leu Ile His Val225 230 235
240Leu His Gly Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro
245 250 255Ser Lys Gln Glu
Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu 260
265 270Glu Leu Phe Thr Phe Gly Gly Gln Asp Ala Asn
Leu Ile Ser Ile Asp 275 280 285Ile
Lys Asn Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile 290
295 300Ala Asn Lys Leu Ser Gln Val Thr Ser Cys
Asn Asp Pro Asn Ile Asp305 310 315
320Ile Asp Ser Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp
Lys 325 330 335Asp Ser Asn
Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu 340
345 350Tyr Asn Ser Ile Met Tyr Gly Phe Thr Glu
Ile Glu Leu Gly Lys Lys 355 360
365Phe Asn Ile Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro 370
375 380Val Lys Ile Pro Asn Leu Leu Asp
Asp Thr Ile Tyr Asn Asp Thr Glu385 390
395 400Gly Phe Asn Ile Glu Ser Lys Asp Leu Lys Ser Glu
Tyr Lys Gly Gln 405 410
415Asn Met Arg Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly
420 425 430Leu Val Ser Lys Leu Ile
Gly Leu Cys Val Asp Gly Ile Ile Thr Ser 435 440
445Lys Thr Lys Ser Leu Ile Glu Gly Arg Phe Gly Gly Phe Thr
Gly Ala 450 455 460Arg Lys Ser Ala Arg
Lys Arg Lys Asn Gln Ala Leu Ala Gly Gly Gly465 470
475 480Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Ala Leu Val Leu 485 490
495Gln Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu
500 505 510Asp Asn Phe Thr Asn
Asp Leu Asn Lys Gly Glu Glu Ile Thr Ser Asp 515
520 525Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu
Asp Leu Ile Gln 530 535 540Gln Tyr Tyr
Leu Thr Phe Asn Phe Asp Asn Glu Pro Glu Asn Ile Ser545
550 555 560Ile Glu Asn Leu Ser Ser Asp
Ile Ile Gly Gln Leu Glu Leu Met Pro 565
570 575Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu
Leu Asp Lys Tyr 580 585 590Thr
Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu His Gly Lys Ser 595
600 605Arg Ile Ala Leu Thr Asn Ser Val Asn
Glu Ala Leu Leu Asn Pro Ser 610 615
620Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys Lys Val Asn Lys625
630 635 640Ala Thr Glu Ala
Ala Met Phe Leu Gly Trp Val Glu Gln Leu Val Tyr 645
650 655Asp Phe Thr Asp Glu Thr Ser Glu Val Ser
Thr Thr Asp Lys Ile Ala 660 665
670Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly
675 680 685Asn Met Leu Tyr Lys Asp Asp
Phe Val Gly Ala Leu Ile Phe Ser Gly 690 695
700Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala Ile Pro Val
Leu705 710 715 720Gly Thr
Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys Val Leu Thr Val
725 730 735Gln Thr Ile Asp Asn Ala Leu
Ser Lys Arg Asn Glu Lys Trp Asp Glu 740 745
750Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys Val Asn
Thr Gln 755 760 765Ile Asp Leu Ile
Arg Lys Lys Met Lys Glu Ala Leu Glu Asn Gln Ala 770
775 780Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn
Gln Tyr Thr Glu785 790 795
800Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu Ser Ser Lys
805 810 815Leu Asn Glu Ser Ile
Asn Lys Ala Met Ile Asn Ile Asn Lys Phe Leu 820
825 830Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met
Ile Pro Tyr Gly 835 840 845Val Lys
Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys Asp Ala Leu Leu 850
855 860Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile
Gly Gln Val Asp Arg865 870 875
880Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp Ile Pro Phe Gln
885 890 895Leu Ser Lys Tyr
Val Asp Asn Gln Arg Leu Leu Ser Thr Leu Asp 900
905 91086180DNAArtificial SequenceSynthetic 86ggatccacgc
acgtcgacgc gattgatggt cgttttggcg gtttcacggg cgcacgcaaa 60tcagcgcgta
aacgtaagaa ccaggcgcta gcgggcggtg gcggtagcgg cggtggcggt 120agcggcggtg
gcggtagcgc actagtgctg cagacgcacg gtctagaatg ataaaagctt
180872715DNAArtificial SequenceSynthetic 87ggatccgaat tcatgccgat
caccatcaac aacttcaact acagcgatcc ggtggataac 60aaaaacatcc tgtacctgga
tacccatctg aataccctgg cgaacgaacc ggaaaaagcg 120tttcgtatca ccggcaacat
ttgggttatt ccggatcgtt ttagccgtaa cagcaacccg 180aatctgaata aaccgccgcg
tgttaccagc ccgaaaagcg gttattacga tccgaactat 240ctgagcaccg atagcgataa
agataccttc ctgaaagaaa tcatcaaact gttcaaacgc 300atcaacagcc gtgaaattgg
cgaagaactg atctatcgcc tgagcaccga tattccgttt 360ccgggcaaca acaacacccc
gatcaacacc tttgatttcg atgtggattt caacagcgtt 420gatgttaaaa cccgccaggg
taacaattgg gtgaaaaccg gcagcattaa cccgagcgtg 480attattaccg gtccgcgcga
aaacattatt gatccggaaa ccagcacctt taaactgacc 540aacaacacct ttgcggcgca
ggaaggtttt ggcgcgctga gcattattag cattagcccg 600cgctttatgc tgacctatag
caacgcgacc aacgatgttg gtgaaggccg tttcagcaaa 660agcgaatttt gcatggaccc
gatcctgatc ctgatgcatg aactgaacca tgcgatgcat 720aacctgtatg gcatcgcgat
tccgaacgat cagaccatta gcagcgtgac cagcaacatc 780ttttacagcc agtacaacgt
gaaactggaa tatgcggaaa tctatgcgtt tggcggtccg 840accattgatc tgattccgaa
aagcgcgcgc aaatacttcg aagaaaaagc gctggattac 900tatcgcagca ttgcgaaacg
tctgaacagc attaccaccg cgaatccgag cagcttcaac 960aaatatatcg gcgaatataa
acagaaactg atccgcaaat atcgctttgt ggtggaaagc 1020agcggcgaag ttaccgttaa
ccgcaataaa ttcgtggaac tgtacaacga actgacccag 1080atcttcaccg aatttaacta
tgcgaaaatc tataacgtgc agaaccgtaa aatctacctg 1140agcaacgtgt ataccccggt
gaccgcgaat attctggatg ataacgtgta cgatatccag 1200aacggcttta acatcccgaa
aagcaacctg aacgttctgt ttatgggcca gaacctgagc 1260cgtaatccgg cgctgcgtaa
agtgaacccg gaaaacatgc tgtacctgtt caccaaattt 1320tgcgtcgacg cgattgatgg
tcgttttggc ggtttcacgg gcgcacgcaa atcagcgcgt 1380aaacgtaaga accaggcgct
agcgggcggt ggcggtagcg gcggtggcgg tagcggcggt 1440ggcggtagcg cactagtgct
gcagtgtcgt gaactgctgg tgaaaaacac cgatctgccg 1500tttattggcg atatcagcga
tgtgaaaacc gatatcttcc tgcgcaaaga tatcaacgaa 1560gaaaccgaag tgatctacta
cccggataac gtgagcgttg atcaggtgat cctgagcaaa 1620aacaccagcg aacatggtca
gctggatctg ctgtatccga gcattgatag cgaaagcgaa 1680attctgccgg gcgaaaacca
ggtgttttac gataaccgta cccagaacgt ggattacctg 1740aacagctatt actacctgga
aagccagaaa ctgagcgata acgtggaaga ttttaccttt 1800acccgcagca ttgaagaagc
gctggataac agcgcgaaag tttacaccta ttttccgacc 1860ctggcgaaca aagttaatgc
gggtgttcag ggcggtctgt ttctgatgtg ggcgaacgat 1920gtggtggaag atttcaccac
caacatcctg cgtaaagata ccctggataa aatcagcgat 1980gttagcgcga ttattccgta
tattggtccg gcgctgaaca ttagcaatag cgtgcgtcgt 2040ggcaatttta ccgaagcgtt
tgcggttacc ggtgtgacca ttctgctgga agcgtttccg 2100gaatttacca ttccggcgct
