Patent application title: COMPOSITIONS AND METHODS FOR ANALYZING HISTIDINE PHOSPHORYLATION
Inventors:
Magda Stankova (Tucson, AZ, US)
Fahad Al-Obeidi (Tucson, AZ, US)
Jacques Mauger (Oro Valley, AZ, US)
Robert A. Binnie (Tucson, AZ, US)
Tony Hunter (Del Mar, CA, US)
Jill Meisenhelder (Vista, CA, US)
Stephen Rush Fuhs (San Diego, CA, US)
IPC8 Class: AC07K706FI
USPC Class:
506 18
Class name: Library, per se (e.g., array, mixture, in silico, etc.) library containing only organic compounds peptides or polypeptides, or derivatives thereof
Publication date: 2015-03-05
Patent application number: 20150065395
Abstract:
A peptide is disclosed of the general structure: Z--W--Y, wherein Z and Y
are independently a one to eight amino acid sequence wherein the amino
acids are selected from glycine and alanine and W is a non-hydrolyzable
pHis analogue. Such peptides can be used to produce sequence-independent
anti-phosphohistidine antibodies. Also provided are antibodies that
specifically bind to a peptide comprising a phosphohistidine (or a
non-hydrolyzable pHis analogue) but fail to specifically bind to an
identical peptide containing histidine instead of phosphohistidine.Claims:
1. A peptide comprising the structure Z--W--Y wherein Z is a sequence
selected from the group consisting of X1, X1X2,
X1X2X3, X1X2X3X4 (SEQ ID NO: 1),
X1X2X3X4X5 (SEQ ID NO: 2),
X1X2X3X4X5X6 (SEQ ID NO: 3),
X1X2X3X4X5X6X7 (SEQ ID NO: 4) and
X1X2X3X4X5X6X7X8 (SEQ ID NO: 5);
W is an amino acid selected from ##STR00012## Y is a sequence selected
from the group consisting of X11, X11X12,
X11X12X13, X11X12X13X14 (SEQ ID NO:
6), X11X12X13X14X15 (SEQ ID NO: 7),
X11X12X13X14X15X16 (SEQ ID NO: 8),
X11X12X13X14X15X16X17 (SEQ ID NO: 9)
and X1X12X13X14X15X16X17X18 (SEQ
ID NO: 10); wherein X1, X2, X3, X4, Xs, X6,
X7, X8, X11, X12, X13, X14, X15,
X16, X17 and X18 are independently alanine or glycine.
2. The peptide of claim 1 further comprising a single cysteine, tyrosine or lysine linked to the amino or carboxy terminus of said peptide.
3. The peptide of claim 1 further comprising an N-terminal cysteine.
4. The peptide of claim 3 wherein the N-terminal amine is acylated and the C-terminal carboxylic acid group is replaced with an amide.
5. The peptide of claim 4 wherein both Z and Y are four amino acids in length.
6-10. (canceled)
11. A composition/library comprising a plurality of different peptides of claim 2.
12. The composition of claim 11 comprising 256 different peptides wherein each of said 256 peptides comprises the structure Cys-Z--W--Y.
13. The composition of claim 11 wherein each of said peptides comprise an N-terminal cysteine, said peptides being covalently coupled to a carrier protein through the side chain of said cysteine amino acid, said composition further comprising an adjuvant, wherein said peptides are emulsified in said adjuvant.
14-33. (canceled)
Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 USC §119(e) of U.S. Provisional Application Ser. Nos. 61/610,180, filed on Mar. 13, 2012, and 61/641,607, filed on May 2, 2012. The disclosure of each provisional application is hereby expressly incorporated by reference in its entirety.
INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0002] Incorporated by reference in its entirety is a computer-readable sequence listing submitted concurrently herewith and identified as follows: one 75,000 byte ASCII (text) file named "seqlist_phosphohis_ST25.txt," created on Mar. 13, 2013.
BACKGROUND
[0003] Protein phosphorylation regulates virtually all cellular processes and aberrant phosphorylation is the underlying cause of numerous cancers. The majority of intracellular proteins are phosphorylated at any given time, and, while 9 of the 20 amino acids can be phosphorylated, Ser, Thr, and Tyr phosphorylation has attracted the majority of attention in eukaryotic organisms. Histidine (His) phosphorylation has long been implicated in signal transduction (e.g. prokaryotic "two-component" systems); however, its role in mammalian cells remains largely unexplored. This is due to the difficulty in studying His phosphorylation by standard biochemical techniques.
[0004] Unlike pTyr, pSer and pThr, phosphohistidine (pHis) is heat and acid labile. Consequently, the importance of His phosphorylation has been greatly underestimated. Despite the brief half-life of pHis under acidic conditions (18-25 sec half-life in 1 M HCl, 49° C.), the high-energy, phosphoramidate bond of pHis is stabilized by basic conditions, and the half-life in protein substrates is strongly influenced by neighboring amino acid residues indicating it is highly context dependent (12 day half-life of histone H4 at RT, pH 7.6). Thus, new tools are needed to bring this under-appreciated post-translational modification to light.
[0005] Despite the paucity of knowledge about His phosphorylation in eukaryotic signal transduction, there is growing evidence implicating His kinases in cancer and tumor metastasis. In fact, the first metastasis suppressor gene identified (by its reduced expression in highly metastatic melanoma cell lines) is one of only two known mammalian His kinases; Nm23-H1 (AKA NME1 or nucleoside diphosphate kinase [NDPK-A]). Nm23 family members are involved in intracellular nucleotide homeostasis as well as in both physiological and pathophysiological cellular processes such as proliferation, differentiation, development, apoptosis, cytokinesis and metastasis, through mechanisms that remain largely unknown.
[0006] Nm23-H1 and the closely related Nm23-H2 (NME2/NDPK-B) catalyze transfer of phosphate from ATP onto nucleoside diphosphates (NDPs) through a pHis enzyme intermediate. Nm23-H1/-H2 also possess His kinase activity, transferring the phosphate from the active site pHis onto a His in a target protein. However, the lack of pHis-specific antibodies (Abs) and pHis's instability under typical conditions used for proteomics have made it difficult to study the role His phosphorylation plays in suppression of metastasis. Phosphospecific Abs exist for pSer, pThr and pTyr, and these, combined with biochemical and proteomic techniques, have proved invaluable in the study of protein phosphorylation in cellular signaling and cancer. Until recently, the difficulties in creating stable pHis peptides have precluded generation of pHis specific Abs. Development of non-cleavable pHis analogues now makes this possible [Kee et al., (2010) J Am Chem Soc. 132, 14327-9 and McAllister et al., (2011) Chem Commun. 47, 1297-9]. These pHis analogues will be used as immunogens to make Abs specific for both biologically relevant pHis isomers, 1- and 3-pHis (FIG. 1). Anti-1- and 3-pHis Abs will serve as novel tools to study His kinase activity of Nm23 proteins as well as yet undiscovered His kinases and their substrates.
[0007] It has been estimated that 6% of phosphorylation in eukaryotes occurs on His and that pHis could be 10-100 times more abundant than pTyr. Despite this, only a handful of mammalian pHis proteins, two His kinases (Nm23-H1/-H2) and a single pHis phosphatase (PHPT1) have been identified. For the few known pHis substrates, this phosphorylation has proved essential to their function and revealed novel signaling pathways. Nm23-H2 phosphorylates KCa3.1 (H358) and is required for potassium channel activation. Phosphorylation of heterotrimeric Gs protein subunit β1 (H266) by Nm23-H2 activates Gs and regulates basal cAMP accumulation. Furthermore, Nm23-H2, G proteins and caveolin expression are mutually dependent for stable localization and caveolae formation. Histone H4 phosphorylation (H18) is associated with enhanced cell proliferation in liver and thymus. The development of pHis-specific Abs combined with improved techniques for pHis peptide enrichment and identification by MS will be used to greatly expand the number of known pHis targets and determine which ones play a role in suppression of tumor metastasis by His kinases. In addition, this protocol may identify novel His kinases, since known His kinases autophosphorylate on His.
SUMMARY
[0008] As disclosed herein peptides are provided that comprise non-hydrolyzable phosphohistidine analogues, 1-pTza & 3-pTza, for use as immunogens (see FIG. 1). Such peptides, comprised only of immunogenically neutral amino acids with small side chains (e.g., alanine and glycine) and a non-hydrolyzable phosphohistidine analogue, are used in generating antibodies that bind to proteins that comprise a phosphohistidine, but fail to bind to proteins of identical sequence wherein the histidine residue is not phosphorylated. In one aspect of the present disclosure, peptides comprising a library of random sequences (Lam, K. et. al., (1991) Nature, 354, 82-4) of immunogenically neutral amino acids with short side chains (e.g., alanine and glycine) in addition to one of the two non-hydrolyzable pHis analogues are used to generate sequence-independent anti-pHis Abs.
[0009] The present disclosure is also directed to methods of generating sequence-independent anti-pHis antibodies as well as the sequence-independent anti-phosphohistidine (pHis) specific poly- and monoclonal anti-phosphohistidine (pHis) antibodies themselves and hybridoma cell lines expressing such antibodies. In accordance with one embodiment an antibody is provided that specifically binds to a peptide or protein comprising a phosphorylated histidine, independent of amino acid sequence, but does not specifically bind to a peptide of identical sequence lacking a phosphorylated histidine. The antibody can be either a polyclonal or monoclonal antibody. Hybridoma cell lines and procaryotic cells producing such antibodies in a recombinant form are also encompassed by the present invention.
[0010] The present invention is also directed to a method of identifying proteins comprising a phosphorylated histidine, comprising the steps of contacting a sample comprising a protein with the sequence-independent anti-phosphohistidine (pHis) specific poly- or monoclonal anti-phosphohistidine (pHis) antibodies disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1. Non-hydrolyzable pHis analogues: 3-pTza and 1-pTza. Structures of phosphohistidine isomers; 3- and 1-phosphohistidine (3-pHis and 1-pHis) are shown in FIG. 1. Non-hydrolyzable 3-pHis and 1-pHis analogues, 1-phosphoryltriazolylalanine (1-pTza) and 3-phosphoryltriazolylalanine (3-pTza), are synthesized via a "click chemistry" reaction between an azidoalanine and an alkyne. The same building blocks with different catalysts are used to make both isomers.
[0012] FIG. 2. Incorporation of the pHis analogues 1-pTza and 3-pTza into immunizing peptides. Three synthetic peptide libraries have been synthesized, consisting of either histidine or a non-hydrolyzable pHis analogue flanked by randomized, non-immunogenic amino acids (glycine [R═H] and alanine [R═CH3]), to promote generation of sequence-independent anti-pHis Abs. Each library is a complex mixture of peptides (28=256) that are acylated at the N-terminus and contain an N-terminal L-cysteine for coupling to a carrier protein such as keyhole limpet hemocyanin (KLH). A. The library incorporating histidine will be used for negative selection of Abs that recognize non-phosphorylated histidine. B. & C. The immunizing peptide libraries, one for each isomer, contain the non-hydrolyzable analogues, 1-pTza or 3-pTza.
[0013] FIG. 3 Amino acid sequence alignment of the histidine kinases Nm23-H1 and Nm23-H2. Human Nm23-H1 (SEQ ID NO: 13) and Nm23-H2 (SEQ ID NO: 11) proteins are 88% identical. Mouse Nm23-H1 (SEQ ID NO: 14) is 98% identical to human Nm23-H1 (SEQ ID NO: 13); and mouse Nm23-H2 (SEQ ID NO: 12) is 94% identical to human Nm23-H2. Non-conserved residues are highlighted in grey. The catalytic, autophosphorylation site at H118 is indicated in bold letter. Nm23-H1 and --H2 autophosphorylate themselves on the catalytic His residue (H118). The kinases themselves can therefore serve as positive and negative controls for testing the specificity of the rabbit antisera in applications such as immunoblotting and immunoprecipitation.
[0014] FIG. 4. Purification of recombinant Nm23-H1 & -H2. Wild type (WT) and the autophosphorylation site catalytic mutant (H118Y) of Nm23-H1 and Nm23-H2 were cloned into a GST fusion vector (pGEX-6P-1), transformed into the BL21 derivate, Rosetta 2(DE3) and expressed by IPTG induction. GST-tagged Nm23 proteins were purified with glutathione resin (GenScript). Nm23 proteins were released from the resin by cleavage of GST tags. Purification and complete cleavage of GST tag was assessed by SDS-PAGE and Coomassie staining. As expected, recombinant Nm23-H1 and Nm23-H2 migrate at 18 kDa and 16 kDa respectively.
[0015] FIGS. 5A-5C. provides the results of immunization of rabbits with the 1-pTza and 3-pTza synthetic peptide libraries. Three New Zealand White rabbits were immunized with the 3-pTza synthetic peptide library (7302, 7303 and 7304), and three rabbits were immunized with the 1-pTza synthetic peptide library (7305, 7306 and 7307). Bleeds were taken at 30 days after initial immunization and 10 days after subsequent immunizations. Dot blots consisting of the two peptide, phosphohistidine analogs (1-pTza and 3-pTza), as well as a non-phosphylated negative control (His) and a phosphorylated tyrosine negative control (pTyr) were analyzed to measure antibody titer in the rabbits. Bleed 1 (FIG. 5A) fails to detect IgG against the phosphorylated peptide analogs as expected, since early antisera contains mostly IgM. Bleed 2 (FIG. 5B) reveals antibodies having specificity for the respective immunogen. Specifically, rabbit serum was used at 1:500 dilution (and not affinity purified) and 5 of 6 rabbits' serum shows reactivity specific to the immunogen, with no cross-reactivity between 1-pTza and 3-pTza isomers or His and pTyr peptides. An antibody that binds phosphorylated tyrosine (4G10) detects the pTyr peptide on the dot blots as indicated in FIGS. 5A and 5C. Similar results were obtained for Bleeds 3 and 4.
[0016] FIGS. 6A & 6B. demonstrate the polyclonal antibodies are capable of binding to proteins autophosphorylated on histidine as well as the 1-pTza and 3-pTza analogs. Recombinant human Nm23 (rNm23) was synthesized and GST-tagged Nm23 proteins purified using a glutathione resin. The column was washed and GST was cleaved on resin with PreScission protease, releasing Nm23 which was then concentrated and allowed to autophosphoryate using standard procedures. Nm23 proteins were incubated for 10 min at room temperature in TMD buffer (20 mM Tris pH 8.0, 5 mM MgCl2, 1 mM DTT) with 1 uM ATP. FIG. 6A shows the results of anti-1-pTza antisera to detect in vitro Nm23 autophosphorylation. Lane 1 indicates a doublet that is not present in lane 3 (the negative control where Nm23 phosphorylation was conducted in the absence of ATP). FIG. 6B shows the results of anti-3-pTza antisera to detect in vitro Nm23 autophosphorylation. Only background/non-specific binding was detected.
[0017] FIGS. 7A & 7B. provide data demonstrating that the detected rNm23 autophosphorylation is reversible. Recombinant Nm23-H1 and Nm23-H2 were autophosphorylated in vitro. 10 min incubation at RT in TMD Buffer (20 mM Tris pH=8.0, 5 mM MgCl2, 1 mM DTT)+/-ATP. After autophosphorylation, the 100 ul reaction was treated with 25 ul 1M HCL at 37° C. for 15 min. The reaction was neutralized with 25 ul 1M NaOH. Acid treatment of the autophosphorylated rNm23 significantly reduces the binding of anti-1-pTza 7305-2 to the pNm23 band as shown in an immuno-(western) blot probed with anti-1-pTza antisera 1:500 (lane 5, see FIG. 7A). Coomassie stained gel demonstrates the presence of the rNm23-H1/H2 and shows a reduction in the phosphorylated pNm23-H1 in the acid treated lane (see FIG. 7B).
[0018] FIGS. 8A & 8B. show the coomassie detection limit vs. the anti-1-pTza antisera detection limit, respectively for rNm23-H1 autophosphorylation. The coomassie limit of detection was approximately 25-50 ng, whereas the anti-1-pTza antisera detection limit was 5 to 10 times higher. Specifically, bleed 3 is approximately 5-10-fold higher titer than bleed 2 and 7305-4 has 2 fold higher titer than 7305-3. 7306-4 only showed non-specific binding (despite a positive result in 1-pTza peptide dot blots). FIG. 8C shows the titer levels continue to increase in the fourth bleed of 7306 and 7305 rabbits. FIG. 8D shows that a polyclonal antibody against Nm23 that detects both phosphorylated & unmodified Nm23-H1 detects both bands on a western blot. FIG. 8E compares the titer of bleed 4 vs. bleed 5 for detecting rNm23-H1 autophosphorylation using 7305 anti-sera with the 5th bleed approximately two fold higher than the 4th bleed.
[0019] FIGS. 9A & 9B. presents data showing stable cell lines expressing FLAG-Nm23-H1. 293T cells were transfected with DNA constructs expression FLAG-Nm23-H1 and stable lines were identified. The clones with the highest expression relative to tubulin were used for all subsequent pHis immunoprecipitation experiments. Screening cell lines (See FIGS. 9A & 9B) revealed that clone 20 exhibited the highest expression of Nm23-H1 relative to tubulin. As shown in FIG. 9B, 7305-5 anti-serum can detect pFLAG-Nm23-H1.
[0020] FIGS. 10A & 10B. show the data for four positive hybridoma multiclones and subclones recovered from the 7305 rabbit and their ability to bind to pGEX-Nm23-H1 transformed E. coli lysates. FIG. 10A shows the results of anti-1-pHis antibodies recovered from four multiclones identified by 1-pTza peptide ELISA used to probe a western blot of pGEX-Nm23-H1 transformed E. coli lysates. Lysates were loaded with or without first being incubated at 95° C. The results show that the multiclonal anti-1-pHis antibodies (with 7 being an example of a 1-pTza ELISA positive, but pHis negative multiclone) bound to the autophosphorylated Nm23-H1 protein, and that binding was heat sensitive. Three hybridoma multiclones from rabbit 7305 were subsequently selected for subcloning and generation of monoclonal hybridoma cell lines. FIG. 10B shows data from a subset of four such subclones derived from multiclones 1, 50 and 77. As in FIG. 10A, lysates from pGEX-Nm23-H1 transformed E. coli were loaded with or without first being incubated at 95° C. Each of the subclones could specifically bind histidine phosphorylated Nm23-H1, as well as other histidine-phosphorylated proteins present in the E. coli lysate.
[0021] FIG. 11. shows the results of the 3-pHis screening with a preparation of phosphoglycerate mutase (PGAM) in vitro phosphorylated in the presence of 2,3-DPG, and using polyclonal antibodies recovered from the 7303 rabbit (bleed 9). Identical samples were heated to 95° C. for 10 min to confirm the detected signal results from binding to 3-pHis. 7303 (3-pTza rabbit) antisera detects only phosphorylated PGAM. Polyclonal antibodies recovered from the 7304 rabbit also produced a signal but much weaker than for 7303.
