Patent application title: PROTEASE STABLE CELL WALL LYSING ENZYMES
Inventors:
Holger Grallert (Weilheim, DE)
Holger Grallert (Weilheim, DE)
Michael Forchheim (Regensburg, DE)
IPC8 Class: AC12N900FI
USPC Class:
424 941
Class name: Drug, bio-affecting and body treating compositions enzyme or coenzyme containing
Publication date: 2013-11-07
Patent application number: 20130295070
Abstract:
The present invention relates to a modified polypeptide with a biological
activity to lyse cell walls of bacteria, wherein the polypeptide has no
caspase, clostripain, enterokinase, factor Xa, granzyme B, staphylococcus
peptidase I (V8 Protease), plasmin, streptopain, bacillolysin and/or
thrombin cleavage site. The invention further relates to nucleic acids
with a sequence encoding a polypeptide according to the present
invention.Claims:
1. A modified polypeptide or variant thereof having the biological
activity of lysing cell walls of bacteria, wherein the polypeptide or the
variant thereof has an amino acid substitution at a protease cleavage
site, thereby preventing a degradation by a protease, wherein the
protease cleavage site is selected from the group consisting of a caspase
cleavage site, a clostripain cleavage site, a enterokinase cleavage site,
a factor Xa cleavage site, a granzyme B cleavage site, a staphylococcus
peptidase I (V8 protease) cleavage site, a plasmin cleavage site, a
streptopain cleavage site, a bacillolysin cleavage site and a thrombin
cleavage site.
2. (canceled)
3. The polypeptide of claim 1, wherein the polypeptide exhibits the biological activity of lysing cell walls of Gram-positive bacteria.
4. The polypeptide of claim 3, wherein the Gram-positive bacteria are selected from the group consisting of clostridia, bacilli, listeria, staphylococci, lactobacilli, enterococci, aerococci, pediococci, streptococci, mycoplasms and/or leuconostoc.
5. The polypeptide of claim 1, wherein the polypeptide is a endolysin, a bacteriophage tail protein, an autolysin, or a bacteriocin.
6. A polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.
7. A nucleic acid molecule comprising a nucleotide sequence coding for a polypeptide or variant thereof having the biological activity of lysing cell walls of bacteria, wherein the polypeptide or the variant thereof has an amino acid substitution at a protease cleavage site, thereby preventing a degradation by a protease, wherein the protease cleavage site is selected from the group consisting of a caspase cleavage site, a clostripain cleavage site, a enterokinase cleavage site, a factor Xa cleavage site, a granzyme B cleavage site, a staphylococcus peptidase I (V8 protease) cleavage site, a plasmin cleavage site, a streptopain cleavage site, a bacillolysin cleavage site and a thrombin cleavage site.
8. An expression vector comprising a nucleic acid molecule according to claim 7.
9. The nucleic acid of claim 7, further comprised within a host cell.
10. A pharmaceutical composition comprising a modified polypeptide or variant thereof having the biological activity of lysing cell walls of bacteria, wherein the polypeptide or the variant thereof has an amino acid substitution at a protease cleavage site, thereby preventing a degradation by a protease, wherein the protease cleavage site is selected from the group consisting of a caspase cleavage site, a clostripain cleavage site, a enterokinase cleavage site, a factor Xa cleavage site, a granzyme B cleavage site, a staphylococcus peptidase I (V8 protease) cleavage site, a plasmin cleavage site, a streptopain cleavage site, a bacillolysin cleavage site and a thrombin cleavage site.
11. A method for prevention or therapy of a disease caused by Gram-positive bacteria, comprising administering to a subject in need thereof a modified polypeptide or variant thereof having the biological activity of lysing cell walls of bacteria, wherein the polypeptide or the variant thereof has an amino acid substitution at a protease cleavage site, thereby preventing a degradation by a protease, wherein the protease cleavage site is selected from the group consisting of a caspase cleavage site, a clostripain cleavage site, a enterokinase cleavage site, a factor Xa cleavage site, a granzyme B cleavage site, a staphylococcus peptidase I (V8 protease) cleavage site, a plasmin cleavage site, a streptopain cleavage site, a bacillolysin cleavage site and a thrombin cleavage site.
12. A method of inhibiting the growth of Gram-positive bacterium comprising contacting said bacterium with a modified polypeptide or variant thereof having the biological activity of lysing cell walls of bacteria, wherein the polypeptide or the variant thereof has an amino acid substitution at a protease cleavage site, thereby preventing a degradation by a protease, wherein the protease cleavage site is selected from the group consisting of a caspase cleavage site, a clostripain cleavage site, a enterokinase cleavage site, a factor Xa cleavage site, a granzyme B cleavage site, a staphylococcus peptidase I (V8 protease) cleavage site, a plasmin cleavage site, a streptopain cleavage site, a bacillolysin cleavage site and a thrombin cleavage site.
13. A method for the detection of a bacterium in a patient sample, an environmental sample, a food sample or a cosmetic sample comprising: (i) contacting said sample with a modified polypeptide or variant thereof having the biological activity of lysing cell walls of bacteria, wherein the polypeptide or the variant thereof has an amino acid substitution at a protease cleavage site, thereby preventing a degradation by a protease, wherein the protease cleavage site is selected from the group consisting of a caspase cleavage site, a clostripain cleavage site, a enterokinase cleavage site, a factor Xa cleavage site, a granzyme B cleavage site, a staphylococcus peptidase I (V8 protease) cleavage site, a plasmin cleavage site, a streptopain cleavage site, a bacillolysin cleavage site and a thrombin cleavage site; and (ii) detecting the polypeptide bound to a bacterium in said sample.
14. The expression vector of claim 8, further comprised within a host cell.
15. The method of claim 11, wherein said disease is caused by Gram-positive bacteria selected from the group consisting of clostridia, bacilli, listeria, staphylococci, lactobacilli, entrococci, aerococci, pediococci, streptococci, mycoplasmas, leuconostoc, or a combination thereof.
16. The method of claim 13, wherein said bacterium is a Gram-positive bacterium.
17. The method of claim 16, wherein said Gram-positive bacterium is selected from the group consisting of clostridia, bacilli, listeria, staphylococci, lactobacilli, entrococci, aerococci, pediococci, streptococci, mycoplasmas, leuconostoc, or a combination thereof.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser. No. 12/674,369, filed as a national phase application under 35 U.S.C. §371 of International Patent Application No. PCT/DE2008/001378, filed Aug. 19, 2008, which claims priority to European Patent Application No. EP 07 114 785.4, filed Aug. 22, 2007, U.S. Provisional Patent Application No. 60/957,351 filed Aug. 22, 2007, German Patent Application No. DE 10 2007 061 929.6, filed Dec. 21, 2007, U.S. Provisional Patent Application No. 61/032,211 filed Feb. 28, 2008, European Patent Application No. EP 08 152 096.7, filed Feb. 28, 2008 and German Patent Application No. DE 10 2008 023 448.6, filed May 14, 2008. The entire text of each of the above-referenced disclosures is specifically incorporated herein by reference without disclaimer.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to a modified polypeptide with a biological activity to lyse cell walls of bacteria, wherein the polypeptide has no caspase, clostripain, enterokinase, factor Xa, granzyme B, staphylococcus peptidase I (V8 Protease), plasmin, streptopain, bacillolysin and/or thrombin cleavage site. The invention further relates to nucleic acids with a sequence encoding a polypeptide according to the present invention.
[0003] Pathogenic bacteria appear at many places and cause enormous health problems by infections as well as high economic costs, e.g. also by unwanted bacteria contamination in the food, cosmetic and environmental industry. In health care the problems are increasing more and more due to germs resistant to antibiotics, so that it is searched hand-wringing for alternatives to antibiotics. In the food and cosmetic industry it is increasingly tried to manage without the classical preservatives, which seek acceptance in the population less and less. As a solution for these problems the use of cell wall lysing enzymes is indicated which prevent naturally the growth of the unexpected bacteria and kill existing germs.
[0004] Examples of cell wall lysing enzymes are endolysins, which were isolated from bacteriophages. Bacteriophages use these enzymes at the end of their cycle of propagation to release the bacteriophages which were produced inside the bacterial cell. Thereby the bacterial shell is lysed and the host bacterium is destroyed in that way. A further example are enzymes, which the bacteriophage requires at the beginning of its cycle of propagation and are localized normally in bacteriophage tail proteins. These enzymes are required for the break up of the bacterial wall in the bacterial infection. A third example are enzymes, which have a similar function and often also a sequence similarity to the endolysins. These enzymes are autolysins produced under certain circumstances by bacteria leading to the self-lysis of the bacteria. A forth example are enzymes, which are also produced by bacteria and are known as bacteriocins. These four groups of cell wall lysing enzymes are already technically deployed and yet gain in importance in the future.
[0005] One field of application is the medical utilisation in the prevention and therapy as well as the diagnostic of bacterial infections in human and animals. A further field of application is the use in the food, environmental and cosmetic industry for prevention of a unexpected bacterial growth and for killing of the germs for example by disinfection, as well as the detection of bacteria in food, environmental and cosmetic samples.
[0006] Virtually all applications, in which bacteria lysing enzymes are used, impose high requirements for the stability of the used enzymes, so that their use is economically profitable. In the U.S. Pat. No. 6,432,444 B1 for stabilization of the cell wall lysing enzymes it is proposed to use for example a stabilizing buffer to assure the optimal enzyme activity. Moreover the addition of stabilizing substances like reducing agents, metal chelators, immune globulins, specific buffer salts, physiological pH values, preservatives or mild detergents are proposed.
[0007] Generally the stability of proteins is often reduced by existing proteases. Strategies for increasing the stability of proteins to protease degradation are, e.g. the addition of metal chelators, to inhibit proteases, which require metal ions for their activity or the addition of special protease inhibitors, as they are commercially available for serine proteases. However protease inhibitors could be only limited used for stabilizing of cell wall lysing enzymes, because they disturb also other essential components at the target site. Furthermore specific inhibitors are also expensive. Also the choice of other environmental conditions, in which the proteases are less active, are normally not possible, because the cell wall lysing enzymes require very similar conditions for their activity as the proteases. Thus, cell wall lysing enzymes operate best in an aqueous environment and under moderate pH values, under which also the protease activity is highest. The known stabilizing methods are not appropriate for the use of cell wall lysing enzymes.
BRIEF SUMMARY OF THE INVENTION
[0008] Thus the object of the present invention is to provide stable cell wall lysing enzymes.
[0009] The object is solved by the subject matter as defined in the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The following figures illustrate the invention.
[0011] FIG. 1 shows the wild type amino acid sequence (SEQ ID NO: 20) of the CHAP domain (amino acid 1-154), the linker region (underlined, amino acid 155-193) and the amidase domain (amino acid 194-393) of an endolysin from PlyPitti26. Potential cleavage sites for V8 protease cleaving after the amino acid residue E, are highlighted in bolt and are located at the amino acid positions 163, 179 and 189. The thrombin cleavage site is highlighted in grey and located at position R167.
[0012] FIG. 2 shows the result of a protease digest and following activity test of non modified PlyPitti26 with the proteases thrombin and V8 protease. FIG. 2A shows a SDS polyacrylamide gel with samples of non modified PlyPitti26 after a digest with thrombin (T), V8 protease (V8) and a control digest with plasmin (P), for which exists no cleavage site. Undigested PlyPitti26 is marked with "-". M describes molecular weight standards with the protein sizes given in kDa in the right-hand margin. In the right-hand margin the position of the bands for the fragment N-terminal of the thrombin cleavage site (CHAP domain), the fragment C-terminal of the cleavage site (ami-CBD) and the band of the added thrombin are given. FIG. 2B shows the result of a liquid lysis test for determination of the specific activities of digested and non-digested non-modified PlyPitti26. The bacteria cell lysis is monitored by a light scattering measurement in the photometer. The lysis curves are illustrated for undigested PlyPitti26 (______) plasmin digested ( - - - ) as well as with thrombin (_._.) or V8 protease (_.._..) digested endolysin.
[0013] FIG. 3 shows the result of a protease digest of modified and non-modified CHAP-AmiPitti26-CBDUSA. FIG. 3A shows the result of a thrombin digest of the non-modified CHAP-AmiPitti26-CBDUSA and of a modified Staphylococcus endolysin CHAP-AmiPitti26-CBDUSA (Mutant 4 in table 3). The non-modified CHAP-AmiPitti26-CBDUSA has a singular thrombin cleavage site between the CHAP- and the amidase domain at position R167, the modified polypeptide has a substitution from R to A at position 167. The FIG. 3A shows a SDS polyacrylamide gel of both enzyme variants before and after the thrombin digest: M-molecular weight marker (molecular weights given in kDa in the margin); 1--non-modified CHAP-AmiPitti26-CBDUSA without thrombin; 2--non-modified CHAP-AmiPitti26-CBDUSA after thrombin digest: 3--modified CHAP-AmiPitti26-CBDUSA without thrombin; 4--modified CHAP-AmiPitti26-CBDUSA after thrombin addition.
[0014] FIG. 3B shows the result of a V8 protease digest of the non-modified CHAP-AmiPitti26-CBDUSA and a modified Staphylococcus endolysin CHAP-AmiPitti26-CBDUSA (mutant 4 in table 3). The non-modified CHAP-AmiPitti26-CBDUSA has a V8 protease cleavage site between the CHAP- and the amidase domain at position R163, the modified polypeptide has a substitution from E to A at position 163. The FIG. 3B shows a SDS polyacrylamide gel of both enzyme variants before and after the V8 protease digest: M-molecular weight marker (molecular weights given in kDa in the margin); 1--non-modified CHAP-AmiPitti26-CBDUSA without V8 protease; 2--non-modified CHAP-AmiPitti26-CBDUSA 15 min V8 protease digest: 3--non-modified CHAP-AmiPitti26-CBDUSA 60 min V8 protease digest; 4--modified CHAP-AmiPitti26-CBDUSA without V8 protease; 5--modified CHAP-AmiPitti26-CBDUSA 15 min V8 protease digest; 6--modified CHAP-AmiPitti26-CBDUSA 60 min V8 protease digest.
[0015] FIG. 4 shows an amino acid sequence comparison of the modified endolysins from the Cl. difficile phage Φ CD119 (CD119) (SEQ ID NO: 5) with the modified endolysin from the Cl. difficile phage Φ 630 (SEQ ID NO: 4). The singular caspase 1 cleavage site at amino acid D214 existing in the Φ CD119 and Φ 630 wild type was modified to E214, the singular thrombin cleavage site R87 existing in the Φ 630 wild type was modified to K87.
[0016] FIG. 5 shows an amino acid sequence comparison of the region of the thrombin cleavage site of various Staphylococcus aureus endolysins (SEQ ID NO: 23). The bolt highlighted amino acid residue R is position R167 in the reference sequence ply_pitti26, described as P26A. The further listed amino acid sequences are aligned with R167 of ply_pitti26. The amino acid sequence comparison was performed with the program BLAST; Altschul et al., 1990, J. Mol: Biol., 215, 403-410. The number to the right of the amino acid sequence comparison describes the amino acid position of the last amino acid in the represented sequence segment of the respective endolysin. The numbers in the left margin stands for different homologous proteins from the amino acid sequence comparison. P26A stands for ply_pitti26, 15 an autolysin (N-acetylmuramoyl-L-alanine amidase) from Staphylococcus aureus (database accession number P24556), 16 stands for an amidase from the bacteriophage 80 alpha (database accession number AAB39699), 3 stands for the endolysin of Staphylococcus phage phi MR11 (database accession number YP 001604156), 4 stands for ORF006 from the Staphylococcus aureus phage 88 (database accession number YP240699), 5 stands for ORF21 from the Staphylococcus aureus phage 85 (database accession number YP239752), 9 stands for the amidase from the Staphylococcus phage 11 (database accession number NP803306), 10 stands for ORF007 from the Staphylococcus phage 52A (database accession number YP240634), 11 stands for the N-acetylmuramoyl-L-alanine amidase from the strain Staphylococcus aureus subsp. aureus JH9 (database accession number YP 001246457), 12 stands for a putative cell wall hydrolase of the phage phiMR25 (database accession number YP 001949866), 13 stands for ORF007 from the Staphylococcus phage 69 (database accession number YP 239596), 14 stands for ORF007 from the Staphylococcus phage 55 (database accession number YP 240484), 2 stands for the N-acetylmuramoyl-L-alanine amidase form the strain Staphylococcus aureus subsp. aureus NCTC 8325 (database accession number YP 500516), 1 stands for the amidase from the Staphylococcus phage phiNM2 (database accession number ABF73160), 6 stands for a phage-related amidase form the strain Staphylococcus aureus RF122 (database accession number YP 417165), 17 stands for ORF006 from the Staphylococcus phage 37 (database accession number YP 240103) and 18 stands for ORF007 from the Staphylococcus phage EW (database accession number YP 240182).
[0017] FIG. 6 represents amino acid sequences of modified and non-modified CHAP-AmiPitti26-CBDUSA. FIG. 6 A to D shows the amino acid sequence of CHAP-AmiPitti26-CBDUSA without amino acid substitutions at protease cleavage sites (A) (SEQ ID NO: 18) and CHAP-AmiPitti26-CBDUSA with amino acid substitutions at protease cleavage sites (B to E), Sequence in FIG. 6B is SEQ ID NO: 2, FIG. 6C is SEQ ID NO: 3, FIG. 6D is SEQ ID NO: 1, FIG. 6E is SEQ ID NO: 11. FIG. 6 F to K shows the amino acid sequence of CHAP-AmiPitti26-CBDUSA-Add2 without amino acid substitutions at protease cleavage sites and an additional amino acid residue at position two (F) (SEQ ID NO: 19) and CHAP-AmiPitti26-CBDUSA-Add2 with amino acid substitutions at protease cleavage sites and an additional amino acid residue at position two (G to K). Sequence in FIG. 6G is SEQ ID NO: 15, FIG. 6H is SEQ ID NO: 16, FIG. 6I is SEQ ID NO: 14, FIG. 6K is SEQ ID NO: 17.
[0018] FIG. 7 A shows the amino acid sequence of the modified endolysin form the Cl. difficile strain 630 with a substitution of D214 to E214 (SEQ ID NO: 4). FIG. 7B shows the amino acid sequence of the modified Cl. difficile endolysin from the phage phi CD119 with a substitution of R87 to K87 and D214 to E214 (SEQ ID NO: 5).
[0019] FIG. 8 shows an amino acid sequence of the endolysin Ply511 (SEQ ID NO: 6). The amino acid positions E7, E40 and E89 printed in bold could be substituted with any other amino acid residue.
[0020] FIG. 9 shows the amino acid sequence of the endolysin Ply511 (SEQ ID NO: 21). The potential cleavage sites (R in P1 position) for the protease clostripain are underlined. The both experimentally determined particularly sensible cleavage sites (R92 and R221) are underlined and printed in bolt.
[0021] FIG. 10 shows the digest of the therapeutically used endolysin CHAP-Amipitti26_CBDUSA-Add2 (L56H, L57T, E164A, R168A, Y201H) by proteases existing in organ extracts. FIG. 10 A shows a SDS gel, in which the protein bands are separated after incubation with liver, kidney and lungs extract. On the left a molecular weight standard is loaded. Lines 1 to 3 show controls (only endolysin, no organ extract), which were incubated not at all (line 1), 18 h at room temperature (line 2) or 18 h at 4° C. (line 3). In the lines 4 to 6 liver extract was added, in line 7 to 9 kidney extract and in the lines 10 to 12 lungs extract. Lines 4, 7 and 10 only contain organ extract, lines 5, 8 and 11 organ extract and endolysin after 18 h incubation at room temperature, lines 6, 9 and 12 organ extract and endolysin after 18 h incubation at 4° C. The arrows with the numbers 1 to 7 beside line 9 mark the bands, which were N-terminally sequenced after transfer on a blot membrane. FIG. 10 B shows a SDS gel, on which the protein bands were separated after incubation with heart or spleen extract. On the left a molecular weight standard is loaded. Lines 1 to 3 as well as 10 show controls (only endolysin, no organ extract), which were incubated not at all (line 1 to 10), 18 h at room temperature (line 2) or 18 h at 4° C. (line 3). In the lines 4 to 6 heart extract was added, in line 7 to 9 spleen extract. Lines 4 and 7 only contain organ extract, lines 5 and 8 organ extract and endolysin after 18 h incubation at room temperature, lines 6 and 9 organ extranct and endolysin after 18 h incubation at 4° C.