gggtgcgttt gtgatctata gcaaagtgca ggaacgcaac 2160gaaatcatca aaaccatcga
taactgcctg gaacagcgta ttaaacgctg gaaagatagc 2220tatgaatgga tgatgggcac
ctggctgagc cgtattatca cccagttcaa caacatcagc 2280taccagatgt acgatagcct
gaactatcag gcgggtgcga ttaaagcgaa aatcgatctg 2340gaatacaaaa aatacagcgg
cagcgataaa gaaaacatca aaagccaggt tgaaaacctg 2400aaaaacagcc tggatgtgaa
aattagcgaa gcgatgaata acatcaacaa attcatccgc 2460gaatgcagcg tgacctacct
gttcaaaaac atgctgccga aagtgatcga tgaactgaac 2520gaatttgatc gcaacaccaa
agcgaaactg atcaacctga tcgatagcca caacattatt 2580ctggtgggcg aagtggataa
actgaaagcg aaagttaaca acagcttcca gaacaccatc 2640ccgtttaaca tcttcagcta
taccaacaac agcctgctga aagatatcat caacgaatac 2700ttcaatctag actag
271588904PRTArtificial
SequenceSynthetic 88Gly Ser Glu Phe Met Pro Ile Thr Ile Asn Asn Phe Asn
Tyr Ser Asp1 5 10 15Pro
Val Asp Asn Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr 20
25 30Leu Ala Asn Glu Pro Glu Lys Ala
Phe Arg Ile Thr Gly Asn Ile Trp 35 40
45Val Ile Pro Asp Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys
50 55 60Pro Pro Arg Val Thr Ser Pro Lys
Ser Gly Tyr Tyr Asp Pro Asn Tyr65 70 75
80Leu Ser Thr Asp Ser Asp Lys Asp Thr Phe Leu Lys Glu
Ile Ile Lys 85 90 95Leu
Phe Lys Arg Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr
100 105 110Arg Leu Ser Thr Asp Ile Pro
Phe Pro Gly Asn Asn Asn Thr Pro Ile 115 120
125Asn Thr Phe Asp Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys
Thr 130 135 140Arg Gln Gly Asn Asn Trp
Val Lys Thr Gly Ser Ile Asn Pro Ser Val145 150
155 160Ile Ile Thr Gly Pro Arg Glu Asn Ile Ile Asp
Pro Glu Thr Ser Thr 165 170
175Phe Lys Leu Thr Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala
180 185 190Leu Ser Ile Ile Ser Ile
Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn 195 200
205Ala Thr Asn Asp Val Gly Glu Gly Arg Phe Ser Lys Ser Glu
Phe Cys 210 215 220Met Asp Pro Ile Leu
Ile Leu Met His Glu Leu Asn His Ala Met His225 230
235 240Asn Leu Tyr Gly Ile Ala Ile Pro Asn Asp
Gln Thr Ile Ser Ser Val 245 250
255Thr Ser Asn Ile Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala
260 265 270Glu Ile Tyr Ala Phe
Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser 275
280 285Ala Arg Lys Tyr Phe Glu Glu Lys Ala Leu Asp Tyr
Tyr Arg Ser Ile 290 295 300Ala Lys Arg
Leu Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn305
310 315 320Lys Tyr Ile Gly Glu Tyr Lys
Gln Lys Leu Ile Arg Lys Tyr Arg Phe 325
330 335Val Val Glu Ser Ser Gly Glu Val Thr Val Asn Arg
Asn Lys Phe Val 340 345 350Glu
Leu Tyr Asn Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn Tyr Ala 355
360 365Lys Ile Tyr Asn Val Gln Asn Arg Lys
Ile Tyr Leu Ser Asn Val Tyr 370 375
380Thr Pro Val Thr Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln385
390 395 400Asn Gly Phe Asn
Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly 405
410 415Gln Asn Leu Ser Arg Asn Pro Ala Leu Arg
Lys Val Asn Pro Glu Asn 420 425
430Met Leu Tyr Leu Phe Thr Lys Phe Cys Val Asp Ala Ile Asp Gly Arg
435 440 445Phe Gly Gly Phe Thr Gly Ala
Arg Lys Ser Ala Arg Lys Arg Lys Asn 450 455
460Gln Ala Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly465 470 475 480Gly Gly
Ser Ala Leu Val Leu Gln Cys Arg Glu Leu Leu Val Lys Asn
485 490 495Thr Asp Leu Pro Phe Ile Gly
Asp Ile Ser Asp Val Lys Thr Asp Ile 500 505
510Phe Leu Arg Lys Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr
Tyr Pro 515 520 525Asp Asn Val Ser
Val Asp Gln Val Ile Leu Ser Lys Asn Thr Ser Glu 530
535 540His Gly Gln Leu Asp Leu Leu Tyr Pro Ser Ile Asp
Ser Glu Ser Glu545 550 555
560Ile Leu Pro Gly Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn
565 570 575Val Asp Tyr Leu Asn
Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser 580
585 590Asp Asn Val Glu Asp Phe Thr Phe Thr Arg Ser Ile
Glu Glu Ala Leu 595 600 605Asp Asn
Ser Ala Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn Lys 610
615 620Val Asn Ala Gly Val Gln Gly Gly Leu Phe Leu
Met Trp Ala Asn Asp625 630 635
640Val Val Glu Asp Phe Thr Thr Asn Ile Leu Arg Lys Asp Thr Leu Asp
645 650 655Lys Ile Ser Asp
Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala Leu 660
665 670Asn Ile Ser Asn Ser Val Arg Arg Gly Asn Phe
Thr Glu Ala Phe Ala 675 680 685Val
Thr Gly Val Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr Ile 690
695 700Pro Ala Leu Gly Ala Phe Val Ile Tyr Ser
Lys Val Gln Glu Arg Asn705 710 715
720Glu Ile Ile Lys Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys
Arg 725 730 735Trp Lys Asp
Ser Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg Ile 740
745 750Ile Thr Gln Phe Asn Asn Ile Ser Tyr Gln
Met Tyr Asp Ser Leu Asn 755 760
765Tyr Gln Ala Gly Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys 770
775 780Tyr Ser Gly Ser Asp Lys Glu Asn
Ile Lys Ser Gln Val Glu Asn Leu785 790
795 800Lys Asn Ser Leu Asp Val Lys Ile Ser Glu Ala Met
Asn Asn Ile Asn 805 810
815Lys Phe Ile Arg Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu
820 825 830Pro Lys Val Ile Asp Glu
Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala 835 840
845Lys Leu Ile Asn Leu Ile Asp Ser His Asn Ile Ile Leu Val
Gly Glu 850 855 860Val Asp Lys Leu Lys
Ala Lys Val Asn Asn Ser Phe Gln Asn Thr Ile865 870
875 880Pro Phe Asn Ile Phe Ser Tyr Thr Asn Asn
Ser Leu Leu Lys Asp Ile 885 890
895Ile Asn Glu Tyr Phe Asn Leu Asp 9008917PRTArtificial
SequenceSynthetic 89Tyr Gly Gly Phe Leu Arg Arg Ile Arg Pro Lys Leu Lys
Trp Asp Asn1 5 10
15Gln902709DNAArtificial SequenceSynthetic 90ggatccatgg agttcgttaa
caaacagttc aactataaag acccagttaa cggtgttgac 60attgcttaca tcaaaatccc
gaacgctggc cagatgcagc cggtaaaggc attcaaaatc 120cacaacaaaa tctgggttat
cccggaacgt gataccttta ctaacccgga agaaggtgac 180ctgaacccgc caccggaagc
gaaacaggtg ccggtatctt actatgactc cacctacctg 240tctaccgata acgaaaagga
caactacctg aaaggtgtta ctaaactgtt cgagcgtatt 300tactccaccg acctgggccg
tatgctgctg actagcatcg ttcgcggtat cccgttctgg 360ggcggttcta ccatcgatac
cgaactgaaa gtaatcgaca ctaactgcat caacgttatt 420cagccggacg gttcctatcg
ttccgaagaa ctgaacctgg tgatcatcgg cccgtctgct 480gatatcatcc agttcgagtg
taagagcttt ggtcacgaag ttctgaacct cacccgtaac 540ggctacggtt ccactcagta