[0022] FIGS. 12A & 12B. shows the results of screening subclones of 3-pHis hybridoma multiclone 7303 MC8 with PGAM+/-heat & acid. FIG. 12A is an initial screen of subclones (8-1 to 8-11) and FIG. 12B is a rescan of the positive 7303 subclones identified in FIG. 12A, wherein the image was optimized to more clearly show the weak signal for phospho-PGAM. Lane 1 was treated with acid and heat, and so no phosphohistidines should be present; lanes 2 and 3 represent different concentrations of in vitro autophosphorylated PGAM (25 and 500 ng, respectively). Positive controls using polyclonal antibodies from the 11th bleed of the rabbit 7303 (7303-11) and affinity-purified 3-pHis antibodies show binding to the autophosphorylated PGAM at both concentrations. 7 of 12 subclones produced a weak signal for phospho-PGAM 500 ng.
DETAILED DESCRIPTION
Definitions
[0023] In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below.
[0024] The term "about" as used herein means greater or lesser than the value or range of values stated by 10 percent, but is not intended to limit any value or range of values to only this broader definition. Each value or range of values preceded by the term "about" is also intended to encompass the embodiment of the stated absolute value or range of values.
[0025] As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.
[0026] As used herein, the term "treating" includes prophylaxis of the specific disorder or condition, or alleviation of the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms.
[0027] The term "antibody" or "antibodies" as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE, including Fab or antigen-recognition fragments thereof. The antibodies may be monoclonal or polyclonal and may be of any species of origin, including (for example) mouse, rat, rabbit, goat, sheep, pig, chicken, horse, or human, or may be chimeric antibodies, or camelid (single chain) antibodies. See, e.g., M. Walker et al., Molec. Immunol. 26: 403-11 (1989); Morrison et al., Proc. Nat'l. Acad. Sci. 81: 6851 (1984); Neuberger et al., Nature 312: 604 (1984)). The antibodies may be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No. 4,474,893 (Reading) or U.S. Pat. No. 4,816,567 (Cabilly et al.), or by recombining variable regions into expression vectors. The antibodies may also be chemically constructed according to the method disclosed in U.S. Pat. No. 4,676,980 (Segel et al.).
[0028] The term "specifically bind" means a special and precise interaction between two molecules which is dependent upon their structure, resulting in a high preference of binding between the two molecules relative to other molecules.
[0029] As used herein the term "patient" without further designation is intended to encompass any warm blooded vertebrate domesticated animal (including for example, but not limited to livestock, horses, cats, dogs and other pets) and humans.
[0030] In the present disclosure, a "chimeric antibody" refers to an antibody obtained by linking a variable region of an antibody of one species with a constant region of an antibody of another species. For example, such a chimeric antibody can be obtained as follows. A mouse is immunized with an antigen. A portion coding an antibody variable part (variable region) which binds to the antigen is cut out from a gene coding a monoclonal antibody of the mouse. The portion is linked with a gene coding a human bone marrow-derived antibody constant part (constant region). These linked genes are incorporated in an expression vector. The expression vector is then introduced into a host which produces a chimeric antibody (Refer to, for example, Japanese Unexamined Patent Application Publication No. Hei 8-280387, U.S. Pat. No. 4,816,397, U.S. Pat. No. 4,816,567, and U.S. Pat. No. 5,807,715).
[0031] In the present disclosure, a "humanized antibody" refers to an antibody obtained by grafting a genome sequence of an antigen-binding site (CDR) of a non-human-derived antibody onto a gene of a human antibody (CDR grafting). Preparation methods of such chimeric antibodies have been known (refer to, for example, EP239400, EP125023, WO90/07861, and WO96/02576). In the present invention, a "functional fragment" of an antibody means a part (a partial fragment) of an antibody, which retains a capability of specifically recognizing an antigen of the antibody from which the part is originated. Specific examples of the functional fragment include Fab, Fab', F(ab')2, a variable region fragment (Fv), a disulfide-linked Fv, a single-chain Fv (scFv), sc(Fv)2, a diabody, a polyspecific antibody, and polymers thereof.
EMBODIMENTS
Detailed Description
[0032] As disclosed herein a library of random peptide sequences is provided wherein each peptide sequence comprises an analog of phosphohistidine wherein the phosphate group is stably linked as a phosphonate to a carbon in the histidine analog. More particularly, two non-hydrolyzable phosphohistidine analogues, 1-pTza & 3-pTza, respectively (see FIG. 1), have been incorporated into synthetic peptides for use in generating antibodies specific for proteins that comprise a phosphohistidine. In one aspect of the present disclosure peptides comprised of only immunogenically neutral amino acids (e.g., alanine and glycine) and a non-hydrolyzable pHis analogue are used to promote generation of sequence-independent anti-pHis Abs. In one embodiment the non-hydrolyzable pHis analogue is 1-pTza or 3-pTza. In a further embodiment the peptide comprises an amino acid that is linked to a hapten carrier protein. In one embodiment the carrier protein is keyhole limpet hemocyanin. In a further embodiment the 1-pTza or 3-pTza containing peptide further comprises a lysine, tyrosine or cysteine amino acid having a carrier protein linked to this amino acid. In one embodiment the 1-pTza or 3-pTza containing peptide comprises a cysteine covalently linked to keyhole limpet hemocyanin through a disulfide bond formed with the cysteine side chain.
[0033] In accordance with one embodiment a peptide is provided with the general formula Z--W--Y, wherein Z and Y are amino acid sequences comprised of immunogenically neutral amino acids having short side chains, and independently ranging in length from about 2 to about 8 amino acids in length, and W is a non-hydrolyzable pHis analogue. In one embodiment the amino acids are either glycine or alanine; however, any amino acid (natural or synthetic) can be used that tends not to induce a specific immune response. In one embodiment the non-hydrolyzable pHis analogue is 1-pTza or 3-pTza. In one embodiment the peptide comprises an amino acid that can be used for covalent attachment of additional compounds including, for example, a carrier protein. In one embodiment an additional amino acid is added at the amino or carboxy terminus of the peptide of the formula Z--W--Y wherein the additional amino acid is cysteine, tyrosine or lysine. In one embodiment the peptide is further modified to include an amino (N)-terminal acylation, and/or a carboxy (C)-terminal amidation.
[0034] In one embodiment the peptide comprises the structure
Cys-Z--W--Y
wherein
[0035] Z is a sequence selected from the group consisting of X1, X1X2, X1X2X3, X1X2X3X4 (SEQ ID NO: 1), X1X2X3X4X5 (SEQ ID NO: 2), X1X2X3X4X5X6 (SEQ ID NO: 3), X1X2X3X4X5X6X7 (SEQ ID NO: 4) and X1X2X3X4X5X6X7X8 (SEQ ID NO: 5);
[0036] W is an amino acid selected from
##STR00001##
[0037] Y is a sequence selected from the group consisting of X11, X11X12, X11X12X13, X11X12X13X14 (SEQ ID NO: 6), X11X12X13X14X15 (SEQ ID NO: 7), X11X12X13X14X15X16 (SEQ ID NO: 8), X11X12X13X14X15X16X17 (SEQ ID NO: 9) and X11X12X13X14X15X16X17X18 (SEQ ID NO: 10); wherein
[0038] X1, X2, X3, X4, X5, X6, X7, X8, X11, X12, X13, X14, X15, X16, X17 and X18 are independently alanine or glycine. In one embodiment the N-terminal amine is acylated and the C-terminal carboxylic acid group is replaced with an amide. In another embodiment Z and Y are each independently 4 to 7 amino acid sequences. In one embodiment both Y and Z are four amino acids in length. In one embodiment one of the amino acids of Z or Y is substituted with an amino acid (e.g. histidine, tyrosine, lysine or cysteine) or such an amino acid is added to the N- or C-terminus of the peptide that allows for covalent attachment of additional moieties, such as carrier proteins. The peptide in some embodiment further comprises a carrier protein linked to the peptide, and in one embodiment the carrier protein is keyhole limpet hemocyanin covalently linked to the side chain of the cysteine of said peptide. In accordance with the present invention any carrier protein known to those skilled in the art can be used in accordance with the embodiment disclosed here.
[0039] In one embodiment a composition is provided comprising a plurality of peptides that differ in their primary sequence but conform to the general structure of Z--W--Y as defined herein. Such a composition is called a library. In one embodiment a library including at least 256 different peptides is provided wherein each of said 256 peptides has the structure Cys-Z--W--Y or Z--W--Y, wherein
[0040] Z is a sequence selected from the group consisting of X1, X1X2, X1X2X3, X1X2X3X4 (SEQ ID NO: 1), X1X2X3X4X5 (SEQ ID NO: 2), X1X2X3X4X5X6 (SEQ ID NO: 3), X1X2X3X4X5X6X7 (SEQ ID NO: 4) and X1X2X3X4X5X6X7X8 (SEQ ID NO: 5);
[0041] W is an amino acid selected from
##STR00002##
[0042] Y is a sequence selected from the group consisting of X11, X11X12, X11X12X13, X11X12X13X14 (SEQ ID NO: 6), X11X12X13X14X15 (SEQ ID NO: 7), X11X12X13X14X15X16 (SEQ ID NO: 8), X11X12X13X14X15X16X17 (SEQ ID NO: 9) and X11X12X13X14X15X16X17X18 (SEQ ID NO: 10); wherein
[0043] X1, X2, X3, X4, X5, X6, X7, X8, X11, X12, X13, X14, X15, X16, X17 and X18 are independently alanine or glycine. In a further embodiment the N-terminal amine is acylated and the C-terminal carboxylic acid group is replaced with an amide.
[0044] In one embodiment two separate libraries of peptides are provided. The first representing a 1-Phosphohistidine Mimetic Peptide Library and having the general structure:
AcCys-A/G-A/G-A/G-A/G-1-pTza-A/G-A/G-A/G-A/G-CONH2
[0045] and the second library representing a 3-Phosphohistidine Mimetic Peptide Library and having the general structure:
AcCys-A/G-A/G-A/G-A/G-3-pTza-A/G-A/G-A/G-A/G-CONH2,
[0046] wherein Ac=acylated N-Terminus, Cys=L-Cysteine for coupling antigens to KLH, A=Alanine, G=Glycine. The designation A/G indicates that either amino acid can be used at that position, thus each library contains a mixture of 28=256 peptides. In one embodiment the peptides are synthesized under conditions where an alanine or glycine is randomly added to the growing peptide chain during synthesis. 1-pTza and 3-pTza=stable phosphohistidine analogues (Kee et al., J. Am. Chem. Soc., 2010).
[0047] In accordance with one embodiment the general structure of the 1-pTza and 3-pTza Peptide Libraries is as follows, wherein X is a non-hydrolyzable pHis analogue
##STR00003##
[0048] wherein R is H or CH3.
[0049] More particularly, in one embodiment the structure of the 1-pTza Peptide Library is
##STR00004##
and the structure of the 3-pTza Peptide Library is
##STR00005##
[0050] The peptides disclosed herein can be used to generate sequence-independent anti-pHis antibodies. In accordance with one embodiment a method for producing sequence-independent anti-pHis antibodies is provided wherein the method comprises
[0051] i) immunizing a host with a peptide of the general structure Z--W--Y as disclosed herein; and
[0052] ii) obtaining an antibody-containing host serum produced as a response to said immunization.
[0053] In one embodiment the host is immunized with a composition comprising a peptide of the general structure Z--W--Y wherein
[0054] Z is a sequence selected from the group consisting of X1, X1X2, X1X2X3, X1X2X3X4 (SEQ ID NO: 1), X1X2X3X4X5 (SEQ ID NO: 2), X1X2X3X4X5X6 (SEQ ID NO: 3), X1X2X3X4X5X6X7 (SEQ ID NO: 4) and X1X2X3X4X5X6X7X8 (SEQ ID NO: 5);
[0055] W is an amino acid selected from
##STR00006##
[0056] Y is a sequence selected from the group consisting of X11, X11X12, X11X12X13, X11X12X13X14 (SEQ ID NO: 6), X11X12X13X14X15 (SEQ ID NO: 7), X11X12X13X14X15X16 (SEQ ID NO: 8), X11X12X13X14X15X16X17 (SEQ ID NO: 9) and X11X12X13X14X15X16X17X18 (SEQ ID NO: 10); wherein
[0057] X1, X2, X3, X4, X5, X6, X7, X8, X11, X12, X13, X14, X15, X16, X17 and X18 are independently alanine or glycine. In a further embodiment each of the peptides of the libraries are covalently linked to a carrier protein such as KLH. In one embodiment each of the peptides of the libraries comprises one or more peptide selected from the group consisting of lysine, cysteine and tyrosine wherein the carrier protein is linked to the side chain of the lysine, cysteine and/or tyrosine amino acid. Composition comprising the libraries (with or without the covalently linked carrier protein) can be optionally combined with an adjuvant to enhance the immunogenic response when the composition is injected into an animal. In one embodiment the adjuvant is Freund's Complete or Incomplete Adjuvant that is mixed/emulsified with the peptide-carrier protein prior to being injected.)
[0058] In one embodiment the host is immunized with a 1-pTza Peptide Library of compounds having the general structure of
##STR00007##
or with a 3-pTza Peptide Library of compounds having the general structure of
##STR00008##
[0059] In one embodiment the peptides comprising the pTza Peptide Library further include a covalently bound carrier protein such as KLH, to create a hapten antigen. Any adjuvant known to those skilled in the art can be used in the method of generating antibodies. In one embodiment the adjuvant is selected from the group consisting of Bacille Calmette-Guerin, Complete Freund's adjuvant, Incomplete Freund's adjuvant and QS-21 (QS-21 is a purified plant extract that enhances the ability of the immune system to respond to vaccine antigens. It is derived from the Soap bark tree.).
[0060] Polyclonal antibodies of the invention may be produced according to standard techniques by immunizing a suitable animal (e.g., rabbit, goat, etc.) with an antigen encompassing a peptide for the general formula Z--W--Y as disclosed herein, covalently linked to a carrier protein, and emulsified with an adjuvant. Immune serum is collected from the animal after immunization using standard techniques, and the polyclonal antibodies are then isolated from the immune serum, in accordance with known procedures of affinity purification. The present invention also encompasses the antibodies produced by applicants' invention. In accordance with one embodiment an affinity column is provided for isolating anti-pHis antibodies, wherein the affinity column comprises a peptide of the general formula Z--W--Y as disclosed herein covalently linked to a solid support.
[0061] Mouse or rabbit monoclonal antibodies of the invention may be produced in a hybridoma cell line according to standard techniques known to those skilled in the art including for example, the well-known technique of Kohler and Milstein. Nature 265: 495-97 (1975); Kohler and Milstein, Eur. J. Immunol. 6: 511 (1976); see also, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al. Eds. (1989). Commercial vendors including, for example, Epitomics can be contracted to undertake splenocyte fusion and hybridoma production after being supplied with spleens form mice or rabbits immunized with an appropriate immunogen. Monoclonal antibodies so produced are highly specific, and improve the selectivity and specificity of diagnostic assay methods provided by the invention. For example, a solution containing the appropriate antigen may be injected into a mouse and, after a sufficient time (in keeping with conventional techniques), the animal is sacrificed and spleen cells obtained. The spleen cells are then immortalized by fusing them with myeloma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells. Alternatively, rabbit fusion hybridomas may be produced as described in U.S. Pat. No. 5,675,063, C. Knight, Issued Oct. 7, 1997. The rabbit spleen cells are fused with a (previously patented) suitable fusion partner to create an immortal cell line which is then grown in a suitable selection media, such as one including hypoxanthine-aminopterin-thymidine (HAT), and the cell supernatant screened for monoclonal antibodies having the desired specificity. The secreted antibody may be recovered from tissue culture supernatant by conventional methods such as precipitation, ion exchange or affinity chromatography, or the like.
[0062] Monoclonal Fab fragments may also be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, Science 246: 1275-81 (1989); Mullinax et al., Proc. Nat'l Acad. Sci. 87: 8095 (1990). If monoclonal antibodies of one isotype are preferred for a particular application, particular isotypes can be prepared directly, by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class-switch variants (Steplewski, et al., Proc. Natl. Acad. Sci., 82: 8653 (1985); Spira et al., J. Immunol. Methods, 74: 307 (1984)). The invention also provides hybridoma clones that produce monoclonal antibodies specific for any protein containing a phosphohistidine. The heavy and light chain sequences from a mouse or rabbit hybridoma can be cloned and sequenced, and introduced into both eukaryotic and prokaryotic expression vectors to produce recombinant antibodies.
[0063] In accordance with the present disclosure the antibodies produced by immunizations with the peptides disclosed herein will specifically bind to peptides comprising a phosphohistidine, but will not specifically bind to an identical peptide that comprises a histidine in place of the phosphohistidine. In one embodiment the antibody is polyclonal, and in another embodiment the antibody is monoclonal. The present invention also encompasses any hybridoma cell line producing such antibody. Accordingly, the antibodies of the present disclosure will specifically bind to histidine bearing amino acid sequences only when the protein is phosphorylated at the histidine (i.e, the protein comprises a 1- or -3-pHis). In one embodiment the antibody will specifically bind to an amino acid sequence of at least 3, 4, 5, 8 or 10 amino acids in length that bears a phosphohistidine, optionally an internal phosphohistidine. In one embodiment the antibody will specifically bind to an amino acid sequence bearing a phosphohistidine, optionally an internal phosphohistidine, wherein the amino acid sequence has a maximum length of 1,000, 800, 600, 500, 400, 300, 200, 100, 50, 25, 15, 10 or 5 amino acids. In one embodiment the antibody will be specific for any and all amino acid sequences bearing a phosphohistidine (i.e., will bind to proteins containing a phosphohistidine independent of amino acid sequence, but will not bind to an amino acid sequence lacking a phosphohistidine).
[0064] In one embodiment an isolated antibody, or an antigen-binding fragment thereof is provided wherein the antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 19, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 20. In one embodiment the antibody is a monoclonal antibody and in a further embodiment the antibody is a rabbit antibody. In one embodiment the antibody is recombinant. In one embodiment the antibody specifically binds to peptides comprising a phosphohistidine, but will not specifically bind to an identical peptide that comprises a histidine in place of the phosphohistidine. In one embodiment the antibody will specifically bind to an amino acid sequence that comprises a 1-pHis amino acid. In one embodiment the antibody is produced by injecting a mammal with an immunogen comprising a 1-pTza Peptide Library of compounds having the general structure of
##STR00009##
or with a 3-pTza Peptide Library of compounds having the general structure of
##STR00010##
[0065] In a further embodiment the antibody is produced by injecting a mammal with the above immunogen, wherein the immunogen further comprises a carrier protein. In one embodiment an isolated polynucleotide is provided encoding an antibody that specifically binds to peptides comprising a phosphohistidine, but will not specifically bind to an identical peptide that comprises a histidine in place of the phosphohistidine. In one embodiment the antibody will specifically bind to an amino acid sequence that comprises a 1-pHis amino acid. In one embodiment the isolated polynucleotide encodes an antibody comprising the amino acid sequences of SEQ ID NO: 19 and 20. In one embodiment the nucleic acid sequence comprises SEQ ID NO: 21 and/or SEQ ID NO: 22.