[0022] FIG. 11 shows the sequence of the endolysin CHAP-Amipitti26_CBDUSA-Add2 (L56H, L57T, E164A, R168A, Y201H) with protease sensitive regions (SEQ ID NO: 22). The cleavage sites (P1 position), which resulted from the incubation with kidney extract, are highlighted in bolt. Amino acid sequences, which resulted from the N-terminal sequencing of the produced fragments, are underlined. Amino acid regions defining the domain linker are presented in italic. This is a domain linker region between the CHAP and amidase 2 domain (amino acids 159 to 198) as well as a second linker between the amidase 2 domain and the CBD (amino acids 347 to 412).
[0023] FIG. 12 shows an amino acid sequence comparison of the region of the caspase I cleavage site of different Staphylococcus endolysins (SEQ ID NO: 24). It is a matter of amidases from the Staphylococcus phage SAP-2, 44AHJD and phage 66. The NCBI database accession numbers for the proteins are YP--001491539, 44AHJD--15 amidase and YP--239469. The position D42, after which it is cleaved, is highlighted in bolt, the conservative recognition sequence of the caspase I is represented each in italic and framed. The three proteins are identical from 89 to 90% from their whole length, not conservative amino acids for the region of the amino acids 1 to 60 are marked with "-". The amino acid sequence comparison was performed with the program BLAST.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The term "polypeptide" or "protein" as used herein describes peptides of at least eight amino acids. The polypeptide may be pharmacologically or immunologically active polypeptides or polypeptides used for diagnostic purposes.
[0025] The term "cell wall lysing enzymes" as used herein describes enzymes, which are capable, to at least partially break up or thus damage the bacterial cell wall that as a result a cell lysis or at least a bacteriostatic effect arises. Particularly the term describes endolysins, autolysins, bacteriophage tail proteins and bacteriocins.
[0026] The term "endolysin" as used herein describes enzymes, which are encoded naturally by bacteriophages and which are produced by them at the end of their host cycle to lyse the host cell and thus, releasing the bacteriophages newly produced in the host cells. Endolysins are built of at least an enzymatically active domain (EAD) and a non-enzymatically active cell binding domain (CBD). The single EADs have different activities, wherein endolysins have at least one EAD selected from the following group: N-acetyl muramoyl-L-alanine amidase (amidase, e.g. Ami--2, Ami--5), (endo)-peptidase (e.g. CHAP, i.e. cysteine, histidine-dependent amidohydrolases/peptidases), transglycosylase, glycosylhydrolase, (N-acetyl)-muramidase (lysozymes) and N-acetyl-glucosaminidase.
[0027] The term "autolysin" as used herein describes bacterial peptidoglycan hydrolases leading to a self-lysis of the bacteria in particular situations. Autolysins and endolysins are often functionally and structurally related. Different domains from endolysins and autolysins may be often modular combined and form active chimera.
[0028] The term "bacteriophage tail protein" as used herein describes structural phage proteins, which in the beginning of the replication cycle of the bacteriophage in the infection fulfil the function to bind receptors on the bacteria surface and thereby lyse components on the cell surface via their enzymatic function. The respective bacteriophage proteins do not always have to be located in the phage tail, but may also sit directly at the phage head in phages without tail.
[0029] The term "bacteriocin" as used herein describes proteinogenic toxins, which are secreted from bacteria to inhibit the size growth of similar bacterial genus. They consist of a cell wall binding domain, a translocation domain and of the "killing factor". The group of the bacteriocins contained in this connection thereby attacks bacterial membranes. Examples are colicin, microcin, nisin, epidermin and lantibiotics (lanthionine-containing antibiotics).
[0030] The term bacterial "cell wall" as used herein describes all components forming the outer cell envelope of the bacteria and thus guarantees their integrity. Particularly it refers to the peptidoglycan, the outer membrane of the Gram-negative bacteria with the lipopolysaccaride, the bacteria cell membrane, but also layers additionally supported on the peptidoglycan like capsules, mucous or outer protein layers.
[0031] The term "protease" as used herein describes an enzyme, which is capable to hydrolytically cleave proteins and/or peptides.
[0032] The term "domain" as used herein describes a part of an amino acid sequence which is defined functionally and/or structurally. Domains often may be predicted very well due to homologies via respective search programs, which are based on respective databases with conserved domains (e.g. Conserved Domain Database (CDD) at the NCBI (Marchler-Bauer et al., 2005, Nucleic Acids Res. 33, D192-6), Pfam (Finn et al., 2006, Nucleic Acids Research 34, D247-D251) or SMART (Schultz et al., 1998, Proc. Natl. Acad. Sci. USA 95, 5857-5864, Letunic et al., 2006, Nucleic Acids Res 34, D257-260).
[0033] The term "wild type" as used herein describes the amino acid sequence, as she occurs in naturally occurring proteins and whose sequence was not modified in regard to protease cleavage sites. These might be amino acid sequences as naturally occurring in a bacteriophage, prophage or bacterium. Additionally the term means all amino acid sequences of cell wall lysing enzymes existing in databases, which contain specific protease cleavage sites yet, even though the amino acid sequences were already modified compared with naturally existing proteins. In connection with nucleotide sequences the term wild type means also the nucleotide sequences encoding a polypeptide, which have an above-described amino acid sequence.
[0034] The present invention relates to a modified polypeptide with the biological activity to lyse cell walls of bacteria, wherein the polypeptide contains no caspase, enterokinase, factor Xa, granzyme B, staphylococcus peptidase I (V8 protease), plasmin, streptopain, bacillolysin or thrombin cleavage site as well as further protease cleavage sites, which were specific for each polypeptide determined by protease digest with subsequent analysis of the formed fragments and afterwards were modified in that way described in the invention. Preferably the present invention relates to modified polypeptides according to the present invention, which lyse Gram-positive bacteria, whereas the bacteria may derive from the group consisting of clostridia, listeria, staphylococci, lactobacilli, enterococci, aerococci, pediococci, streptococci, mycoplasms and/or leuconostoc. Preferably the polypeptide of the invention is an endolysin, a bacteriophage tail protein, an autolysin or a bacteriocin.
[0035] The present invention further relates to a recombinantly produced polypeptide with a biological activity to lyse cell walls of bacteria, wherein the amino acid sequence of the polypeptide compared to the wild type sequence has modifications at amino acid positions, which are recognized by proteases and/or after which the proteases cleave the polypeptides.
[0036] Surprisingly it turned out that the cell wall lysing enzymes may be modified in such a way in their amino acid sequence, that an increased protease stability may be achieved, without stabilizing substances have to be added. It is generally known that different proteases cleave the polypeptide chain at a specific amino acid residue or require amino acid sequence motifs for their enzymatic activity. Protease cleavage sites in proteins may be predicted with respective sequence analysis programs in case of existing amino acid sequence (e.g. ExPASy, PeptideCutter, Gasteiger et al., Protein Identification and Analysis Tools on the ExPASy server in John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005)). Thus, it results normally in far more than 100 potential protease cleavage sites for a protein of medium size with approximately 300 to 400 amino acid residues, so that a stabilization of proteins is only to be achieved by modifying the cleavage sites of a plurality of proteases. But this is impracticable because the protein looses its effect and activity if to much amino acid modifications are introduced. A further possibility for determination of protease sensitive regions beside the prediction by means of the amino acid sequence, especially for proteases, in which no recognition sequence is known or their identity is not known, is the experimental determination of the actually cleavage sites by a digest of the proteins with commercially available proteases. Further isolated proteases from organisms or samples may be used therefore. Particularly the samples, which contain the proteases such as food samples, environmental samples, organ extracts, medical samples may be used without isolating the proteases before. Subsequent the assays are subjected to an analysis of the formed degradation products by means of sequence determination (e.g. N- or C-terminal sequencing, peptide mapping in connection with mass spectrometry) and size determination (e.g. mass spectrometry, band analysis of SDS gels, analytical gel filtration). Actually the inventors have found cleavage sites and amino acid sequence motifs for proteases in many cell wall lysing enzymes. This occurs surprisingly, whether it is assumed that the cell wall lysing enzymes must be evolved to a high stability. Surprisingly it became apparent, however, that the cleavage sites and amino acid sequence motifs for the proteases are in small number, often even in singular form in the cell wall lysing enzymes and often also are conserved in homologous proteins. As a result a modification of just a few or even only one protease cleavage site is sufficient to increase the stability of the cell wall lysing enzymes.
[0037] In the present invention preferably only such protease cleavage sites are modified, which are recognized by specific proteases potentially existing at the target site of the cell wall lysing enzymes. Thereby the amino acid sequences of the cell wall lysing enzymes are modified, that they are no longer recognized by the specific proteases and thus are no longer cleaved. This leads to an increased stability of the cell wall lysing enzymes towards proteases existing at the target site. Thus, a protease caused loss of activity of the cell wall lysing enzymes is prevented and a longer effectiveness is achieved.
[0038] There are a series of proteases, which require a specific recognition sequence in their substrate proteins for their enzymatic activity. Several examples therefore are presented in the following table. A plurality of further proteases is known by the person skilled in the art like they are described for example in the Merops peptidase database (Rawlings et al., 2008, Nucleic Acids Res 36, D320-D325). The following, however, also applies to protease cleavage sites of unknown proteases, which may be experimentally determined. P1 describes the position of the amino acid, after which it is cleaved, P4, P3 and P2 are the positions N-terminal before the cleavage site P1. The description P1' und P2' are the positions C-terminal following P1. This means that the proteases cleave the polypeptide chain between P1 and P1'. The capitals presented instead of the amino acid residues represent the international used description of the amino acid residues and are generally known. If the capitals are used in the description of the present invention, the respective amino acid residues are meant.
TABLE-US-00001 TABLE 1 Cleavage site Protease P4 P3 P2 P1 P1{grave over ( )} P2{grave over ( )} Caspase 1 F, W, Y -- H, A oder T D not P, E, -- or L D, Q, K or R Caspase 2 D V A D not P, E, -- D, Q, K or R Caspase 3 D M Q D not P, E, -- D, Q, K or R Caspase 4 L E V D not P, E, -- D, Q, K or R Caspase 5 L or W E H D -- -- Caspase 6 V E H or I D not P, E, -- D, Q, K or R Caspase 7 D E V D not P, E, -- D, Q, K or R Caspase 8 I or L E T D not P, E, -- D, Q, K or R Caspase 9 L E H D -- -- Caspase 10 I E A D -- -- Clostripain -- -- -- R -- -- (Clostridiopeptidase B) Enterokinase D or N D or N D or N K -- -- Factor Xa A, F, G, I, D or E G R -- -- L, T, V or M Plasmin (Fibrinolysin, -- -- -- K or -- -- Fibrinase) R Streptopain (Streptococcus -- -- F F Y -- Peptidase A) Bacillolysin (Bacillus subtilis -- G or F G or F L or A or G or -- neutral Protease) or A F L Granzyme B I E P D -- -- Staphylococcus peptidase I -- -- not E E -- -- (V8 Protease) Thrombin -- -- G R G -- -- A, F, G, I, A, F, G, I, P R not D, E not D, E L, T, V or L, T, V, W M or A
[0039] Preferably the present invention relates to a modified polypeptide with a biological activity to lyse cell walls of bacteria, wherein the polypeptide according to the present invention has compared to the naturally occurring polypeptide one or more modifications in its amino acid sequence at one or more positions shown in table 1 or in experimentally determined protease cleavage sites, wherein the one modification or the several modifications occur at amino acids of protease cleavage sites shown in table 1 or experimentally determined. Thus the stability of the polypeptide increases towards the protease or proteases, whose recognition sequence was changed respectively.
[0040] In a preferred embodiment of the present invention the amino acid residue is substituted by another appropriate amino acid residue at the position, after which it is cleaved (P1), so that the polypeptide of the invention is no longer cleaved by the protease whose recognition site was modified respectively. Instead of a substitution of the amino acid residue at position P1 or also additional to a substitution at position P1 further amino acid residues in the recognition site may be substituted, preferably the positions at P4, P3, P2, P1' and/or P2' presented in table 1, by other amino acid residues. Preferably 6, 5, 4, 3, 2, 1 substitutions per recognition site are performed, more preferably in particular 1 to 3 substitutions.
[0041] In a further embodiment of the present invention one or more protease cleavage sites in a polypeptide, as previously described, may be modified.
[0042] It is important, that the protein variant according to the present invention after the mutation do not only have a higher stability to protease degradation, but also further retain its cell wall lysing activity. Thus, the amino acid substitution is performed as described below.
[0043] In the state of the art a series of essential amino acid residues are known, which are known for the activity of the cell wall lysing enzymes, e.g. conserved cysteines and histidines in different amidases or CHAP domains. The respective essential residues may also be found with sequence analysis programs, which specifically search for conserved domains (e.g. Conserved Domain Database (CDD) via NCBI (Marchler-Bauer et al., 2005, Nucleic Acids Res. 33, D192-6), Pfam (Finn et al., 2006, Nucleic Acids Research 34, D247-D251) or SMART (Schultz et al., 1998, Proc. Natl. Acad. Sci. USA 95, 5857-5864, Letunic et al., 2006, Nucleic Acids Res 34, D257-D260).
[0044] To little destabilize the stability, structure and function of the polypeptides according to the present invention as possible, the amino acid residues of the protease recognition sequence to be substituted are substituted by amino acid residues, which are closely related by their structure and biochemistry as possible and however are not recognized by the protease. In the meantime it is general specialist knowledge to assign the 20 naturally occurring amino acids in specific groups, which differ in their biochemistry (e.g. acid, basic, hydrophobic, polar, aromatic amino acids), but within which the amino acids are related to each other. Preferred substitutions for the amino acid residues in the polypeptides of the invention are summarized in table 2. The substitution possibilities mentioned in column 2 are conservative amino acid alternatives, which are especially similar to the amino acid residue in the wild type either by their structure and/or their function. These are more preferred substitution variants. However, further substitution variants are also possible for the cell wall lysing polypeptides of the present invention, which are mentioned in column 3.
TABLE-US-00002 TABLE 2 Amino acid in Conservative Further substitution the wild type substitution possibilities A (Ala) S T, G, V, E, D N (Asn) Q, D K, H, I, T, S, A D (Asp) E, N A, G, S R (Arg) K S, Q, A C (Cys) S M, G, A E (Glu) D, Q A, G, S, A Q (Gln) N, E H, L, R, A G (Gly) A S, N, D H (His) Q, N R, A I (Ile) V, L T, A L (Leu) V, I M, F, A K (Lys) R N, T, S, Q, A M (Met) L K, V, I, C, A F (Phe) Y L, I, M, A P (Pro) A S, Q S (Ser) A, T G, N, K T (Thr) S, A V, K, N W (Trp) Y F, S, R, A Y (Tyr) F H, N, C, A V (Val) I, A L, T
[0045] A further possibility to determine, which amino acid residue is integrated instead of the residue to be substituted, represents the Dayhoff-Matrix; see for example in Creighton (Proteins: Structure and Molecular Properties, 2nd Ed., 1984, Freeman, New York). From studies of homologous proteins it is known, that the mere statistically random mutation on nucleic acid level is not reflected in the amino acid sequence of the respective proteins. Thus, it seems to exist a selection pressure on specific amino acid residues for specific amino acid positions in the homologous proteins, what is given in the Dayhoff-Matrix.
[0046] A further possibility to determine which amino acid residue is integrated instead of the residue to be substituted, employs the assistance of sequence analysis programs, e.g. the program BLAST, see Altschul et al., 1990, J. Mol. Biol., 215, 403-410. So, it may be searched for proteins related to the cell wall lysing enzymes to be mutated. Thereby the search algorithm finds similarities on sequence level. If it is searched on amino acid sequence level, the program "Protein BLAST" is suitable to show functional and/or evolutionary relationships. Whether amino acid variants with at least about 60% or at least about 80% or at least about 90% identity are found in a region of about 100 or 50 or 20 amino acid residues as limit around the potential protease cleavage site, the amino acid sequence, which differs from the protease recognition sequence may be integrated in the polypeptide of the present invention. Thereby the amino acid substitutions may differ from the substitution possibilities represented in table 2.
[0047] There are a series of known endolysins having protease cleavage sites, as represented in table 1, in particular cleavage sites for the proteases thrombin, caspase, clostripain or V8 protease. Particularly thrombin- or caspase cleavage sites are often singular and may be substituted according to the present invention resulting in a stabilized endolysin. In a preferred embodiment the polypeptide of the present invention is modified in one or more thrombin recognition sequences. Thereby the amino acid residue at position P1 described by R is substituted with the amino acid residue described by K. A substitution against S, Q or A is also preferred. In the first variant of the thrombin recognition sequence (G; R; G) both G or only one thereof may be substituted with preferably A additionally or alternative to the substitution of the R. In the second variant of the thrombin recognition sequence (P; R; not D, E; not D, E) preferably also the R is substituted with a K or with a S, Q or A. Further one or both positions P1' and P2' may be substituted with a D or E additionally or as alternative.
[0048] In a further preferred embodiment the amino acid residue D at the position P1 of a caspase cleavage site is substituted with N or A. The amino acid residue R at the P1 position of a clostripain cleavage site is preferably substituted with K or A, the amino acid residue E at the P1 position of a V8 protease cleavage site with Q or A. Further preferred substitutions are summarized in table 2, stabilizing substitution in the recognition site beyond the P1 position may also be performed.
[0049] More preferred embodiments are given in SEQ ID NO: 1, 2, 3, 4 and 5.
[0050] The present invention further relates to a recombinantly produced polypeptide with the biological activity to lyse cell walls of bacteria, wherein the amino acid sequence of the polypeptide has amino acid additions compared to the wild type sequence. Preferably the polypeptide of the invention has one, two, three or four additions of amino acid residues, wherein the amino acid residues may be introduced coherently or independently of each other. Preferred is the addition of one or two amino acid residues, particularly preferred is the addition of an amino acid residue at position two. The amino acid additions may be introduced in polypeptides, which have modifications at amino acid positions, which are recognized by proteases and/or after which the proteases cleave the polypeptides or also in polypeptides, in which such modifications were not integrated. Particularly preferred embodiments are given in SEQ ID NO: 12-17, 19 and 22.
[0051] In a further preferred embodiment the polypeptide of the invention is modified in one or more V8 protease recognition sequences. Thereby the amino acid residue described by E at position P1 is substituted with another amino acid residue preferably with the amino acid residue described by A or Q. A particularly preferred embodiment is given in SEQ ID NO: 6.
[0052] In a further preferred embodiment the polypeptide of the invention is modified in one or more clostripain cleavage sites. Thereby the amino acid residue described by R at position P1 is substituted with another amino acid residue preferably with the amino acid residue described by K or A. Particularly preferred embodiments are given in SEQ ID NO: 7 and 8.
[0053] In a further preferred embodiment the polypeptide of the invention is modified in one or more caspase recognition sequences. Thereby the amino acid residue described by D at position P1 is substituted with another amino acid residue preferably with the amino acid residue described by E or A. Particularly preferred embodiments are given in SEQ ID NO: 9 and 10.
[0054] In a further preferred embodiment the polypeptide of the invention is modified in one or more V8 protease recognition sequences and one or more thrombin recognition sequences. Thereby the amino acid residue at position P1 described by E is substituted with another amino acid residue preferably against the amino acid residue described by Q or A and the amino acid residue at position P1 described by R is substituted with another amino acid residue preferably against the amino acid residue described by K or A. A particularly preferred embodiment is given in SEQ ID NO: 11.
[0055] In a further preferred embodiment the polypeptide of the invention is modified in one or more protease recognition sequences, which were identified after a digest with kidney extract. Thereby the amino acid residue at position P1 described by Q is substituted with another amino acid residue preferably against the amino acid residue described by N or A and the amino acid residue at position P1 described by S is substituted with another amino acid residue preferably against the amino acid residue described by T or A. Particularly preferred embodiments are given in SEQ ID NO: 12 and 13.