catccgtttc tctccggact tcaccttcgg ttttgaagaa 600tccctggaag tagacacgaa
cccactgctg ggcgctggta aattcgcaac tgatcctgcg 660gttaccctgg ctcacgaact
gattcatgca ggccaccgcc tgtacggtat cgccatcaat 720ccgaaccgtg tcttcaaagt
taacaccaac gcgtattacg agatgtccgg tctggaagtt 780agcttcgaag aactgcgtac
ttttggcggt cacgacgcta aattcatcga ctctctgcaa 840gaaaacgagt tccgtctgta
ctactataac aagttcaaag atatcgcatc caccctgaac 900aaagcgaaat ccatcgtggg
taccactgct tctctccagt acatgaagaa cgtttttaaa 960gaaaaatacc tgctcagcga
agacacctcc ggcaaattct ctgtagacaa gttgaaattc 1020gataaacttt acaaaatgct
gactgaaatt tacaccgaag acaacttcgt taagttcttt 1080aaagttctga accgcaaaac
ctatctgaac ttcgacaagg cagtattcaa aatcaacatc 1140gtgccgaaag ttaactacac
tatctacgat ggtttcaacc tgcgtaacac caacctggct 1200gctaatttta acggccagaa
cacggaaatc aacaacatga acttcacaaa actgaaaaac 1260ttcactggtc tgttcgagtt
ttacaagctg ctgtgcgtcg acggcatcat tacctccaaa 1320actaaatctg acgatgacga
taaatatgga ggttttttga gaaggatacg accaaaatta 1380aagtgggata atcaagcgct
agcgggcggt ggcggtagcg gcggtggcgg tagcggcggt 1440ggcggtagcg cactagtgct
gcagtgtatc aaggttaaca actgggattt attcttcagc 1500ccgagtgaag acaacttcac
caacgacctg aacaaaggtg aagaaatcac ctcagatact 1560aacatcgaag cagccgaaga
aaacatctcg ctggacctga tccagcagta ctacctgacc 1620tttaatttcg acaacgagcc
ggaaaacatt tctatcgaaa acctgagctc tgatatcatc 1680ggccagctgg aactgatgcc
gaacatcgaa cgtttcccaa acggtaaaaa gtacgagctg 1740gacaaatata ccatgttcca
ctacctgcgc gcgcaggaat ttgaacacgg caaatcccgt 1800atcgcactga ctaactccgt
taacgaagct ctgctcaacc cgtcccgtgt atacaccttc 1860ttctctagcg actacgtgaa
aaaggtcaac aaagcgactg aagctgcaat gttcttgggt 1920tgggttgaac agcttgttta
tgattttacc gacgagacgt ccgaagtatc tactaccgac 1980aaaattgcgg atatcactat
catcatcccg tacatcggtc cggctctgaa cattggcaac 2040atgctgtaca aagacgactt
cgttggcgca ctgatcttct ccggtgcggt gatcctgctg 2100gagttcatcc cggaaatcgc
catcccggta ctgggcacct ttgctctggt ttcttacatt 2160gcaaacaagg ttctgactgt
acaaaccatc gacaacgcgc tgagcaaacg taacgaaaaa 2220tgggatgaag tttacaaata
tatcgtgacc aactggctgg ctaaggttaa tactcagatc 2280gacctcatcc gcaaaaaaat
gaaagaagca ctggaaaacc aggcggaagc taccaaggca 2340atcattaact accagtacaa
ccagtacacc gaggaagaaa aaaacaacat caacttcaac 2400atcgacgatc tgtcctctaa
actgaacgaa tccatcaaca aagctatgat caacatcaac 2460aagttcctga accagtgctc
tgtaagctat ctgatgaact ccatgatccc gtacggtgtt 2520aaacgtctgg aggacttcga
tgcgtctctg aaagacgccc tgctgaaata catttacgac 2580aaccgtggca ctctgatcgg
tcaggttgat cgtctgaagg acaaagtgaa caatacctta 2640tcgaccgaca tcccttttca
gctcagtaaa tatgtcgata accaacgcct tttgtccact 2700ctagactag
270991902PRTArtificial
SequenceSynthetic 91Gly Ser Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys
Asp Pro Val1 5 10 15Asn
Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met 20
25 30Gln Pro Val Lys Ala Phe Lys Ile
His Asn Lys Ile Trp Val Ile Pro 35 40
45Glu Arg Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro
50 55 60Pro Glu Ala Lys Gln Val Pro Val
Ser Tyr Tyr Asp Ser Thr Tyr Leu65 70 75
80Ser Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val
Thr Lys Leu 85 90 95Phe
Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser
100 105 110Ile Val Arg Gly Ile Pro Phe
Trp Gly Gly Ser Thr Ile Asp Thr Glu 115 120
125Leu Lys Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp
Gly 130 135 140Ser Tyr Arg Ser Glu Glu
Leu Asn Leu Val Ile Ile Gly Pro Ser Ala145 150
155 160Asp Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly
His Glu Val Leu Asn 165 170
175Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro
180 185 190Asp Phe Thr Phe Gly Phe
Glu Glu Ser Leu Glu Val Asp Thr Asn Pro 195 200
205Leu Leu Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr
Leu Ala 210 215 220His Glu Leu Ile His
Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn225 230
235 240Pro Asn Arg Val Phe Lys Val Asn Thr Asn
Ala Tyr Tyr Glu Met Ser 245 250
255Gly Leu Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp
260 265 270Ala Lys Phe Ile Asp
Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr 275
280 285Tyr Asn Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn
Lys Ala Lys Ser 290 295 300Ile Val Gly
Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys305
310 315 320Glu Lys Tyr Leu Leu Ser Glu
Asp Thr Ser Gly Lys Phe Ser Val Asp 325
330 335Lys Leu Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr
Glu Ile Tyr Thr 340 345 350Glu
Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr 355
360 365Leu Asn Phe Asp Lys Ala Val Phe Lys
Ile Asn Ile Val Pro Lys Val 370 375
380Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala385
390 395 400Ala Asn Phe Asn
Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr 405
410 415Lys Leu Lys Asn Phe Thr Gly Leu Phe Glu
Phe Tyr Lys Leu Leu Cys 420 425
430Val Asp Gly Ile Ile Thr Ser Lys Thr Lys Ser Asp Asp Asp Asp Lys
435 440 445Tyr Gly Gly Phe Leu Arg Arg
Ile Arg Pro Lys Leu Lys Trp Asp Asn 450 455
460Gln Ala Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly465 470 475 480Gly Gly
Ser Ala Leu Val Leu Gln Cys Ile Lys Val Asn Asn Trp Asp
485 490 495Leu Phe Phe Ser Pro Ser Glu
Asp Asn Phe Thr Asn Asp Leu Asn Lys 500 505
510Gly Glu Glu Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu
Glu Asn 515 520 525Ile Ser Leu Asp
Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp 530
535 540Asn Glu Pro Glu Asn Ile Ser Ile Glu Asn Leu Ser
Ser Asp Ile Ile545 550 555
560Gly Gln Leu Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys
565 570 575Lys Tyr Glu Leu Asp
Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln 580
585 590Glu Phe Glu His Gly Lys Ser Arg Ile Ala Leu Thr
Asn Ser Val Asn 595 600 605Glu Ala
Leu Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp 610
615 620Tyr Val Lys Lys Val Asn Lys Ala Thr Glu Ala
Ala Met Phe Leu Gly625 630 635
640Trp Val Glu Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val
645 650 655Ser Thr Thr Asp
Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile 660
665 670Gly Pro Ala Leu Asn Ile Gly Asn Met Leu Tyr
Lys Asp Asp Phe Val 675 680 685Gly
Ala Leu Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro 690
695 700Glu Ile Ala Ile Pro Val Leu Gly Thr Phe
Ala Leu Val Ser Tyr Ile705 710 715
720Ala Asn Lys Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser
Lys 725 730 735Arg Asn Glu
Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp 740
745 750Leu Ala Lys Val Asn Thr Gln Ile Asp Leu
Ile Arg Lys Lys Met Lys 755 760
765Glu Ala Leu Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr 770
775 780Gln Tyr Asn Gln Tyr Thr Glu Glu
Glu Lys Asn Asn Ile Asn Phe Asn785 790
795 800Ile Asp Asp Leu Ser Ser Lys Leu Asn Glu Ser Ile
Asn Lys Ala Met 805 810
815Ile Asn Ile Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met
820 825 830Asn Ser Met Ile Pro Tyr
Gly Val Lys Arg Leu Glu Asp Phe Asp Ala 835 840
845Ser Leu Lys Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg
Gly Thr 850 855 860Leu Ile Gly Gln Val
Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu865 870
875 880Ser Thr Asp Ile Pro Phe Gln Leu Ser Lys
Tyr Val Asp Asn Gln Arg 885 890
895Leu Leu Ser Thr Leu Asp 90092892PRTArtificial
SequenceSynthetic 92Gly Ser Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys
Asp Pro Val1 5 10 15Asn
Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met 20
25 30Gln Pro Val Lys Ala Phe Lys Ile
His Asn Lys Ile Trp Val Ile Pro 35 40
45Glu Arg Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro
50 55 60Pro Glu Ala Lys Gln Val Pro Val
Ser Tyr Tyr Asp Ser Thr Tyr Leu65 70 75
80Ser Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val
Thr Lys Leu 85 90 95Phe
Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser
100 105 110Ile Val Arg Gly Ile Pro Phe
Trp Gly Gly Ser Thr Ile Asp Thr Glu 115 120
125Leu Lys Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp
Gly 130 135 140Ser Tyr Arg Ser Glu Glu
Leu Asn Leu Val Ile Ile Gly Pro Ser Ala145 150
155 160Asp Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly
His Glu Val Leu Asn 165 170
175Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro
180 185 190Asp Phe Thr Phe Gly Phe
Glu Glu Ser Leu Glu Val Asp Thr Asn Pro 195 200
205Leu Leu Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr
Leu Ala 210 215 220His Glu Leu Ile His
Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn225 230
235 240Pro Asn Arg Val Phe Lys Val Asn Thr Asn
Ala Tyr Tyr Glu Met Ser 245 250
255Gly Leu Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp
260 265 270Ala Lys Phe Ile Asp
Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr 275
280 285Tyr Asn Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn
Lys Ala Lys Ser 290 295 300Ile Val Gly
Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys305
310 315 320Glu Lys Tyr Leu Leu Ser Glu
Asp Thr Ser Gly Lys Phe Ser Val Asp 325
330 335Lys Leu Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr
Glu Ile Tyr Thr 340 345 350Glu
Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr 355
360 365Leu Asn Phe Asp Lys Ala Val Phe Lys
Ile Asn Ile Val Pro Lys Val 370 375
380Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala385
390 395 400Ala Asn Phe Asn
Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr 405
410 415Lys Leu Lys Asn Phe Thr Gly Leu Phe Glu
Phe Tyr Lys Leu Leu Cys 420 425
430Val Asp Gly Ile Ile Thr Ser Lys Thr Lys Ser Asp Asp Asp Asp Lys
435 440 445Tyr Gly Gly Phe Leu Arg Arg
Ile Arg Pro Lys Leu Lys Trp Asp Asn 450 455
460Gln Ala Leu Ala Gly Gly Gly Gly Ser Ala Leu Val Leu Gln Cys
Ile465 470 475 480Lys Val
Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu Asp Asn Phe
485 490 495Thr Asn Asp Leu Asn Lys Gly
Glu Glu Ile Thr Ser Asp Thr Asn Ile 500 505
510Glu Ala Ala Glu Glu Asn Ile Ser Leu Asp Leu Ile Gln Gln
Tyr Tyr 515 520 525Leu Thr Phe Asn
Phe Asp Asn Glu Pro Glu Asn Ile Ser Ile Glu Asn 530
535 540Leu Ser Ser Asp Ile Ile Gly Gln Leu Glu Leu Met
Pro Asn Ile Glu545 550 555
560Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu Asp Lys Tyr Thr Met Phe
565 570 575His Tyr Leu Arg Ala
Gln Glu Phe Glu His Gly Lys Ser Arg Ile Ala 580
585 590Leu Thr Asn Ser Val Asn Glu Ala Leu Leu Asn Pro
Ser Arg Val Tyr 595 600 605Thr Phe
Phe Ser Ser Asp Tyr Val Lys Lys Val Asn Lys Ala Thr Glu 610
615 620Ala Ala Met Phe Leu Gly Trp Val Glu Gln Leu
Val Tyr Asp Phe Thr625 630 635
640Asp Glu Thr Ser Glu Val Ser Thr Thr Asp Lys Ile Ala Asp Ile Thr
645 650 655Ile Ile Ile Pro
Tyr Ile Gly Pro Ala Leu Asn Ile Gly Asn Met Leu 660
665 670Tyr Lys Asp Asp Phe Val Gly Ala Leu Ile Phe
Ser Gly Ala Val Ile 675 680 685Leu
Leu Glu Phe Ile Pro Glu Ile Ala Ile Pro Val Leu Gly Thr Phe 690
695 700Ala Leu Val Ser Tyr Ile Ala Asn Lys Val
Leu Thr Val Gln Thr Ile705 710 715
720Asp Asn Ala Leu Ser Lys Arg Asn Glu Lys Trp Asp Glu Val Tyr
Lys 725 730 735Tyr Ile Val
Thr Asn Trp Leu Ala Lys Val Asn Thr Gln Ile Asp Leu 740
745 750Ile Arg Lys Lys Met Lys Glu Ala Leu Glu
Asn Gln Ala Glu Ala Thr 755 760
765Lys Ala Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr Thr Glu Glu Glu Lys 770
775 780Asn Asn Ile Asn Phe Asn Ile Asp
Asp Leu Ser Ser Lys Leu Asn Glu785 790
795 800Ser Ile Asn Lys Ala Met Ile Asn Ile Asn Lys Phe
Leu Asn Gln Cys 805 810
815Ser Val Ser Tyr Leu Met Asn Ser Met Ile Pro Tyr Gly Val Lys Arg
820 825 830Leu Glu Asp Phe Asp Ala
Ser Leu Lys Asp Ala Leu Leu Lys Tyr Ile 835 840
845Tyr Asp Asn Arg Gly Thr Leu Ile Gly Gln Val Asp Arg Leu
Lys Asp 850 855 860Lys Val Asn Asn Thr
Leu Ser Thr Asp Ile Pro Phe Gln Leu Ser Lys865 870
875 880Tyr Val Asp Asn Gln Arg Leu Leu Ser Thr
Leu Asp 885 89093897PRTArtificial
SequenceSynthetic 93Gly Ser Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys
Asp Pro Val1 5 10 15Asn
Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met 20
25 30Gln Pro Val Lys Ala Phe Lys Ile
His Asn Lys Ile Trp Val Ile Pro 35 40
45Glu Arg Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro
50 55 60Pro Glu Ala Lys Gln Val Pro Val
Ser Tyr Tyr Asp Ser Thr Tyr Leu65 70 75
80Ser Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val
Thr Lys Leu 85 90 95Phe
Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser
100 105 110Ile Val Arg Gly Ile Pro Phe
Trp Gly Gly Ser Thr Ile Asp Thr Glu 115 120
125Leu Lys Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp
Gly 130 135 140Ser Tyr Arg Ser Glu Glu
Leu Asn Leu Val Ile Ile Gly Pro Ser Ala145 150
155 160Asp Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly
His Glu Val Leu Asn 165 170
175Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro
180 185 190Asp Phe Thr Phe Gly Phe
Glu Glu Ser Leu Glu Val Asp Thr Asn Pro 195 200
205Leu Leu Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr
Leu Ala 210 215 220His Glu Leu Ile His
Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn225 230
235 240Pro Asn Arg Val Phe Lys Val Asn Thr Asn
Ala Tyr Tyr Glu Met Ser 245 250
255Gly Leu Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp
260 265 270Ala Lys Phe Ile Asp
Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr 275
280 285Tyr Asn Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn
Lys Ala Lys Ser 290 295 300Ile Val Gly
Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys305
310 315 320Glu Lys Tyr Leu Leu Ser Glu
Asp Thr Ser Gly Lys Phe Ser Val Asp 325
330 335Lys Leu Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr
Glu Ile Tyr Thr 340 345 350Glu
Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr 355
360 365Leu Asn Phe Asp Lys Ala Val Phe Lys
Ile Asn Ile Val Pro Lys Val 370 375
380Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala385
390 395 400Ala Asn Phe Asn
Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr 405
410 415Lys Leu Lys Asn Phe Thr Gly Leu Phe Glu
Phe Tyr Lys Leu Leu Cys 420 425
430Val Asp Gly Ile Ile Thr Ser Lys Thr Lys Ser Asp Asp Asp Asp Lys
435 440 445Tyr Gly Gly Phe Leu Arg Arg
Ile Arg Pro Lys Leu Lys Trp Asp Asn 450 455
460Gln Ala Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
Leu465 470 475 480Val Leu
Gln Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe Ser Pro
485 490 495Ser Glu Asp Asn Phe Thr Asn
Asp Leu Asn Lys Gly Glu Glu Ile Thr 500 505
510Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu
Asp Leu 515 520 525Ile Gln Gln Tyr
Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro Glu Asn 530
535 540Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly
Gln Leu Glu Leu545 550 555
560Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu Asp
565 570 575Lys Tyr Thr Met Phe
His Tyr Leu Arg Ala Gln Glu Phe Glu His Gly 580
585 590Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu
Ala Leu Leu Asn 595 600 605Pro Ser
Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys Lys Val 610
615 620Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly
Trp Val Glu Gln Leu625 630 635
640Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr Asp Lys
645 650 655Ile Ala Asp Ile
Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn 660
665 670Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val
Gly Ala Leu Ile Phe 675 680 685Ser
Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala Ile Pro 690
695 700Val Leu Gly Thr Phe Ala Leu Val Ser Tyr
Ile Ala Asn Lys Val Leu705 710 715
720Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu Lys
Trp 725 730 735Asp Glu Val
Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys Val Asn 740
745 750Thr Gln Ile Asp Leu Ile Arg Lys Lys Met
Lys Glu Ala Leu Glu Asn 755 760
765Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr 770
775 780Thr Glu Glu Glu Lys Asn Asn Ile
Asn Phe Asn Ile Asp Asp Leu Ser785 790
795 800Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile
Asn Ile Asn Lys 805 810
815Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met Ile Pro
820 825 830Tyr Gly Val Lys Arg Leu
Glu Asp Phe Asp Ala Ser Leu Lys Asp Ala 835 840
845Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly
Gln Val 850 855 860Asp Arg Leu Lys Asp
Lys Val Asn Asn Thr Leu Ser Thr Asp Ile Pro865 870
875 880Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln
Arg Leu Leu Ser Thr Leu 885 890
895Asp94907PRTArtificial SequenceSynthetic 94Gly Ser Met Glu Phe Val
Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val1 5
10 15Asn Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn
Ala Gly Gln Met 20 25 30Gln
Pro Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro 35
40 45Glu Arg Asp Thr Phe Thr Asn Pro Glu
Glu Gly Asp Leu Asn Pro Pro 50 55
60Pro Glu Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu65
70 75 80Ser Thr Asp Asn Glu
Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu 85
90 95Phe Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg
Met Leu Leu Thr Ser 100 105
110Ile Val Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu
115 120 125Leu Lys Val Ile Asp Thr Asn
Cys Ile Asn Val Ile Gln Pro Asp Gly 130 135
140Ser Tyr Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser
Ala145 150 155 160Asp Ile
Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn
165 170 175Leu Thr Arg Asn Gly Tyr Gly
Ser Thr Gln Tyr Ile Arg Phe Ser Pro 180 185
190Asp Phe Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr
Asn Pro 195 200 205Leu Leu Gly Ala
Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala 210
215 220His Glu Leu Ile His Ala Gly His Arg Leu Tyr Gly
Ile Ala Ile Asn225 230 235
240Pro Asn Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser
245 250 255Gly Leu Glu Val Ser
Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp 260
265 270Ala Lys Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe
Arg Leu Tyr Tyr 275 280 285Tyr Asn
Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser 290
295 300Ile Val Gly Thr Thr Ala Ser Leu Gln Tyr Met
Lys Asn Val Phe Lys305 310 315
320Glu Lys Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp
325 330 335Lys Leu Lys Phe
Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr 340
345 350Glu Asp Asn Phe Val Lys Phe Phe Lys Val Leu
Asn Arg Lys Thr Tyr 355 360 365Leu
Asn Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val 370
375 380Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu
Arg Asn Thr Asn Leu Ala385 390 395
400Ala Asn Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe
Thr 405 410 415Lys Leu Lys
Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys 420
425 430Val Asp Gly Ile Ile Thr Ser Lys Thr Lys
Ser Asp Asp Asp Asp Lys 435 440
445Tyr Gly Gly Phe Leu Arg Arg Ile Arg Pro Lys Leu Lys Trp Asp Asn 450
455 460Gln Ala Leu Ala Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly465 470
475 480Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Val Leu