[0066] In accordance with another embodiment the disclosure also provides a method for screening biological samples to detect proteins comprising a phosphohistidine. The method comprises the steps of:
[0067] (a) incubating/mixing a biological sample with at least one antibody that specifically binds to peptides comprising a phosphohistidine (i.e., will not bind to an identical peptide that comprises a histidine in place of the phosphohistidine) under conditions suitable for formation of a reagent-antibody complex (phosphohistidine-antibody complex). Since phosphohistidine is heat and acid labile, biological samples will be buffered at pH 8.0 or above and handled in such a manner that will preserve histidine phosphorylation (i.e. the avoidance of heat and acidic conditions whenever possible.) In addition, the phosphohistidine specific phosphatase PHPT1 may be inhibited or depleted from cells through RNA interference, either transiently or stably in a cell line expressing PHPT1 shRNAs.
[0068] (b) detecting the presence of said complex in said sample, wherein the presence of said complex indicates the presence of a protein comprising a phosphohistidine in said sample. In one embodiment the antibody is labeled. In one embodiment the anti-pHis antibody is not directly labeled but is detected via a labeled secondary antibody. Means for producing a detectable signal include the use of radioactive labels, fluorescent labels, enzyme labels, and so forth. Examples of suitable immunoassays are the radioimmunoassay, immunofluorescence methods, enzyme-linked immunoassays, immuno-(western) blotting, immunoprecipitation, and the like.
[0069] The peptides and antibodies disclosed herein may be conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques. Antibodies of the invention, may likewise be conjugated to detectable groups such as radiolabels (e.g., 3H, 14C, 33P, 35S, 125I, 131I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), far-red laser-activated dyes, and fluorescent labels (e.g., fluorescein) in accordance with known techniques.
[0070] In accordance with one embodiment an isolated or purified antibody is provided that specifically binds to a peptide or protein comprising a phosphorylated histidine independent of amino acid sequence. However, the antibody does not bind to an identical amino acid that lacks a phosphorylated histidine and only contains non-phosphorylated histidine. In one embodiment the antibody is a polyclonal antibody, and in another embodiment the antibody is a monoclonal antibody. The present disclosure is also directed to antibodies are humanized or are chimeric. Nucleic acids and vectors encoding the antibodies or portions thereof, recombinant cells that contain the nucleic acids, and compositions comprising the antibodies or antigen-binding fragments are also part of the present disclosure.
[0071] In one embodiment a kit is provided for detecting proteins that comprise a phosphohistidine. In one embodiment the kit comprises a polyclonal or monoclonal antibody that specifically binds to peptides/proteins comprising a phosphohistidine independent of the sequence of the peptide/protein. In one embodiment the kit further comprises a secondary antibody conjugated to a detectable label. The kit may alternatively or in addition include one or more containers, e.g., vials, tubes, bottles, and the like, optionally containing the antibody and assay reagents in a lyophilized form or in an aqueous solution. Preferably, the kits will also include instructions for use.
Example 1
Generation of Sequence Independent pHis Antibodies
[0072] Histidine exists as two tautomers and undergoes rapid tautomerization, with a hydrogen present at either N1 or N3 of the imidazole ring. Consequently, phosphorylation can occur at either N1 (1-pHis) or N3 (3-pHis) of the imidazole ring and isomerization can occur between N1 and N3. The local hydrogen-bonding environment of target His residues likely determines specificity of pHis at N1 or N3 in proteins.
[0073] Unsuccessful attempts have been made to generate pHis antibodies--pHis containing peptide immunogens are hydrolyzed too rapidly to elicit an immune response. In 2010, two groups (Muir-Rockefeller & Webb-Leeds) successfully synthesized stable analogs of pHis (pTza). The Muir group successfully incorporated the analogs into synthetic peptides, and raised the first sequence-specific pHis antibody (histone H4). pHis substrates lack an obvious pattern/motif in flanking amino acids and so it has been difficult to generate pHis antibodies that recognize more than one single kinase target sequence.
[0074] To generate sequence independent pHis antibodies, non-hydrolyzable phosphohistidine analogs were synthesized and then incorporated into synthetic peptides containing these pHis analogs flanked by randomized Gly and Ala. These peptide libraries were coupled to KLH, emulsified with Freund's adjuvant, and injected into New Zealand White rabbits. The rabbits were bled and antibodies recovered by affinity purification. Validation of the recovered anti-1-pHis and anti-3-pHis polyclonal antibodies was conducted based on standard immunoblot and immunoprecipitation procedures using purified pHis proteins and cell lysates.
Materials and Methods
[0075] Reagents and their sources were as follows: KLH for peptide conjugation (Calbiochem; catalog no. 374817), Rosetta 2 (DE3) Competent Cells (Novagen; Catalog no. 71397), Glutathione Resin (GenScript; catalog no. L00206), PreScission Protease (GE Healthcare; catalog no. 27-0843-01), PBS blocking buffer with 1% Casein (BioRad; catalog no. 161-0783). Freund's Complete Adjuvant and Freund's Incomplete Adjuvant (Calbiochem; catalog no. 344289 and 344291), Adenosine 5'-triphosphate disodium salt solution (Sigma; catalog no. A6559), 2,3-diphosphoglycerate (Sigma; catalog no. D5746).
Immunizing Peptide Library Synthesis
[0076] Three synthetic peptide libraries have been synthesized, consisting of either histidine or a non-hydrolyzable pHis analogue flanked by randomized amino acids (glycine [R═H] and alanine [R═CH3]), to promote generation of sequence-independent anti-pHis Abs. Each library is a complex mixture of peptides that are acylated at the N-terminus and contain L-cysteine for coupling to KLH. The three libraries each comprise polypeptides 10 amino acid in length and of the general structure:
##STR00011##
wherein
[0077] R is independently Gly or Ala (randomly assigned);
[0078] X is His, 1-pTza or 3-pTza (pTza=phosphoryltriazolylalanine);
[0079] each polypeptide is acylated at the N-terminus; and each polypeptide comprises an L-Cysteine (e.g., at or near the N- or C-terminus) for chemical ligation to a carrier protein (e.g., KLH). The total number of polypeptide combinations is 28×3=768, and thus each of the three libraries (His, 1-pTza and 3-pTza contain 256 unique polypeptides
[0080] The library incorporating histidine was used for negative selection of Abs that recognize non-phosphorylated histidine. The immunizing peptide libraries, one for each isomer, contain the non-hydrolyzable analogues, 1-pTza & 3-pTza, respectively. The pTza peptide libraries were coupled to KLH through their L-cysteine by the heterobifunctional linker m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) according to previously published methods (Using Antibodies: A Laboratory Manual by Ed Harlow, Ch 5) wherein the carrier KLH is activated and purified prior to the coupling.
Rabbit Immunization
[0081] In general, rabbits were immunized every three to four weeks and serum was collected ten days after each immunization. Each rabbit was injected intradermally with 0.5 ml antigen mixed with 0.5 ml adjuvant where Complete Freund's Adjuvant was used for the initial injection, and each subsequent injection was made with Freund's Incomplete Adjuvant.
Peptide Dot Blots
[0082] Each of the three peptide libraries (0.5 mg) was dissolved in PBS and used to make a 1 ug/ul stock solution. Six fold, 1:5 serial dilutions were performed with a starting concentration of 500 ng/ul for each peptide library. 1 ul of each peptide dilution was spotted on nitrocellulose membranes (Whatman Protran BA85) and allowed to dry for 30 min. Membranes were then blocked for 1 hr at room temperature in blocking buffer (0.1% Casein Block [BioRad] in 0.2×PBS) prior to overnight incubation at 4° C. with rabbit antisera diluted 1:1000 in blocking buffer supplemented with 0.1% Tween-20. Membranes were washed with 0.1% TBST and incubated Alexa Fluor 680 goat anti-rabbit IgG secondary antibodies (Invitrogen, Cat. A21109) diluted at 1:20,000 in 0.1% TBST for 45 min at room temperature. Membranes were then washed and imaged using the LI-COR Odyssey Infrared Imaging System. FIG. 5 (A-E) shows the results of such dot blots for the first four bleeds of the six rabbits used. Rabbits are identified by numbers (7302-7307). Rabbits 7302-7304 were injected with the 3-pTza analog; rabbits 7305-7307 were injected with the 1-pTza analog.
Recombinant Nm23 and PGAM Expression and Purification
[0083] The pGEX-Nm23-H1 plasmid was created by inserting Nm23-H1 into the BamHI/EcoRI restriction sites of the GST fusion vector pGEX-6-P1 using the following primers; Forward 3'-GATCGGATCCATGGCCAACTGTGAGCGTAC-5' (SEQ ID NO: 15) and Reverse 3' GATCGAATTCTCATTCATAGATCCAGTTCTC-5' (SEQ ID NO: 16). The pGEX-PGAM plasmid was created by inserting PGAM into the BamHI/EcoRI restriction sites of the same vector using the following primers; Forward 3'-GATCGGATCCATGGCCGCCTACAAACTGGTG-5' (SEQ ID NO: 17) and Reverse 3' GATCGAATTCTCACTTCTTGGCCTTGCCCTG-5' (SEQ ID NO: 18). Rosetta 2 (DE3) competent cells were transformed with pGEX-Nm23-H1 and an overnight culture of LB Amp [50]/Chl [34] was inoculated with a single colony for protein expression. The next day, a 1:100 dilution of overnight culture was used to inoculate 200 mL LB Amp [50]/Chl [34] to an OD600 of 0.2 and protein expression was induced at OD600=0.6 with 1 mM IPTG for 3 hrs at 30° C. E. coli were pelleted at 5,000×g for 10 min at 4° C. and stored at -80° C. until protein purification.
[0084] Purification of Nm23-H1 and PGAM: bacterial pellets were thawed on ice, resuspended in 1 mL GST Lysis Buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM DTT)/50 mL culture and sonicated to lyse cells (8 sec. pulse, rest 1 min, 4× (on ice)). Sonicated lysates were spun at 14,000×g for 30 min at 4° C. to pellet insoluble material. The Glutathione Resin (1.5 mL slurry per 200 mL culture) was equilibrated by washing with GST Lysis Buffer. The washed resin was resuspended with 2 mL GST Lysis Buffer/200 mL culture and the supernatant from bacterial lysates was transferred from ultracentrifuge tubes to fresh 15 mL conical tubes, combined with the washed resin and rotated 1.5 hrs at 4° C. The bound resin was pelleted (1000×g for 2 min at 4° C.) and washed 3× with 10 mL GST Lysis buffer.
[0085] Ice-cold PreScission Protease Buffer (50 mM Tris-HCl pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) was used to resuspend the washed, pelleted resin and 2.5 ul PreScission Protease (5 U/200 mL culture) was added and incubated overnight at 4° C. to cleave GST and release purified Nm23-H1 and PGAM from the resin. The resin was then pelleted (1000×g for 5 min at 4° C.) and the supernatant was carefully removed, transferred to a concentrator/desalting column (Millipore, Ultrafree 0.5-5K MWCO) in 500 ul increments and spun at 12,000×g, 4° C. until the volume reached 50 ul. Buffer exchange was performed by adding 3×450 ul Storage Buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM DTT) to the 50 ul concentrated sample. Purified, concentrated Nm23-H1 and PGAM proteins were brought to 10% Glycerol in Buffer B, aliquoted, frozen in liquid nitrogen and stored at -80° C. until use.
Nm23 Autophosphorylation Assay
[0086] Autophosphorylation of recombinant Nm23-H1 was performed using ATP as the phosphate donor for Nm23-H1; as a negative control, reactions were performed lacking ATP. 1 ul recombinant Nm23-H1 [1 ug/ul] was added per 50 ul TMD buffer (20 mM Tris-HCl pH 8.0, 5 mM MgCl2, 1 mM DTT). ATP (0.1-1 mM) was added fresh from aliquots of a 10 mM stock solution. Reactions were incubated at room temperature for 10 min and immediately stopped by addition of 5× pH 8.8 Sample Buffer (50 mM Tris-HCl pH 8.8, 2% SDS, 100 mM DTT, 10% Glycerol, 10 mM EDTA, 0.01% bromophenol blue).
[0087] Reactions were not heated prior to loading on 12.5% polyacrylamide gels (made with minimal pH 6.8 stacking gels) to preserve phosphorylation of histidine by limiting exposure to heat and low pH. The acid treated controls were performed by adding 15 ul 1M HCL to completed autophosphorylation reactions with incubation at 37° C. for 15 min, followed by neutralization with 15 ul 1M NaOH. All gels were run at 80-100V for approximately 3 hrs at 4° C. to prevent generation of heat. Proteins were transferred at 30V to PVDF membranes, overnight at 4° C. for immunoblotting by standard procedures. The detection limit for anti-pHis antisera was assayed by making a 1:2 serial dilutions (200, 100, 50, 25, 12 and 6 ng) of autophosphorylated Nm23-H1 kinase in TMD buffer. 5× pH 8.8 Sample Buffer was added to each dilution followed by analysis by SDS-PAGE (without heating) and immunoblotting with the pHis antisera diluted at 1:1000 in blocking buffer supplemented with 0.1% Tween-20. Membranes were washed and imaged as described above using the LI-COR Odyssey Infrared Imaging System.
PGAM Autophosphorylation Assay
[0088] Autophosphorylation of recombinant PGAM was performed using 2,3-diphosphoglycerate (2,3-DPG) as the phosphate donor; as a negative control, reactions were performed lacking 2,3-DPG and/or treated with heat and/or acid for 10 min. 1 ul recombinant PGAM [1 ug/ul] was added per 50 ul TMD buffer and reactions were incubated at 30° C. for 10 min followed by addition of 5× pH 8.8 Sample Buffer. SDS-PAGE and immunoblotting were performed as described for Nm23-H1.
[0089] FIGS. 7-9 show preliminary tests (western blots) of the antisera's abilities to recognize the autophosphorylated Nm23-H1.
Results
[0090] The peptide libraries have been coupled to KLH via an N-terminal Cys present in the pHis analog containing peptides and an immunization protocol has been initiated for three rabbits per immunogen. Bleeds were screened for pHis detection by dot blot using the immunizing peptides. Unphosphorylated versions of the peptide libraries (with His in place of the pHis analog) were used as negative selection of Abs and served as a negative control for dot blot screening of antisera prior to purification. Anti-1- or -3-pHis-specific Abs were purified from antisera using first an affinity column consisting of immobilized unphosphorylated peptides followed by using affinity columns consisting of the immobilized libraries, which include one/either of the non-hydrolyzable analogues.
[0091] To remove Abs crossreactive with other phosphoamino acids, the column was washed with sodium phosphate followed by pSer, pThr and pTyr. That some anti-pTyr Abs cross-react with pHis demonstrates that pHis is indeed antigenic and using the approach outlined above generated Abs that specifically recognize pHis and not other phosphoamino acids. See FIGS. 5A-5E demonstrating polyclonal antibodies specific for the 3-pHis library polypeptides (7306 and 7305) or 3-pHis library polypeptide (7302, 7303 and 7304) were generated.
[0092] To validate Ab specificity, autophosphorylated pH118 Nm23-H1/-H2 and dot blots with in vitro phosphorylated pHis substrates (e.g. histone H4) were used as positive controls. Recombinant GST-Nm23 proteins were expressed and purified using glutathione resin with subsequent cleavage of the GST using PreScission protease (See FIG. 4). In vitro autophosphorylation was conducted on the purified recombinant Nm23-H1 as described in the material and method section. Autophosphorylated rNm23 is detected by anti-1-pTza 7305 antisera in an immunoblot (see FIG. 5A); but anti-3-pTza 7305 antisera only produced background/non-specific binding to rNm23 (See FIG. 5B). Presumably this is due to a conserved Glu-NH hydrogen bond present at E129 in human Nm23 restricting autophosphorylation to only N1.
[0093] The detected rNm23 autophosphorylation is reversible. Acid treatment (1M HCl at 37° C. for 15 min followed by neutralization with NaOH) of the autophosphorylated rNm23 significantly reduces the binding of anti-1-pTza 7305-2 to the pNm23 band (see FIG. 7A). Coomassie stained gel demonstrates the presence of the rNm23-H1/H2 and shows a reduction in the phosphorylated pNm23-H1 in the acid treated lane (See FIG. 7B). FIGS. 8A and 8B show the coomassie detection limit vs the anti-1-pTza antisera detection limit, respectively for rNm23-H1 autophosphorylation. FIG. 8C shows the titer levels continue to increase in the fourth bleed of 7306 and 7305 rabbits, with FIG. 8D showing the 7306 and 7305 fourth bleed detection limits. FIG. 8E compares the titer of bleed 4 vs bleed 5 for detecting rNm23-H1 autophosphorylation using 7305 anti-sera.
Stable Cell Lines Expressing FLAG-Nm23-H1
[0094] 293T cell were transfected with DNA constructs expression FLAG-Nm23-H1 and stable lines were identified. The clones with the highest expression relative to tubulin was then used for all subsequent pHis immunoprecipitation experiments. Screening cell lines (See FIGS. 9A and 9B) revealed that clone 20 exhibited the highest expression of Nm23-H1 relative to tubulin. As shown in FIG. 9B 7305-5 anti-sera can detect pFLAG-Nm23-H1.
[0095] In general, pHis substrates will be identified by immunoprecipitation (IP) and immunoblotting (IB) with anti-pHis Abs, or by pHis peptide enrichment via an immobilized anti-pHis Ab affinity column and mass spectroscopy (MS) analysis. pHis substrates with relevance to cancer and metastasis will be identified by comparing cells with high and low Nm23 expression, followed by annotation using GO and pathway analysis to prioritize which substrates to study further.
Example 2
Identification and Analysis of pHis Substrates
[0096] Endogenous pHis Substrates from Cancer Cells.