[0056] The amino acid substitutions introduced within the scope of the present invention may be performed by means of molecular biological standard techniques which are known by the person skilled in the art and are given for example in Sambrook et al., Molecular cloning. A laboratory manual; 2nd ed. Cold Spring Harbor Laboratory Press 1989. Thus, by means of DNA primers the desired mutations may be integrated into the nucleic acid sequence encoding the polypeptide of the invention. The modified nucleotide sequences thereby obtained may be then expressed in an appropriate host, preferably E. coli, and the polypeptides are purified. The nucleotide sequence encoding the polypeptide of the invention further may be amended with the so-called codon usage to the respective host cell to achieve a better expression. The present invention relates to the nucleotide sequence encoding the polypeptide of the invention, respective expression vectors and host cells for the production of the polypeptide according to the invention.
[0057] The polypeptides of the invention may be investigated with different tests for protease sensitivity and/or enzyme activity. Examples for such tests are protease digest and subsequent separation of the fragments by SDS gel electrophoresis, mass spectrometry, cell lysis test on agar plates, liquid lysis test by measuring the light scattering in the photometer, zymogram assay for activity test on gels.
[0058] The polypeptides of the invention may be used in medical, therapeutical, diagnostical, environmental, cosmetic or food sector.
[0059] The present invention further relates to a nucleic acid molecule, comprising a nucleotide sequence encoding a polypeptide of the invention. The present invention further relates to a vector, comprising a nucleic acid molecule of the invention.
[0060] Further the present invention relates to appropriate host cells for the expression of the polypeptides according to the invention. Preferably an appropriate host cell for the expression of the polypeptides according to the invention comprises a nucleic acid molecule according to the invention or a vector according to the invention. Preferably an appropriate host cell for the expression of the polypeptides according to the invention is transformed with a nucleic acid molecule according to the invention.
[0061] In the modification of the recognition sites thereby preferably the recognition site of such proteases is modified, which occur from experience in the desired field of application. Thus, it is known, that in human or in animals for example the caspases, thrombin, granzyme B, enterokinase, factor Xa occur. Tissue specific proteases, e.g. the proteases meprin and rennin, occurring in the kidney are known by the person skilled in the art. Further proteases from pathogenic bacteria may be additionally available, which cause an infection in human or animal or which are present as secondary flora. These include for example the V8 protease from Staphylococcus or clostridiopeptidase B from clostridia. If the polypeptides of the invention should be used in the therapy of such infection, preferably the recognition sites for the proteases are modified, which are present in bacteria being responsible for the infection to be treated.
[0062] Preferably the protease sensitive regions of the cell wall lysing enzymes are determined experimentally by a digest of the proteins with commercially available proteases, isolated proteases from organisms or samples containing proteases, such as food samples, environmental samples, organ extracts, medical samples and a subsequent analysis of the resulting degradation products by means of sequence determination (e.g. N- or C-terminal sequencing, peptide mapping in connection with mass spectrometry) and determination of the size (e.g. mass spectrometry, analysis of bands of SDS gels, analytical gel filtration). One field of application for cell wall lysing enzymes is the topical application in wounds in form of ointments, creams, tinctures or wound dressings like dressings and bandages, e.g. in case of infections by staphylococci or clostridia. Additional stability problems for the cell wall lysing enzymes may occur by the fact that, in the wound care partially medicaments or medical products are used, which also contain proteases. For example it is tried in wounds of the skin to achieve a gentle wound cleaning by fibrinolysis. Examples for proteases which are used in the wound care, are fibrinolysin, plasmin, streptokinase, clostridiopeptidase A and Bacillus subtilis protease. Preferably the polypeptides of the invention have also modifications of the recognition sites for these proteases.
[0063] In the food, environmental and cosmetic industry a plurality of proteases may also be present, which are produced either by bacteria used for example in the food production, like lactobacilli, lactococci or different bacilli strains, or which are excreted by undesirable germs, which should be lysed (e.g. B. cereus, B. subtilis, Cl. perfringens, Cl. botulinum). In bacteria used in the food production, such as Lactobacillus acidophilus, L. casei, L. delbrueckii, L. brevis, L. helveticus or Lactococcus lactis for example each between 60 and 130 proteases are known. Additionally proteases are also present in the food itself. Preferably the polypeptides of the invention have such modifications of the recognition sites of proteases, which occur in the food, environmental and cosmetic industry.
[0064] The present invention further relates to the polypeptide of the invention as medicament or a pharmaceutical composition as well as the use of the polypeptide of the invention as medicament or pharmaceutical composition for prevention or therapy of diseases, which are caused by Gram-positive bacteria, particularly clostridia, bacilli, listeria, staphylococci, lactobacilli, enterococci, aerococci, pediococci, streptococci, mycoplasms and/or leuconostoc.
[0065] The present invention further relates to the polypeptide of the invention as a diagnostic agent or diagnostic composition as well as the use of the polypeptide of the invention as a diagnostic agent or diagnostic composition in the medicine for detection of diseases, in particular which are caused by Gram-positive bacteria, particularly clostridia, bacilli, listeria, staphylococci, lactobacilli, enterococci, aerococci, pediococci, streptococci, mycoplasms and/or leuconostoc.
[0066] The polypeptide of the invention is used in the diagnostic to specifically lyse the bacteria to be detected, so that one or more detection steps may follow, which detect specific cell components of the bacteria, like DNA, RNA, enzymes, cell wall components. The appropriate methods therefore, e.g. PCR, NASBA, hybridization, antibody based detections like ELISA, biochemical detections for certain specific enzymes, colorimetric detections are known in the state of the art.
[0067] Furthermore the present invention relates to the use of the polypeptide of the invention for inhibiting the growth of the detection of Gram-positive bacteria in the environmental, food or cosmetic industry.
[0068] The following examples are provided merely by way of explanation, but in no sense restrict the scope of invention.
Example 1
Protease Digest of PlyPitti26
[0069] It was performed a digest of PlyPitti26 with the proteases thrombin and V8. Each 90 μl protein solution (protein concentration 1.35 mg/ml) were tampered with 10 μl thrombin and in control samples only tempered with plasmin. For the V8 protease digest 99 μl protein solution and 1 protease solution were used. The concentration of the protease stock solutions was each 500 μg/ml. The samples were incubated in a 20 mM Tris/HCl pH 7.5, 10 mM DTE, 0.1 mM ZnSO4 Buffer over night at 25° C. A part of the samples was tempered with SDS gel-application buffer and was analysed on SDS polyacrylamide gels after boiling.
[0070] It was found that PlyPitti26 is degraded completely by thrombin, wherein two relative large protein fragments emerged, of which the one contained the CHAP-domain and the other the amidase domain and the CBD. In a digest with V8 protease several smaller, but also defined protein fragments emerged. In a control digest with plasmin no fragmentation of the protein occurs under the given conditions. The results are shown in FIG. 2A.
Example 2
Activity Test of Untreated PlyPitti26 and PlyPitti26 Treated with Proteases
[0071] In further experiments the samples were tested in the liquid lysis test on the lysis activity towards Staphylococcus cells. The liquid lysis test is an activity test for cell wall lysing enzymes, in which the bacterial cell lysis was measured in real time via light scattering in the photometer. The absorption at a wave length of 600 nm is thereby a measure for the number of existing bacteria cells. If the bacteria lyse the sample solution is getting clear by degrees and the measured absorption decreases in the ideal case down to a value of zero. Staphylococcus bacteria were cultivated in BHI medium to an OD600 of about 0.8. The not heat activated cells were harvested and resuspended to an OD600 of about 1 in TBST buffer (20 mM Tris/HCl pH 7.5, 60 mM NaCl, 0.1% Tween, 2 mM CaCl2). 990 μl cell suspension was tempered each with 10 μl enzyme solution (enzyme concentration in the test 10 μg/ml) and the decrease in absorption at 600 nm at 30° C. was monitored.
[0072] It was found, that a complete lysis of the staphylococci was occurred in non-digested PlyPitti26 and in the plasmin control after about 1000 s, whereas no visual cell lysis has taken place anymore in the samples digested with thrombin or V8 protease, the cell wall lysing enzyme was inactivated completely. The results are shown in FIG. 2B.
Example 3
Stabilization of CHAP-AmiPitti26-CBDUSA
[0073] A synthetically produced endolysin variant active against staphylococci, which consists of the enzymatically active domains CHAP and Ami of the endolysins of PlyPitti26 and of the cell wall binding domain (CBD) of the endolysin of the prophage Φ SA2USA deriving from the genome of the MRSA strain USA300 (Diep et al., The Lancet, 2006, 367, 731-739; database accession number NC--007793) is described as CHAP-AmiPitti26-CBDUSA.
[0074] To stabilize this protein against thrombin digest and digest by V8 protease, both the thrombin cleavage site and V8 cleavage sites lying in the linker between the CHAP- and amidase domains were modified by amino acid substitutions by means of site-directed mutagenesis. The single, double, triple and quadruple mutations performed for that purpose are summarized in table 3. For all mutations given in table 3, endolysin activity against staphylococci was still detected in the plate lysis test, as described below after mutation. The amino acid positions given in the table refer to the respective positions of the wild type sequence and the positions in modified polypeptides respectively, in which no amino acid residues were added. For example the position E163 is in one embodiment of a polypeptide according to the invention with one amino acid addition at position two E164, then the position E167 is E168. In case of more than one amino acid addition the position shifts respectively. If an amino acid residue is introduced only at position 174, the position E163 remains equal, but the position E189 changes to E190.
TABLE-US-00003 TABLE 3 Cleavage site V8: V8 protease Mutant T: thrombin substitution 1 E163 (V8) 163Q R167 (T) 167K 2 E163 (V8) 163Q R167 (T) 167A 3 E163 (V8) 163A R167 (T) 167K 4 E163 (V8) 163A R167 (T) 167A 5 E179 (V8) 179Q 6 E179 (V8) 179A 7 E189 (V8) 189Q 8 E189 (V8) 179A 9 E163 (V8) 163Q R167 (T) 167A E179 (V8) 179Q E189 (V8) 189Q 10 E163 (V8) 163Q R167 (T) 167A E179 (V8) 179A E189 (V8) 189Q 11 E163 (V8) 163A R167 (T) 167A E179 (V8) 179Q E189 (V8) 189Q 12 E163 (V8) 163A R167 (T) 167A E179 (V8) 179A E189 (V8) 189Q 13 R167 (T) 167A 14 E163 (V8) 163A 15 E163 (V8) 163Q 16 R167 (T) 167K 17 E179 (V8) 179A E189 (V8) 189Q 18 E179 (V8) 179Q E189 (V8) 189Q 19 E163 (V8) 163Q R167 (T) 167A E189 (V8) 189Q 20 E163 (V8) 163A R167 (T) 167A E189 (V8) 189Q
[0075] The endolysins were applied in a concentration of 1 mg/ml into the digest. For the thrombin digest 90 μl protein and 10 μl human thrombin (stock solution with 50 μg/ml) were applied. The samples were incubated over night at 25° C. in buffer (20 mM Tris/HCl pH 7.5, 10 mM DTE, 0.1 mM ZnSO4). A part of the samples were tempered with SDS gel application buffer and were analysed after boiling on SDS polyacrylamide gels. The mutation of R at position 167 against A leads to a complete insensitization against thrombin. The same was true for the mutation of R at position 168 against A, after addition of A at amino acid position two (CHAP-Ami_Pitti26-CBD-USA-Add2).
[0076] Non-modified CHAP-Ami_Pitti26-CBD-USA, CHAP-Ami_Pitti26-CBD-USA-Add2 and the respective mutant 4 (E163A, R167A and E164A, R1678 respectively) were also digested with V8 protease. The endolysins were applied in a concentration of 1 mg/ml in the digest. For the V8 protease digest 99 μl protein and 1 μl V8 protease (stock solution with 500 μg/ml) were applied. The samples were incubated for 15 min and 60 min respectively in 20 mM Tris/HCl pH 7.5, 10 mM DTE, 0.1 mM ZnSO4 buffer. A part of the samples were tempered with SDS gel application buffer and were analysed after boiling on SDS polyacrylamide gels. Although, the mutation of E at position 163 and 164 respectively against A does not lead to a complete insensitization against V8 protease, but the mutant of the invention is considerably stabilized, compared to the wild type, in which a complete digest occurs. The modified thrombin cleavage site at position 167/168 plays no role in the performed V8 protease digest. In the modified polypeptides two fragments of about 40 kDa are still available both after 15 min and also after 60 min, which are after 15 min only weak and after 60 min no longer available in the non modified polypeptides.
Example 4
Lysis Test with Protease Sensitive and Protease Resistant Endolysins
[0077] To investigate, if the polypeptides of the invention already have the desired activity after the amino acid substitution, they were tested in a plate lysis test with different Staphylococcus strains. The bacteria strains were cultivated over night in 5 ml cultures in BHI medium and the bacteria cells were centrifuged for 5 min at 4000 UpM in a table centrifuge. The cell pellet was resuspended afterwards in 200 μl PBS buffer (10 mM sodium phosphate, 144 mM NaCl, 50 mM KCl, pH 7.4) and the cells were heat inactivated for 30 min at 80° C. The heat inactivated cells were poured with 15 ml top agar in petri dishes and dried. Then each 5 μg protein was spotted and the agar plates were incubated for 1 h at 30° C. Occurring cell lysis area at the positions of the proteins spotted proteins were evaluated visually and valued depending on size with +++, ++, and + or with -, if no lysis occurred.
[0078] The test result for several polypeptides of the invention is summarized in table 4.
TABLE-US-00004 Mutant 10 from table Mutant 4 CHAP- 3 (E163Q, from AmiPitti26- PlyPitti26 R167A, table 3 CBDUSA Staphylococcus (non- E179A, (E163A, (non- species modified) E189Q) R167A) modified) Staphylococcus +++ +++ +++ +++ aureus MRSA Staphylococcus +++ +++ +++ +++ aureus MRSA Staphylococcus ++ +++ +++ +++ aureus MRSA Staphylococcus +++ +++ +++ +++ epidermis Staphylococcus + ++ ++ ++ equorum Staphylococcus sciuri - ++ + ++ Staphylococcus - +++ ++ +++ saprophyticus Staphylococcus sciuri - - - - Staphylococcus +++ +++ +++ +++ aureus MRSA Staphylococcus +++ +++ +++ +++ epidermidis Staphylococcus ++ ++ ++ ++ epidermidis koagulase-negative Staphylococcus ++ ++ ++ ++ haemolyticus koagulase-negative Staphylococcus +++ +++ +++ +++ epidermidis koagulase-negative
[0079] Both the non-modified PlyPitti26 and also CHAP-AmiPitti26-CBDUSA had a broad lysis spectrum towards different Staphylococcus strains, wherein the CHAP-AmiPitti26-CBDUSA showed a slightly better lysis activity. The modified polypeptides of the invention mutant 4 and mutant 10 both shown in the table with the amino acid substitutions at the thrombin and different V8 protease cleavage sites respectively had a practically identical activity as the non-modified protein CHAP-AmiPitti26-CBDUSA. This showed that the mutations of the invention did not negatively influence the activity of the cell wall lysing enzymes. The same was true for non-modified CHAP-Ami_Pitti26-CBD-USA-Add2 as well as the respective modified CHAP-Ami_Pitti26-CBD-USA-Add2 polypeptides.
Example 5
Modified Cell Wall Binding Enzymes Against Clostridium difficile
[0080] Two endolysins from different Cl. difficile phages (accession numbers YP--529586 for the endolysin from the phage Φ CD119 and YP--001087453 for the endolysin from the strain Cl. difficile 630) had a singular caspase 1 cleavage site after amino acid D214, whereas a singular thrombin cleavage site only in endolysin from the phage Φ CD119 at position 87 occurred. Because of the high sequence similarity of both endolysins, besides the modifications of the caspase 1 cleavage sites from D214 to E214, R at position 87 in Φ CD119 endolysin was substituted with K, which was present in the homologous endolysin from the strain Cl. difficile 630 at this position and is not recognized by thrombin as cleavage site. Further modifications of the invention are presented in table 2.
Example 6
Determination of Protease Sensitive Regions within the Ply511 Sequence
[0081] Ply511 has 6 potential cleavage sites for Staphylococcus peptidase I (6 glutamates) because of its amino acid sequence. To find out, which regions of the endolysins Ply511 are especially protease sensitive, protease digest experiments were performed with staphylococcus peptidase I. For the staphylococcus peptidase I degradation 99 μl protein solution (concentration about 1 mg/ml) and 1 μl protease solution (concentration about 0.5 mg/ml) were applied. The samples were incubated for different time intervals (minutes up to several hours) at 37° C. in a buffer with 20 mM Tris/HCl, 100 mM NaCl, pH 8.0. The protein bands were separated on SDS gels. The occurring degradation fragments were blotted on PVDF membranes (polyvinylidenfluoride), well separable bands were cut and N-terminal sequenced. There were three preferred cleavage sites for staphylococcus peptidase I after the amino acids E7, E40 and E89.
Example 7
Removal of Staphylococcus Peptidase I Cleavage Sites in Ply511
[0082] At the positions E7, E40 and E89 substitutions were performed either as single substitution or in combination, so that in the occurring mutants other amino acids except glutamate substitute the respective positions, which are no longer cleaved by staphylococcus peptidase I. The mutants thus obtained were applied in the protease digest described in example 6 and were afterwards analysed on SDS gels, whether the respective degradation bands still occurred or not. The mutations E7A and E7Q as well as E40A and E40Q turned out as being particularly suitable.
Example 8
Resistance to Thrombin and V8 Protease of Different Endolysin Variants of PlyPitti26 and CHAP-AmiPitti26-CBDUSA
[0083] Different mutation combinations of the invention from table 3 were introduced into the Plypitti26 variant CHAP-AmiPitti26-CBDUSA and CHAP-AmiPitti26-CBDUSA-Add2 and partially combined with further mutations. Afterwards these endolysin variants were tested for their resistance to thrombin and V8 protease as well as the enzyme activity (detection as described in example 3). The results are summarized in table 5 for CHAP-AmiPitti26-CBDUSA and the modifications thereof. The results for CHAP-AmiPitti26-CBDUSA-Add2 and the modifications thereof (amino acid positions are each shifted by one position in the direction to the C-terminus towards the positions in the table) are identical and not described again in table 5.
TABLE-US-00005 TABLE 5 Resistance to thrombin und V8 protease of different mutants of CHAP-Amipitti26_CBDUSA (described as EADpitti26_CBDUSA in the table) in comparison to Plypitti26-wild type Endolysin construct EADpitti26-- EADpitti26-- EADpitti26-- EADpitti26-- EADpitti26-- EADpitti26-- EADpitti26-- plypitti26 CBDUSA CBDUSA CBDUSA CBDUSA CBDUSA CBDUSA CBDUSA Mutations E163A, L55H, R167A, L56T, L55H, L56T, E163A, E179A, E163A, E163A, R167A, Prop- R167A, E189Q, R167A, E179A, E189Q, L55H, E163A, erty Assay Y200H Y200H Y200H Y200H L56T, R167A Throm- SDS- - +++ ++ +++ +++ - +++ + bin PAGE resis- tance Throm- liquid - - - ++ ++ - - + bin lysis resis- tance V8 SDS- - - + - + - - - pro- PAGE tease resis- tance
[0084] It was shown that the endolysin variants of the invention CHAP-Amipitti26_CBDUSA (E163A, R167A, Y200H), CHAP-Amipitti26_CBDUSA (E163A, R167A, E179A, E189Q, Y200H), CHAP-Amipitti26_CBDUSA (L55H, L56T, E163A, R167A, Y200H), CHAP-Amipitti26_CBDUSA (L55H, L56T, E163A, R167A, E179A, E189Q, Y200H) and CHAP-Amipitti26_CBDUSA (E163A, R167A) as well as CHAP-Amipitti26_CBDUSA-Add2 (E164A, R168A, Y201H), CHAP-Amipitti26_CBDUSA-Add2 (E164A, R168A, E180A, E190Q, Y201H), CHAP-Amipitti26_CBDUSA-Add2 (L56H, L57T, E164A, R168A, Y201H), CHAP-Amipitti26_CBDUSA-Add2 (L56H, L57T, E164A, R168A, E180A, E190Q, Y201H) and CHAP-Amipitti26_CBDUSA-Add2 (E164A, R168A) had a good to very good resistance to thrombin digest. The endolysin variants CHAP-Amipitti26_CBDUSA (L55H, L56T, E163A, R167A, Y200H) and CHAP-Amipitti26_CBDUSA (L55H, L56T, E163A, R167A, E179A, E189Q, Y200H) as well as CHAP-Amipitti26_CBDUSA-Add2 (L56H, L57T, E164A, R168A, Y201H) and CHAP-Amipitti26_CBDUSA-Add2 (L56H, L57T, E164A, R168A, E180A, E190Q, Y201H) also showed a high enzyme activity in the liquid lysis test after thrombin digest. The endolysin variants CHAP-Amipitti26_CBDUSA (E163A, R167A, E179A, E189Q, Y200H) and CHAP-Amipitti26_CBDUSA (L55H, L56T, E163A, R167A, E179A, E189Q, Y200H), as well as CHAP-Amipitti26_CBDUSA-Add2 (E164A, R168A, E180A, E190Q, Y201H) and CHAP-Amipitti26_CBDUSA-Add2 (L56H, L57T, E164A, R168A, E180A, E190Q, Y201H) had an improved resistance to V8 protease digest. Particularly the substitutions E179A and E189Q and E180A and E190Q, respectively proved beneficial here.