Gln Cys Ile Lys 485 490
495Val Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu Asp Asn Phe Thr
500 505 510Asn Asp Leu Asn Lys Gly
Glu Glu Ile Thr Ser Asp Thr Asn Ile Glu 515 520
525Ala Ala Glu Glu Asn Ile Ser Leu Asp Leu Ile Gln Gln Tyr
Tyr Leu 530 535 540Thr Phe Asn Phe Asp
Asn Glu Pro Glu Asn Ile Ser Ile Glu Asn Leu545 550
555 560Ser Ser Asp Ile Ile Gly Gln Leu Glu Leu
Met Pro Asn Ile Glu Arg 565 570
575Phe Pro Asn Gly Lys Lys Tyr Glu Leu Asp Lys Tyr Thr Met Phe His
580 585 590Tyr Leu Arg Ala Gln
Glu Phe Glu His Gly Lys Ser Arg Ile Ala Leu 595
600 605Thr Asn Ser Val Asn Glu Ala Leu Leu Asn Pro Ser
Arg Val Tyr Thr 610 615 620Phe Phe Ser
Ser Asp Tyr Val Lys Lys Val Asn Lys Ala Thr Glu Ala625
630 635 640Ala Met Phe Leu Gly Trp Val
Glu Gln Leu Val Tyr Asp Phe Thr Asp 645
650 655Glu Thr Ser Glu Val Ser Thr Thr Asp Lys Ile Ala
Asp Ile Thr Ile 660 665 670Ile
Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly Asn Met Leu Tyr 675
680 685Lys Asp Asp Phe Val Gly Ala Leu Ile
Phe Ser Gly Ala Val Ile Leu 690 695
700Leu Glu Phe Ile Pro Glu Ile Ala Ile Pro Val Leu Gly Thr Phe Ala705
710 715 720Leu Val Ser Tyr
Ile Ala Asn Lys Val Leu Thr Val Gln Thr Ile Asp 725
730 735Asn Ala Leu Ser Lys Arg Asn Glu Lys Trp
Asp Glu Val Tyr Lys Tyr 740 745
750Ile Val Thr Asn Trp Leu Ala Lys Val Asn Thr Gln Ile Asp Leu Ile
755 760 765Arg Lys Lys Met Lys Glu Ala
Leu Glu Asn Gln Ala Glu Ala Thr Lys 770 775
780Ala Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr Thr Glu Glu Glu Lys
Asn785 790 795 800Asn Ile
Asn Phe Asn Ile Asp Asp Leu Ser Ser Lys Leu Asn Glu Ser
805 810 815Ile Asn Lys Ala Met Ile Asn
Ile Asn Lys Phe Leu Asn Gln Cys Ser 820 825
830Val Ser Tyr Leu Met Asn Ser Met Ile Pro Tyr Gly Val Lys
Arg Leu 835 840 845Glu Asp Phe Asp
Ala Ser Leu Lys Asp Ala Leu Leu Lys Tyr Ile Tyr 850
855 860Asp Asn Arg Gly Thr Leu Ile Gly Gln Val Asp Arg
Leu Lys Asp Lys865 870 875
880Val Asn Asn Thr Leu Ser Thr Asp Ile Pro Phe Gln Leu Ser Lys Tyr
885 890 895Val Asp Asn Gln Arg
Leu Leu Ser Thr Leu Asp 900
90595913PRTArtificial SequenceSynthetic 95Gly Ser Glu Phe Met Pro Ile Thr
Ile Asn Asn Phe Asn Tyr Ser Asp1 5 10
15Pro Val Asp Asn Lys Asn Ile Leu Tyr Leu Asp Thr His Leu
Asn Thr 20 25 30Leu Ala Asn
Glu Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp 35
40 45Val Ile Pro Asp Arg Phe Ser Arg Asn Ser Asn
Pro Asn Leu Asn Lys 50 55 60Pro Pro
Arg Val Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr65
70 75 80Leu Ser Thr Asp Ser Asp Lys
Asp Thr Phe Leu Lys Glu Ile Ile Lys 85 90
95Leu Phe Lys Arg Ile Asn Ser Arg Glu Ile Gly Glu Glu
Leu Ile Tyr 100 105 110Arg Leu
Ser Thr Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile 115
120 125Asn Thr Phe Asp Phe Asp Val Asp Phe Asn
Ser Val Asp Val Lys Thr 130 135 140Arg
Gln Gly Asn Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val145
150 155 160Ile Ile Thr Gly Pro Arg
Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr 165
170 175Phe Lys Leu Thr Asn Asn Thr Phe Ala Ala Gln Glu
Gly Phe Gly Ala 180 185 190Leu
Ser Ile Ile Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn 195
200 205Ala Thr Asn Asp Val Gly Glu Gly Arg
Phe Ser Lys Ser Glu Phe Cys 210 215
220Met Asp Pro Ile Leu Ile Leu Met His Glu Leu Asn His Ala Met His225
230 235 240Asn Leu Tyr Gly
Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val 245
250 255Thr Ser Asn Ile Phe Tyr Ser Gln Tyr Asn
Val Lys Leu Glu Tyr Ala 260 265
270Glu Ile Tyr Ala Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser
275 280 285Ala Arg Lys Tyr Phe Glu Glu
Lys Ala Leu Asp Tyr Tyr Arg Ser Ile 290 295
300Ala Lys Arg Leu Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe
Asn305 310 315 320Lys Tyr
Ile Gly Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe
325 330 335Val Val Glu Ser Ser Gly Glu
Val Thr Val Asn Arg Asn Lys Phe Val 340 345
350Glu Leu Tyr Asn Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn
Tyr Ala 355 360 365Lys Ile Tyr Asn
Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr 370
375 380Thr Pro Val Thr Ala Asn Ile Leu Asp Asp Asn Val
Tyr Asp Ile Gln385 390 395
400Asn Gly Phe Asn Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly
405 410 415Gln Asn Leu Ser Arg
Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn 420
425 430Met Leu Tyr Leu Phe Thr Lys Phe Cys Val Asp Gly
Ile Ile Thr Ser 435 440 445Lys Thr
Lys Ser Asp Asp Asp Asp Lys Tyr Gly Gly Phe Leu Arg Arg 450
455 460Ile Arg Pro Lys Leu Lys Trp Asp Asn Gln Ala
Leu Ala Gly Gly Gly465 470 475
480Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Val Leu
485 490 495Gln Cys Arg Glu
Leu Leu Val Lys Asn Thr Asp Leu Pro Phe Ile Gly 500
505 510Asp Ile Ser Asp Val Lys Thr Asp Ile Phe Leu
Arg Lys Asp Ile Asn 515 520 525Glu
Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser Val Asp Gln 530
535 540Val Ile Leu Ser Lys Asn Thr Ser Glu His
Gly Gln Leu Asp Leu Leu545 550 555
560Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly Glu Asn
Gln 565 570 575Val Phe Tyr
Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu Asn Ser Tyr 580
585 590Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp
Asn Val Glu Asp Phe Thr 595 600
605Phe Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser Ala Lys Val Tyr 610
615 620Thr Tyr Phe Pro Thr Leu Ala Asn
Lys Val Asn Ala Gly Val Gln Gly625 630
635 640Gly Leu Phe Leu Met Trp Ala Asn Asp Val Val Glu
Asp Phe Thr Thr 645 650
655Asn Ile Leu Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp Val Ser Ala
660 665 670Ile Ile Pro Tyr Ile Gly
Pro Ala Leu Asn Ile Ser Asn Ser Val Arg 675 680
685Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val Thr
Ile Leu 690 695 700Leu Glu Ala Phe Pro
Glu Phe Thr Ile Pro Ala Leu Gly Ala Phe Val705 710
715 720Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu
Ile Ile Lys Thr Ile Asp 725 730
735Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser Tyr Glu Trp
740 745 750Met Met Gly Thr Trp
Leu Ser Arg Ile Ile Thr Gln Phe Asn Asn Ile 755
760 765Ser Tyr Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala
Gly Ala Ile Lys 770 775 780Ala Lys Ile
Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser Asp Lys Glu785