[0097] pHis-containing proteins from whole cell lysates (buffered to pH 8.0 to stabilize pHis) made from cancer cell lines or primary tumor samples with high metastatic potential (i.e. low Nm23 expression; breast, ovarian, hepatocellular, cervical and gastric carcinomas or low metastatic potential (i.e. high Nm23 expression) will be immunoprecipitated using anti-pHis Abs. The immunoprecipitated proteins will be digested into peptides and enriched for pHis prior to MS analysis. Protocols for the selective extraction of peptides containing phosphomonoester residues (pSer, pThr and pTyr) using immobilized Fe(III) or Ga(III) ion affinity chromatography are well established. A similar protocol has been developed for enrichment and identification of pHis peptides based on use of appropriate pH conditions, and immobilized Cu(II) ion affinity chromatography (Cu(II)-IMAC) followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The synthetic pHis immunogen peptides and recombinant, autophosphorylated Nm23 proteins will be used to optimize identification of pHis-containing peptides by MS. His phosphorylation levels due to Nm23 His kinase activity are expected to be reduced in highly metastatic cell lines relative to non-metastatic cell lines, reflecting the reduced Nm23-H1/-H2 expression in these cells. We will also deplete PHPT1 pHis phosphatase using shRNA in an attempt to increase the levels of pHis in proteins, analogous to using pervanadate to enhance pTyr levels.
Overexpression of Histidine Kinases in Cells.
[0098] Autophosphorylation of His118 (H118) on Nm23-H1 and Nm23-H2 is required for their kinase activity. Line IV human melanoma cells have high metastatic potential and low Nm23 expression. Line IV cell lines stably expressing Nm23-H1, Nm23-H2 and their respective, catalytically inactive mutants (H118Y) have been obtained. Cell lysates or tryptic digests will be probed for pHis with Abs and analyzed by MS as described in above. In vitro kinase assays using purified proteins will be used to validate selected substrates of interest identified by MS. Mutagenesis of specific His residues to generate pHis-deficient mutants will be used to investigate the in vivo function of pHis for each respective target by expression in a null background (e.g. knockout MEFs) or by re-expression in depleted cells (e.g. si/shRNA-treated cells) to determine if the pHis-deficient mutants rescue WT protein function.
[0099] Nm23-H1 and Nm23-H2 genes are 88% identical and yet have distinct substrates and functions. Nm23 genes are highly conserved between human and rodents and have similar organization and tissue expression. Nm23-H1-/- and Nm23-H2-/- mice are viable; however, H1/H2-deficient mice are not, suggesting some functional overlap and that compensation occurs. Tissues from Nm23-H1-/- and H2-/- mice will be analyzed for pHis (using methods outlined above, as well as straight IB) and compared with tissues of WT mice to determine substrates specific for Nm23-H1, Nm23-H2 or both. Selected targets identified in this way will be further investigated through the use of si/shRNA knockdown and re-expression of WT or pHis-deficient mutants to examine effects of His phosphorylation on tumor metastasis (e.g. migration, invasion, anchorage-independent colony formation, and tumorigenesis in nude mice).
Example 3
Monoclonal Antibodies
[0100] For 1-pHis, rabbit 7305 was chosen for hyperimmunization and rabbit monoclonal antibody (RabMab) production. The spleen was harvested and the rabbit spleenic B cells were fused with rabbit partner cells (240E-W2 cells; U.S. Pat. No. 7,429,487, the disclosure of which is incorporated herein by reference). Culture supernatants from the hybridoma cells were screened by ELISA for recognition of 1-pHis and the identified antibodies having specificity for the immunogen were further characterized. For 1-pHis antibodies, 48 positive hybridoma multiclones were identified and screened against genuine pHis: E. coli expressing GST-Nm23-H1, a 293T-FLAG-Nm23-H1 stable cell line and immunoprecipitated FLAG-Nm23-H1 (See FIGS. 10A & 10B). The top 3 were selected for subcloning (clones 1, 50 and 77). Further analysis revealed the loss of pHis signal on Nm23 and other proteins after heating at 95° C. The three selected multiclones were subcloned and the best 9 subclones were selected: SC1-1, 1-3, 1-7, 1-9, 1-11, 50-3, 50-8, 50-11 & 77-11.
[0101] For 3-pHis, rabbit 7303 was chosen for Hyperimmunization and RabMab production. The spleen was harvested and the rabbit B cells were fused with rabbit partner cells (240E-W2 cells; U.S. Pat. No. 7,429,487, the disclosure of which is incorporated herein by reference). Culture supernatants from the hybridoma cells were screened by ELISA for recognition of 3-pHis and the identified antibodies having specificity for the immunogen were further characterized. ELISA screening was hindered by 3-pTza antigen solubility or lack of adherence to ELISA plates. Improved solubility was achieved by coupling antigen to BSA for screening and 20 ELISA positive multiclones were obtained. A 3-pHis screening assay was developed based on the 2,3-DPG dependent PGAM phosphorylation. PGAM catalyzes step 8 of glycolysis: The internal transfer of phosphate to convert 3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG) through a 2,3-diphosphoglycerate (2,3-DPG) intermediate. In vitro phosphorylated PGAM was used as the assay for identifying antibodies specific for 3-pHis. As shown in FIG. 11, polyclonal antibodies recovered from rabbit 7303 were able to bind to phosphorylated PGAM (but not heat-treated PGAM). Only 6-7 PGAM positive multiclones were identified from the 20 ELISA positive multiclones. The top 3 hybridoma multiclones were selected for subcloning--MC6, MC8 and MC17, but only MC8 and MC17 were stable. None of the MC17 subclones could detect genuine 3-pHis in the PGAM assay, however, MC8 subclones could, though weakly, with 7 of 12 subclones producing antibodies that give a weak signal for pPGAM 500 ng (see FIGS. 12A & 12B). This is likely due to very low IgG concentration and should be overcome by purification and concentrating the antibody. Accordingly, subclones 8-1, 8-2, 8-3, 8-4, 8-5, 8-6 and 8-9 were selected.
Example 4
Sequencing of SC1 Heavy and Light Variable Region Chains
[0102] The heavy chain variable regions of the SC1-1, SC1-3 and SC50-3 hybridomas were sequenced using either a pooled set of primers or a single primer. Results are provided below.
TABLE-US-00001 Pooled VH primers-VHP RVH1_A (SEQ ID NO: 19) 5' AGTCGGTGGAGGAGTCCAGG 3' RVH1_G (SEQ ID NO: 20) 5' AGTCGGTGGAGGAGTCCGGG 3' RVH2 (SEQ ID NO: 21) 5' AGTCGGTGAAGGAGTCCGAG 3' RVH3_C (SEQ ID NO: 22) 5' AGTCGCTGGAGGAGTCCGGG 3' RVH3_T (SEQ ID NO: 23) 5' AGTCGTTGGAGGAGTCCGGG 3' RVH4_CA (SEQ ID NO: 24) 5' AGCAGCAGCTGATGGAGTCCGG 3' RVH4_GA (SEQ ID NO: 25) 5' AGGAGCAGCTGATGGAGTCCGG 3' RVH4_CG (SEQ ID NO: 26) 5' AGCAGCAGCTGGTGGAGTCCGG 3' RVH4_GG (SEQ ID NO: 27) 5' AGGAGCAGCTGGTGGAGTCCGG 3' >HI-SF3-VHP_C08 SC1-1 (SEQ ID NO: 29) GNNCAAAGTATGATCATCTTCTCTTAGGCAGATCACGACGGTTACTCCTCATCTGCT CCTAGTCTCTGGGTCCTCCTCGCTCCATGTCAGGGGCTGGAATGGATCGGAAGTGC TGGCGCTTATTGTAGAATATCCTACGCGAGCTGGGCGAAAAGCCGATCCACCATCA CCAGAAACACCAACCTGAACACGGTGACTCTGAAAATGACCAGTCTGACATCCGC GGACACGGCCACTTATTTCTGTGCGAGAAGAAGTGATGTTGGTACTTCTGTGGGTT TTGATTCCTGGGGCCCAGGCACCCTGGTCACCGTCTCTCTACCTTTTATCTGCCTGA CTCCACCCGCTGCtTCCTAAAAATCGAAAAGGGGGGGGCCGGTACAGTTTGGGGTC GGGAGGGGGGGGTCTGTGGGGATGTCGGCCAGGTAGGCCATTGTCCGCTGCCCGA CGAGGGTTTTATGGGTCGGTGTTAGTCGGGAATTGGGGTATCTACATAGGCGCAAT GAGTAGAATCTCTGATTTTTCTTTTGGGTTGTTGCGGTGCGAGAAAGAATAGTCCAC CGGATCGATCCTGTGTCATTGAGTTTGGGTGAGTCGAGGTGCTTCCGATACTGCTCC CCTAGGCATCTCA Single Primer: RVH2 (SEQ ID NO: 30) 5' AGTCGGTGAAGGAGTCCGAG 3' >HI-SF12-VH3_D09 SC1-1 (SEQ ID NO: 28) GNATCGCATCTGTACCTAGGTCCTCAGCAGGAAGGCTCATGTGTCCTCCCTCGATG GGGTAGGCTTGGACTCTGGCCGGGGGGGCCTGGGGGGAGCCTTGGCCCGGGACAA CTAGCTGCAGGGTGGACACCACTTTGCCTAGCGGCACCCTGGTCACCGTCTCTTCC ACCATCACCAGAAACACCAACCTGAACACGGTGACTCTGAAAATGACCAGTCTGA CAGCCGCGGACACGGCCACTTATTTCTGTGCGAGAAGAAGTGATGTTGGTACTTCT GTGGGTTTTGATTCCTGGGGCCCAGGCACCCTGGCACCGTCTCTCTATTGACCATTC CCCTACTCTTGCCGCCTCTCTCTACGTCCTGGGCACTGCCACCCGCTGGCCCCTGGC CGTCCGCTAATGGACCACGACCGCACTCGGGCTGCTGGCCATCCCTGTTATCATCC ACCCCATCGACAGGTCGGTGGACTTCCTGCTGAACTCCAGCCTGCGTAAGCTCTAC CCATCCGTGGGGAAGCCCAGCTCCTCCTGAGCCCACCCTGGTACCTGGCCTGCGAG TTGGCTCACTCCCACCATGGGAAGTGGGGGCCAGGCTCCCCGGGGTCTGAGGCCCT GGCACCTGTGTGGATTAGAACTGGATGGAAGCTTCTAGATGGGGGTTTCTGGGAGC ATCAGCTGCAGCGAGACCCATACGGAGGCGGGACCTGACCCCCAACTGTCTCCACG GACACTGTGACAGTGGCGGGGAGTGCGGGGATGGCGCCATCCATAGCATGGAAGT CCTGTGCTCCCGCGGGGATACATATCTTATGCCTGGTATGGAG SC1-3 Heavy Chain Pooled VH primers-VHP: >HI-SF15-VHP_G09 SC1-3 (SEQ ID NO: 31) GGCGTACGTATGATCATCACGCTCTTAGGTAGATCACGGCGTTACTCCTCATCGGCT ACAAAATGTCCTGGGTCCTGCTCGCTCCATGTAAGGGGCTGGAATGGATCGGAAGT GCTGGCGCTTATGGTAGAATATACTACGCGAGCTGGGCGAAAAGCCGATCCACCAT CACCAGAAACACCAACCTGAACACGGTGACTCTGAAAATGACCAGTCTGACAGCC GCGGACACGGCCACTTATTTCTGTGCGAGAAGAAGTGATGTTGGTACTTCTGTGGG TTTTGATTCCTGGGGCCCAGGCACCCTGGCACCGTCTCCCTCCCTCTTATCCAGCGT CTGGGGGAGGGCACGGGGAGTAGGAGCTGCGGGCCTGTGAGATGTTCGGCAGGGG CTCTTTCTACCGGGGTGCTGTGGGAGAGATGTGAAGTCGGCACGCACATTGAGTGG CTGGAGGTTATACCGTCCTGGTGAGTGAGTTATGCTGGAATTGGAACTCCTCCACC GACTAATAGACTATA Single Primer: RVH2 (SEQ ID NO: 33) 5' AGTCGGTGAAGGAGTCCGAG 3' >HI-SF23-VH2_G10 SC1-3 (SEQ ID NO: 32) AANCACGCATACACTGACCTCACCTGTACAGCCTCTGGGTTCTCCCTCAGTGAGGC ATGGAGTGATCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAAG TGCTGGCGCTTATGGTAGAATATACTACGCGAGCTGGGCGAAAAGCCGATCCACCA TCACCAGAAACACCAACCTGAACACGGTGACTCTGAAAATGACCAGTCTGACAGC CGCGGACACGGCCACTTATTTCTGTGCGAGAAGAAGTGATGTTGGTACTTCTGTGG GTTTTGATTCCTGGGGCCCAGGCACCCTGCCTCCGTCTCTGAGATGGCTCCGTCGGG GACTTCGGTGGGGTAGGATAAAGGACCAGGTGGTCAAGCAGTTTGTTGTCGATTCT GTAGGGGACGGCGGAAGGAGTTGGGTGGGCACGAGGCGGATCTTGGGGGGGGGG ACCAGCTGGGGATCGGGCGGGGGGATCCCTTGATTGAGCGCCCAAGTAGGATTGA GATAAATTCTGTGTATGTAGAAGCGACTTCTAGCCTCCAGCACCCGTGCTCGATTTT AAGATTGAAGCTGAGCTTTTTGGTG SC50-3 Heavy Chain Pooled VH primers-VHP: >HI-SF27-VHP_C11 (SEQ ID NO: 34) NNCGCCTGGGATCGTAGAGCATCATCTCTCAGGCAGATGACGGGGTTACTCCTGAT GTGCTTCTAGAGACTGGGCCCTGCTCGCTCCCGGGGAGGGCCTGGAATGGATCGGA AGTGCTGGTGCATATGGTAGAATATATTACCCAAACTGGGCGAGAAGCCGAGCCA CCATCACCAGAAACACCAACCTCAACACGGTGTCTCTGAAAATGACCAGTCTGACA GCCGCGGACACGGCCACTTATTTCTGTGCGAGAAGGACGAATGTTAGTACTTCTGT GGGTTTTGATTCCTGGGGACCAGGCACCCTGGTCTCCGTCTCTTAAA Single Primer: RVH2 (SEQ ID NO: 36) 5' AGTCGGTGAAGGAGTCCGAG 3' >HI-SF35-VH2_C12 (SEQ ID NO: 35) GGGCTGAGCACGCATACACTGACCTCACCTGCACAGCCTCTGGGTTCTCCCTCAAT GGCTATGGAGTGATCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAATGGATCG GAAGTGCTGGTGCATATGGTAGAATATATTACCCAAACTGGGCGAGAAGCCGAGC CACCATCACCAGAAACACCAACCTCAACACGGTGTCTCTGAAAATGACCAGTCTGA CAGCCGCGGACACGGCCACTTATTTCTGTGCGAGAAGGACGAATGTTAGTACTTCT GTGGGTTTTGATTCCTGGGGACCAGGCACCCTGCACCCGTCTCCCATACCTACTCAG TGTCTGTAACCGTCGGGGGGGGGGGGGATGGAACCAGGCGGCAAAGCGGAACGTA ACTCGAAAAGTGTACGGCCGGTGGTGCAGGCAGTCCCGGCAGCAGTGGAACCGCT GGGAGGGACCAAGATGCTGGCTAAGGGAAAGGTTCCCGGTCATGGACAGCGCTGT GTTCTACTCCCTGGTCACCATCTCCTAGAGAGG SC1-1 Light Chain kappa Pooled VL kappa primers-VKP RHFabVκ1_A (SEQ ID NO: 37) 5' GTGATGACCCAGACTCCA 3' RHFabVκ1_C (SEQ ID NO: 38) 5' GTGCTGACCCAGACTCCA 3' RHFabVκ2_A (SEQ ID NO: 39) 5' GATATGACCCAGACTCCA 3' RHFabVκ2_C (SEQ ID NO: 40) 5' GATCTGACCCAGACTCCA 3' >HI-SF1-VKP_A08 SC1-1 (SEQ ID NO: 41) CCTATCAGCTGGTAGACAGTCACCATCATTGCCAGGCCAGTGAGAGCGTTTATAGT AACAACCGCTTATCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAACTCCTGAT CTATCTGGTATCCACTCTGGCATCTGGGGTCCCATCGCGATTCAAAGGCAGTGGAT CTGGGACACAATTCACTCTCACCATTAGCGATGTGGTGTGTGACGATGCTGCCACT TACTACTGTGTAGGCTATAAAAGTAGTACCACTGATGGTTTGGCTTTCGGCGGAGG GACCGATGGATTGGTCAAAAAACTGTGGTGGTGCGGGTATGTTGCCGGCGGGTAGT