Example 9
Endolysins with Protease Cleavage Sites According to the Invention
[0085] Whether a substitution of an amino acid residue proves beneficial, may be detected with the detection for protease stability and endolysin activity as described above. A series of examples for endolysins, which lyse members from different genera of bacteria and which could be stabilized according to the invention against protease degradation, are shown in table 6. The protease cleavage sites were determined with the program PeptideCutter (Gasteiger et al., Protein Identification and Analysis Tools on the ExPASy Server in John M. Walker (ed): John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005)).
TABLE-US-00006 TABLE 6 Endolysins with protease cleavage sites of table 1 Source for Protease cleavage sites for the amino Thrombin; Caspase1; acid number of number of sequence amino acid amino acid P1 Endolysin (lysis (NCBI P1 and and V8 of bacteria of accession recognition recognition Clostripain; Protease; the genera) number) sequence sequence number number PlyPitti26 New 167 (TAPR) 20 24 (Staphylococcus) CHAP- New 167 (TAPR) 21 23 AmiPitti26- CBDUSA (Staphylococcus) Amidase of (YP_001491539) 42 (YITDG) 7 9 Staphylococcus phage SAP-2 E1 Endolysin (NP_814147) 106 (FLPR) 358 (YGTDY) 6 15 (Enterococcus) E2 Endolysin (NP_815016) 330 (YIADG) 13 13 (Enterococcus) and Caspase 10 31 (IEAD) E5 Endolysin (NP_815749) 147 (WLADY) 8 13 (Enterococcus) Fab25 Neu 112 (LLHDL) 14 9 (Enterococus) B30-PlyGBS (AAR99416) 388 (LSTDY) 12 18 (Streptococcus) Pal amidase (CAB07986) 14 13 (Streptococcus) Cp1 Lysin (2IU_A) 133 (FTHDN) 7 12 (Streptococcus) LysK (YP_024461) 60 (LITDY) 16 18 (Staphylococcus) and 67 (WLTDN) Ply187 (CAA69022) 596 (YATDI) 23 20 (Staphylococcus) Lysostaphin (AAB53783) 9 51 (Staphylococcus) Ply511 (Listeria) (Q38653) 6 6 Ply118 (Listeria) EP0781349 262(GTPR) 129 (YGTDT) 8 6 B1; SEQ ID NO: 4 PlyA500 (AAY52812) 12 12 (Listeria) Ply21 (Bacillus) (CAA72267) 82 (GRG) 15 13 PlyBA (Bacillus) (CAA72266) 11 25 Ply12 (Bacillus) (CAA72264) 14 10 PlyCD119 New 87 (GRG) 214 (LVTDI) 7 13 (Clostridium) Ply3626 U.S. Pat. No. 7,371,375 17 27 (Clostridium) B2; SEQ ID NO: 2
Example 10
Determination of Clostripain Cleavage Sites in Listeria Endolysins
[0086] As can be seen from table 6, potential cleavage sites for clostripain in endolysins are often present in higher number (6 to 23 in the described examples). Since a substitution of all potential cleavage sites may influence negatively the activity of the endolysin, it is useful to determine the cleavage sites available for the proteases and to modify only such. The listeria endolysin Ply511 contains six potential cleavage sites for clostripain. It was performed a clostripain digest of Ply511, to determine the sensitive regions for clostripain of the endolysin. Ply511 (0.1 mg/ml) was digested for 3 h and over night respectively at room temperature with 5 units clostripain (unit definition according to manufacture's data, Sigma) in 60 μl sample volume with the following composition: 25 mM sodium phosphate, 1 mM calcium acetate, 2.5 mM DTT, pH 7.6. The occurring protein fragments were separated by SDS gel electrophoresis (gradient gel 10-20% acryl amide). Three bands occurred (molecular weights about 25 kDa, about 14 kDa, about 10 kDa), which were blotted on PVDF membranes, afterwards they were cut and sequenced N-terminal via Edman-degradation.
[0087] There were the following N-terminal sequences for the fragments:
[0088] 1. (M) V K Y T V E N K; the N-terminal methionine was partly cleaved
[0089] 2. DKLAK
[0090] 3. TSNATTF
[0091] This result shows, that from the six potential clostripain cleavage sites (R46, R62, R92, R221, R312, R326) two are recognized by the protease, namely R92 and R221. Stabilized variants of Ply511 according to the invention have at these positions substitutions from R to other amino acid residues, in particular R62K or R62A as well as, R221K or R221A.
Example 11
Protease Degradation of a Therapeutically Applicable Protein in Different Organs and Determination of the Protease Sensitive Regions
[0092] In pharmacokinetic studies it was found out, that the endolysin CHAP-Amipitti26_CBDUSA-Add2 (L56H, L57T, E164A, R168A, Y201H) used against staphylococci was no longer detectable at different speeds in different organs. To test, whether this is attributed to protease degradation, the endolysin was incubated with organ extracts and potential degradation bands were analysed. Tissue samples from liver, heart, spleen, kidney and lungs of rats were deep-frozen, were again thawed and homogenized in the same volume PBS buffer. Per sample 40 μl CHAP-Amipitti26_CBDUSA-Add2 (L56H, L57T, E164A, R168A, Y201H) (1 mg/ml in 20 mM Tris, pH 8.0) and 10 μl organ extract (1:20 diluted with PBS) were mixed and incubated for 18 h at room temperature and at 4° C. respectively. In the protein controls instead of organ extract only PBS buffer was added and incubated for the same time as well as one protein control was prepared without incubation time. As further controls each organ extract without addition of endolysin was analysed. The samples were mixed with sample buffer and applied on 12% SDS gels.
[0093] It was shown, that only in the samples, to which kidney extract was added, a significant protein degradation took place during the experimental period, in all other organ extracts as well as the controls the endolysin remains stable. By incubation with the kidney extract larger protein fragments with defined size occurred, so that it was likely, that the endolysin was degraded by one or a few kidney specific proteases. To determine the sensitive region for this/these protease(s) the occurring bands (marked with arrows and the numbers 1 to 7) were blotted on a PVDF membrane and the polypeptides were N-terminally sequenced by Edman-degradation. The approximate size of the occurring fragments was derived by means of a standard curve, which was created from the running distances of the bands of the molecular weight standard, from which the molecular weight was known. The following fragments occurred, which are summarized in table 7. The occurring protein sections were determined starting from the determined N-terminal sequences in combination with the fragment sizes.
TABLE-US-00007 TABLE 7 Fragments of endolysin CHAP-Amipitti26_CBDUSA-Add2 (L56H, L57T, E164A, R168A, Y201H) after degradation by kidney specific protease(s) size (calculated size from the (calculated running from the distance in amino acid Band the gel) N-terminal sequence) no. kDa sequence protein sector kDa 1 58 ASIIMEV complete protein, N-terminal M 55 cleaved 2 43 ASIIM N-terminal fragment, CHAP- and 44 amidase domain, complete EAD to the cleavage site before band 5 (ASSNTV) 3 25 ASKKETAPQ Amidase domain, to the cleavage 24 site before band 5 (ASSNTV). 4 19 ASIIMEVATMQ N-terminal fragment, CHAP- 19 domain to the cleavage site before band 3 (ASKKETAPQ). 5 15 ASSNTV C-terminal fragment, CBD. 12 6 20 ASIIMEVAT N-terminal fragment, CHAP- 20 domain to the cleavage site before band 7 (XPTQA). 7 41 XPTQA C-terminal fragment, amidase 36 domain and CBD. X: amino acid, which could not have been defined.
[0094] From the determination of the N-terminal sequences in combination with the size determination of the occurring fragments it resulted that the proteases present in the kidney extract cleave the endolysin CHAP-Amipitti26_CBDUSA-Add2 at 3 positions, namely after the amino acids Q171, Q175 and S384. A search for conservative domains within the endolysins CHAP-Amipitti26_CBDUSA-Add2 in the CDD (conserved domain database) resulted in 3 conserved domains, namely N-terminal a CHAP-domain (amino acids 28 to 158), followed by an amidase--2 domain (amino acids 199 to 346) as well as a C-terminally occurring SH3--5 domain (amino acids 413 to 478). The CHAP- and amidase 2 domain are the EADs, as the SH3--5 domain forms the CBD. Between the CHAP and amidase 2 domain occurs a domain linker (amino acids 159 to 198), as well as a second linker between the amidase 2 domain and the CBD (amino acids 347 to 412). All three found cleavage sites occurred in the middle of the domain linker regions, which proved less stable than the conserved domains. The proteases from the kidney, which were responsible for the degradation, could not be identified, but it struck, that at least one protease requires a Q at position P1 as well as that all three protease recognition sites were rich in serines and alanines.
Sequence CWU
1
1
241496PRTArtificialSynthetic peptide 1Met Ser Ile Ile Met Glu Val Ala Thr
Met Gln Ala Lys Leu Thr Lys 1 5 10
15 Lys Glu Phe Ile Glu Trp Leu Lys Thr Ser Glu Gly Lys Gln
Phe Asn 20 25 30
Val Asp Leu Trp Tyr Gly Phe Gln Cys Phe Asp Tyr Ala Asn Ala Gly
35 40 45 Trp Lys Val Leu
Phe Gly Leu Leu Leu Lys Gly Leu Gly Ala Lys Asp 50
55 60 Ile Pro Phe Ala Asn Asn Phe Asp
Gly Leu Ala Thr Val Tyr Gln Asn 65 70
75 80 Thr Pro Asp Phe Leu Ala Gln Pro Gly Asp Met Val
Val Phe Gly Ser 85 90
95 Asn Tyr Gly Ala Gly Tyr Gly His Val Ala Trp Val Ile Glu Ala Thr
100 105 110 Leu Asp Tyr
Ile Ile Val Tyr Glu Gln Asn Trp Leu Gly Gly Gly Trp 115
120 125 Thr Asp Arg Ile Glu Gln Pro Gly
Trp Gly Trp Glu Lys Val Thr Arg 130 135
140 Arg Gln His Ala Tyr Asp Phe Pro Met Trp Phe Ile Arg
Pro Asn Phe 145 150 155
160 Lys Ser Glu Thr Ala Pro Ala Ser Ile Gln Ser Pro Thr Gln Ala Ser
165 170 175 Lys Lys Glu Thr
Ala Lys Pro Gln Pro Lys Ala Val Glu Leu Lys Ile 180
185 190 Ile Lys Asp Val Val Lys Gly Tyr Asp
Leu Pro Lys Arg Gly Gly Asn 195 200
205 Pro Lys Gly Ile Val Ile His Asn Asp Ala Gly Ser Lys Gly
Ala Thr 210 215 220
Ala Glu Ala Tyr Arg Asn Gly Leu Val Asn Ala Pro Ser Ser Arg Leu 225
230 235 240 Glu Ala Gly Ile Ala
His Ser Tyr Val Ser Gly Asn Thr Val Trp Gln 245
250 255 Ala Leu Asp Glu Ser Gln Val Gly Trp His
Thr Ala Asn Gln Leu Gly 260 265
270 Asn Lys Tyr Tyr Tyr Gly Ile Glu Val Cys Gln Ser Met Gly Ala
Asp 275 280 285 Asn
Ala Thr Phe Leu Lys Asn Glu Gln Ala Thr Phe Gln Glu Cys Ala 290
295 300 Arg Leu Leu Lys Lys Trp
Gly Leu Pro Ala Asn Arg Asn Thr Ile Arg 305 310
315 320 Leu His Asn Glu Phe Thr Ser Thr Ser Cys Pro
His Arg Ser Ser Val 325 330
335 Leu His Thr Gly Phe Asp Pro Val Thr Arg Gly Leu Leu Pro Glu Asp
340 345 350 Lys Arg
Leu Gln Leu Lys Asp Tyr Phe Ile Lys Gln Ile Arg Ala Tyr 355
360 365 Met Asp Gly Lys Ile Pro Val
Ala Thr Val Ser Asn Glu Ser Ser Ala 370 375
380 Ser Ser Asn Thr Val Lys Pro Val Ala Glu Leu Met
Pro Pro Val Pro 385 390 395
400 Ala Gly Tyr Thr Leu Asp Lys Asn Asn Val Pro Tyr Lys Lys Glu Gln
405 410 415 Gly Asn Tyr
Thr Val Ala Asn Val Lys Gly Asn Asn Val Arg Asp Gly 420
425 430 Tyr Ser Thr Asn Ser Arg Ile Thr
Gly Val Leu Pro Asn Asn Thr Thr 435 440
445 Ile Thr Tyr Asp Gly Ala Tyr Cys Ile Asn Gly Tyr Arg
Trp Ile Thr 450 455 460
Tyr Ile Ala Asn Ser Gly Gln Arg Arg Tyr Ile Ala Thr Gly Glu Val 465
470 475 480 Asp Ile Ala Gly
Asn Arg Ile Ser Ser Phe Gly Lys Phe Ser Ala Val 485
490 495 2496PRTArtificialSynthetic peptide
2Met Ser Ile Ile Met Glu Val Ala Thr Met Gln Ala Lys Leu Thr Lys 1
5 10 15 Lys Glu Phe Ile
Glu Trp Leu Lys Thr Ser Glu Gly Lys Gln Phe Asn 20
25 30 Val Asp Leu Trp Tyr Gly Phe Gln Cys
Phe Asp Tyr Ala Asn Ala Gly 35 40
45 Trp Lys Val Leu Phe Gly Leu Leu Leu Lys Gly Leu Gly Ala
Lys Asp 50 55 60
Ile Pro Phe Ala Asn Asn Phe Asp Gly Leu Ala Thr Val Tyr Gln Asn 65
70 75 80 Thr Pro Asp Phe Leu
Ala Gln Pro Gly Asp Met Val Val Phe Gly Ser 85
90 95 Asn Tyr Gly Ala Gly Tyr Gly His Val Ala
Trp Val Ile Glu Ala Thr 100 105
110 Leu Asp Tyr Ile Ile Val Tyr Glu Gln Asn Trp Leu Gly Gly Gly
Trp 115 120 125 Thr
Asp Arg Ile Glu Gln Pro Gly Trp Gly Trp Glu Lys Val Thr Arg 130
135 140 Arg Gln His Ala Tyr Asp
Phe Pro Met Trp Phe Ile Arg Pro Asn Phe 145 150
155 160 Lys Ser Ala Thr Ala Pro Ala Ser Ile Gln Ser
Pro Thr Gln Ala Ser 165 170
175 Lys Lys Glu Thr Ala Lys Pro Gln Pro Lys Ala Val Glu Leu Lys Ile
180 185 190 Ile Lys
Asp Val Val Lys Gly Tyr Asp Leu Pro Lys Arg Gly Gly Asn 195
200 205 Pro Lys Gly Ile Val Ile His
Asn Asp Ala Gly Ser Lys Gly Ala Thr 210 215