790 795 800Asn Ile Lys Ser Gln Val Glu
Asn Leu Lys Asn Ser Leu Asp Val Lys 805
810 815Ile Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile
Arg Glu Cys Ser 820 825 830Val
Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile Asp Glu Leu 835
840 845Asn Glu Phe Asp Arg Asn Thr Lys Ala
Lys Leu Ile Asn Leu Ile Asp 850 855
860Ser His Asn Ile Ile Leu Val Gly Glu Val Asp Lys Leu Lys Ala Lys865
870 875 880Val Asn Asn Ser
Phe Gln Asn Thr Ile Pro Phe Asn Ile Phe Ser Tyr 885
890 895Thr Asn Asn Ser Leu Leu Lys Asp Ile Ile
Asn Glu Tyr Phe Asn Leu 900 905
910Asp961432PRTArtificial SequenceSynthetic 96Gly Ser Leu Val Arg Asp
Asp Val Asp Tyr Gln Ile Phe Arg Asp Phe1 5
10 15Ala Glu Asn Lys Gly Lys Phe Phe Val Gly Ala Thr
Asp Leu Ser Val 20 25 30Lys
Asn Lys Arg Gly Gln Asn Ile Gly Asn Ala Leu Ser Asn Val Pro 35
40 45Met Ile Asp Phe Ser Val Ala Asp Val
Asn Lys Arg Ile Ala Thr Val 50 55
60Val Asp Pro Gln Tyr Ala Val Ser Val Lys His Ala Lys Ala Glu Val65
70 75 80His Thr Phe Tyr Tyr
Gly Gln Tyr Asn Gly His Asn Asp Val Ala Asp 85
90 95Lys Glu Asn Glu Tyr Arg Val Val Glu Gln Asn
Asn Tyr Glu Pro His 100 105
110Lys Ala Trp Gly Ala Ser Asn Leu Gly Arg Leu Glu Asp Tyr Asn Met
115 120 125Ala Arg Phe Asn Lys Phe Val
Thr Glu Val Ala Pro Ile Ala Pro Thr 130 135
140Asp Ala Gly Gly Gly Leu Asp Thr Tyr Lys Asp Lys Asn Arg Phe
Ser145 150 155 160Ser Phe
Val Arg Ile Gly Ala Gly Arg Gln Leu Val Tyr Glu Lys Gly
165 170 175Val Tyr His Gln Glu Gly Asn
Glu Lys Gly Tyr Asp Leu Arg Asp Leu 180 185
190Ser Gln Ala Tyr Arg Tyr Ala Ile Ala Gly Thr Pro Tyr Lys
Asp Ile 195 200 205Asn Ile Asp Gln
Thr Met Asn Thr Glu Gly Leu Ile Gly Phe Gly Asn 210
215 220His Asn Lys Gln Tyr Ser Ala Glu Glu Leu Lys Gln
Ala Leu Ser Gln225 230 235
240Asp Ala Leu Thr Asn Tyr Gly Val Leu Gly Asp Ser Gly Ser Pro Leu
245 250 255Phe Ala Phe Asp Lys
Gln Lys Asn Gln Trp Val Phe Leu Gly Thr Tyr 260
265 270Asp Tyr Trp Ala Gly Tyr Gly Lys Lys Ser Trp Gln
Glu Trp Asn Ile 275 280 285Tyr Lys
Lys Glu Phe Ala Asp Lys Ile Lys Gln His Asp Asn Ala Gly 290
295 300Thr Val Lys Gly Asn Gly Glu His His Trp Lys
Thr Thr Gly Thr Asn305 310 315
320Ser His Ile Gly Ser Thr Ala Val Arg Leu Ala Asn Asn Glu Gly Asp
325 330 335Ala Asn Asn Gly
Gln Asn Val Thr Phe Glu Asp Asn Gly Thr Leu Val 340
345 350Leu Asn Gln Asn Ile Asn Gln Gly Ala Gly Gly
Leu Phe Phe Lys Gly 355 360 365Asp
Tyr Thr Val Lys Gly Ala Asn Asn Asp Ile Thr Trp Leu Gly Ala 370
375 380Gly Ile Asp Val Ala Asp Gly Lys Lys Val
Val Trp Gln Val Lys Asn385 390 395
400Pro Asn Gly Asp Arg Leu Ala Lys Ile Gly Lys Gly Thr Leu Glu
Ile 405 410 415Asn Gly Thr
Gly Val Asn Gln Gly Gln Leu Lys Val Gly Asp Gly Thr 420
425 430Val Ile Leu Asn Gln Lys Ala Asp Ala Asp
Lys Lys Val Gln Ala Phe 435 440
445Ser Gln Val Gly Ile Val Ser Gly Arg Gly Thr Leu Val Leu Asn Ser 450
455 460Ser Asn Gln Ile Asn Pro Asp Asn
Leu Tyr Phe Gly Phe Arg Gly Gly465 470
475 480Arg Leu Asp Ala Asn Gly Asn Asp Leu Thr Phe Glu
His Ile Arg Asn 485 490
495Val Asp Glu Gly Ala Arg Ile Val Asn His Asn Thr Asp His Ala Ser
500 505 510Thr Ile Thr Leu Thr Gly
Lys Ser Leu Ile Thr Asn Pro Asn Ser Leu 515 520
525Ser Val His Ser Ile Gln Asn Asp Tyr Asp Glu Asp Asp Tyr
Ser Tyr 530 535 540Tyr Tyr Arg Pro Arg
Arg Pro Ile Pro Gln Gly Lys Asp Leu Tyr Tyr545 550
555 560Lys Asn Tyr Arg Tyr Tyr Ala Leu Lys Ser
Gly Gly Arg Leu Asn Ala 565 570
575Pro Met Pro Glu Asn Gly Val Ala Glu Asn Asn Asp Trp Ile Phe Met
580 585 590Gly Tyr Thr Gln Glu
Glu Ala Arg Lys Asn Ala Met Asn His Lys Asn 595
600 605Asn Arg Arg Ile Gly Asp Phe Gly Gly Phe Phe Asp
Glu Glu Asn Gly 610 615 620Lys Gly His
Asn Gly Ala Leu Asn Leu Asn Phe Asn Gly Lys Ser Ala625
630 635 640Gln Lys Arg Phe Leu Leu Thr
Gly Gly Ala Asn Leu Asn Gly Lys Ile 645
650 655Ser Val Thr Gln Gly Asn Val Leu Leu Ser Gly Arg
Pro Thr Pro His 660 665 670Ala
Arg Asp Phe Val Asn Lys Ser Ser Ala Arg Lys Asp Ala His Phe 675
680 685Ser Lys Asn Asn Glu Val Val Phe Glu
Asp Asp Trp Ile Asn Arg Thr 690 695
700Phe Lys Ala Ala Glu Ile Ala Val Asn Gln Ser Ala Ser Phe Ser Ser705
710 715 720Gly Arg Asn Val
Ser Asp Ile Thr Ala Asn Ile Thr Ala Thr Asp Asn 725
730 735Ala Lys Val Asn Leu Gly Tyr Lys Asn Gly
Asp Glu Val Cys Val Arg 740 745
750Ser Asp Tyr Thr Gly Tyr Val Thr Cys Asn Thr Gly Asn Leu Ser Asp
755 760 765Lys Ala Leu Asn Ser Phe Asp
Ala Thr Arg Ile Asn Gly Asn Val Asn 770 775
780Leu Asn Gln Asn Ala Ala Leu Val Leu Gly Lys Ala Ala Leu Trp
Gly785 790 795 800Lys Ile
Gln Gly Gln Gly Asn Ser Arg Val Ser Leu Asn Gln His Ser
805 810 815Lys Trp His Leu Thr Gly Asp
Ser Gln Val His Asn Leu Ser Leu Ala 820 825
830Asp Ser His Ile His Leu Asn Asn Ala Ser Asp Ala Gln Ser
Ala Asn 835 840 845Lys Tyr His Thr
Ile Lys Ile Asn His Leu Ser Gly Asn Gly His Phe 850
855 860His Tyr Leu Thr Asp Leu Ala Lys Asn Leu Gly Asp
Lys Val Leu Val865 870 875
880Lys Glu Ser Ala Ser Gly His Tyr Gln Leu His Val Gln Asn Lys Thr
885 890 895Gly Glu Pro Asn Gln
Glu Gly Leu Asp Leu Phe Asp Ala Ser Ser Val 900
905 910Gln Asp Arg Ser Arg Leu Phe Val Ser Leu Ala Asn
His Tyr Val Asp 915 920 925Leu Gly
Ala Leu Arg Tyr Thr Ile Lys Thr Glu Asn Gly Ile Thr Arg 930
935 940Leu Tyr Asn Pro Tyr Ala Gly Asn Gly Arg Pro
Val Lys Pro Ala Pro945 950 955
960Cys Val Asp Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Asp Asp
965 970 975Asp Lys Tyr Gly
Gly Phe Leu Arg Arg Ile Arg Pro Lys Leu Lys Trp 980
985 990Asp Asn Gln Ala Leu Ala Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser 995 1000
1005Gly Gly Gly Gly Ser Ala Leu Val Leu Gln Cys Ile Lys Val Asn
1010 1015 1020Asn Trp Asp Leu Phe Phe
Ser Pro Ser Glu Asp Asn Phe Thr Asn 1025 1030
1035Asp Leu Asn Lys Gly Glu Glu Ile Thr Ser Asp Thr Asn Ile
Glu 1040 1045 1050Ala Ala Glu Glu Asn
Ile Ser Leu Asp Leu Ile Gln Gln Tyr Tyr 1055 1060
1065Leu Thr Phe Asn Phe Asp Asn Glu Pro Glu Asn Ile Ser
Ile Glu 1070 1075 1080Asn Leu Ser Ser
Asp Ile Ile Gly Gln Leu Glu Leu Met Pro Asn 1085
1090 1095Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu
Leu Asp Lys Tyr 1100 1105 1110Thr Met
Phe His Tyr Leu Arg Ala Gln Glu Phe Glu His Gly Lys 1115
1120 1125Ser Arg Ile Ala Leu Thr Asn Ser Val Asn
Glu Ala Leu Leu Asn 1130 1135 1140Pro
Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys Lys 1145
1150 1155Val Asn Lys Ala Thr Glu Ala Ala Met
Phe Leu Gly Trp Val Glu 1160 1165
1170Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr
1175 1180 1185Thr Asp Lys Ile Ala Asp
Ile Thr Ile Ile Ile Pro Tyr Ile Gly 1190 1195
1200Pro Ala Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe
Val 1205 1210 1215Gly Ala Leu Ile Phe
Ser Gly Ala Val Ile Leu Leu Glu Phe Ile 1220 1225
1230Pro Glu Ile Ala Ile Pro Val Leu Gly Thr Phe Ala Leu
Val Ser 1235 1240 1245Tyr Ile Ala Asn
Lys Val Leu Thr Val Gln Thr Ile Asp Asn Ala 1250
1255 1260Leu Ser Lys Arg Asn Glu Lys Trp Asp Glu Val
Tyr Lys Tyr Ile 1265 1270 1275Val Thr
Asn Trp Leu Ala Lys Val Asn Thr Gln Ile Asp Leu Ile 1280
1285 1290Arg Lys Lys Met Lys Glu Ala Leu Glu Asn
Gln Ala Glu Ala Thr 1295 1300 1305Lys
Ala Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr Thr Glu Glu Glu 1310
1315 1320Lys Asn Asn Ile Asn Phe Asn Ile Asp
Asp Leu Ser Ser Lys Leu 1325 1330
1335Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile Asn Lys Phe Leu
1340 1345 1350Asn Gln Cys Ser Val Ser
Tyr Leu Met Asn Ser Met Ile Pro Tyr 1355 1360
1365Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys Asp
Ala 1370 1375 1380Leu Leu Lys Tyr Ile
Tyr Asp Asn Arg Gly Thr Leu Ile Gly Gln 1385 1390
1395Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser
Thr Asp 1400 1405 1410Ile Pro Phe Gln
Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu 1415
1420 1425Ser Thr Leu Asp 143097911PRTArtificial
SequenceSynthetic 97Gly Ser Met Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser
Asp Pro Val1 5 10 15Asn
Asn Asp Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu 20
25 30Asp Ile Tyr Tyr Lys Ala Phe Lys
Ile Thr Asp Arg Ile Trp Ile Val 35 40
45Pro Glu Arg Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro
50 55 60Ser Ser Leu Ile Glu Gly Ala Ser
Glu Tyr Tyr Asp Pro Asn Tyr Leu65 70 75
80Arg Thr Asp Ser Asp Lys Asp Arg Phe Leu Gln Thr Met
Val Lys Leu 85 90 95Phe
Asn Arg Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys
100 105 110Ile Ile Asn Ala Ile Pro Tyr
Leu Gly Asn Ser Tyr Ser Leu Leu Asp 115 120
125Lys Phe Asp Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu
Gln 130 135 140Asp Pro Ser Gly Ala Thr
Thr Lys Ser Ala Met Leu Thr Asn Leu Ile145 150
155 160Ile Phe Gly Pro Gly Pro Val Leu Asn Lys Asn
Glu Val Arg Gly Ile 165 170
175Val Leu Arg Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe
180 185 190Gly Ser Ile Met Gln Met
Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe 195 200
205Asp Asn Val Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys
Ser Lys 210 215 220Tyr Phe Gln Asp Pro
Ala Leu Leu Leu Met His Glu Leu Ile His Val225 230
235 240Leu His Gly Leu Tyr Gly Met Gln Val Ser
Ser His Glu Ile Ile Pro 245 250
255Ser Lys Gln Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu
260 265 270Glu Leu Phe Thr Phe
Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp 275
280 285Ile Lys Asn Asp Leu Tyr Glu Lys Thr Leu Asn Asp
Tyr Lys Ala Ile 290 295 300Ala Asn Lys
Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp305
310 315 320Ile Asp Ser Tyr Lys Gln Ile
Tyr Gln Gln Lys Tyr Gln Phe Asp Lys 325
330 335Asp Ser Asn Gly Gln Tyr Ile Val Asn Glu Asp Lys
Phe Gln Ile Leu 340 345 350Tyr
Asn Ser Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys 355
360 365Phe Asn Ile Lys Thr Arg Leu Ser Tyr
Phe Ser Met Asn His Asp Pro 370 375
380Val Lys Ile Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu385
390 395 400Gly Phe Asn Ile
Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln 405
410 415Asn Met Arg Val Asn Thr Asn Ala Phe Arg
Asn Val Asp Gly Ser Gly 420 425
430Leu Val Ser Lys Leu Ile Gly Leu Cys Val Asp Gly Ile Ile Thr Ser
435 440 445Lys Thr Lys Ser Asp Asp Asp
Asp Lys Tyr Gly Gly Phe Leu Arg Arg 450 455
460Ile Arg Pro Lys Leu Lys Trp Asp Asn Gln Ala Leu Ala Gly Gly
Gly465 470 475 480Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Val Leu
485 490 495Gln Cys Ile Lys Val Asn Asn
Trp Asp Leu Phe Phe Ser Pro Ser Glu 500 505
510Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu Ile Thr
Ser Asp 515 520 525Thr Asn Ile Glu
Ala Ala Glu Glu Asn Ile Ser Leu Asp Leu Ile Gln 530
535 540Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro
Glu Asn Ile Ser545 550 555
560Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu Glu Leu Met Pro
565 570 575Asn Ile Glu Arg Phe
Pro Asn Gly Lys Lys Tyr Glu Leu Asp Lys Tyr 580
585 590Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu
His Gly Lys Ser 595 600 605Arg Ile
Ala Leu Thr Asn Ser Val Asn Glu Ala Leu Leu Asn Pro Ser 610
615 620Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val
Lys Lys Val Asn Lys625 630 635
640Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu Gln Leu Val Tyr
645 650 655Asp Phe Thr Asp
Glu Thr Ser Glu Val Ser Thr Thr Asp Lys Ile Ala 660
665 670Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro
Ala Leu Asn Ile Gly 675 680 685Asn
Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu Ile Phe Ser Gly 690
695 700Ala Val Ile Leu Leu Glu Phe Ile Pro Glu
Ile Ala Ile Pro Val Leu705 710 715
720Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys Val Leu Thr
Val 725 730 735Gln Thr Ile
Asp Asn Ala Leu Ser Lys Arg Asn Glu Lys Trp Asp Glu 740
745 750Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu
Ala Lys Val Asn Thr Gln 755 760
765Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu Glu Asn Gln Ala 770
775 780Glu Ala Thr Lys Ala Ile Ile Asn
Tyr Gln Tyr Asn Gln Tyr Thr Glu785 790
795 800Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp
Leu Ser Ser Lys 805 810
815Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile Asn Lys Phe Leu
820 825 830Asn Gln Cys Ser Val Ser
Tyr Leu Met Asn Ser Met Ile Pro Tyr Gly 835 840
845Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys Asp Ala
Leu Leu 850 855 860Lys Tyr Ile Tyr Asp
Asn Arg Gly Thr Leu Ile Gly Gln Val Asp Arg865 870
875 880Leu Lys Asp Lys Val Asn Asn Thr Leu Ser
Thr Asp Ile Pro Phe Gln 885 890
895Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser Thr Leu Asp
900 905 910
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