AGATGGCAGGGTCGTGAGCACCAGACAGAAGGAAAGGGAAATTAAGGGAAGGAC ACGCCAAAGGGAGGGGCGGGTATGGAGGGCAGAAAGGAGGTAGATTATATGTGG ATGGGGAGTAATGGGTTTGTTTGTAGTGAAAAAAGGAGGAGTCTTTTTTTTTTTAA GAAAGCCACCTGCCCGAATTGGGGTGTGGTCGTTTTTCGCCCGGGGACGGGATGTT GAAAGAGGAGTGCACCGGCTCCAAAAAAGCTGGATAGAGGATGTTTGTTTTCGAA GTGTGGCGCTAGGAGACT Primers VK2: RHFabVκ2_A (SEQ ID NO: 42) 5' GATATGACCCAGACTCCA 3' RHFabVκ2_C (SEQ ID NO: 43) 5' GATCTGACCCAGACTCCA 3' >HI-SF8-VK2_H08 SC1-1 (SEQ ID NO: 44) GNCNGNCTGTGGTAGACAGTCACCATCATTGCCAGGCCAGTGAGAGCGTTTATAGT AACAACCGCTTATCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAACTCCTGAT CTATCTGGTATCCACTCTGGCATCTGGGGTCCCATCGCGATTCAAAGGCAGTGGAT CTGGGACACAATTCACTCTCACCATTAGCGATGTGGTGTGTGACGATGCTGCCACT TACTACTGTGTAGGCTATAAAAGTAGTACCACTGATGGTTTGGCTTTCGGCGGAGG GACCGACGGCGGATCCTAAAAGCGGATGAGGCGACGGCATCTACACTTGCTCCGG TTAGTCAGAGGCGGTTACCTTAGAAAACCAAACATTACGAGCTGGCGACTAAGGA CAAGGGTTGTGCTCGGCGCGGGACTTAGCCCACCAGCTCAGGACACCAGTATGACG ACGCCCATGCTGCATCTGTGTTCGAGTGCACCAAATGCACTAATCCATCTCTTGAA AGTTATCGACATGTGAAAGGCCAGGTAACGTTATACGGCTTGCAACACAAATAGAC TCGCGCACGCCGCGGCTCGTGCCGTCACATCTTAATAACTTTCAGATACAGTATTGC GACCGTAGCTCAGCTGACGCCAAGTTAACCATGCTAGCTGCGAAACTGAGGTGCAT AATATGATCGCGACATCCACTTCGGGATCGGTTAGGGGTGGACTACGAGCTCAAGT CTAAATCCAGCGTTGTAACCCCACATGT SC1-3 Light Chain kappa Pooled VL kappa primers-VKP >HI-SF13-VKP_E09 SC1-3 (SEQ ID NO: 45) TNTTTCTGTGGTAGACAGTCACCATCAATTGCCAGGCCAGTGAGAGCGTTTATAGT AACAACCGCTTATCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAACTCCTGAT CTATCTGGTATCCACTCTGGCATCTGGGGTCCCATCGCGATTCAAAGGCAGTGGAT CTGGGACACAATTCACTCTCACCATTAGCGATGTGGTGTGTGACGATGCTGCCACT TACTACTGTGTAGGCTATAAAAGTAGTACCACTGATGGTTTGGCTTTCGGCGGAGG GACCGAGGGAGGGGTCAAAAAAACATGTGGGCTACTTTTTATACAGTTCGGTAGTA AGTGGCATCATCTGACACACCAGACTAATGTTTGAATGTTAATTGTGTCTAATACG ATGGTTTAAGGGGGGGGGCCCCATTCGGAACGTAAAAAAAGAGCATGAGGAGTTT GAGAAGGTGAATGGTTATTTCATTAACCGAGAAAGGCGTCGGTTACTTAAA Primers VK2: RHFabVκ2_A (SEQ ID NO: 46) 5' GATATGACCCAGACTCCA 3' RHFabVκ2_C (SEQ ID NO: 47) 5' GATCTGACCCAGACTCCA 3' >HI-SF21-VK2_E10 (SEQ ID NO: 48) GACCTGTTCTCTGGTAGACAGTCACCATCAATTGCCAGGCCAGTGAGAGCGTTTAT AGTAACAACCGCTTATCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAACTCCT GATCTATCTGGTATCCACTCTGGCATCTGGGGTCCCATCGCGATTCAAAGGCAGTG GATCTGGGACACAATTCACTCTCACCATTAGCGATGTGGTGTGTGACGATGCTGCC ACTTACTACTGTGTAGGCTATAAAAGTAGTACCACTGATGGTTTGGCTTTCGGCGG AGGGACCGAGGGGGTGGTCAAAAAAGGTTCCGCCC SC50-3 Light Chain kappa Pooled VL kappa primers-VKP >HI-SF25-VKP_A11 (SEQ ID NO: 49) ACTGTCATGTGGTAGTCAGTCACCATCAATTGCCAGGCCAGTGAGACCGTTTATAA TAATGACCGCTTAGCCTGGTTTCAACAGAAACCAGGGCAGCCTCCCAAGCTCCTGA TCTATCTGGCATCCACTCTGCCATCTGGGGTCCCATCGCGATTCAAAGGCAGTGGA TCTGGGACACAGTTCACTCTCACCATTAGTGATGTGGGGTGTGATGATGCTGCCAC TTATTATTGTGTAGGCTATAAAAGTAGTACGACTGATGGTTTGGCTTTCGGCGGAG GGACCGAGGGGTTGGTCAAAACAACACGTTGTGTACTACTTTTATAACCTACACAA TAATAAGTGGCAGCATCATCACACACGTAAAAATGGTGAGAGGGAACTGGGGTCC AAAACCTGCCTTTGAATCGGATGGGACCAGATTGAGAGTGGAAGCAGATGAGATA GGAGGGGGGGAAGGCGCTCGCTTTTTTTTTAAACCGGCCCGGGACCTTTTATATAT AAAAAAATTCTTTCACGGGGAATTGTGATGGGGAAAGAAACCCCCCCGGGACAAA ACTGTGGAGGAGGGAACTTGGGGGGCCCACCCAAGG Primers VK1:
RHFabVκ1_A (SEQ ID NO: 50) 5' GTGATGACCCAGACTCCA 3' RHFabVκ1_C (SEQ ID NO: 51) 5' GTGCTGACCCAGACTCCA 3' >HI-SF29-VK1_E11 (SEQ ID NO: 52) GNCTNTTCTGTGGTAGTCAGTCACCATCAATTGCCAGGCCAGTGAGACCGTTTATA ATAATGACCGCTTAGCCTGGTTTCAACAGAAACCAGGGCAGCCTCCCAAGCTCCTG ATCTATCTGGCATCCACTCTGCCATCTGGGGTCCCATCGCGATTCAAAGGCAGTGG ATCTGGGACACAGTTCACTCTCACCATTAGTGATGTGGGGTGTGATGATGCTGCCA CTTATTATTGTGTAGGCTATAAAAGTAGTACGACTGATGGTTTGGCTTTCGGCGGA GGGACCGAGTGGCAGATCCTAAAACAGCCCTGCAGGAGGGGGGAACGTTGGTCAT GCTCGGACGGGGAGTGGGGCTGCCAAAGGAGTCTGATAGAGGAAAGGCTCCTGTA AAGGGGGAATTGGCGTGGGAAGTGGGTAAGCCATCTGACGGGAATGACAAGAAGT TTCTTATGAAGGGGAGTGAAGTGTTGTAATTGAAGCATCGGCAAAATCACCCTAAG ATTATAGAGCTGATGAGAGAGTTGTAGAGTAGGCTCGCCGGG EXAMPLE 4 VH Chain Translations >HI-SF3-VHP_C08 SC1-1 GOOD SEQ (SEQ ID NO: 53) QSMIIFS*ADHDGYSSSAPSLWVLLAPCQGLEWIGSAGAYCRISYASWAKSRSTITRNTN LNTVTLKMTSLTSADTATYFCARRSDVGTSVGFDSWGPGTLVTVSLPFICLTPPAAPKN RKGGGRYSLGSGGGGLWGCRPGRPLSAARRGFYGSVLVGNWGIYIGAMSRISDFSFGL LRCEKE*STGSILCH*VWVSRGASDTAPLGIS >HI-SF11-VH2_C09 (SEQ ID NO: 54) L*HGYTDLTCTASGFSLSGHGVIWVRQAPGKGLEWIGSAGAYGRIYYASWAKSRSTIT RNTNLNTVTLKMTSLTAADTATYFCARRSDVGTSVGFDSWGPGTLVTVSL >HI-SF15-VHP_G09 SC1-3 GOOD SEQ (SEQ ID NO: 55) RTYDHHALR*ITALLLIGYKMSWVLLAPCKGLEWIGSAGAYGRIYYASWAKSRSTITR NTNLNTVTLKMTSLTAADTATYFCARRSDVGTSVGFDSWGPGTLAPSPSLLSSVWGRA RGVGAAGL*DVRQGLFLPGCCGRDVKSARTLSGWRLYRPGE*VMLELELLHRLIDY >HI-SF23-VH2_G10 SC1-3 GOOD SEQ (SEQ ID NO: 56) THTLTSPVQPLGSPSVRHGVIWVRQAPGKGLEWIGSAGAYGRIYYASWAKSRSTITRNT NLNTVTLKMTSLTAADTATYFCARRSDVGTSVGFDSWGPGTLPPSLRWLRRGLRWGR IKDQVVKQFVVDSVGDGGRSWVGTRRILGGGTSWGSGGGIP*LSAQVGLR*ILCM*KR LLASSTRARF*D*S*AFW >HI-SF27-VHP_C11 SC50-3 GOOD SEQ (SEQ ID NO: 57) PGIVEHHLSGR*RGYS*CASRDWALLAPGEGLEWIGSAGAYGRIYYPNWARSRATITRN TNLNTVSLKMTSLTAADTATYFCARRTNVSTSVGFDSWGPGTLVSVS* >HI-SF35-VH2_C12 SC50-3 GOOD SEQ (SEQ ID NO: 58) AEHAYTDLTCTASGFSLNGYGVIWVRQAPGKGLEWIGSAGAYGRIYYPNWARSRATIT RNTNLNTVSLKMTSLTAADTATYFCARRTNVSTSVGFDSWGPGTLHPSPIPTQCL*PSG GGGMEPGGKAERNSKSVRPVVQAVPAAVEPLGGTKMLAKGKVPGHGQRCVLLPGHH LLER >HI-SF10-VH1_B09 (SEQ ID NO: 59) SGDHSAYEQQHDVDQDGPLDLRHPGHRLSRLHDVLLCWHFVLNITHAAGKGGNGPRL LGPVTLVTLSLTSLTAADTATYFCARRSDVGTSVGFDSWGPGTLVTVSLGPPLADVQV RQGHQLLGLQDVPQGHFQPRLPVHQVWAPRAQGVPGGDASLQDHISCRPGLLPRLTTT TK >HI-SF12-VH3_D09 (SEQ ID NO: 60) IASVPRSSAGRLMCPPSMG*AWTLAGGAWGEPWPGTTSCRVDTTLPSGTLVTVSSTITR NTNLNTVTLKMTSLTAADTATYFCARRSDVGTSVGFDSWGPGTLAPSLY*PFPYSCRLS LRPGHCHPLAPGRPLMDHDRTRAAGHPCYHPPHRQVGGLPAELQPA*ALPIRGEAQLL LSPPWYLACELAHSHHGKWGPGSPGSEALAPVWIRTGWKLLDGGFWEHQLQRDPYG GGT*PPTVSTDTVTVAGSAGMAPSIAWKSCAPAGIHILCLVW >HI-SF22-VH1_F10 SC1-3 (SEQ ID NO: 61) MSCVTKLMSSSTTLTRYGPLDLRHPGHRLSKLHDVLLFVHRVLIMTHAAVMGGNGPR LLGPGTLVTVSLTSLTAADTATYFCARRSDVGTSVGFDSWGPGTLVTVSLSPSLLDVHV RQGHHLLGLQHVPQGHFQPRLPVHQV*ARRAQGVPGGDANLQDHISCWPGLHHLLTT >HI-SF24-VH3_H10 SC1-3 (SEQ ID NO: 62) ALGSLILTSQEEGPPWIG*TWEMAGGPWGKPLTGSQAWGGHHFA*RHPGHHLIRITRN TNLNTVTLKMTSLTAADTATYFCARRSDVGTSVGFD SWGPGTLVTVSLLTPPLLLPHLS TSCCCHPLAPGSPLMDLDRTRAAGRPCYHPRVRRSVDFLLNSRLRKHYPSVGNPLLLSR PWYLACDLAHSHHGKWGPGSPGSDALLPVCIIKGWKPRDGGFREPSSA >HI-SF34-VH1_B12 SC50-3 (SEQ ID NO: 63) VFAHVSLAFEQQHDVDQVWSSGSQAPWSPSPKAWCGAHVLAPRA*HDPRCLDGRKQ PHATGPCQPGVPQLTSLTAADTATYFCARRTNVSTSVGFDSWGPGTLVTVSYTTTASR CTRSTRPPPARAATCSSRALSTTATCAPSVSSARTRSAWR*CQPAGSHLLQTWTPPPTEY VL Chain Translations >HI-SF1-VKP_A08 SC1-1 (SEQ ID NO: 64) YQLVDSHHHCQASESVYSNNRLSWFQQKPGQPPKLLIYLVSTLASGVPSRFKGSGSGT QFTLTISDVVCDDAATYYCVGYKSSTTDGLAFGGGTDGLVKKLWWCGYVAGG**MA GS*APDRRKGKLREGHAKGRGGYGGQKGGRLYVDGE*WVCL**KKEESFFF*ESHLPE LGCGRFSPGDGMLKEECTGSKKAG*RMFVFEVWR*ET >HI-SF5-VK1_E08 (SEQ ID NO: 65) S*WKTVTINCQASESVYSNNRLSWFQQKPGQPPKLLIYLVSTLASGVPSRFKGSGSGTQ FTLTISDVVCDDAATYYCVGYKSSTTDGLAFGGGTDGNPKMKDMGDGSRRCSGCRRR *ARVPGLPVATKDKGKVVAGVSRVDRNSSGRLDAVLCVLVDDCQIHILKMFHLSSFVS FVC >HI-SF6-VK1_F08 (SEQ ID NO: 66) VVTVTINCQASESVYSNNRLSWFQQKPGQPPKLLIYLVSTLASGVPSRFKGSGSGTQFT LTISDVVCDDAATYYCVGYKSSTTDGLAFGGGTEGMVKKSGGGALMPYG*SGRIVTL TVQDRAGHADDKGHAVALAAAVGS*DPDSAKDGNSALVILRRKDRSLS*DVRRLHRV LDSVTTRRPARPARPHPMSLKSFPVPTRLAYAAVDKSPCLLHG >HI-SF8-VK2_H08 (SEQ ID NO: 67) VVDSHHHCQASESVYSNNRLSWFQQKPGQPPKLLIYLVSTLASGVPSRFKGSGSGTQFT LTISDVVCDDAATYYCVGYKSSTTDGLAFGGGTDG#GS*KRMRRRHLHLLRLVRGGY LRKPNITSWRLRTRVVLGAGLSPPAQDTSMTTPMLHLCSSAPNALIHLLKVIDM*KAR* RYTACNTNRLAHAAARAVTS**LSDTVLRP*LS*RQVNHASCETEVHNMIATSTSGSVR GGLRAQV*IQRCNPTC >HI-SF9-VK2_A09 (SEQ ID NO: 68) QMGDSHHHCQASESVYSNNRLSWFQQKPGQPPKLLIYLVSTLASGVPSRFKGSGSGTQ FTLTISDVVCDDAATYYCVGYKSSTTDGLAFGGGTEGWSKKRVPCQ*SPAGTKWAHR YTHQ*GWRKCV >HI-SF13-VKP_E09 SC1-3 (SEQ ID NO: 69) FLW*TVTINCQASESVYSNNRLSWFQQKPGQPPKWYLVSTLASGVPSRFKGSGSGTQ FTLTISDVVCDDAATYYCVGYKSSTTDGLAFGGGTEGGVKKTCGLLFIQFGSKWHHLT HQTNV*MLIVSNTMV*GGGPHSERKKRA*GV*EGEWLFH*PRKASVT* >HI-SF17-VK1_A10 (SEQ ID NO: 70) S*WETVTINCQASESVYSNNRLSWFQQKPGQPPKWYLVSTLASGVPSRFKGSGSGTQ FTLTISDVVCDDAATYYCVGYKSSTTDGLAFGGGTDGIS*N >HI-SF18-VK1_B10 (SEQ ID NO: 71) LVLRGASHHHCQASESVYSNNRLSWFQQKPGQPPKLLIYLVSTLASGVPSRFKGSGSGT QFTLTISDVVCDDAATYYCVGYKSNTTDGLAFGGGTEVVVKKD >HI-SF20-VK2_D10 (SEQ ID NO: 72) LFMLGDSHHHCQASESVYSNNRLSWFQQKPGQPPKLLIYLVSTLASGVPSRFKGSGSGT QFTLTISDVVCDDAATYYCVGYKSSTTDGLAFGGGTELEILKAPP >HI-SF21-VK2_E10 (SEQ ID NO: 73) PVLW*TVTINCQASESVYSNNRLSWFQQKPGQPPKWYLVSTLASGVPSRFKGSGSGT QFTLTISDVVCDDAATYYCVGYKSSTTDGLAFGGGTEGVVKKGSA >HI-SF25-VKP_A11 SC50-3 (SEQ ID NO: 74) TVMW*SVTINCQASETVYNNDRLAWFQQKPGQPPKWYLASTLPSGVPSRFKGSGSGT QFTLTISDVGCDDAATYYCVGYKSSTTDGLAFGGGTEGLVKTTRCVLLL*PTQ**VAAS SHT*KW*EGTGVQNLPLNRMGPD*EWKQMR*EGGKALAFFLNRPGTFYI*KNSFTGNC DGERNPPGTKLWRRELGGPTQ >HI-SF29-VK1_E11 SC50-3 (SEQ ID NO: 75) LW*SVTINCQASETVYNNDRLAWFQQKPGQPPKWYLASTLPSGVPSRFKGSGSGTQF TLTISDVGCDDAATYYCVGYKSSTTDGLAFGGGTEWQILKQPCRRGERWSCSDGEWG CQRSLIEERLL*RGNWRGKWVSHLTGMTRSFL*RGVKCCN*SIGKITLRL*S**ESCRVG SP >HI-SF30-VK1_F11 SC50-3 (SEQ ID NO: 76) CLWQSVTINCQASETVYNNDRLAWFQQKPGQPPKWYLASTLPSGVPSRFKGSGSGTQ FTLTISDVGCDDAATYYCVGYKSSTTDGLAFGGGTDGVVKTIWSVWGGGLCIILGSGV TPQ*SLTGLMP*S*GGNGLGRECAR*HERKKK*VHRHCIVWSQAGDLLYNEAIPYDREV QYAPGGNCYKRNLVSKQHH >HI-SF33-VK2_A12 SC50-3 (SEQ ID NO: 77) TVQWESVTINCQASETVYNNDRLAWFQQKPGQPPKWYLASTLPSGVPSRFKGSGSGT QFTLTISDVGCDDAATYYCVGYKSSTTDGLAFGGGTEGVVKK >HI-SF32-VK2_H11 SC50-3 (SEQ ID NO: 78) CS*WESVTINCQASETVYNNDRLPGFNRNQGSLPSS*SIWHPLCHLGSHRDSKAVDLGH SSLSPLVMWGVMMLPLIIV*AIKVVRLMVWLSAEGPTADPKTGV*GSRWEWGQGERS GETRL*GGL
Sequence CWU
1
1
8214PRTArtificial Sequenceamino acid sequence for antibody generation 1Xaa
Xaa Xaa Xaa 1 25PRTArtificial Sequenceamino acid sequence
for antibody generation 2Xaa Xaa Xaa Xaa Xaa 1 5
36PRTArtificial Sequenceamino acid sequence for antibody generation 3Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 47PRTArtificial Sequenceamino
acid sequence for antibody generation 4Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1
5 58PRTArtificial Sequenceamino acid sequence for
antibody generation 5Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
64PRTArtificial Sequenceamino acid sequence for antibody
generation 6Xaa Xaa Xaa Xaa 1 75PRTArtificial Sequenceamino
acid sequence for antibody generation 7Xaa Xaa Xaa Xaa Xaa 1
5 86PRTArtificial Sequenceamino acid sequence for antibody generation
8Xaa Xaa Xaa Xaa Xaa Xaa 1 5 97PRTArtificial
Sequenceamino acid sequence for antibody generation 9Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 108PRTArtificial Sequenceamino acid
sequence for antibody generation 10Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1
5 11152PRTHomo sapiens 11Met Ala Asn Leu Glu Arg
Thr Phe Ile Ala Ile Lys Pro Asp Gly Val 1 5
10 15 Gln Arg Gly Leu Val Gly Glu Ile Ile Lys Arg
Phe Glu Gln Lys Gly 20 25
30 Phe Arg Leu Val Ala Met Lys Phe Leu Arg Ala Ser Glu Glu His
Leu 35 40 45 Lys
Gln His Tyr Ile Asp Leu Lys Asp Arg Pro Phe Phe Pro Gly Leu 50
55 60 Val Lys Tyr Met Asn Ser
Gly Pro Val Val Ala Met Val Trp Glu Gly 65 70
75 80 Leu Asn Val Val Lys Thr Gly Arg Val Met Leu
Gly Glu Thr Asn Pro 85 90
95 Ala Asp Ser Lys Pro Gly Thr Ile Arg Gly Asp Phe Cys Ile Gln Val
100 105 110 Gly Arg
Asn Ile Ile His Gly Ser Asp Ser Val Lys Ser Ala Glu Lys 115
120 125 Glu Ile Ser Leu Trp Phe Lys
Pro Glu Glu Leu Val Asp Tyr Lys Ser 130 135
140 Cys Ala His Asp Trp Val Tyr Glu 145
150 12152PRTMus musculus 12Met Ala Asn Leu Glu Arg Thr Phe
Ile Ala Ile Lys Pro Asp Gly Val 1 5 10
15 Gln Arg Gly Leu Val Gly Glu Ile Ile Lys Arg Phe Glu
Gln Lys Gly 20 25 30
Phe Arg Leu Val Ala Met Lys Phe Leu Arg Ala Ser Glu Glu His Leu
35 40 45 Lys Gln His Tyr
Ile Asp Leu Lys Asp Arg Pro Phe Phe Pro Gly Leu 50
55 60 Val Lys Tyr Met Asn Ser Gly Pro
Val Val Ala Met Val Trp Glu Gly 65 70
75 80 Leu Asn Val Val Lys Thr Gly Arg Val Met Leu Gly
Glu Thr Asn Pro 85 90
95 Ala Asp Ser Lys Pro Gly Thr Ile Arg Gly Asp Phe Cys Ile Gln Val
100 105 110 Gly Arg Asn
Ile Ile His Gly Ser Asp Ser Val Glu Ser Ala Glu Lys 115