220 Ala Glu Ala Tyr Arg Asn Gly Leu Val Asn Ala Pro
Ser Ser Arg Leu 225 230 235
240 Glu Ala Gly Ile Ala His Ser Tyr Val Ser Gly Asn Thr Val Trp Gln
245 250 255 Ala Leu Asp
Glu Ser Gln Val Gly Trp His Thr Ala Asn Gln Leu Gly 260
265 270 Asn Lys Tyr Tyr Tyr Gly Ile Glu
Val Cys Gln Ser Met Gly Ala Asp 275 280
285 Asn Ala Thr Phe Leu Lys Asn Glu Gln Ala Thr Phe Gln
Glu Cys Ala 290 295 300
Arg Leu Leu Lys Lys Trp Gly Leu Pro Ala Asn Arg Asn Thr Ile Arg 305
310 315 320 Leu His Asn Glu
Phe Thr Ser Thr Ser Cys Pro His Arg Ser Ser Val 325
330 335 Leu His Thr Gly Phe Asp Pro Val Thr
Arg Gly Leu Leu Pro Glu Asp 340 345
350 Lys Arg Leu Gln Leu Lys Asp Tyr Phe Ile Lys Gln Ile Arg
Ala Tyr 355 360 365
Met Asp Gly Lys Ile Pro Val Ala Thr Val Ser Asn Glu Ser Ser Ala 370
375 380 Ser Ser Asn Thr Val
Lys Pro Val Ala Glu Leu Met Pro Pro Val Pro 385 390
395 400 Ala Gly Tyr Thr Leu Asp Lys Asn Asn Val
Pro Tyr Lys Lys Glu Gln 405 410
415 Gly Asn Tyr Thr Val Ala Asn Val Lys Gly Asn Asn Val Arg Asp
Gly 420 425 430 Tyr
Ser Thr Asn Ser Arg Ile Thr Gly Val Leu Pro Asn Asn Thr Thr 435
440 445 Ile Thr Tyr Asp Gly Ala
Tyr Cys Ile Asn Gly Tyr Arg Trp Ile Thr 450 455
460 Tyr Ile Ala Asn Ser Gly Gln Arg Arg Tyr Ile
Ala Thr Gly Glu Val 465 470 475
480 Asp Ile Ala Gly Asn Arg Ile Ser Ser Phe Gly Lys Phe Ser Ala Val
485 490 495
3496PRTArtificialSynthetic peptide 3Met Ser Ile Ile Met Glu Val Ala Thr
Met Gln Ala Lys Leu Thr Lys 1 5 10
15 Lys Glu Phe Ile Glu Trp Leu Lys Thr Ser Glu Gly Lys Gln
Phe Asn 20 25 30
Val Asp Leu Trp Tyr Gly Phe Gln Cys Phe Asp Tyr Ala Asn Ala Gly
35 40 45 Trp Lys Val Leu
Phe Gly Leu Leu Leu Lys Gly Leu Gly Ala Lys Asp 50
55 60 Ile Pro Phe Ala Asn Asn Phe Asp
Gly Leu Ala Thr Val Tyr Gln Asn 65 70
75 80 Thr Pro Asp Phe Leu Ala Gln Pro Gly Asp Met Val
Val Phe Gly Ser 85 90
95 Asn Tyr Gly Ala Gly Tyr Gly His Val Ala Trp Val Ile Glu Ala Thr
100 105 110 Leu Asp Tyr
Ile Ile Val Tyr Glu Gln Asn Trp Leu Gly Gly Gly Trp 115
120 125 Thr Asp Arg Ile Glu Gln Pro Gly
Trp Gly Trp Glu Lys Val Thr Arg 130 135
140 Arg Gln His Ala Tyr Asp Phe Pro Met Trp Phe Ile Arg
Pro Asn Phe 145 150 155
160 Lys Ser Gln Thr Ala Pro Ala Ser Ile Gln Ser Pro Thr Gln Ala Ser
165 170 175 Lys Lys Ala Thr
Ala Lys Pro Gln Pro Lys Ala Val Gln Leu Lys Ile 180
185 190 Ile Lys Asp Val Val Lys Gly Tyr Asp
Leu Pro Lys Arg Gly Gly Asn 195 200
205 Pro Lys Gly Ile Val Ile His Asn Asp Ala Gly Ser Lys Gly
Ala Thr 210 215 220
Ala Glu Ala Tyr Arg Asn Gly Leu Val Asn Ala Pro Ser Ser Arg Leu 225
230 235 240 Glu Ala Gly Ile Ala
His Ser Tyr Val Ser Gly Asn Thr Val Trp Gln 245
250 255 Ala Leu Asp Glu Ser Gln Val Gly Trp His
Thr Ala Asn Gln Leu Gly 260 265
270 Asn Lys Tyr Tyr Tyr Gly Ile Glu Val Cys Gln Ser Met Gly Ala
Asp 275 280 285 Asn
Ala Thr Phe Leu Lys Asn Glu Gln Ala Thr Phe Gln Glu Cys Ala 290
295 300 Arg Leu Leu Lys Lys Trp
Gly Leu Pro Ala Asn Arg Asn Thr Ile Arg 305 310
315 320 Leu His Asn Glu Phe Thr Ser Thr Ser Cys Pro
His Arg Ser Ser Val 325 330
335 Leu His Thr Gly Phe Asp Pro Val Thr Arg Gly Leu Leu Pro Glu Asp
340 345 350 Lys Arg
Leu Gln Leu Lys Asp Tyr Phe Ile Lys Gln Ile Arg Ala Tyr 355
360 365 Met Asp Gly Lys Ile Pro Val
Ala Thr Val Ser Asn Glu Ser Ser Ala 370 375
380 Ser Ser Asn Thr Val Lys Pro Val Ala Glu Leu Met
Pro Pro Val Pro 385 390 395
400 Ala Gly Tyr Thr Leu Asp Lys Asn Asn Val Pro Tyr Lys Lys Glu Gln
405 410 415 Gly Asn Tyr
Thr Val Ala Asn Val Lys Gly Asn Asn Val Arg Asp Gly 420
425 430 Tyr Ser Thr Asn Ser Arg Ile Thr
Gly Val Leu Pro Asn Asn Thr Thr 435 440
445 Ile Thr Tyr Asp Gly Ala Tyr Cys Ile Asn Gly Tyr Arg
Trp Ile Thr 450 455 460
Tyr Ile Ala Asn Ser Gly Gln Arg Arg Tyr Ile Ala Thr Gly Glu Val 465
470 475 480 Asp Ile Ala Gly
Asn Arg Ile Ser Ser Phe Gly Lys Phe Ser Ala Val 485
490 495 4271PRTArtificialSynthetic peptide
4Met Lys Ile Gly Ile Asn Cys Gly His Thr Lys Thr Gly Ala Gly Ser 1
5 10 15 Gly Ala Ile Gly
Lys Ile Asn Glu Ser Ile Glu Thr Arg Asn Val Gly 20
25 30 Tyr Lys Val Ile Asp Lys Leu Lys Lys
Leu Gly Asn Asn Val Val Asp 35 40
45 Cys Thr Ile Asp Lys Ala Ser Thr Gln Ser Glu Cys Leu Ser
Lys Ile 50 55 60
Thr Ala Gln Ala Asn Arg Gln Asp Leu Asp Trp Phe Ile Ser Ile His 65
70 75 80 Phe Asn Ala Gly Gly
Gly Lys Gly Cys Glu Val Tyr Thr Tyr Lys Gly 85
90 95 Lys Gln Tyr Gln Asp Ala Ile Asp Val Cys
Lys Lys Ile Ser Asp Leu 100 105
110 Gly Phe Thr Asn Arg Gly Val Lys Asp Gly Ser Gly Leu Tyr Val
Val 115 120 125 Lys
Lys Thr Lys Ala Lys Ser Met Leu Ile Glu Val Cys Phe Val Asp 130
135 140 Thr Glu Asp Ala Asn Lys
Tyr Leu Ser Leu Gly Ala Asp Lys Leu Ala 145 150
155 160 Thr Ala Ile Val Glu Ala Ile Thr Lys His Ile
Ser Ser Ala Glu Glu 165 170
175 Asn Asn Tyr Asn Arg Tyr Lys His Thr Ile Val Tyr Ser Gly Asp Asp
180 185 190 Lys Val
Ser Ala Asp Ile Leu Gly Leu Tyr Tyr Lys Arg Lys Lys Glu 195
200 205 Ser Tyr Leu Val Thr Glu Ile
Lys Asp Tyr Lys Pro His Arg Thr Gln 210 215
220 Asn Leu Tyr Val Ile Gly Gly Val Thr Cys Asn Lys
Met Lys Glu Met 225 230 235
240 Ser Lys Thr Thr Gly Glu Lys Phe Thr Gln Leu Tyr Gly Asn Asp Val
245 250 255 Trp Ser Thr
Met Asp Lys Ala Ile Glu Phe Val Lys Glu Lys Leu 260
265 270 5271PRTArtificialSynthetic peptide 5Met
Lys Ile Gly Val Asn Cys Gly His Thr Lys Thr Gly Ala Gly Ser 1
5 10 15 Gly Ala Ile Gly Lys Ile
Asn Glu Ser Ile Glu Thr Arg Asn Val Gly 20
25 30 Tyr Lys Val Ile Asp Lys Leu Lys Thr Leu
Gly Asn Asn Val Val Asp 35 40
45 Cys Thr Ile Asp Lys Ala Ser Thr Gln Ser Glu Cys Leu Ser
Lys Ile 50 55 60
Ala Thr Gln Ala Asn Arg Gln Asp Leu Asp Trp Phe Ile Ser Ile His 65
70 75 80 Phe Asn Ala Gly Lys
Gly Lys Gly Cys Glu Val Tyr Thr Tyr Lys Gly 85
90 95 Lys Gln Tyr Gln Asp Ala Ile Asp Val Cys
Lys Lys Ile Ser Asp Leu 100 105
110 Gly Phe Thr Asn Arg Gly Val Lys Asp Gly Ser Gly Leu Tyr Val
Val 115 120 125 Lys
Lys Thr Lys Ala Lys Ser Met Leu Ile Glu Val Cys Phe Val Asp 130
135 140 Ser Glu Asp Ala Asn Lys
Tyr Leu Ser Leu Gly Ala Asp Lys Leu Ala 145 150
155 160 Thr Ala Ile Val Glu Ala Ile Thr Lys His Ile
Ser Ser Ala Glu Glu 165 170
175 Asn Asn Tyr Asn Arg Tyr Lys His Thr Ile Val Tyr Ser Gly Asp Asp
180 185 190 Lys Val
Ser Ala Asp Ile Leu Gly Leu Tyr Tyr Lys Arg Lys Lys Glu 195
200 205 Ser Tyr Leu Val Thr Glu Ile
Lys Asp Tyr Lys Pro His Arg Thr Gln 210 215
220 Asn Leu Tyr Val Ile Gly Gly Val Thr Cys Asn Lys
Met Lys Glu Met 225 230 235
240 Ser Lys Thr Thr Gly Glu Lys Phe Thr Gln Leu Tyr Gly Asn Asp Val
245 250 255 Trp Ser Thr
Met Asp Lys Ala Ile Glu Phe Val Lys Glu Lys Leu 260
265 270 6341PRTArtificialSynthetic peptide 6Met
Val Lys Tyr Thr Val Ala Asn Lys Ile Ile Ala Gly Leu Pro Lys 1
5 10 15 Gly Lys Leu Lys Gly Ala
Asn Phe Val Ile Ala His Glu Thr Ala Asn 20
25 30 Ser Lys Ser Thr Ile Asp Asn Gln Val Ser
Tyr Met Thr Arg Asn Trp 35 40
45 Lys Asn Ala Phe Val Thr His Phe Val Gly Gly Gly Gly Arg
Val Val 50 55 60
Gln Val Ala Asn Val Asn Tyr Val Ser Trp Gly Ala Gly Gln Tyr Ala 65
70 75 80 Asn Ser Tyr Ser Tyr
Ala Gln Val Glu Leu Cys Arg Thr Ser Asn Ala 85
90 95 Thr Thr Phe Lys Lys Asp Tyr Glu Val Tyr
Cys Gln Leu Leu Val Asp 100 105
110 Leu Ala Lys Lys Ala Gly Ile Pro Ile Thr Leu Asp Ser Gly Ser
Lys 115 120 125 Thr
Ser Asp Lys Gly Ile Lys Ser His Lys Trp Val Ala Asp Lys Leu 130
135 140 Gly Gly Thr Thr His Gln
Asp Pro Tyr Ala Tyr Leu Ser Ser Trp Gly 145 150
155 160 Ile Ser Lys Ala Gln Phe Ala Ser Asp Leu Ala
Lys Val Ser Gly Gly 165 170
175 Gly Asn Thr Gly Thr Ala Pro Ala Lys Pro Ser Thr Pro Ala Pro Lys
180 185 190 Pro Ser
Thr Pro Ser Thr Asn Leu Asp Lys Leu Gly Leu Val Asp Tyr 195
200 205 Met Asn Ala Lys Lys Met Asp
Ser Ser Tyr Ser Asn Arg Asp Lys Leu 210 215
220 Ala Lys Gln Tyr Gly Ile Ala Asn Tyr Ser Gly Thr
Ala Ser Gln Asn 225 230 235
240 Thr Thr Leu Leu Ser Lys Ile Lys Gly Gly Ala Pro Lys Pro Ser Thr
245 250 255 Pro Ala Pro
Lys Pro Ser Thr Ser Thr Ala Lys Lys Ile Tyr Phe Pro 260
265 270 Pro Asn Lys Gly Asn Trp Ser Val
Tyr Pro Thr Asn Lys Ala Pro Val 275 280
285 Lys Ala Asn Ala Ile Gly Ala Ile Asn Pro Thr Lys Phe
Gly Gly Leu 290 295 300
Thr Tyr Thr Ile Gln Lys Asp Arg Gly Asn Gly Val Tyr Glu Ile Gln 305
310 315 320 Thr Asp Gln Phe
Gly Arg Val Gln Val Tyr Gly Ala Pro Ser Thr Gly 325
330 335 Ala Val Ile Lys Lys 340
7341PRTartificialSynthetic peptide 7Met Val Lys Tyr Thr Val Glu Asn
Lys Ile Ile Ala Gly Leu Pro Lys 1 5 10
15 Gly Lys Leu Lys Gly Ala Asn Phe Val Ile Ala His Glu
Thr Ala Asn 20 25 30
Ser Lys Ser Thr Ile Asp Asn Glu Val Ser Tyr Met Thr Arg Asn Trp
35 40 45 Lys Asn Ala Phe
Val Thr His Phe Val Gly Gly Gly Gly Arg Val Val 50
55 60 Gln Val Ala Asn Val Asn Tyr Val
Ser Trp Gly Ala Gly Gln Tyr Ala 65 70
75 80 Asn Ser Tyr Ser Tyr Ala Gln Val Glu Leu Cys Lys
Thr Ser Asn Ala 85 90
95 Thr Thr Phe Lys Lys Asp Tyr Glu Val Tyr Cys Gln Leu Leu Val Asp
100 105 110 Leu Ala Lys
Lys Ala Gly Ile Pro Ile Thr Leu Asp Ser Gly Ser Lys 115
120 125 Thr Ser Asp Lys Gly Ile Lys Ser
His Lys Trp Val Ala Asp Lys Leu 130 135
140 Gly Gly Thr Thr His Gln Asp Pro Tyr Ala Tyr Leu Ser
Ser Trp Gly 145 150 155
160 Ile Ser Lys Ala Gln Phe Ala Ser Asp Leu Ala Lys Val Ser Gly Gly
165 170 175 Gly Asn Thr Gly
Thr Ala Pro Ala Lys Pro Ser Thr Pro Ala Pro Lys 180
185 190 Pro Ser Thr Pro Ser Thr Asn Leu Asp
Lys Leu Gly Leu Val Asp Tyr 195 200
205 Met Asn Ala Lys Lys Met Asp Ser Ser Tyr Ser Asn Lys Asp
Lys Leu 210 215 220
Ala Lys Gln Tyr Gly Ile Ala Asn Tyr Ser Gly Thr Ala Ser Gln Asn 225
230 235 240 Thr Thr Leu Leu Ser
Lys Ile Lys Gly Gly Ala Pro Lys Pro Ser Thr 245
250 255 Pro Ala Pro Lys Pro Ser Thr Ser Thr Ala
Lys Lys Ile Tyr Phe Pro 260 265
270 Pro Asn Lys Gly Asn Trp Ser Val Tyr Pro Thr Asn Lys Ala Pro
Val 275 280 285 Lys
Ala Asn Ala Ile Gly Ala Ile Asn Pro Thr Lys Phe Gly Gly Leu 290
295 300 Thr Tyr Thr Ile Gln Lys
Asp Arg Gly Asn Gly Val Tyr Glu Ile Gln 305 310
315 320 Thr Asp Gln Phe Gly Arg Val Gln Val Tyr Gly
Ala Pro Ser Thr Gly 325 330
335 Ala Val Ile Lys Lys 340
8341PRTartificialSynthetic peptide 8Met Val Lys Tyr Thr Val Glu Asn Lys
Ile Ile Ala Gly Leu Pro Lys 1 5 10
15 Gly Lys Leu Lys Gly Ala Asn Phe Val Ile Ala His Glu Thr
Ala Asn 20 25 30
Ser Lys Ser Thr Ile Asp Asn Glu Val Ser Tyr Met Thr Arg Asn Trp
35 40 45 Lys Asn Ala Phe
Val Thr His Phe Val Gly Gly Gly Gly Arg Val Val 50
55 60 Gln Val Ala Asn Val Asn Tyr Val
Ser Trp Gly Ala Gly Gln Tyr Ala 65 70
75 80 Asn Ser Tyr Ser Tyr Ala Gln Val Glu Leu Cys Ala
Thr Ser Asn Ala 85 90
95 Thr Thr Phe Lys Lys Asp Tyr Glu Val Tyr Cys Gln Leu Leu Val Asp
100 105 110 Leu Ala Lys
Lys Ala Gly Ile Pro Ile Thr Leu Asp Ser Gly Ser Lys 115
120 125 Thr Ser Asp Lys Gly Ile Lys Ser
His Lys Trp Val Ala Asp Lys Leu 130 135
140 Gly Gly Thr Thr His Gln Asp Pro Tyr Ala Tyr Leu Ser
Ser Trp Gly 145 150 155
160 Ile Ser Lys Ala Gln Phe Ala Ser Asp Leu Ala Lys Val Ser Gly Gly
165 170 175 Gly Asn Thr Gly
Thr Ala Pro Ala Lys Pro Ser Thr Pro Ala Pro Lys 180
185 190 Pro Ser Thr Pro Ser Thr Asn Leu Asp
Lys Leu Gly Leu Val Asp Tyr 195 200
205 Met Asn Ala Lys Lys Met Asp Ser Ser Tyr Ser Asn Ala Asp
Lys Leu 210 215 220
Ala Lys Gln Tyr Gly Ile Ala Asn Tyr Ser Gly Thr Ala Ser Gln Asn 225
230 235 240 Thr Thr Leu Leu Ser
Lys Ile Lys Gly Gly Ala Pro Lys Pro Ser Thr 245
250 255 Pro Ala Pro Lys Pro Ser Thr Ser Thr Ala
Lys Lys Ile Tyr Phe Pro 260 265
270 Pro Asn Lys Gly Asn Trp Ser Val Tyr Pro Thr Asn Lys Ala Pro
Val 275 280 285 Lys
Ala Asn Ala Ile Gly Ala Ile Asn Pro Thr Lys Phe Gly Gly Leu 290
295 300 Thr Tyr Thr Ile Gln Lys
Asp Arg Gly Asn Gly Val Tyr Glu Ile Gln 305 310
315 320 Thr Asp Gln Phe Gly Arg Val Gln Val Tyr Gly
Ala Pro Ser Thr Gly 325 330
335 Ala Val Ile Lys Lys 340
9317PRTartificialSynthetic peptide 9Met Ala Tyr Lys Val Glu Arg Arg Ile
Arg Pro Gly Leu Pro Gln Val 1 5 10
15 Gly Tyr Ala Pro Tyr Gly Gln Val His Ala His Ser Thr Gly
Asn Pro 20 25 30
Arg Ser Thr Ala Gln Asn Glu Ala Asp Tyr Phe Gln Thr Lys Asp Ile
35 40 45 Thr Thr Gly Phe
Tyr Thr His Leu Val Gly Asn Gly Arg Val Ile Gln 50
55 60 Leu Ala Glu Val Asn Arg Gly Ala
Trp Asp Val Gly Gly Gly Trp Asn 65 70
75 80 Gln Trp Gly Tyr Ala Ser Val Glu Leu Ile Glu Ser
His Ser Asn Arg 85 90
95 Asp Glu Phe Met Arg Asp Tyr Lys Ile Tyr Cys Glu Leu Leu His Glu
100 105 110 Leu Ala Lys
Gln Ala Gly Leu Pro Thr Thr Val Asp Gln Gly Asn Thr 115
120 125 Gly Ile Ile Thr His Asn Tyr Ala
Thr His Asn Gln Pro Asn Asn Gly 130 135