120 125 Glu Ile His Leu Trp Phe Lys Pro
Glu Glu Leu Ile Asp Tyr Lys Ser 130 135
140 Cys Ala His Asp Trp Val Tyr Glu 145
150 13152PRTHomo sapiens 13Met Ala Asn Cys Glu Arg Thr Phe Ile
Ala Ile Lys Pro Asp Gly Val 1 5 10
15 Gln Arg Gly Leu Val Gly Glu Ile Ile Lys Arg Phe Glu Gln
Lys Gly 20 25 30
Phe Arg Leu Val Gly Leu Lys Phe Met Gln Ala Ser Glu Asp Leu Leu
35 40 45 Lys Glu His Tyr
Val Asp Leu Lys Asp Arg Pro Phe Phe Ala Gly Leu 50
55 60 Val Lys Tyr Met His Ser Gly Pro
Val Val Ala Met Val Trp Glu Gly 65 70
75 80 Leu Asn Val Val Lys Thr Gly Arg Val Met Leu Gly
Glu Thr Asn Pro 85 90
95 Ala Asp Ser Lys Pro Gly Thr Ile Arg Gly Asp Phe Cys Ile Gln Val
100 105 110 Gly Arg Asn
Ile Ile His Gly Ser Asp Ser Val Glu Ser Ala Glu Lys 115
120 125 Glu Ile Gly Leu Trp Phe His Pro
Glu Glu Leu Val Asp Tyr Thr Ser 130 135
140 Cys Ala Gln Asn Trp Ile Tyr Glu 145
150 14152PRTMus musculus 14Met Ala Asn Ser Glu Arg Thr Phe Ile
Ala Ile Lys Pro Asp Gly Val 1 5 10
15 Gln Arg Gly Leu Val Gly Glu Ile Ile Lys Arg Phe Glu Gln
Lys Gly 20 25 30
Phe Arg Leu Val Gly Leu Lys Phe Leu Gln Ala Ser Glu Asp Leu Leu
35 40 45 Lys Glu His Tyr
Thr Asp Leu Lys Asp Arg Pro Phe Phe Thr Gly Leu 50
55 60 Val Lys Tyr Met His Ser Gly Pro
Val Val Ala Met Val Trp Glu Gly 65 70
75 80 Leu Asn Val Val Lys Thr Gly Arg Val Met Leu Gly
Glu Thr Asn Pro 85 90
95 Ala Asp Ser Lys Pro Gly Thr Ile Arg Gly Asp Phe Cys Ile Gln Val
100 105 110 Gly Arg Asn
Ile Ile His Gly Ser Asp Ser Val Lys Ser Ala Glu Lys 115
120 125 Glu Ile Ser Leu Trp Phe Gln Pro
Glu Glu Leu Val Glu Tyr Lys Ser 130 135
140 Cys Ala Gln Asn Trp Ile Tyr Glu 145
150 1530DNAArtificial SequencePCR primer 15gatcggatcc atggccaact
gtgagcgtac 301631DNAArtificial
SequencePCR primer 16gatcgaattc tcattcatag atccagttct c
311731DNAArtificial SequencePCR primer 17gatcggatcc
atggccgcct acaaactggt g
311831DNAArtificial SequencePCR primer 18gatcgaattc tcacttcttg gccttgccct
g 311920DNAArtificial Sequencepcr
primer 19agtcggtgga ggagtccagg
202020DNAArtificial Sequencepcr primer 20agtcggtgga ggagtccggg
202120DNAArtificial Sequencepcr
primer 21agtcggtgaa ggagtccgag
202220DNAArtificial Sequencepcr primer 22agtcgctgga ggagtccggg
202320DNAArtificial Sequencepcr
primer 23agtcgttgga ggagtccggg
202422DNAArtificial Sequencepcr primer 24agcagcagct gatggagtcc gg
222522DNAArtificial Sequencepcr
primer 25aggagcagct gatggagtcc gg
222622DNAArtificial Sequencepcr primer 26agcagcagct ggtggagtcc gg
222722DNAArtificial Sequencepcr
primer 27aggagcagct ggtggagtcc gg
2228742DNAOryctolagus cuniculus 28cctgggggga gccttggccc gggacaacta
gctgcagggt ggacaccact ttgcctagcg 60gcaccctggt caccgtctct tccaccatca
ccagaaacac caacctgaac acggtgactc 120tgaaaatgac cagtctgaca gccgcggaca
cggccactta tttctgtgcg agaagaagtg 180atgttggtac ttctgtgggt tttgattcct
ggggcccagg caccctggca ccgtctctct 240attgaccatt cccctactct tgccgcctct
ctctacgtcc tgggcactgc cacccgctgg 300cccctggccg tccgctaatg gaccacgacc
gcactcgggc tgctggccat ccctgttatc 360atccacccca tcgacaggtc ggtggacttc
ctgctgaact ccagcctgcg taagctctac 420ccatccgtgg ggaagcccag ctcctcctga
gcccaccctg gtacctggcc tgcgagttgg 480ctcactccca ccatgggaag tgggggccag
gctccccggg gtctgaggcc ctggcacctg 540tgtggattag aactggatgg aagcttctag
atgggggttt ctgggagcat cagctgcagc 600gagacccata cggaggcggg acctgacccc
caactgtctc cacggacact gtgacagtgg 660cggggagtgc ggggatggcg ccatccatag
catggaagtc ctgtgctccc gcggggatac 720atatcttatg cctggtatgg ag
74229631DNAOryctolagus
cuniculusmisc_feature(2)..(3)n is a, c, g, or t 29gnncaaagta tgatcatctt
ctcttaggca gatcacgacg gttactcctc atctgctcct 60agtctctggg tcctcctcgc
tccatgtcag gggctggaat ggatcggaag tgctggcgct 120tattgtagaa tatcctacgc
gagctgggcg aaaagccgat ccaccatcac cagaaacacc 180aacctgaaca cggtgactct
gaaaatgacc agtctgacat ccgcggacac ggccacttat 240ttctgtgcga gaagaagtga
tgttggtact tctgtgggtt ttgattcctg gggcccaggc 300accctggtca ccgtctctct
accttttatc tgcctgactc cacccgctgc ttcctaaaaa 360tcgaaaaggg gggggccggt
acagtttggg gtcgggaggg gggggtctgt ggggatgtcg 420gccaggtagg ccattgtccg
ctgcccgacg agggttttat gggtcggtgt tagtcgggaa 480ttggggtatc tacataggcg
caatgagtag aatctctgat ttttcttttg ggttgttgcg 540gtgcgagaaa gaatagtcca
ccggatcgat cctgtgtcat tgagtttggg tgagtcgagg 600tgcttccgat actgctcccc
taggcatctc a 6313020DNAOryctolagus
cuniculus 30agtcggtgaa ggagtccgag
2031519DNAOryctolagus cuniculus 31ggcgtacgta tgatcatcac
gctcttaggt agatcacggc gttactcctc atcggctaca 60aaatgtcctg ggtcctgctc
gctccatgta aggggctgga atggatcgga agtgctggcg 120cttatggtag aatatactac
gcgagctggg cgaaaagccg atccaccatc accagaaaca 180ccaacctgaa cacggtgact
ctgaaaatga ccagtctgac agccgcggac acggccactt 240atttctgtgc gagaagaagt
gatgttggta cttctgtggg ttttgattcc tggggcccag 300gcaccctggc accgtctccc
tccctcttat ccagcgtctg ggggagggca cggggagtag 360gagctgcggg cctgtgagat
gttcggcagg ggctctttct accggggtgc tgtgggagag 420atgtgaagtc ggcacgcaca
ttgagtggct ggaggttata ccgtcctggt gagtgagtta 480tgctggaatt ggaactcctc
caccgactaa tagactata 51932582DNAOryctolagus
cuniculusmisc_feature(3)..(3)n is a, c, g, or t 32aancacgcat acactgacct
cacctgtaca gcctctgggt tctccctcag tgaggcatgg 60agtgatctgg gtccgccagg
ctccagggaa ggggctggaa tggatcggaa gtgctggcgc 120ttatggtaga atatactacg
cgagctgggc gaaaagccga tccaccatca ccagaaacac 180caacctgaac acggtgactc
tgaaaatgac cagtctgaca gccgcggaca cggccactta 240tttctgtgcg agaagaagtg
atgttggtac ttctgtgggt tttgattcct ggggcccagg 300caccctgcct ccgtctctga
gatggctccg tcggggactt cggtggggta ggataaagga 360ccaggtggtc aagcagtttg
ttgtcgattc tgtaggggac ggcggaagga gttgggtggg 420cacgaggcgg atcttggggg
gggggaccag ctggggatcg ggcgggggga tcccttgatt 480gagcgcccaa gtaggattga
gataaattct gtgtatgtag aagcgacttc tagcctccag 540cacccgtgct cgattttaag
attgaagctg agctttttgg tg 5823320DNAOryctolagus
cuniculus 33agtcggtgaa ggagtccgag
2034326DNAOryctolagus cuniculusmisc_feature(1)..(2)n is a, c, g,
or t 34nncgcctggg atcgtagagc atcatctctc aggcagatga cggggttact cctgatgtgc
60ttctagagac tgggccctgc tcgctcccgg ggagggcctg gaatggatcg gaagtgctgg
120tgcatatggt agaatatatt acccaaactg ggcgagaagc cgagccacca tcaccagaaa
180caccaacctc aacacggtgt ctctgaaaat gaccagtctg acagccgcgg acacggccac
240ttatttctgt gcgagaagga cgaatgttag tacttctgtg ggttttgatt cctggggacc
300aggcaccctg gtctccgtct cttaaa
32635533DNAOryctolagus cuniculus 35gggctgagca cgcatacact gacctcacct
gcacagcctc tgggttctcc ctcaatggct 60atggagtgat ctgggtccgc caggctccag
ggaagggcct ggaatggatc ggaagtgctg 120gtgcatatgg tagaatatat tacccaaact
gggcgagaag ccgagccacc atcaccagaa 180acaccaacct caacacggtg tctctgaaaa
tgaccagtct gacagccgcg gacacggcca 240cttatttctg tgcgagaagg acgaatgtta
gtacttctgt gggttttgat tcctggggac 300caggcaccct gcacccgtct cccataccta
ctcagtgtct gtaaccgtcg gggggggggg 360ggatggaacc aggcggcaaa gcggaacgta
actcgaaaag tgtacggccg gtggtgcagg 420cagtcccggc agcagtggaa ccgctgggag
ggaccaagat gctggctaag ggaaaggttc 480ccggtcatgg acagcgctgt gttctactcc
ctggtcacca tctcctagag agg 5333620DNAOryctolagus cuniculus
36agtcggtgaa ggagtccgag
203718DNAArtificial Sequencesequence primer 37gtgatgaccc agactcca
183818DNAArtificial
Sequencesequence primer 38gtgctgaccc agactcca
183918DNAArtificial Sequencesequence primer
39gatatgaccc agactcca
184018DNAArtificial Sequencesequence primer 40gatctgaccc agactcca
1841629DNAOryctolagus cuniculus
41cctatcagct ggtagacagt caccatcatt gccaggccag tgagagcgtt tatagtaaca
60accgcttatc ctggtttcag cagaaaccag ggcagcctcc caaactcctg atctatctgg
120tatccactct ggcatctggg gtcccatcgc gattcaaagg cagtggatct gggacacaat
180tcactctcac cattagcgat gtggtgtgtg acgatgctgc cacttactac tgtgtaggct
240ataaaagtag taccactgat ggtttggctt tcggcggagg gaccgatgga ttggtcaaaa
300aactgtggtg gtgcgggtat gttgccggcg ggtagtagat ggcagggtcg tgagcaccag
360acagaaggaa agggaaatta agggaaggac acgccaaagg gaggggcggg tatggagggc
420agaaaggagg tagattatat gtggatgggg agtaatgggt ttgtttgtag tgaaaaaagg
480aggagtcttt ttttttttaa gaaagccacc tgcccgaatt ggggtgtggt cgtttttcgc
540ccggggacgg gatgttgaaa gaggagtgca ccggctccaa aaaagctgga tagaggatgt
600ttgttttcga agtgtggcgc taggagact
6294218DNAArtificial Sequencesequence primer 42gatatgaccc agactcca
184318DNAArtificial
Sequencesequence primer 43gatctgaccc agactcca
1844755DNAOryctolagus
cuniculusmisc_feature(2)..(2)n is a, c, g, or t 44gncngnctgt ggtagacagt
caccatcatt gccaggccag tgagagcgtt tatagtaaca 60accgcttatc ctggtttcag
cagaaaccag ggcagcctcc caaactcctg atctatctgg 120tatccactct ggcatctggg
gtcccatcgc gattcaaagg cagtggatct gggacacaat 180tcactctcac cattagcgat
gtggtgtgtg acgatgctgc cacttactac tgtgtaggct 240ataaaagtag taccactgat
ggtttggctt tcggcggagg gaccgacggc ggatcctaaa 300agcggatgag gcgacggcat
ctacacttgc tccggttagt cagaggcggt taccttagaa 360aaccaaacat tacgagctgg
cgactaagga caagggttgt gctcggcgcg ggacttagcc 420caccagctca ggacaccagt
atgacgacgc ccatgctgca tctgtgttcg agtgcaccaa 480atgcactaat ccatctcttg
aaagttatcg acatgtgaaa ggccaggtaa cgttatacgg 540cttgcaacac aaatagactc
gcgcacgccg cggctcgtgc cgtcacatct taataacttt 600cagatacagt attgcgaccg
tagctcagct gacgccaagt taaccatgct agctgcgaaa 660ctgaggtgca taatatgatc
gcgacatcca cttcgggatc ggttaggggt ggactacgag 720ctcaagtcta aatccagcgt
tgtaacccca catgt 75545498DNAOryctolagus
cuniculusmisc_feature(2)..(2)n is a, c, g, or t 45tntttctgtg gtagacagtc
accatcaatt gccaggccag tgagagcgtt tatagtaaca 60accgcttatc ctggtttcag
cagaaaccag ggcagcctcc caaactcctg atctatctgg 120tatccactct ggcatctggg
gtcccatcgc gattcaaagg cagtggatct gggacacaat 180tcactctcac cattagcgat
gtggtgtgtg acgatgctgc cacttactac tgtgtaggct 240ataaaagtag taccactgat
ggtttggctt tcggcggagg gaccgaggga ggggtcaaaa 300aaacatgtgg gctacttttt
atacagttcg gtagtaagtg gcatcatctg acacaccaga 360ctaatgtttg aatgttaatt
gtgtctaata cgatggttta aggggggggg ccccattcgg 420aacgtaaaaa aagagcatga
ggagtttgag aaggtgaatg gttatttcat taaccgagaa 480aggcgtcggt tacttaaa
4984618DNAArtificial
Sequencesequence primer 46gatatgaccc agactcca
184718DNAArtificial SequenceSequence primer
47gatctgaccc agactcca
1848315DNAOryctolagus cuniculus 48gacctgttct ctggtagaca gtcaccatca
attgccaggc cagtgagagc gtttatagta 60acaaccgctt atcctggttt cagcagaaac
cagggcagcc tcccaaactc ctgatctatc 120tggtatccac tctggcatct ggggtcccat
cgcgattcaa aggcagtgga tctgggacac 180aattcactct caccattagc gatgtggtgt
gtgacgatgc tgccacttac tactgtgtag 240gctataaaag tagtaccact gatggtttgg
ctttcggcgg agggaccgag ggggtggtca 300aaaaaggttc cgccc
31549593DNAOryctolagus cuniculus
49actgtcatgt ggtagtcagt caccatcaat tgccaggcca gtgagaccgt ttataataat
60gaccgcttag cctggtttca acagaaacca gggcagcctc ccaagctcct gatctatctg
120gcatccactc tgccatctgg ggtcccatcg cgattcaaag gcagtggatc tgggacacag
180ttcactctca ccattagtga tgtggggtgt gatgatgctg ccacttatta ttgtgtaggc
240tataaaagta gtacgactga tggtttggct ttcggcggag ggaccgaggg gttggtcaaa
300acaacacgtt gtgtactact tttataacct acacaataat aagtggcagc atcatcacac
360acgtaaaaat ggtgagaggg aactggggtc caaaacctgc ctttgaatcg gatgggacca
420gattgagagt ggaagcagat gagataggag ggggggaagg cgctcgcttt ttttttaaac
480cggcccggga ccttttatat ataaaaaaat tctttcacgg ggaattgtga tggggaaaga
540aacccccccg ggacaaaact gtggaggagg gaacttgggg ggcccaccca agg
5935018DNAArtificial Sequencesequence primer 50gtgatgaccc agactcca
185118DNAArtificial
Sequencesequence primer 51gtgctgaccc agactcca
1852543DNAOryctolagus
cuniculusmisc_feature(2)..(2)n is a, c, g, or t 52gnctnttctg tggtagtcag
tcaccatcaa ttgccaggcc agtgagaccg tttataataa 60tgaccgctta gcctggtttc
aacagaaacc agggcagcct cccaagctcc tgatctatct 120ggcatccact ctgccatctg
gggtcccatc gcgattcaaa ggcagtggat ctgggacaca 180gttcactctc accattagtg
atgtggggtg tgatgatgct gccacttatt attgtgtagg 240ctataaaagt agtacgactg
atggtttggc tttcggcgga gggaccgagt ggcagatcct 300aaaacagccc tgcaggaggg
gggaacgttg gtcatgctcg gacggggagt ggggctgcca 360aaggagtctg atagaggaaa
ggctcctgta aagggggaat tggcgtggga agtgggtaag 420ccatctgacg ggaatgacaa
gaagtttctt atgaagggga gtgaagtgtt gtaattgaag 480catcggcaaa atcaccctaa
gattatagag ctgatgagag agttgtagag taggctcgcc 540ggg
54353104PRTOryctolagus
cuniculus 53Leu Phe Met Leu Gly Asp Ser His His His Cys Gln Ala Ser Glu
Ser 1 5 10 15 Val
Tyr Ser Asn Asn Arg Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln
20 25 30 Pro Pro Lys Leu Leu
Ile Tyr Leu Val Ser Thr Leu Ala Ser Gly Val 35
40 45 Pro Ser Arg Phe Lys Gly Ser Gly Ser
Gly Thr Gln Phe Thr Leu Thr 50 55
60 Ile Ser Asp Val Val Cys Asp Asp Ala Ala Thr Tyr Tyr
Cys Val Gly 65 70 75
80 Tyr Lys Ser Ser Thr Thr Asp Gly Leu Ala Phe Gly Gly Gly Thr Glu
85 90 95 Leu Glu Ile Leu
Lys Ala Pro Pro 100 54209PRTOryctolagus
cuniculusmisc_feature(8)..(8)Xaa can be any naturally occurring amino
acid 54Gln Ser Met Ile Ile Phe Ser Xaa Ala Asp His Asp Gly Tyr Ser Ser 1
5 10 15 Ser Ala Pro
Ser Leu Trp Val Leu Leu Ala Pro Cys Gln Gly Leu Glu 20
25 30 Trp Ile Gly Ser Ala Gly Ala Tyr
Cys Arg Ile Ser Tyr Ala Ser Trp 35 40
45 Ala Lys Ser Arg Ser Thr Ile Thr Arg Asn Thr Asn Leu
Asn Thr Val 50 55 60
Thr Leu Lys Met Thr Ser Leu Thr Ser Ala Asp Thr Ala Thr Tyr Phe 65
70 75 80 Cys Ala Arg Arg
Ser Asp Val Gly Thr Ser Val Gly Phe Asp Ser Trp 85
90 95 Gly Pro Gly Thr Leu Val Thr Val Ser
Leu Pro Phe Ile Cys Leu Thr 100 105
110 Pro Pro Ala Ala Pro Lys Asn Arg Lys Gly Gly Gly Arg Tyr
Ser Leu 115 120 125
Gly Ser Gly Gly Gly Gly Leu Trp Gly Cys Arg Pro Gly Arg Pro Leu 130
135 140 Ser Ala Ala Arg Arg
Gly Phe Tyr Gly Ser Val Leu Val Gly Asn Trp 145 150
155 160 Gly Ile Tyr Ile Gly Ala Met Ser Arg Ile
Ser Asp Phe Ser Phe Gly 165 170
175 Leu Leu Arg Cys Glu Lys Glu Xaa Ser Thr Gly Ser Ile Leu Cys
His 180 185 190 Xaa
Val Trp Val Ser Arg Gly Ala Ser Asp Thr Ala Pro Leu Gly Ile 195
200 205 Ser
55108PRTOryctolagus cuniculusmisc_feature(2)..(2)Xaa can be any naturally
occurring amino acid 55Leu Xaa His Gly Tyr Thr Asp Leu Thr Cys Thr Ala
Ser Gly Phe Ser 1 5 10
15 Leu Ser Gly His Gly Val Ile Trp Val Arg Gln Ala Pro Gly Lys Gly
20 25 30 Leu Glu Trp
Ile Gly Ser Ala Gly Ala Tyr Gly Arg Ile Tyr Tyr Ala 35
40 45 Ser Trp Ala Lys Ser Arg Ser Thr
Ile Thr Arg Asn Thr Asn Leu Asn 50 55
60 Thr Val Thr Leu Lys Met Thr Ser Leu Thr Ala Ala Asp
Thr Ala Thr 65 70 75
80 Tyr Phe Cys Ala Arg Arg Ser Asp Val Gly Thr Ser Val Gly Phe Asp
85 90 95 Ser Trp Gly Pro
Gly Thr Leu Val Thr Val Ser Leu 100 105
56172PRTOryctolagus cuniculusmisc_feature(10)..(10)Xaa can be any
naturally occurring amino acid 56Arg Thr Tyr Asp His His Ala Leu Arg Xaa
Ile Thr Ala Leu Leu Leu 1 5 10
15 Ile Gly Tyr Lys Met Ser Trp Val Leu Leu Ala Pro Cys Lys Gly
Leu 20 25 30 Glu
Trp Ile Gly Ser Ala Gly Ala Tyr Gly Arg Ile Tyr Tyr Ala Ser 35
40 45 Trp Ala Lys Ser Arg Ser
Thr Ile Thr Arg Asn Thr Asn Leu Asn Thr 50 55
60 Val Thr Leu Lys Met Thr Ser Leu Thr Ala Ala
Asp Thr Ala Thr Tyr 65 70 75
80 Phe Cys Ala Arg Arg Ser Asp Val Gly Thr Ser Val Gly Phe Asp Ser
85 90 95 Trp Gly
Pro Gly Thr Leu Ala Pro Ser Pro Ser Leu Leu Ser Ser Val 100
105 110 Trp Gly Arg Ala Arg Gly Val
Gly Ala Ala Gly Leu Xaa Asp Val Arg 115 120
125 Gln Gly Leu Phe Leu Pro Gly Cys Cys Gly Arg Asp
Val Lys Ser Ala 130 135 140
Arg Thr Leu Ser Gly Trp Arg Leu Tyr Arg Pro Gly Glu Xaa Val Met 145
150 155 160 Leu Glu Leu
Glu Leu Leu His Arg Leu Ile Asp Tyr 165
170 57192PRTOryctolagus cuniculusmisc_feature(158)..(158)Xaa can
be any naturally occurring amino acid 57Thr His Thr Leu Thr Ser Pro Val
Gln Pro Leu Gly Ser Pro Ser Val 1 5 10
15 Arg His Gly Val Ile Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu 20 25 30
Trp Ile Gly Ser Ala Gly Ala Tyr Gly Arg Ile Tyr Tyr Ala Ser Trp
35 40 45 Ala Lys Ser Arg
Ser Thr Ile Thr Arg Asn Thr Asn Leu Asn Thr Val 50
55 60 Thr Leu Lys Met Thr Ser Leu Thr
Ala Ala Asp Thr Ala Thr Tyr Phe 65 70
75 80 Cys Ala Arg Arg Ser Asp Val Gly Thr Ser Val Gly
Phe Asp Ser Trp 85 90
95 Gly Pro Gly Thr Leu Pro Pro Ser Leu Arg Trp Leu Arg Arg Gly Leu
100 105 110 Arg Trp Gly
Arg Ile Lys Asp Gln Val Val Lys Gln Phe Val Val Asp 115
120 125 Ser Val Gly Asp Gly Gly Arg Ser
Trp Val Gly Thr Arg Arg Ile Leu 130 135
140 Gly Gly Gly Thr Ser Trp Gly Ser Gly Gly Gly Ile Pro
Xaa Leu Ser 145 150 155
160 Ala Gln Val Gly Leu Arg Xaa Ile Leu Cys Met Xaa Lys Arg Leu Leu
165 170 175 Ala Ser Ser Thr
Arg Ala Arg Phe Xaa Asp Xaa Ser Xaa Ala Phe Trp 180
185 190 58107PRTOryctolagus
cuniculusmisc_feature(12)..(12)Xaa can be any naturally occurring amino
acid 58Pro Gly Ile Val Glu His His Leu Ser Gly Arg Xaa Arg Gly Tyr Ser 1
5 10 15 Xaa Cys Ala
Ser Arg Asp Trp Ala Leu Leu Ala Pro Gly Glu Gly Leu 20
25 30 Glu Trp Ile Gly Ser Ala Gly Ala
Tyr Gly Arg Ile Tyr Tyr Pro Asn 35 40
45 Trp Ala Arg Ser Arg Ala Thr Ile Thr Arg Asn Thr Asn
Leu Asn Thr 50 55 60
Val Ser Leu Lys Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr 65
70 75 80 Phe Cys Ala Arg
Arg Thr Asn Val Ser Thr Ser Val Gly Phe Asp Ser 85
90 95 Trp Gly Pro Gly Thr Leu Val Ser Val
Ser Xaa 100 105 59177PRTOryctolagus
cuniculusmisc_feature(114)..(114)Xaa can be any naturally occurring amino
acid 59Ala Glu His Ala Tyr Thr Asp Leu Thr Cys Thr Ala Ser Gly Phe Ser 1
5 10 15 Leu Asn Gly
Tyr Gly Val Ile Trp Val Arg Gln Ala Pro Gly Lys Gly 20
25 30 Leu Glu Trp Ile Gly Ser Ala Gly
Ala Tyr Gly Arg Ile Tyr Tyr Pro 35 40
45 Asn Trp Ala Arg Ser Arg Ala Thr Ile Thr Arg Asn Thr
Asn Leu Asn 50 55 60
Thr Val Ser Leu Lys Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr 65
70 75 80 Tyr Phe Cys Ala
Arg Arg Thr Asn Val Ser Thr Ser Val Gly Phe Asp 85
90 95 Ser Trp Gly Pro Gly Thr Leu His Pro
Ser Pro Ile Pro Thr Gln Cys 100 105
110 Leu Xaa Pro Ser Gly Gly Gly Gly Met Glu Pro Gly Gly Lys
Ala Glu 115 120 125
Arg Asn Ser Lys Ser Val Arg Pro Val Val Gln Ala Val Pro Ala Ala 130
135 140 Val Glu Pro Leu Gly
Gly Thr Lys Met Leu Ala Lys Gly Lys Val Pro 145 150
155 160 Gly His Gly Gln Arg Cys Val Leu Leu Pro
Gly His His Leu Leu Glu 165 170
175 Arg 60175PRTOryctolagus cuniculus 60Ser Gly Asp His Ser Ala
Tyr Glu Gln Gln His Asp Val Asp Gln Asp 1 5
10 15 Gly Pro Leu Asp Leu Arg His Pro Gly His Arg
Leu Ser Arg Leu His 20 25
30 Asp Val Leu Leu Cys Trp His Phe Val Leu Asn Ile Thr His Ala
Ala 35 40 45 Gly
Lys Gly Gly Asn Gly Pro Arg Leu Leu Gly Pro Val Thr Leu Val 50
55 60 Thr Leu Ser Leu Thr Ser
Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe 65 70
75 80 Cys Ala Arg Arg Ser Asp Val Gly Thr Ser Val
Gly Phe Asp Ser Trp 85 90
95 Gly Pro Gly Thr Leu Val Thr Val Ser Leu Gly Pro Pro Leu Ala Asp
100 105 110 Val Gln
Val Arg Gln Gly His Gln Leu Leu Gly Leu Gln Asp Val Pro 115
120 125 Gln Gly His Phe Gln Pro Arg
Leu Pro Val His Gln Val Trp Ala Pro 130 135
140 Arg Ala Gln Gly Val Pro Gly Gly Asp Ala Ser Leu
Gln Asp His Ile 145 150 155
160 Ser Cys Arg Pro Gly Leu Leu Pro Arg Leu Thr Thr Thr Thr Lys
165 170 175 61274PRTOryctolagus
cuniculusmisc_feature(20)..(20)Xaa can be any naturally occurring amino
acid 61Ile Ala Ser Val Pro Arg Ser Ser Ala Gly Arg Leu Met Cys Pro Pro 1
5 10 15 Ser Met Gly
Xaa Ala Trp Thr Leu Ala Gly Gly Ala Trp Gly Glu Pro 20
25 30 Trp Pro Gly Thr Thr Ser Cys Arg
Val Asp Thr Thr Leu Pro Ser Gly 35 40
45 Thr Leu Val Thr Val Ser Ser Thr Ile Thr Arg Asn Thr
Asn Leu Asn 50 55 60
Thr Val Thr Leu Lys Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr 65
70 75 80 Tyr Phe Cys Ala
Arg Arg Ser Asp Val Gly Thr Ser Val Gly Phe Asp 85
90 95 Ser Trp Gly Pro Gly Thr Leu Ala Pro
Ser Leu Tyr Xaa Pro Phe Pro 100 105
110 Tyr Ser Cys Arg Leu Ser Leu Arg Pro Gly His Cys His Pro
Leu Ala 115 120 125
Pro Gly Arg Pro Leu Met Asp His Asp Arg Thr Arg Ala Ala Gly His 130
135 140 Pro Cys Tyr His Pro
Pro His Arg Gln Val Gly Gly Leu Pro Ala Glu 145 150
155 160 Leu Gln Pro Ala Xaa Ala Leu Pro Ile Arg
Gly Glu Ala Gln Leu Leu 165 170
175 Leu Ser Pro Pro Trp Tyr Leu Ala Cys Glu Leu Ala His Ser His
His 180 185 190 Gly
Lys Trp Gly Pro Gly Ser Pro Gly Ser Glu Ala Leu Ala Pro Val 195
200 205 Trp Ile Arg Thr Gly Trp
Lys Leu Leu Asp Gly Gly Phe Trp Glu His 210 215
220 Gln Leu Gln Arg Asp Pro Tyr Gly Gly Gly Thr
Xaa Pro Pro Thr Val 225 230 235
240 Ser Thr Asp Thr Val Thr Val Ala Gly Ser Ala Gly Met Ala Pro Ser
245 250 255 Ile Ala
Trp Lys Ser Cys Ala Pro Ala Gly Ile His Ile Leu Cys Leu 260
265 270 Val Trp 62173PRTOryctolagus
cuniculusmisc_feature(143)..(143)Xaa can be any naturally occurring amino
acid 62Met Ser Cys Val Thr Lys Leu Met Ser Ser Ser Thr Thr Leu Thr Arg 1
5 10 15 Tyr Gly Pro
Leu Asp Leu Arg His Pro Gly His Arg Leu Ser Lys Leu 20
25 30 His Asp Val Leu Leu Phe Val His
Arg Val Leu Ile Met Thr His Ala 35 40
45 Ala Val Met Gly Gly Asn Gly Pro Arg Leu Leu Gly Pro
Gly Thr Leu 50 55 60
Val Thr Val Ser Leu Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr 65
70 75 80 Phe Cys Ala Arg
Arg Ser Asp Val Gly Thr Ser Val Gly Phe Asp Ser 85
90 95 Trp Gly Pro Gly Thr Leu Val Thr Val
Ser Leu Ser Pro Ser Leu Leu 100 105
110 Asp Val His Val Arg Gln Gly His His Leu Leu Gly Leu Gln
His Val 115 120 125
Pro Gln Gly His Phe Gln Pro Arg Leu Pro Val His Gln Val Xaa Ala 130
135 140 Arg Arg Ala Gln Gly
Val Pro Gly Gly Asp Ala Asn Leu Gln Asp His 145 150
155 160 Ile Ser Cys Trp Pro Gly Leu His His Leu
Leu Thr Thr 165 170
63224PRTOryctolagus cuniculusmisc_feature(19)..(19)Xaa can be any
naturally occurring amino acid 63Ala Leu Gly Ser Leu Ile Leu Thr Ser Gln
Glu Glu Gly Pro Pro Trp 1 5 10
15 Ile Gly Xaa Thr Trp Glu Met Ala Gly Gly Pro Trp Gly Lys Pro
Leu 20 25 30 Thr
Gly Ser Gln Ala Trp Gly Gly His His Phe Ala Xaa Arg His Pro 35
40 45 Gly His His Leu Ile Arg
Ile Thr Arg Asn Thr Asn Leu Asn Thr Val 50 55
60 Thr Leu Lys Met Thr Ser Leu Thr Ala Ala Asp
Thr Ala Thr Tyr Phe 65 70 75
80 Cys Ala Arg Arg Ser Asp Val Gly Thr Ser Val Gly Phe Asp Ser Trp
85 90 95 Gly Pro
Gly Thr Leu Val Thr Val Ser Leu Leu Thr Pro Pro Leu Leu 100
105 110 Leu Pro His Leu Ser Thr Ser
Cys Cys Cys His Pro Leu Ala Pro Gly 115 120
125 Ser Pro Leu Met Asp Leu Asp Arg Thr Arg Ala Ala
Gly Arg Pro Cys 130 135 140
Tyr His Pro Arg Val Arg Arg Ser Val Asp Phe Leu Leu Asn Ser Arg 145
150 155 160 Leu Arg Lys
His Tyr Pro Ser Val Gly Asn Pro Leu Leu Leu Ser Arg 165
170 175 Pro Trp Tyr Leu Ala Cys Asp Leu
Ala His Ser His His Gly Lys Trp 180 185
190 Gly Pro Gly Ser Pro Gly Ser Asp Ala Leu Leu Pro Val
Cys Ile Ile 195 200 205
Lys Gly Trp Lys Pro Arg Asp Gly Gly Phe Arg Glu Pro Ser Ser Ala 210
215 220
64174PRTOryctolagus cuniculusmisc_feature(45)..(45)Xaa can be any
naturally occurring amino acid 64Val Phe Ala His Val Ser Leu Ala Phe Glu
Gln Gln His Asp Val Asp 1 5 10
15 Gln Val Trp Ser Ser Gly Ser Gln Ala Pro Trp Ser Pro Ser Pro
Lys 20 25 30 Ala
Trp Cys Gly Ala His Val Leu Ala Pro Arg Ala Xaa His Asp Pro 35
40 45 Arg Cys Leu Asp Gly Arg
Lys Gln Pro His Ala Thr Gly Pro Cys Gln 50 55
60 Pro Gly Val Pro Gln Leu Thr Ser Leu Thr Ala
Ala Asp Thr Ala Thr 65 70 75
80 Tyr Phe Cys Ala Arg Arg Thr Asn Val Ser Thr Ser Val Gly Phe Asp
85 90 95 Ser Trp
Gly Pro Gly Thr Leu Val Thr Val Ser Tyr Thr Thr Thr Ala 100
105 110 Ser Arg Cys Thr Arg Ser Thr
Arg Pro Pro Pro Ala Arg Ala Ala Thr 115 120
125 Cys Ser Ser Arg Ala Leu Ser Thr Thr Ala Thr Cys
Ala Pro Ser Val 130 135 140
Ser Ser Ala Arg Thr Arg Ser Ala Trp Arg Xaa Cys Gln Pro Ala Gly 145
150 155 160 Ser His Leu
Leu Gln Thr Trp Thr Pro Pro Pro Thr Glu Tyr 165
170 65209PRTOryctolagus
cuniculusmisc_feature(111)..(112)Xaa can be any naturally occurring amino
acid 65Tyr Gln Leu Val Asp Ser His His His Cys Gln Ala Ser Glu Ser Val 1
5 10 15 Tyr Ser Asn
Asn Arg Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro 20
25 30 Pro Lys Leu Leu Ile Tyr Leu Val
Ser Thr Leu Ala Ser Gly Val Pro 35 40
45 Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr
Leu Thr Ile 50 55 60
Ser Asp Val Val Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Val Gly Tyr 65
70 75 80 Lys Ser Ser Thr
Thr Asp Gly Leu Ala Phe Gly Gly Gly Thr Asp Gly 85
90 95 Leu Val Lys Lys Leu Trp Trp Cys Gly
Tyr Val Ala Gly Gly Xaa Xaa 100 105
110 Met Ala Gly Ser Xaa Ala Pro Asp Arg Arg Lys Gly Lys Leu
Arg Glu 115 120 125
Gly His Ala Lys Gly Arg Gly Gly Tyr Gly Gly Gln Lys Gly Gly Arg 130