140 Ser Asp His Val Asp Pro Ile Pro Tyr Leu Ala Lys Trp
Gly Ile Asn 145 150 155
160 Leu Ala Gln Phe Arg Ser Asp Val Ala Asn Ala Lys Ser Asn Ser Lys
165 170 175 Pro Val Thr Pro
Ser Lys Pro Val Ser His Asp Lys Ala Ile Ala Lys 180
185 190 Ser Pro Ala Lys Thr Val Asn Gly Tyr
Thr Gly Lys Met Asp Lys Phe 195 200
205 Asn Val Gln Gly Asn Lys Phe Arg Val Ala Gly Trp Met Leu
Pro Thr 210 215 220
Ala Gly Gly Gln Pro Tyr Asn Tyr Gly Tyr Val Phe Leu Leu Asp Ala 225
230 235 240 Lys Thr Gly Lys Glu
Ile Ala Arg Gln Leu Ala Gly Ala Val Ser Arg 245
250 255 Pro Asp Val Thr Lys Ala Tyr Gly Val Lys
Gly Gly Thr Asn Tyr Gly 260 265
270 Leu Asp Val Thr Phe Asp Val Lys Lys Leu Lys Gly Lys Lys Phe
Tyr 275 280 285 Ala
Met Phe Arg Arg Thr Asn Asp Lys Ala Gly Asn Thr Ala Gly Gly 290
295 300 His Lys Asp Ile Gly Phe
Asn Glu Phe Tyr Phe Thr Leu 305 310 315
10317PRTartificialSynthetic peptide 10Met Ala Tyr Lys Val Glu Arg
Arg Ile Arg Pro Gly Leu Pro Gln Val 1 5
10 15 Gly Tyr Ala Pro Tyr Gly Gln Val His Ala His
Ser Thr Gly Asn Pro 20 25
30 Arg Ser Thr Ala Gln Asn Glu Ala Asp Tyr Phe Gln Thr Lys Asp
Ile 35 40 45 Thr
Thr Gly Phe Tyr Thr His Leu Val Gly Asn Gly Arg Val Ile Gln 50
55 60 Leu Ala Glu Val Asn Arg
Gly Ala Trp Asp Val Gly Gly Gly Trp Asn 65 70
75 80 Gln Trp Gly Tyr Ala Ser Val Glu Leu Ile Glu
Ser His Ser Asn Arg 85 90
95 Asp Glu Phe Met Arg Asp Tyr Lys Ile Tyr Cys Glu Leu Leu His Ala
100 105 110 Leu Ala
Lys Gln Ala Gly Leu Pro Thr Thr Val Asp Gln Gly Asn Thr 115
120 125 Gly Ile Ile Thr His Asn Tyr
Ala Thr His Asn Gln Pro Asn Asn Gly 130 135
140 Ser Asp His Val Asp Pro Ile Pro Tyr Leu Ala Lys
Trp Gly Ile Asn 145 150 155
160 Leu Ala Gln Phe Arg Ser Asp Val Ala Asn Ala Lys Ser Asn Ser Lys
165 170 175 Pro Val Thr
Pro Ser Lys Pro Val Ser His Asp Lys Ala Ile Ala Lys 180
185 190 Ser Pro Ala Lys Thr Val Asn Gly
Tyr Thr Gly Lys Met Asp Lys Phe 195 200
205 Asn Val Gln Gly Asn Lys Phe Arg Val Ala Gly Trp Met
Leu Pro Thr 210 215 220
Ala Gly Gly Gln Pro Tyr Asn Tyr Gly Tyr Val Phe Leu Leu Asp Ala 225
230 235 240 Lys Thr Gly Lys
Glu Ile Ala Arg Gln Leu Ala Gly Ala Val Ser Arg 245
250 255 Pro Asp Val Thr Lys Ala Tyr Gly Val
Lys Gly Gly Thr Asn Tyr Gly 260 265
270 Leu Asp Val Thr Phe Asp Val Lys Lys Leu Lys Gly Lys Lys
Phe Tyr 275 280 285
Ala Met Phe Arg Arg Thr Asn Asp Lys Ala Gly Asn Thr Ala Gly Gly 290
295 300 His Lys Asp Ile Gly
Phe Asn Glu Phe Tyr Phe Thr Leu 305 310
315 11496PRTartificialSynthetic peptide 11Met Ser Ile Ile Met Glu
Val Ala Thr Met Gln Ala Lys Leu Thr Lys 1 5
10 15 Lys Glu Phe Ile Glu Trp Leu Lys Thr Ser Glu
Gly Lys Gln Phe Asn 20 25
30 Val Asp Leu Trp Tyr Gly Phe Gln Cys Phe Asp Tyr Ala Asn Ala
Gly 35 40 45 Trp
Lys Val Leu Phe Gly Leu Leu Leu Lys Gly Leu Gly Ala Lys Asp 50
55 60 Ile Pro Phe Ala Asn Asn
Phe Asp Gly Leu Ala Thr Val Tyr Gln Asn 65 70
75 80 Thr Pro Asp Phe Leu Ala Gln Pro Gly Asp Met
Val Val Phe Gly Ser 85 90
95 Asn Tyr Gly Ala Gly Tyr Gly His Val Ala Trp Val Ile Glu Ala Thr
100 105 110 Leu Asp
Tyr Ile Ile Val Tyr Glu Gln Asn Trp Leu Gly Gly Gly Trp 115
120 125 Thr Asp Arg Ile Glu Gln Pro
Gly Trp Gly Trp Glu Lys Val Thr Arg 130 135
140 Arg Gln His Ala Tyr Asp Phe Pro Met Trp Phe Ile
Arg Pro Asn Phe 145 150 155
160 Lys Ser Gln Thr Ala Pro Ala Ser Ile Gln Ser Pro Thr Gln Ala Ser
165 170 175 Lys Lys Glu
Thr Ala Lys Pro Gln Pro Lys Ala Val Glu Leu Lys Ile 180
185 190 Ile Lys Asp Val Val Lys Gly Tyr
Asp Leu Pro Lys Arg Gly Gly Asn 195 200
205 Pro Lys Gly Ile Val Ile His Asn Asp Ala Gly Ser Lys
Gly Ala Thr 210 215 220
Ala Glu Ala Tyr Arg Asn Gly Leu Val Asn Ala Pro Ser Ser Arg Leu 225
230 235 240 Glu Ala Gly Ile
Ala His Ser Tyr Val Ser Gly Asn Thr Val Trp Gln 245
250 255 Ala Leu Asp Glu Ser Gln Val Gly Trp
His Thr Ala Asn Gln Leu Gly 260 265
270 Asn Lys Tyr Tyr Tyr Gly Ile Glu Val Cys Gln Ser Met Gly
Ala Asp 275 280 285
Asn Ala Thr Phe Leu Lys Asn Glu Gln Ala Thr Phe Gln Glu Cys Ala 290
295 300 Arg Leu Leu Lys Lys
Trp Gly Leu Pro Ala Asn Arg Asn Thr Ile Arg 305 310
315 320 Leu His Asn Glu Phe Thr Ser Thr Ser Cys
Pro His Arg Ser Ser Val 325 330
335 Leu His Thr Gly Phe Asp Pro Val Thr Arg Gly Leu Leu Pro Glu
Asp 340 345 350 Lys
Arg Leu Gln Leu Lys Asp Tyr Phe Ile Lys Gln Ile Arg Ala Tyr 355
360 365 Met Asp Gly Lys Ile Pro
Val Ala Thr Val Ser Asn Glu Ser Ser Ala 370 375
380 Ser Ser Asn Thr Val Lys Pro Val Ala Glu Leu
Met Pro Pro Val Pro 385 390 395
400 Ala Gly Tyr Thr Leu Asp Lys Asn Asn Val Pro Tyr Lys Lys Glu Gln
405 410 415 Gly Asn
Tyr Thr Val Ala Asn Val Lys Gly Asn Asn Val Arg Asp Gly 420
425 430 Tyr Ser Thr Asn Ser Arg Ile
Thr Gly Val Leu Pro Asn Asn Thr Thr 435 440
445 Ile Thr Tyr Asp Gly Ala Tyr Cys Ile Asn Gly Tyr
Arg Trp Ile Thr 450 455 460
Tyr Ile Ala Asn Ser Gly Gln Arg Arg Tyr Ile Ala Thr Gly Glu Val 465
470 475 480 Asp Ile Ala
Gly Asn Arg Ile Ser Ser Phe Gly Lys Phe Ser Ala Val 485
490 495 12497PRTartificialSynthetic
peptide 12Met Ala Ser Ile Ile Met Glu Val Ala Thr Met Gln Ala Lys Leu Thr
1 5 10 15 Lys Lys
Glu Phe Ile Glu Trp Leu Lys Thr Ser Glu Gly Lys Gln Phe 20
25 30 Asn Val Asp Leu Trp Tyr Gly
Phe Gln Cys Phe Asp Tyr Ala Asn Ala 35 40
45 Gly Trp Lys Val Leu Phe Gly His Thr Leu Lys Gly
Leu Gly Ala Lys 50 55 60
Asp Ile Pro Phe Ala Asn Asn Phe Asp Gly Leu Ala Thr Val Tyr Gln 65
70 75 80 Asn Thr Pro
Asp Phe Leu Ala Gln Pro Gly Asp Met Val Val Phe Gly 85
90 95 Ser Asn Tyr Gly Ala Gly Tyr Gly
His Val Ala Trp Val Ile Glu Ala 100 105
110 Thr Leu Asp Tyr Ile Ile Val Tyr Glu Gln Asn Trp Leu
Gly Gly Gly 115 120 125
Trp Thr Asp Arg Ile Glu Gln Pro Gly Trp Gly Trp Glu Lys Val Thr 130
135 140 Arg Arg Gln His
Ala Tyr Asp Phe Pro Met Trp Phe Ile Arg Pro Asn 145 150
155 160 Phe Lys Ser Ala Thr Ala Pro Ala Ser
Ile Asn Ser Pro Thr Asn Ala 165 170
175 Ser Lys Lys Glu Thr Ala Lys Pro Gln Pro Lys Ala Val Glu
Leu Lys 180 185 190
Ile Ile Lys Asp Val Val Lys Gly His Asp Leu Pro Lys Arg Gly Gly
195 200 205 Asn Pro Lys Gly
Ile Val Ile His Asn Asp Ala Gly Ser Lys Gly Ala 210
215 220 Thr Ala Glu Ala Tyr Arg Asn Gly
Leu Val Asn Ala Pro Ser Ser Arg 225 230
235 240 Leu Glu Ala Gly Ile Ala His Ser Tyr Val Ser Gly
Asn Thr Val Trp 245 250
255 Gln Ala Leu Asp Glu Ser Gln Val Gly Trp His Thr Ala Asn Gln Leu
260 265 270 Gly Asn Lys
Tyr Tyr Tyr Gly Ile Glu Val Cys Gln Ser Met Gly Ala 275
280 285 Asp Asn Ala Thr Phe Leu Lys Asn
Glu Gln Ala Thr Phe Gln Glu Cys 290 295
300 Ala Arg Leu Leu Lys Lys Trp Gly Leu Pro Ala Asn Arg
Asn Thr Ile 305 310 315
320 Arg Leu His Asn Glu Phe Thr Ser Thr Ser Cys Pro His Arg Ser Ser
325 330 335 Val Leu His Thr
Gly Phe Asp Pro Val Thr Arg Gly Leu Leu Pro Glu 340
345 350 Asp Lys Arg Leu Gln Leu Lys Asp Tyr
Phe Ile Lys Gln Ile Arg Ala 355 360
365 Tyr Met Asp Gly Lys Ile Pro Val Ala Thr Val Ser Asn Glu
Ser Thr 370 375 380
Ala Ser Ser Asn Thr Val Lys Pro Val Ala Glu Leu Met Pro Pro Val 385
390 395 400 Pro Ala Gly Tyr Thr
Leu Asp Lys Asn Asn Val Pro Tyr Lys Lys Glu 405
410 415 Gln Gly Asn Tyr Thr Val Ala Asn Val Lys
Gly Asn Asn Val Arg Asp 420 425
430 Gly Tyr Ser Thr Asn Ser Arg Ile Thr Gly Val Leu Pro Asn Asn
Thr 435 440 445 Thr
Ile Thr Tyr Asp Gly Ala Tyr Cys Ile Asn Gly Tyr Arg Trp Ile 450
455 460 Thr Tyr Ile Ala Asn Ser
Gly Gln Arg Arg Tyr Ile Ala Thr Gly Glu 465 470
475 480 Val Asp Ile Ala Gly Asn Arg Ile Ser Ser Phe
Gly Lys Phe Ser Ala 485 490
495 Val 13497PRTartificialSynthetic peptide 13Met Ala Ser Ile Ile
Met Glu Val Ala Thr Met Gln Ala Lys Leu Thr 1 5
10 15 Lys Lys Glu Phe Ile Glu Trp Leu Lys Thr
Ser Glu Gly Lys Gln Phe 20 25
30 Asn Val Asp Leu Trp Tyr Gly Phe Gln Cys Phe Asp Tyr Ala Asn
Ala 35 40 45 Gly
Trp Lys Val Leu Phe Gly His Thr Leu Lys Gly Leu Gly Ala Lys 50
55 60 Asp Ile Pro Phe Ala Asn
Asn Phe Asp Gly Leu Ala Thr Val Tyr Gln 65 70
75 80 Asn Thr Pro Asp Phe Leu Ala Gln Pro Gly Asp
Met Val Val Phe Gly 85 90
95 Ser Asn Tyr Gly Ala Gly Tyr Gly His Val Ala Trp Val Ile Glu Ala
100 105 110 Thr Leu
Asp Tyr Ile Ile Val Tyr Glu Gln Asn Trp Leu Gly Gly Gly 115
120 125 Trp Thr Asp Arg Ile Glu Gln
Pro Gly Trp Gly Trp Glu Lys Val Thr 130 135
140 Arg Arg Gln His Ala Tyr Asp Phe Pro Met Trp Phe
Ile Arg Pro Asn 145 150 155
160 Phe Lys Ser Ala Thr Ala Pro Ala Ser Ile Ala Ser Pro Thr Ala Ala
165 170 175 Ser Lys Lys
Glu Thr Ala Lys Pro Gln Pro Lys Ala Val Glu Leu Lys 180
185 190 Ile Ile Lys Asp Val Val Lys Gly
His Asp Leu Pro Lys Arg Gly Gly 195 200
205 Asn Pro Lys Gly Ile Val Ile His Asn Asp Ala Gly Ser
Lys Gly Ala 210 215 220
Thr Ala Glu Ala Tyr Arg Asn Gly Leu Val Asn Ala Pro Ser Ser Arg 225
230 235 240 Leu Glu Ala Gly
Ile Ala His Ser Tyr Val Ser Gly Asn Thr Val Trp 245
250 255 Gln Ala Leu Asp Glu Ser Gln Val Gly
Trp His Thr Ala Asn Gln Leu 260 265
270 Gly Asn Lys Tyr Tyr Tyr Gly Ile Glu Val Cys Gln Ser Met
Gly Ala 275 280 285
Asp Asn Ala Thr Phe Leu Lys Asn Glu Gln Ala Thr Phe Gln Glu Cys 290
295 300 Ala Arg Leu Leu Lys
Lys Trp Gly Leu Pro Ala Asn Arg Asn Thr Ile 305 310
315 320 Arg Leu His Asn Glu Phe Thr Ser Thr Ser
Cys Pro His Arg Ser Ser 325 330
335 Val Leu His Thr Gly Phe Asp Pro Val Thr Arg Gly Leu Leu Pro
Glu 340 345 350 Asp
Lys Arg Leu Gln Leu Lys Asp Tyr Phe Ile Lys Gln Ile Arg Ala 355
360 365 Tyr Met Asp Gly Lys Ile
Pro Val Ala Thr Val Ser Asn Glu Ser Ala 370 375
380 Ala Ser Ser Asn Thr Val Lys Pro Val Ala Glu
Leu Met Pro Pro Val 385 390 395
400 Pro Ala Gly Tyr Thr Leu Asp Lys Asn Asn Val Pro Tyr Lys Lys Glu
405 410 415 Gln Gly
Asn Tyr Thr Val Ala Asn Val Lys Gly Asn Asn Val Arg Asp 420
425 430 Gly Tyr Ser Thr Asn Ser Arg
Ile Thr Gly Val Leu Pro Asn Asn Thr 435 440
445 Thr Ile Thr Tyr Asp Gly Ala Tyr Cys Ile Asn Gly
Tyr Arg Trp Ile 450 455 460
Thr Tyr Ile Ala Asn Ser Gly Gln Arg Arg Tyr Ile Ala Thr Gly Glu 465
470 475 480 Val Asp Ile
Ala Gly Asn Arg Ile Ser Ser Phe Gly Lys Phe Ser Ala 485
490 495 Val 14497PRTartificialSynthetic
peptide 14Met Ala Ser Ile Ile Met Glu Val Ala Thr Met Gln Ala Lys Leu Thr
1 5 10 15 Lys Lys
Glu Phe Ile Glu Trp Leu Lys Thr Ser Glu Gly Lys Gln Phe 20
25 30 Asn Val Asp Leu Trp Tyr Gly
Phe Gln Cys Phe Asp Tyr Ala Asn Ala 35 40
45 Gly Trp Lys Val Leu Phe Gly Leu Leu Leu Lys Gly
Leu Gly Ala Lys 50 55 60
Asp Ile Pro Phe Ala Asn Asn Phe Asp Gly Leu Ala Thr Val Tyr Gln 65
70 75 80 Asn Thr Pro
Asp Phe Leu Ala Gln Pro Gly Asp Met Val Val Phe Gly 85
90 95 Ser Asn Tyr Gly Ala Gly Tyr Gly
His Val Ala Trp Val Ile Glu Ala 100 105
110 Thr Leu Asp Tyr Ile Ile Val Tyr Glu Gln Asn Trp Leu
Gly Gly Gly 115 120 125
Trp Thr Asp Arg Ile Glu Gln Pro Gly Trp Gly Trp Glu Lys Val Thr 130
135 140 Arg Arg Gln His
Ala Tyr Asp Phe Pro Met Trp Phe Ile Arg Pro Asn 145 150
155 160 Phe Lys Ser Glu Thr Ala Pro Ala Ser
Ile Gln Ser Pro Thr Gln Ala 165 170
175 Ser Lys Lys Glu Thr Ala Lys Pro Gln Pro Lys Ala Val Glu
Leu Lys 180 185 190
Ile Ile Lys Asp Val Val Lys Gly Tyr Asp Leu Pro Lys Arg Gly Gly
195 200 205 Asn Pro Lys Gly
Ile Val Ile His Asn Asp Ala Gly Ser Lys Gly Ala 210
215 220 Thr Ala Glu Ala Tyr Arg Asn Gly
Leu Val Asn Ala Pro Ser Ser Arg 225 230
235 240 Leu Glu Ala Gly Ile Ala His Ser Tyr Val Ser Gly
Asn Thr Val Trp 245 250
255 Gln Ala Leu Asp Glu Ser Gln Val Gly Trp His Thr Ala Asn Gln Leu
260 265 270 Gly Asn Lys
Tyr Tyr Tyr Gly Ile Glu Val Cys Gln Ser Met Gly Ala 275
280 285 Asp Asn Ala Thr Phe Leu Lys Asn
Glu Gln Ala Thr Phe Gln Glu Cys 290 295
300 Ala Arg Leu Leu Lys Lys Trp Gly Leu Pro Ala Asn Arg
Asn Thr Ile 305 310 315
320 Arg Leu His Asn Glu Phe Thr Ser Thr Ser Cys Pro His Arg Ser Ser
325 330 335 Val Leu His Thr
Gly Phe Asp Pro Val Thr Arg Gly Leu Leu Pro Glu 340
345 350 Asp Lys Arg Leu Gln Leu Lys Asp Tyr
Phe Ile Lys Gln Ile Arg Ala 355 360
365 Tyr Met Asp Gly Lys Ile Pro Val Ala Thr Val Ser Asn Glu
Ser Ser 370 375 380
Ala Ser Ser Asn Thr Val Lys Pro Val Ala Glu Leu Met Pro Pro Val 385
390 395 400 Pro Ala Gly Tyr Thr
Leu Asp Lys Asn Asn Val Pro Tyr Lys Lys Glu 405
410 415 Gln Gly Asn Tyr Thr Val Ala Asn Val Lys
Gly Asn Asn Val Arg Asp 420 425
430 Gly Tyr Ser Thr Asn Ser Arg Ile Thr Gly Val Leu Pro Asn Asn
Thr 435 440 445 Thr
Ile Thr Tyr Asp Gly Ala Tyr Cys Ile Asn Gly Tyr Arg Trp Ile 450
455 460 Thr Tyr Ile Ala Asn Ser
Gly Gln Arg Arg Tyr Ile Ala Thr Gly Glu 465 470
475 480 Val Asp Ile Ala Gly Asn Arg Ile Ser Ser Phe
Gly Lys Phe Ser Ala 485 490
495 Val 15497PRTartificialSynthetic peptide 15Met Ala Ser Ile Ile
Met Glu Val Ala Thr Met Gln Ala Lys Leu Thr 1 5
10 15 Lys Lys Glu Phe Ile Glu Trp Leu Lys Thr
Ser Glu Gly Lys Gln Phe 20 25
30 Asn Val Asp Leu Trp Tyr Gly Phe Gln Cys Phe Asp Tyr Ala Asn
Ala 35 40 45 Gly
Trp Lys Val Leu Phe Gly Leu Leu Leu Lys Gly Leu Gly Ala Lys 50
55 60 Asp Ile Pro Phe Ala Asn
Asn Phe Asp Gly Leu Ala Thr Val Tyr Gln 65 70
75 80 Asn Thr Pro Asp Phe Leu Ala Gln Pro Gly Asp
Met Val Val Phe Gly 85 90
95 Ser Asn Tyr Gly Ala Gly Tyr Gly His Val Ala Trp Val Ile Glu Ala
100 105 110 Thr Leu
Asp Tyr Ile Ile Val Tyr Glu Gln Asn Trp Leu Gly Gly Gly 115
120 125 Trp Thr Asp Arg Ile Glu Gln
Pro Gly Trp Gly Trp Glu Lys Val Thr 130 135
140 Arg Arg Gln His Ala Tyr Asp Phe Pro Met Trp Phe
Ile Arg Pro Asn 145 150 155
160 Phe Lys Ser Ala Thr Ala Pro Ala Ser Ile Gln Ser Pro Thr Gln Ala
165 170 175 Ser Lys Lys
Glu Thr Ala Lys Pro Gln Pro Lys Ala Val Glu Leu Lys 180
185 190 Ile Ile Lys Asp Val Val Lys Gly
Tyr Asp Leu Pro Lys Arg Gly Gly 195 200
205 Asn Pro Lys Gly Ile Val Ile His Asn Asp Ala Gly Ser
Lys Gly Ala 210 215 220
Thr Ala Glu Ala Tyr Arg Asn Gly Leu Val Asn Ala Pro Ser Ser Arg 225
230 235 240 Leu Glu Ala Gly
Ile Ala His Ser Tyr Val Ser Gly Asn Thr Val Trp 245
250 255 Gln Ala Leu Asp Glu Ser Gln Val Gly
Trp His Thr Ala Asn Gln Leu 260 265
270 Gly Asn Lys Tyr Tyr Tyr Gly Ile Glu Val Cys Gln Ser Met
Gly Ala 275 280 285
Asp Asn Ala Thr Phe Leu Lys Asn Glu Gln Ala Thr Phe Gln Glu Cys 290
295 300 Ala Arg Leu Leu Lys
Lys Trp Gly Leu Pro Ala Asn Arg Asn Thr Ile 305 310
315 320 Arg Leu His Asn Glu Phe Thr Ser Thr Ser
Cys Pro His Arg Ser Ser 325 330
335 Val Leu His Thr Gly Phe Asp Pro Val Thr Arg Gly Leu Leu Pro
Glu 340 345 350 Asp
Lys Arg Leu Gln Leu Lys Asp Tyr Phe Ile Lys Gln Ile Arg Ala 355
360 365 Tyr Met Asp Gly Lys Ile
Pro Val Ala Thr Val Ser Asn Glu Ser Ser 370 375
380 Ala Ser Ser Asn Thr Val Lys Pro Val Ala Glu
Leu Met Pro Pro Val 385 390 395
400 Pro Ala Gly Tyr Thr Leu Asp Lys Asn Asn Val Pro Tyr Lys Lys Glu
405 410 415 Gln Gly
Asn Tyr Thr Val Ala Asn Val Lys Gly Asn Asn Val Arg Asp 420
425 430 Gly Tyr Ser Thr Asn Ser Arg
Ile Thr Gly Val Leu Pro Asn Asn Thr 435 440
445 Thr Ile Thr Tyr Asp Gly Ala Tyr Cys Ile Asn Gly
Tyr Arg Trp Ile 450 455 460
Thr Tyr Ile Ala Asn Ser Gly Gln Arg Arg Tyr Ile Ala Thr Gly Glu 465
470 475 480 Val Asp Ile
Ala Gly Asn Arg Ile Ser Ser Phe Gly Lys Phe Ser Ala 485
490 495 Val 16497PRTartificialSynthetic
peptide 16Met Ala Ser Ile Ile Met Glu Val Ala Thr Met Gln Ala Lys Leu Thr
1 5 10 15 Lys Lys
Glu Phe Ile Glu Trp Leu Lys Thr Ser Glu Gly Lys Gln Phe 20
25 30 Asn Val Asp Leu Trp Tyr Gly
Phe Gln Cys Phe Asp Tyr Ala Asn Ala 35 40
45 Gly Trp Lys Val Leu Phe Gly Leu Leu Leu Lys Gly
Leu Gly Ala Lys 50 55 60
Asp Ile Pro Phe Ala Asn Asn Phe Asp Gly Leu Ala Thr Val Tyr Gln 65
70 75 80 Asn Thr Pro
Asp Phe Leu Ala Gln Pro Gly Asp Met Val Val Phe Gly 85
90 95 Ser Asn Tyr Gly Ala Gly Tyr Gly
His Val Ala Trp Val Ile Glu Ala 100 105
110 Thr Leu Asp Tyr Ile Ile Val Tyr Glu Gln Asn Trp Leu
Gly Gly Gly 115 120 125
Trp Thr Asp Arg Ile Glu Gln Pro Gly Trp Gly Trp Glu Lys Val Thr 130
135 140 Arg Arg Gln His
Ala Tyr Asp Phe Pro Met Trp Phe Ile Arg Pro Asn 145 150
155 160 Phe Lys Ser Gln Thr Ala Pro Ala Ser
Ile Gln Ser Pro Thr Gln Ala 165 170
175 Ser Lys Lys Ala Thr Ala Lys Pro Gln Pro Lys Ala Val Gln
Leu Lys 180 185 190
Ile Ile Lys Asp Val Val Lys Gly Tyr Asp Leu Pro Lys Arg Gly Gly
195 200 205 Asn Pro Lys Gly
Ile Val Ile His Asn Asp Ala Gly Ser Lys Gly Ala 210
215 220 Thr Ala Glu Ala Tyr Arg Asn Gly
Leu Val Asn Ala Pro Ser Ser Arg 225 230
235 240 Leu Glu Ala Gly Ile Ala His Ser Tyr Val Ser Gly
Asn Thr Val Trp 245 250
255 Gln Ala Leu Asp Glu Ser Gln Val Gly Trp His Thr Ala Asn Gln Leu
260 265 270 Gly Asn Lys
Tyr Tyr Tyr Gly Ile Glu Val Cys Gln Ser Met Gly Ala 275
280 285 Asp Asn Ala Thr Phe Leu Lys Asn
Glu Gln Ala Thr Phe Gln Glu Cys 290 295
300 Ala Arg Leu Leu Lys Lys Trp Gly Leu Pro Ala Asn Arg
Asn Thr Ile 305 310 315
320 Arg Leu His Asn Glu Phe Thr Ser Thr Ser Cys Pro His Arg Ser Ser
325 330 335 Val Leu His Thr
Gly Phe Asp Pro Val Thr Arg Gly Leu Leu Pro Glu 340
345 350 Asp Lys Arg Leu Gln Leu Lys Asp Tyr
Phe Ile Lys Gln Ile Arg Ala 355 360
365 Tyr Met Asp Gly Lys Ile Pro Val Ala Thr Val Ser Asn Glu
Ser Ser 370 375 380
Ala Ser Ser Asn Thr Val Lys Pro Val Ala Glu Leu Met Pro Pro Val 385
390 395 400 Pro Ala Gly Tyr Thr
Leu Asp Lys Asn Asn Val Pro Tyr Lys Lys Glu 405
410 415 Gln Gly Asn Tyr Thr Val Ala Asn Val Lys
Gly Asn Asn Val Arg Asp 420 425
430 Gly Tyr Ser Thr Asn Ser Arg Ile Thr Gly Val Leu Pro Asn Asn
Thr 435 440 445 Thr
Ile Thr Tyr Asp Gly Ala Tyr Cys Ile Asn Gly Tyr Arg Trp Ile 450
455 460 Thr Tyr Ile Ala Asn Ser
Gly Gln Arg Arg Tyr Ile Ala Thr Gly Glu 465 470
475 480 Val Asp Ile Ala Gly Asn Arg Ile Ser Ser Phe
Gly Lys Phe Ser Ala 485 490
495 Val 17497PRTartificialSynthetic peptide 17Met Ala Ser Ile Ile
Met Glu Val Ala Thr Met Gln Ala Lys Leu Thr 1 5
10 15 Lys Lys Glu Phe Ile Glu Trp Leu Lys Thr
Ser Glu Gly Lys Gln Phe 20 25
30 Asn Val Asp Leu Trp Tyr Gly Phe Gln Cys Phe Asp Tyr Ala Asn
Ala 35 40 45 Gly
Trp Lys Val Leu Phe Gly Leu Leu Leu Lys Gly Leu Gly Ala Lys 50
55 60 Asp Ile Pro Phe Ala Asn
Asn Phe Asp Gly Leu Ala Thr Val Tyr Gln 65 70
75 80 Asn Thr Pro Asp Phe Leu Ala Gln Pro Gly Asp
Met Val Val Phe Gly 85 90
95 Ser Asn Tyr Gly Ala Gly Tyr Gly His Val Ala Trp Val Ile Glu Ala
100 105 110 Thr Leu
Asp Tyr Ile Ile Val Tyr Glu Gln Asn Trp Leu Gly Gly Gly 115
120 125 Trp Thr Asp Arg Ile Glu Gln
Pro Gly Trp Gly Trp Glu Lys Val Thr 130 135
140 Arg Arg Gln His Ala Tyr Asp Phe Pro Met Trp Phe
Ile Arg Pro Asn 145 150 155
160 Phe Lys Ser Gln Thr Ala Pro Ala Ser Ile Gln Ser Pro Thr Gln Ala
165 170 175 Ser Lys Lys
Glu Thr Ala Lys Pro Gln Pro Lys Ala Val Glu Leu Lys 180
185 190 Ile Ile Lys Asp Val Val Lys Gly
Tyr Asp Leu Pro Lys Arg Gly Gly 195 200
205 Asn Pro Lys Gly Ile Val Ile His Asn Asp Ala Gly Ser
Lys Gly Ala 210 215 220
Thr Ala Glu Ala Tyr Arg Asn Gly Leu Val Asn Ala Pro Ser Ser Arg 225
230 235 240 Leu Glu Ala Gly
Ile Ala His Ser Tyr Val Ser Gly Asn Thr Val Trp 245
250 255 Gln Ala Leu Asp Glu Ser Gln Val Gly
Trp His Thr Ala Asn Gln Leu 260 265
270 Gly Asn Lys Tyr Tyr Tyr Gly Ile Glu Val Cys Gln Ser Met
Gly Ala 275 280 285
Asp Asn Ala Thr Phe Leu Lys Asn Glu Gln Ala Thr Phe Gln Glu Cys 290
295 300 Ala Arg Leu Leu Lys
Lys Trp Gly Leu Pro Ala Asn Arg Asn Thr Ile 305 310
315 320 Arg Leu His Asn Glu Phe Thr Ser Thr Ser
Cys Pro His Arg Ser Ser 325 330
335 Val Leu His Thr Gly Phe Asp Pro Val Thr Arg Gly Leu Leu Pro
Glu 340 345 350 Asp
Lys Arg Leu Gln Leu Lys Asp Tyr Phe Ile Lys Gln Ile Arg Ala 355
360 365 Tyr Met Asp Gly Lys Ile
Pro Val Ala Thr Val Ser Asn Glu Ser Ser 370 375
380 Ala Ser Ser Asn Thr Val Lys Pro Val Ala Glu
Leu Met Pro Pro Val 385 390 395
400 Pro Ala Gly Tyr Thr Leu Asp Lys Asn Asn Val Pro Tyr Lys Lys Glu
405 410 415 Gln Gly
Asn Tyr Thr Val Ala Asn Val Lys Gly Asn Asn Val Arg Asp 420
425 430 Gly Tyr Ser Thr Asn Ser Arg
Ile Thr Gly Val Leu Pro Asn Asn Thr 435 440
445 Thr Ile Thr Tyr Asp Gly Ala Tyr Cys Ile Asn Gly
Tyr Arg Trp Ile 450 455 460
Thr Tyr Ile Ala Asn Ser Gly Gln Arg Arg Tyr Ile Ala Thr Gly Glu 465
470 475 480 Val Asp Ile
Ala Gly Asn Arg Ile Ser Ser Phe Gly Lys Phe Ser Ala 485
490 495 Val 18496PRTartificialSynthetic
peptide 18Met Ser Ile Ile Met Glu Val Ala Thr Met Gln Ala Lys Leu Thr Lys
1 5 10 15 Lys Glu
Phe Ile Glu Trp Leu Lys Thr Ser Glu Gly Lys Gln Phe Asn 20
25 30 Val Asp Leu Trp Tyr Gly Phe
Gln Cys Phe Asp Tyr Ala Asn Ala Gly 35 40
45 Trp Lys Val Leu Phe Gly Leu Leu Leu Lys Gly Leu
Gly Ala Lys Asp 50 55 60
Ile Pro Phe Ala Asn Asn Phe Asp Gly Leu Ala Thr Val Tyr Gln Asn 65
70 75 80 Thr Pro Asp
Phe Leu Ala Gln Pro Gly Asp Met Val Val Phe Gly Ser 85
90 95 Asn Tyr Gly Ala Gly Tyr Gly His
Val Ala Trp Val Ile Glu Ala Thr 100 105
110 Leu Asp Tyr Ile Ile Val Tyr Glu Gln Asn Trp Leu Gly
Gly Gly Trp 115 120 125
Thr Asp Arg Ile Glu Gln Pro Gly Trp Gly Trp Glu Lys Val Thr Arg 130
135 140 Arg Gln His Ala
Tyr Asp Phe Pro Met Trp Phe Ile Arg Pro Asn Phe 145 150
155 160 Lys Ser Glu Thr Ala Pro Arg Ser Ile
Gln Ser Pro Thr Gln Ala Ser 165 170
175 Lys Lys Glu Thr Ala Lys Pro Gln Pro Lys Ala Val Glu Leu
Lys Ile 180 185 190
Ile Lys Asp Val Val Lys Gly Tyr Asp Leu Pro Lys Arg Gly Gly Asn
195 200 205 Pro Lys Gly Ile
Val Ile His Asn Asp Ala Gly Ser Lys Gly Ala Thr 210
215 220 Ala Glu Ala Tyr Arg Asn Gly Leu
Val Asn Ala Pro Ser Ser Arg Leu 225 230
235 240 Glu Ala Gly Ile Ala His Ser Tyr Val Ser Gly Asn
Thr Val Trp Gln 245 250
255 Ala Leu Asp Glu Ser Gln Val Gly Trp His Thr Ala Asn Gln Leu Gly
260 265 270 Asn Lys Tyr
Tyr Tyr Gly Ile Glu Val Cys Gln Ser Met Gly Ala Asp 275
280 285 Asn Ala Thr Phe Leu Lys Asn Glu
Gln Ala Thr Phe Gln Glu Cys Ala 290 295
300 Arg Leu Leu Lys Lys Trp Gly Leu Pro Ala Asn Arg Asn
Thr Ile Arg 305 310 315
320 Leu His Asn Glu Phe Thr Ser Thr Ser Cys Pro His Arg Ser Ser Val
325 330 335 Leu His Thr Gly
Phe Asp Pro Val Thr Arg Gly Leu Leu Pro Glu Asp 340
345 350 Lys Arg Leu Gln Leu Lys Asp Tyr Phe
Ile Lys Gln Ile Arg Ala Tyr 355 360
365 Met Asp Gly Lys Ile Pro Val Ala Thr Val Ser Asn Glu Ser
Ser Ala 370 375 380
Ser Ser Asn Thr Val Lys Pro Val Ala Glu Leu Met Pro Pro Val Pro 385
390 395 400 Ala Gly Tyr Thr Leu
Asp Lys Asn Asn Val Pro Tyr Lys Lys Glu Gln 405
410 415 Gly Asn Tyr Thr Val Ala Asn Val Lys Gly
Asn Asn Val Arg Asp Gly 420 425
430 Tyr Ser Thr Asn Ser Arg Ile Thr Gly Val Leu Pro Asn Asn Thr
Thr 435 440 445 Ile
Thr Tyr Asp Gly Ala Tyr Cys Ile Asn Gly Tyr Arg Trp Ile Thr 450
455 460 Tyr Ile Ala Asn Ser Gly
Gln Arg Arg Tyr Ile Ala Thr Gly Glu Val 465 470
475 480 Asp Ile Ala Gly Asn Arg Ile Ser Ser Phe Gly
Lys Phe Ser Ala Val 485 490
495 19497PRTartificialSynthetic peptide 19Met Ala Ser Ile Ile Met
Glu Val Ala Thr Met Gln Ala Lys Leu Thr 1 5
10 15 Lys Lys Glu Phe Ile Glu Trp Leu Lys Thr Ser
Glu Gly Lys Gln Phe 20 25
30 Asn Val Asp Leu Trp Tyr Gly Phe Gln Cys Phe Asp Tyr Ala Asn
Ala 35 40 45 Gly
Trp Lys Val Leu Phe Gly Leu Leu Leu Lys Gly Leu Gly Ala Lys 50
55 60 Asp Ile Pro Phe Ala Asn
Asn Phe Asp Gly Leu Ala Thr Val Tyr Gln 65 70
75 80 Asn Thr Pro Asp Phe Leu Ala Gln Pro Gly Asp
Met Val Val Phe Gly 85 90
95 Ser Asn Tyr Gly Ala Gly Tyr Gly His Val Ala Trp Val Ile Glu Ala
100 105 110 Thr Leu
Asp Tyr Ile Ile Val Tyr Glu Gln Asn Trp Leu Gly Gly Gly 115
120 125 Trp Thr Asp Arg Ile Glu Gln
Pro Gly Trp Gly Trp Glu Lys Val Thr 130 135
140 Arg Arg Gln His Ala Tyr Asp Phe Pro Met Trp Phe
Ile Arg Pro Asn 145 150 155
160 Phe Lys Ser Glu Thr Ala Pro Arg Ser Ile Gln Ser Pro Thr Gln Ala
165 170 175 Ser Lys Lys
Glu Thr Ala Lys Pro Gln Pro Lys Ala Val Glu Leu Lys 180
185 190 Ile Ile Lys Asp Val Val Lys Gly
Tyr Asp Leu Pro Lys Arg Gly Gly 195 200
205 Asn Pro Lys Gly Ile Val Ile His Asn Asp Ala Gly Ser
Lys Gly Ala 210 215 220
Thr Ala Glu Ala Tyr Arg Asn Gly Leu Val Asn Ala Pro Ser Ser Arg 225
230 235 240 Leu Glu Ala Gly
Ile Ala His Ser Tyr Val Ser Gly Asn Thr Val Trp 245
250 255 Gln Ala Leu Asp Glu Ser Gln Val Gly
Trp His Thr Ala Asn Gln Leu 260 265
270 Gly Asn Lys Tyr Tyr Tyr Gly Ile Glu Val Cys Gln Ser Met
Gly Ala 275 280 285
Asp Asn Ala Thr Phe Leu Lys Asn Glu Gln Ala Thr Phe Gln Glu Cys 290
295 300 Ala Arg Leu Leu Lys
Lys Trp Gly Leu Pro Ala Asn Arg Asn Thr Ile 305 310
315 320 Arg Leu His Asn Glu Phe Thr Ser Thr Ser
Cys Pro His Arg Ser Ser 325 330
335 Val Leu His Thr Gly Phe Asp Pro Val Thr Arg Gly Leu Leu Pro
Glu 340 345 350 Asp
Lys Arg Leu Gln Leu Lys Asp Tyr Phe Ile Lys Gln Ile Arg Ala 355
360 365 Tyr Met Asp Gly Lys Ile
Pro Val Ala Thr Val Ser Asn Glu Ser Ser 370 375
380 Ala Ser Ser Asn Thr Val Lys Pro Val Ala Glu
Leu Met Pro Pro Val 385 390 395
400 Pro Ala Gly Tyr Thr Leu Asp Lys Asn Asn Val Pro Tyr Lys Lys Glu
405 410 415 Gln Gly
Asn Tyr Thr Val Ala Asn Val Lys Gly Asn Asn Val Arg Asp 420
425 430 Gly Tyr Ser Thr Asn Ser Arg
Ile Thr Gly Val Leu Pro Asn Asn Thr 435 440
445 Thr Ile Thr Tyr Asp Gly Ala Tyr Cys Ile Asn Gly
Tyr Arg Trp Ile 450 455 460
Thr Tyr Ile Ala Asn Ser Gly Gln Arg Arg Tyr Ile Ala Thr Gly Glu 465
470 475 480 Val Asp Ile
Ala Gly Asn Arg Ile Ser Ser Phe Gly Lys Phe Ser Ala 485
490 495 Val 20393PRTartificialSynthetic
peptide 20Met Ser Ile Ile Met Glu Val Ala Thr Met Gln Ala Lys Leu Thr Lys
1 5 10 15 Lys Glu
Phe Ile Glu Trp Leu Lys Thr Ser Glu Gly Lys Gln Phe Asn 20
25 30 Val Asp Leu Trp Tyr Gly Phe
Gln Cys Phe Asp Tyr Ala Asn Ala Gly 35 40
45 Trp Lys Val Leu Phe Gly Leu Leu Leu Lys Gly Leu
Gly Ala Lys Asp 50 55 60
Ile Pro Phe Ala Asn Asn Phe Asp Gly Leu Ala Thr Val Tyr Gln Asn 65
70 75 80 Thr Pro Asp
Phe Leu Ala Gln Pro Gly Asp Met Val Val Phe Gly Ser 85
90 95 Asn Tyr Gly Ala Gly Tyr Gly His
Val Ala Trp Val Ile Glu Ala Thr 100 105
110 Leu Asp Tyr Ile Ile Val Tyr Glu Gln Asn Trp Leu Gly
Gly Gly Trp 115 120 125
Thr Asp Arg Ile Glu Gln Pro Gly Trp Gly Trp Glu Lys Val Thr Arg 130
135 140 Arg Gln His Ala
Tyr Asp Phe Pro Met Trp Phe Ile Arg Pro Asn Phe 145 150
155 160 Lys Ser Glu Thr Ala Pro Arg Ser Ile
Gln Ser Pro Thr Gln Ala Ser 165 170
175 Lys Lys Glu Thr Ala Lys Pro Gln Pro Lys Ala Val Glu Leu
Lys Ile 180 185 190
Ile Lys Asp Val Val Lys Gly Tyr Asp Leu Pro Lys Arg Gly Gly Asn
195 200 205 Pro Lys Gly Ile
Val Ile His Asn Asp Ala Gly Ser Lys Gly Ala Thr 210
215 220 Ala Glu Ala Tyr Arg Asn Gly Leu
Val Asn Ala Pro Ser Ser Arg Leu 225 230
235 240 Glu Ala Gly Ile Ala His Ser Tyr Val Ser Gly Asn
Thr Val Trp Gln 245 250
255 Ala Leu Asp Glu Ser Gln Val Gly Trp His Thr Ala Asn Gln Leu Gly
260 265 270 Asn Lys Tyr
Tyr Tyr Gly Ile Glu Val Cys Gln Ser Met Gly Ala Asp 275
280 285 Asn Ala Thr Phe Leu Lys Asn Glu
Gln Ala Thr Phe Gln Glu Cys Ala 290 295
300 Arg Leu Leu Lys Lys Trp Gly Leu Pro Ala Asn Arg Asn
Thr Ile Arg 305 310 315
320 Leu His Asn Glu Phe Thr Ser Thr Ser Cys Pro His Arg Ser Ser Val
325 330 335 Leu His Thr Gly
Phe Asp Pro Val Thr Arg Gly Leu Leu Pro Glu Asp 340
345 350 Lys Arg Leu Gln Leu Lys Asp Tyr Phe
Ile Lys Gln Ile Arg Ala Tyr 355 360
365 Met Asp Gly Lys Ile Pro Val Ala Thr Val Ser Asn Glu Ser
Ser Ala 370 375 380
Ser Ser Asn Thr Val Lys Pro Val Ala 385 390
21341PRTArtificialSynthetic peptide 21Met Val Lys Tyr Thr Val Glu Asn Lys
Ile Ile Ala Gly Leu Pro Lys 1 5 10
15 Gly Lys Leu Lys Gly Ala Asn Phe Val Ile Ala His Glu Thr
Ala Asn 20 25 30
Ser Lys Ser Thr Ile Asp Asn Glu Val Ser Tyr Met Thr Arg Asn Trp
35 40 45 Lys Asn Ala Phe
Val Thr His Phe Val Gly Gly Gly Gly Arg Val Val 50
55 60 Gln Val Ala Asn Val Asn Tyr Val
Ser Trp Gly Ala Gly Gln Tyr Ala 65 70
75 80 Asn Ser Tyr Ser Tyr Ala Gln Val Glu Leu Cys Arg
Thr Ser Asn Ala 85 90
95 Thr Thr Phe Lys Lys Asp Tyr Glu Val Tyr Cys Gln Leu Leu Val Asp
100 105 110 Leu Ala Lys
Lys Ala Gly Ile Pro Ile Thr Leu Asp Ser Gly Ser Lys 115
120 125 Thr Ser Asp Lys Gly Ile Lys Ser
His Lys Trp Val Ala Asp Lys Leu 130 135
140 Gly Gly Thr Thr His Gln Asp Pro Tyr Ala Tyr Leu Ser
Ser Trp Gly 145 150 155
160 Ile Ser Lys Ala Gln Phe Ala Ser Asp Leu Ala Lys Val Ser Gly Gly
165 170 175 Gly Asn Thr Gly
Thr Ala Pro Ala Lys Pro Ser Thr Pro Ala Pro Lys 180
185 190 Pro Ser Thr Pro Ser Thr Asn Leu Asp
Lys Leu Gly Leu Val Asp Tyr 195 200
205 Met Asn Ala Lys Lys Met Asp Ser Ser Tyr Ser Asn Arg Asp
Lys Leu 210 215 220
Ala Lys Gln Tyr Gly Ile Ala Asn Tyr Ser Gly Thr Ala Ser Gln Asn 225
230 235 240 Thr Thr Leu Leu Ser
Lys Ile Lys Gly Gly Ala Pro Lys Pro Ser Thr 245
250 255 Pro Ala Pro Lys Pro Ser Thr Ser Thr Ala
Lys Lys Ile Tyr Phe Pro 260 265
270 Pro Asn Lys Gly Asn Trp Ser Val Tyr Pro Thr Asn Lys Ala Pro
Val 275 280 285 Lys
Ala Asn Ala Ile Gly Ala Ile Asn Pro Thr Lys Phe Gly Gly Leu 290
295 300 Thr Tyr Thr Ile Gln Lys
Asp Arg Gly Asn Gly Val Tyr Glu Ile Gln 305 310
315 320 Thr Asp Gln Phe Gly Arg Val Gln Val Tyr Gly
Ala Pro Ser Thr Gly 325 330
335 Ala Val Ile Lys Lys 340
22497PRTartificialSynthetic peptide 22Met Ala Ser Ile Ile Met Glu Val Ala
Thr Met Gln Ala Lys Leu Thr 1 5 10
15 Lys Lys Glu Phe Ile Glu Trp Leu Lys Thr Ser Glu Gly Lys
Gln Phe 20 25 30
Asn Val Asp Leu Trp Tyr Gly Phe Gln Cys Phe Asp Tyr Ala Asn Ala
35 40 45 Gly Trp Lys Val
Leu Phe Gly His Thr Leu Lys Gly Leu Gly Ala Lys 50
55 60 Asp Ile Pro Phe Ala Asn Asn Phe
Asp Gly Leu Ala Thr Val Tyr Gln 65 70
75 80 Asn Thr Pro Asp Phe Leu Ala Gln Pro Gly Asp Met
Val Val Phe Gly 85 90
95 Ser Asn Tyr Gly Ala Gly Tyr Gly His Val Ala Trp Val Ile Glu Ala
100 105 110 Thr Leu Asp
Tyr Ile Ile Val Tyr Glu Gln Asn Trp Leu Gly Gly Gly 115
120 125 Trp Thr Asp Arg Ile Glu Gln Pro
Gly Trp Gly Trp Glu Lys Val Thr 130 135
140 Arg Arg Gln His Ala Tyr Asp Phe Pro Met Trp Phe Ile
Arg Pro Asn 145 150 155
160 Phe Lys Ser Ala Thr Ala Pro Ala Ser Ile Gln Ser Pro Thr Gln Ala
165 170 175 Ser Lys Lys Glu
Thr Ala Lys Pro Gln Pro Lys Ala Val Glu Leu Lys 180
185 190 Ile Ile Lys Asp Val Val Lys Gly His
Asp Leu Pro Lys Arg Gly Gly 195 200
205 Asn Pro Lys Gly Ile Val Ile His Asn Asp Ala Gly Ser Lys
Gly Ala 210 215 220
Thr Ala Glu Ala Tyr Arg Asn Gly Leu Val Asn Ala Pro Ser Ser Arg 225
230 235 240 Leu Glu Ala Gly Ile
Ala His Ser Tyr Val Ser Gly Asn Thr Val Trp 245
250 255 Gln Ala Leu Asp Glu Ser Gln Val Gly Trp
His Thr Ala Asn Gln Leu 260 265
270 Gly Asn Lys Tyr Tyr Tyr Gly Ile Glu Val Cys Gln Ser Met Gly
Ala 275 280 285 Asp
Asn Ala Thr Phe Leu Lys Asn Glu Gln Ala Thr Phe Gln Glu Cys 290
295 300 Ala Arg Leu Leu Lys Lys
Trp Gly Leu Pro Ala Asn Arg Asn Thr Ile 305 310
315 320 Arg Leu His Asn Glu Phe Thr Ser Thr Ser Cys
Pro His Arg Ser Ser 325 330
335 Val Leu His Thr Gly Phe Asp Pro Val Thr Arg Gly Leu Leu Pro Glu
340 345 350 Asp Lys
Arg Leu Gln Leu Lys Asp Tyr Phe Ile Lys Gln Ile Arg Ala 355
360 365 Tyr Met Asp Gly Lys Ile Pro
Val Ala Thr Val Ser Asn Glu Ser Ser 370 375
380 Ala Ser Ser Asn Thr Val Lys Pro Val Ala Glu Leu
Met Pro Pro Val 385 390 395
400 Pro Ala Gly Tyr Thr Leu Asp Lys Asn Asn Val Pro Tyr Lys Lys Glu
405 410 415 Gln Gly Asn
Tyr Thr Val Ala Asn Val Lys Gly Asn Asn Val Arg Asp 420
425 430 Gly Tyr Ser Thr Asn Ser Arg Ile
Thr Gly Val Leu Pro Asn Asn Thr 435 440
445 Thr Ile Thr Tyr Asp Gly Ala Tyr Cys Ile Asn Gly Tyr
Arg Trp Ile 450 455 460
Thr Tyr Ile Ala Asn Ser Gly Gln Arg Arg Tyr Ile Ala Thr Gly Glu 465
470 475 480 Val Asp Ile Ala
Gly Asn Arg Ile Ser Ser Phe Gly Lys Phe Ser Ala 485
490 495 Val 231063PRTArtificial
SequenceSynthetic peptide 23Gln Asn Trp Leu Gly Gly Gly Trp Thr Asp Gly
Ile Glu Gln Pro Ala 1 5 10
15 Gly Val Gly Lys Lys Leu Gln Asp Asp Asn Met Leu Met Ile Ser Leu
20 25 30 Cys Gly
Leu Ser Val Arg Ile Leu Lys Val Arg Gln Arg His Asp Gln 35
40 45 Phe Asn Leu Leu His Lys His
Pro Lys Lys Glu Gln Asn Trp Leu Gly 50 55
60 Gly Gly Trp Thr Asp Gly Ile Glu Gln Pro Ala Gly
Val Gly Lys Lys 65 70 75
80 Leu Gln Asp Asp Asn Met Leu Met Ile Ser Leu Cys Gly Leu Ser Val
85 90 95 Arg Ile Leu
Lys Val Arg Gln Arg His Asp Gln Phe Asn Leu Leu His 100
105 110 Lys His Pro Lys Lys Glu Gln Asn
Trp Leu Gly Gly Gly Trp Thr Asp 115 120
125 Arg Ile Glu Gln Pro Gly Trp Gly Trp Glu Lys Val Thr
Arg Arg Gln 130 135 140
His Ala Tyr Asp Phe Pro Met Trp Phe Ile Arg Pro Asn Phe Lys Ser 145
150 155 160 Glu Thr Ala Pro
Arg Ser Ile Gln Ser Pro Thr Gln Ala Ser Lys Lys 165
170 175 Glu Gln Asn Trp Leu Gly Gly Gly Trp
Thr Asp Arg Ile Glu Gln Pro 180 185
190 Gly Trp Gly Trp Glu Lys Val Thr Arg Arg Gln His Ala Tyr
Asp Phe 195 200 205
Pro Met Trp Phe Ile Arg Pro Asn Phe Lys Ser Glu Thr Ala Pro Arg 210
215 220 Ser Ile Gln Ser Pro
Thr Gln Ala Ser Lys Lys Glu Gln Asn Trp Leu 225 230
235 240 Gly Gly Gly Trp Thr Asp Arg Ile Glu Gln
Pro Gly Trp Gly Trp Glu 245 250
255 Lys Val Thr Arg Arg Gln His Ala Tyr Asp Phe Pro Met Trp Phe
Ile 260 265 270 Arg
Pro Asn Phe Lys Ser Glu Thr Ala Pro Arg Ser Ile Gln Ser Pro 275
280 285 Thr Gln Ala Ser Lys Lys
Glu Gln Asn Trp Leu Gly Gly Gly Trp Thr 290 295
300 Asp Arg Ile Glu Gln Pro Gly Trp Gly Trp Glu
Lys Val Thr Arg Arg 305 310 315
320 Gln His Ala Tyr Asp Phe Pro Met Trp Phe Ile Arg Pro Asn Phe Lys
325 330 335 Ser Glu
Thr Ala Pro Arg Ser Ile Gln Ser Pro Thr Gln Ala Ser Lys 340
345 350 Lys Glu Gln Asn Trp Leu Gly
Gly Gly Trp Thr Asp Gly Ile Glu Gln 355 360
365 Pro Gly Trp Gly Trp Glu Lys Val Thr Arg Arg Gln
His Ala Tyr Asp 370 375 380
Phe Pro Met Trp Phe Ile Arg Pro Asn Phe Lys Ser Glu Thr Ala Pro 385
390 395 400 Arg Ser Val
Gln Ser Pro Thr Gln Ala Pro Lys Lys Glu Gln Asn Trp 405
410 415 Leu Gly Gly Gly Trp Thr Asp Gly
Ile Glu Gln Pro Gly Trp Gly Trp 420 425
430 Glu Lys Val Thr Arg Arg Gln His Ala Tyr Asp Phe Pro
Met Trp Phe 435 440 445
Ile Arg Pro Asn Phe Lys Ser Glu Ile Ala Pro Arg Ser Val Gln Ser 450
455 460 Pro Thr Gln Ala
Pro Lys Lys Glu Gln Asn Trp Leu Gly Gly Gly Trp 465 470
475 480 Thr Asp Gly Ile Glu Gln Pro Gly Trp
Gly Trp Glu Lys Val Thr Arg 485 490
495 Arg Gln His Ala Tyr Asp Phe Pro Met Trp Phe Ile Arg Pro
Asn Phe 500 505 510
Lys Ser Glu Ile Thr Pro Arg Ser Val Gln Ser Pro Thr Gln Ala Pro
515 520 525 Lys Lys Glu Gln
Asn Trp Leu Gly Gly Gly Trp Thr Asp Gly Ile Glu 530
535 540 Gln Pro Gly Trp Gly Trp Glu Lys
Val Thr Arg Arg Gln His Ala Tyr 545 550
555 560 Asp Phe Pro Met Trp Phe Ile Arg Pro Asn Phe Lys
Ser Glu Thr Ala 565 570
575 Pro Arg Ser Val Gln Ser Pro Thr Gln Ala Pro Lys Lys Glu Gln Asn
580 585 590 Trp Leu Gly
Gly Gly Trp Thr Asp Gly Ile Glu Gln Pro Gly Trp Gly 595
600 605 Trp Glu Lys Val Thr Arg Arg Gln
His Ala Tyr Asp Phe Pro Met Trp 610 615
620 Phe Ile Arg Pro Asn Phe Lys Ser Glu Ile Ala Pro Arg
Ser Val Gln 625 630 635
640 Ser Pro Thr Gln Ala Pro Lys Lys Glu Gln Asn Trp Leu Gly Gly Gly
645 650 655 Trp Thr Asp Gly
Ile Glu Gln Pro Gly Trp Gly Trp Glu Lys Val Thr 660
665 670 Arg Arg Gln His Ala Tyr Asp Phe Pro
Met Trp Phe Ile Arg Pro Asn 675 680
685 Phe Lys Ser Glu Thr Ala Pro Arg Ser Val Gln Ser Pro Thr
Gln Ala 690 695 700
Pro Lys Lys Glu Gln Asn Trp Leu Gly Gly Gly Trp Thr Asp Gly Ile 705
710 715 720 Glu Gln Pro Gly Trp
Gly Trp Glu Lys Val Thr Arg Arg Gln His Ala 725
730 735 Tyr Asp Phe Pro Met Trp Phe Ile Arg Pro
Asn Phe Lys Ser Glu Thr 740 745
750 Ala Pro Arg Ser Val Gln Ser Pro Thr Gln Ala Pro Lys Lys Glu
Gln 755 760 765 Asn
Trp Leu Gly Gly Gly Trp Thr Asp Gly Ile Glu Gln Pro Gly Trp 770
775 780 Gly Trp Glu Lys Val Thr
Arg Arg Gln His Ala Tyr Asp Phe Pro Met 785 790
795 800 Trp Phe Ile Arg Pro Asn Phe Lys Ser Glu Thr
Ala Pro Arg Ser Val 805 810
815 Gln Ser Pro Thr Gln Ala Pro Lys Lys Glu Gln Asn Trp Leu Gly Gly
820 825 830 Gly Trp
Thr Asp Gly Ile Glu Gln Pro Gly Trp Gly Trp Glu Lys Val 835
840 845 Thr Arg Arg Gln His Ala Tyr
Asp Phe Pro Met Trp Phe Ile Arg Pro 850 855
860 Asn Phe Lys Ser Glu Thr Ala Pro Arg Ser Val Gln
Ser Pro Thr Gln 865 870 875
880 Ala Pro Lys Lys Glu Gln Asn Trp Leu Gly Gly Gly Trp Thr Asp Gly
885 890 895 Val Gln Gln
Pro Gly Ser Gly Trp Glu Lys Val Thr Arg Arg Gln His 900
905 910 Ala Tyr Asp Phe Pro Met Trp Phe
Ile Arg Pro Asn Phe Lys Ser Glu 915 920
925 Thr Ala Pro Arg Ser Val Gln Ser Pro Thr Gln Ala Ser
Lys Lys Glu 930 935 940
Gln Asn Trp Leu Gly Gly Gly Trp Thr Asp Gly Ile Glu Gln Pro Gly 945
950 955 960 Trp Gly Trp Glu
Lys Val Thr Arg Arg Gln His Ala Tyr Asp Phe Pro 965
970 975 Met Phe Phe Ile Arg Pro Lys Phe Lys
Thr Ala Thr Ala Thr Arg Ser 980 985
990 Ala Gln Ser Pro Thr Gln Ser Val Lys Lys Ala Gln Asn
Trp Leu Gly 995 1000 1005
Gly Gly Trp Thr Asn Gly Pro Glu Gln Gly Gly Thr Gly Trp Glu
1010 1015 1020 Lys Ala Thr
Arg Arg Thr His Gly Tyr Asp Phe Pro Met Trp Phe 1025
1030 1035 Ile Arg Pro Asn Phe Lys Gln Thr
Asp Val Thr Val Lys Ser Ser 1040 1045
1050 Gln Ser Ala Thr Val Gly Asp Lys Lys Ser 1055
1060 24180PRTArtificial SequenceSynthetic peptide
24Met Lys Ser Gln Gln Gln Ala Lys Asp Trp Ile Tyr Lys His Glu Gly 1
5 10 15 Val Gly Val Asp
Phe Asp Gly Ala Tyr Gly Phe Gln Cys Met Asp Leu 20
25 30 Ala Val Ala Tyr Ile Tyr Tyr Ile Thr
Asp Gly Lys Val Arg Met Trp 35 40
45 Gly Asn Ala Lys Asp Ala Ile Asn Asn Asp Phe Lys Met Lys
Ser Gln 50 55 60
Gln Gln Ala Lys Glu Trp Ile Tyr Lys His Glu Gly Ala Gly Val Asp 65
70 75 80 Phe Asp Gly Ala Tyr
Gly Phe Gln Cys Met Asp Leu Ser Val Ala Tyr 85
90 95 Val Tyr Tyr Ile Thr Asp Gly Lys Val Arg
Met Trp Gly Asn Ala Lys 100 105
110 Asp Ala Ile Asn Asn Asp Phe Lys Met Lys Ser Gln Gln Gln Ala
Lys 115 120 125 Glu
Trp Ile Tyr Asn His Glu Gly Ala Gly Val Asp Phe Asp Gly Ala 130
135 140 Tyr Gly Phe Gln Cys Met
Asp Leu Ala Val Ala Tyr Val Tyr Tyr Ile 145 150
155 160 Thr Asp Gly Lys Val Arg Met Trp Gly Asn Ala
Lys Asp Ala Ile Asn 165 170
175 Asn Asp Phe Lys 180
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