135 140 Leu Tyr Val Asp Gly
Glu Xaa Trp Val Cys Leu Xaa Xaa Lys Lys Glu 145 150
155 160 Glu Ser Phe Phe Phe Xaa Glu Ser His Leu
Pro Glu Leu Gly Cys Gly 165 170
175 Arg Phe Ser Pro Gly Asp Gly Met Leu Lys Glu Glu Cys Thr Gly
Ser 180 185 190 Lys
Lys Ala Gly Xaa Arg Met Phe Val Phe Glu Val Trp Arg Xaa Glu 195
200 205 Thr 66177PRTOryctolagus
cuniculusmisc_feature(2)..(2)Xaa can be any naturally occurring amino
acid 66Ser Xaa Trp Lys Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Ser Val 1
5 10 15 Tyr Ser Asn
Asn Arg Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro 20
25 30 Pro Lys Leu Leu Ile Tyr Leu Val
Ser Thr Leu Ala Ser Gly Val Pro 35 40
45 Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr
Leu Thr Ile 50 55 60
Ser Asp Val Val Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Val Gly Tyr 65
70 75 80 Lys Ser Ser Thr
Thr Asp Gly Leu Ala Phe Gly Gly Gly Thr Asp Gly 85
90 95 Asn Pro Lys Met Lys Asp Met Gly Asp
Gly Ser Arg Arg Cys Ser Gly 100 105
110 Cys Arg Arg Arg Xaa Ala Arg Val Pro Gly Leu Pro Val Ala
Thr Lys 115 120 125
Asp Lys Gly Lys Val Val Ala Gly Val Ser Arg Val Asp Arg Asn Ser 130
135 140 Ser Gly Arg Leu Asp
Ala Val Leu Cys Val Leu Val Asp Asp Cys Gln 145 150
155 160 Ile His Ile Leu Lys Met Phe His Leu Ser
Ser Phe Val Ser Phe Val 165 170
175 Cys 67216PRTOryctolagus
cuniculusmisc_feature(109)..(109)Xaa can be any naturally occurring amino
acid 67Val Val Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Ser Val Tyr Ser 1
5 10 15 Asn Asn Arg
Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys 20
25 30 Leu Leu Ile Tyr Leu Val Ser Thr
Leu Ala Ser Gly Val Pro Ser Arg 35 40
45 Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr
Ile Ser Asp 50 55 60
Val Val Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Val Gly Tyr Lys Ser 65
70 75 80 Ser Thr Thr Asp
Gly Leu Ala Phe Gly Gly Gly Thr Glu Gly Met Val 85
90 95 Lys Lys Ser Gly Gly Gly Ala Leu Met
Pro Tyr Gly Xaa Ser Gly Arg 100 105
110 Ile Val Thr Leu Thr Val Gln Asp Arg Ala Gly His Ala Asp
Asp Lys 115 120 125
Gly His Ala Val Ala Leu Ala Ala Ala Val Gly Ser Xaa Asp Pro Asp 130
135 140 Ser Ala Lys Asp Gly
Asn Ser Ala Leu Val Ile Leu Arg Arg Lys Asp 145 150
155 160 Arg Ser Leu Ser Xaa Asp Val Arg Arg Leu
His Arg Val Leu Asp Ser 165 170
175 Val Thr Thr Arg Arg Pro Ala Arg Pro Ala Arg Pro His Pro Met
Ser 180 185 190 Leu
Lys Ser Phe Pro Val Pro Thr Arg Leu Ala Tyr Ala Ala Val Asp 195
200 205 Lys Ser Pro Cys Leu Leu
His Gly 210 215 68249PRTOryctolagus
cuniculusmisc_feature(97)..(97)Xaa can be any naturally occurring amino
acid 68Val Val Asp Ser His His His Cys Gln Ala Ser Glu Ser Val Tyr Ser 1
5 10 15 Asn Asn Arg
Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys 20
25 30 Leu Leu Ile Tyr Leu Val Ser Thr
Leu Ala Ser Gly Val Pro Ser Arg 35 40
45 Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr
Ile Ser Asp 50 55 60
Val Val Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Val Gly Tyr Lys Ser 65
70 75 80 Ser Thr Thr Asp
Gly Leu Ala Phe Gly Gly Gly Thr Asp Gly Gly Ser 85
90 95 Xaa Lys Arg Met Arg Arg Arg His Leu
His Leu Leu Arg Leu Val Arg 100 105
110 Gly Gly Tyr Leu Arg Lys Pro Asn Ile Thr Ser Trp Arg Leu
Arg Thr 115 120 125
Arg Val Val Leu Gly Ala Gly Leu Ser Pro Pro Ala Gln Asp Thr Ser 130
135 140 Met Thr Thr Pro Met
Leu His Leu Cys Ser Ser Ala Pro Asn Ala Leu 145 150
155 160 Ile His Leu Leu Lys Val Ile Asp Met Xaa
Lys Ala Arg Xaa Arg Tyr 165 170
175 Thr Ala Cys Asn Thr Asn Arg Leu Ala His Ala Ala Ala Arg Ala
Val 180 185 190 Thr
Ser Xaa Xaa Leu Ser Asp Thr Val Leu Arg Pro Xaa Leu Ser Xaa 195
200 205 Arg Gln Val Asn His Ala
Ser Cys Glu Thr Glu Val His Asn Met Ile 210 215
220 Ala Thr Ser Thr Ser Gly Ser Val Arg Gly Gly
Leu Arg Ala Gln Val 225 230 235
240 Xaa Ile Gln Arg Cys Asn Pro Thr Cys 245
69126PRTOryctolagus cuniculusmisc_feature(105)..(105)Xaa can be
any naturally occurring amino acid 69Gln Met Gly Asp Ser His His His Cys
Gln Ala Ser Glu Ser Val Tyr 1 5 10
15 Ser Asn Asn Arg Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln
Pro Pro 20 25 30
Lys Leu Leu Ile Tyr Leu Val Ser Thr Leu Ala Ser Gly Val Pro Ser
35 40 45 Arg Phe Lys Gly
Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser 50
55 60 Asp Val Val Cys Asp Asp Ala Ala
Thr Tyr Tyr Cys Val Gly Tyr Lys 65 70
75 80 Ser Ser Thr Thr Asp Gly Leu Ala Phe Gly Gly Gly
Thr Glu Gly Trp 85 90
95 Ser Lys Lys Arg Val Pro Cys Gln Xaa Ser Pro Ala Gly Thr Lys Trp
100 105 110 Ala His Arg
Tyr Thr His Gln Xaa Gly Trp Arg Lys Cys Val 115
120 125 70165PRTOryctolagus
cuniculusmisc_feature(4)..(4)Xaa can be any naturally occurring amino
acid 70Phe Leu Trp Xaa Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Ser Val 1
5 10 15 Tyr Ser Asn
Asn Arg Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro 20
25 30 Pro Lys Leu Leu Ile Tyr Leu Val
Ser Thr Leu Ala Ser Gly Val Pro 35 40
45 Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr
Leu Thr Ile 50 55 60
Ser Asp Val Val Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Val Gly Tyr 65
70 75 80 Lys Ser Ser Thr
Thr Asp Gly Leu Ala Phe Gly Gly Gly Thr Glu Gly 85
90 95 Gly Val Lys Lys Thr Cys Gly Leu Leu
Phe Ile Gln Phe Gly Ser Lys 100 105
110 Trp His His Leu Thr His Gln Thr Asn Val Xaa Met Leu Ile
Val Ser 115 120 125
Asn Thr Met Val Xaa Gly Gly Gly Pro His Ser Glu Arg Lys Lys Arg 130
135 140 Ala Xaa Gly Val Xaa
Glu Gly Glu Trp Leu Phe His Xaa Pro Arg Lys 145 150
155 160 Ala Ser Val Thr Xaa 165
71100PRTOryctolagus cuniculusmisc_feature(2)..(2)Xaa can be any naturally
occurring amino acid 71Ser Xaa Trp Glu Thr Val Thr Ile Asn Cys Gln Ala
Ser Glu Ser Val 1 5 10
15 Tyr Ser Asn Asn Arg Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro
20 25 30 Pro Lys Leu
Leu Ile Tyr Leu Val Ser Thr Leu Ala Ser Gly Val Pro 35
40 45 Ser Arg Phe Lys Gly Ser Gly Ser
Gly Thr Gln Phe Thr Leu Thr Ile 50 55
60 Ser Asp Val Val Cys Asp Asp Ala Ala Thr Tyr Tyr Cys
Val Gly Tyr 65 70 75
80 Lys Ser Ser Thr Thr Asp Gly Leu Ala Phe Gly Gly Gly Thr Asp Gly
85 90 95 Ile Ser Xaa Asn
100 72102PRTOryctolagus cuniculus 72Leu Val Leu Arg Gly Ala
Ser His His His Cys Gln Ala Ser Glu Ser 1 5
10 15 Val Tyr Ser Asn Asn Arg Leu Ser Trp Phe Gln
Gln Lys Pro Gly Gln 20 25
30 Pro Pro Lys Leu Leu Ile Tyr Leu Val Ser Thr Leu Ala Ser Gly
Val 35 40 45 Pro
Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr 50
55 60 Ile Ser Asp Val Val Cys
Asp Asp Ala Ala Thr Tyr Tyr Cys Val Gly 65 70
75 80 Tyr Lys Ser Asn Thr Thr Asp Gly Leu Ala Phe
Gly Gly Gly Thr Glu 85 90
95 Val Val Val Lys Lys Asp 100
73104PRTOryctolagus cuniculusmisc_feature(5)..(5)Xaa can be any naturally
occurring amino acid 73Pro Val Leu Trp Xaa Thr Val Thr Ile Asn Cys Gln
Ala Ser Glu Ser 1 5 10
15 Val Tyr Ser Asn Asn Arg Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln
20 25 30 Pro Pro Lys
Leu Leu Ile Tyr Leu Val Ser Thr Leu Ala Ser Gly Val 35
40 45 Pro Ser Arg Phe Lys Gly Ser Gly
Ser Gly Thr Gln Phe Thr Leu Thr 50 55
60 Ile Ser Asp Val Val Cys Asp Asp Ala Ala Thr Tyr Tyr
Cys Val Gly 65 70 75
80 Tyr Lys Ser Ser Thr Thr Asp Gly Leu Ala Phe Gly Gly Gly Thr Glu
85 90 95 Gly Val Val Lys
Lys Gly Ser Ala 100 74197PRTOryctolagus
cuniculusmisc_feature(5)..(5)Xaa can be any naturally occurring amino
acid 74Thr Val Met Trp Xaa Ser Val Thr Ile Asn Cys Gln Ala Ser Glu Thr 1
5 10 15 Val Tyr Asn
Asn Asp Arg Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln 20
25 30 Pro Pro Lys Leu Leu Ile Tyr Leu
Ala Ser Thr Leu Pro Ser Gly Val 35 40
45 Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe
Thr Leu Thr 50 55 60
Ile Ser Asp Val Gly Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Val Gly 65
70 75 80 Tyr Lys Ser Ser
Thr Thr Asp Gly Leu Ala Phe Gly Gly Gly Thr Glu 85
90 95 Gly Leu Val Lys Thr Thr Arg Cys Val
Leu Leu Leu Xaa Pro Thr Gln 100 105
110 Xaa Xaa Val Ala Ala Ser Ser His Thr Xaa Lys Trp Xaa Glu
Gly Thr 115 120 125
Gly Val Gln Asn Leu Pro Leu Asn Arg Met Gly Pro Asp Xaa Glu Trp 130
135 140 Lys Gln Met Arg Xaa
Glu Gly Gly Lys Ala Leu Ala Phe Phe Leu Asn 145 150
155 160 Arg Pro Gly Thr Phe Tyr Ile Xaa Lys Asn
Ser Phe Thr Gly Asn Cys 165 170
175 Asp Gly Glu Arg Asn Pro Pro Gly Thr Lys Leu Trp Arg Arg Glu
Leu 180 185 190 Gly
Gly Pro Thr Gln 195 75178PRTOryctolagus
cuniculusmisc_feature(3)..(3)Xaa can be any naturally occurring amino
acid 75Leu Trp Xaa Ser Val Thr Ile Asn Cys Gln Ala Ser Glu Thr Val Tyr 1
5 10 15 Asn Asn Asp
Arg Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 20
25 30 Lys Leu Leu Ile Tyr Leu Ala Ser
Thr Leu Pro Ser Gly Val Pro Ser 35 40
45 Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu
Thr Ile Ser 50 55 60
Asp Val Gly Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Val Gly Tyr Lys 65
70 75 80 Ser Ser Thr Thr
Asp Gly Leu Ala Phe Gly Gly Gly Thr Glu Trp Gln 85
90 95 Ile Leu Lys Gln Pro Cys Arg Arg Gly
Glu Arg Trp Ser Cys Ser Asp 100 105
110 Gly Glu Trp Gly Cys Gln Arg Ser Leu Ile Glu Glu Arg Leu
Leu Xaa 115 120 125
Arg Gly Asn Trp Arg Gly Lys Trp Val Ser His Leu Thr Gly Met Thr 130
135 140 Arg Ser Phe Leu Xaa
Arg Gly Val Lys Cys Cys Asn Xaa Ser Ile Gly 145 150
155 160 Lys Ile Thr Leu Arg Leu Xaa Ser Xaa Xaa
Glu Ser Cys Arg Val Gly 165 170
175 Ser Pro 76195PRTOryctolagus
cuniculusmisc_feature(121)..(121)Xaa can be any naturally occurring amino
acid 76Cys Leu Trp Gln Ser Val Thr Ile Asn Cys Gln Ala Ser Glu Thr Val 1
5 10 15 Tyr Asn Asn
Asp Arg Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro 20
25 30 Pro Lys Leu Leu Ile Tyr Leu Ala
Ser Thr Leu Pro Ser Gly Val Pro 35 40
45 Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr
Leu Thr Ile 50 55 60
Ser Asp Val Gly Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Val Gly Tyr 65
70 75 80 Lys Ser Ser Thr
Thr Asp Gly Leu Ala Phe Gly Gly Gly Thr Asp Gly 85
90 95 Val Val Lys Thr Ile Trp Ser Val Trp
Gly Gly Gly Leu Cys Ile Ile 100 105
110 Leu Gly Ser Gly Val Thr Pro Gln Xaa Ser Leu Thr Gly Leu
Met Pro 115 120 125
Xaa Ser Xaa Gly Gly Asn Gly Leu Gly Arg Glu Cys Ala Arg Xaa His 130
135 140 Glu Arg Lys Lys Lys
Xaa Val His Arg His Cys Ile Val Trp Ser Gln 145 150
155 160 Ala Gly Asp Leu Leu Tyr Asn Glu Ala Ile
Pro Tyr Asp Arg Glu Val 165 170
175 Gln Tyr Ala Pro Gly Gly Asn Cys Tyr Lys Arg Asn Leu Val Ser
Lys 180 185 190 Gln
His His 195 77101PRTOryctolagus cuniculus 77Thr Val Gln Trp Glu
Ser Val Thr Ile Asn Cys Gln Ala Ser Glu Thr 1 5
10 15 Val Tyr Asn Asn Asp Arg Leu Ala Trp Phe
Gln Gln Lys Pro Gly Gln 20 25
30 Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Thr Leu Pro Ser Gly
Val 35 40 45 Pro
Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr 50
55 60 Ile Ser Asp Val Gly Cys
Asp Asp Ala Ala Thr Tyr Tyr Cys Val Gly 65 70
75 80 Tyr Lys Ser Ser Thr Thr Asp Gly Leu Ala Phe
Gly Gly Gly Thr Glu 85 90
95 Gly Val Val Lys Lys 100 78125PRTOryctolagus
cuniculusmisc_feature(3)..(3)Xaa can be any naturally occurring amino
acid 78Cys Ser Xaa Trp Glu Ser Val Thr Ile Asn Cys Gln Ala Ser Glu Thr 1
5 10 15 Val Tyr Asn
Asn Asp Arg Leu Pro Gly Phe Asn Arg Asn Gln Gly Ser 20
25 30 Leu Pro Ser Ser Xaa Ser Ile Trp
His Pro Leu Cys His Leu Gly Ser 35 40
45 His Arg Asp Ser Lys Ala Val Asp Leu Gly His Ser Ser
Leu Ser Pro 50 55 60
Leu Val Met Trp Gly Val Met Met Leu Pro Leu Ile Ile Val Xaa Ala 65
70 75 80 Ile Lys Val Val
Arg Leu Met Val Trp Leu Ser Ala Glu Gly Pro Thr 85
90 95 Ala Asp Pro Lys Thr Gly Val Xaa Gly
Ser Arg Trp Glu Trp Gly Gln 100 105
110 Gly Glu Arg Ser Gly Glu Thr Arg Leu Xaa Gly Gly Leu
115 120 125 7985PRTOryctolagus
cuniculusMISC_FEATURE(8)..(8)Xaa at position 8 is glutamine or lysine
79Trp Val Leu Leu Ala Pro Cys Xaa Gly Leu Glu Trp Ile Gly Ser Ala 1
5 10 15 Gly Ala Tyr Cys
Arg Ile Xaa Tyr Ala Ser Trp Ala Lys Ser Arg Ser 20
25 30 Thr Ile Thr Arg Asn Thr Asn Leu Asn
Thr Val Thr Leu Lys Met Thr 35 40
45 Ser Leu Thr Xaa Ala Asp Thr Ala Thr Tyr Phe Cys Ala Arg
Arg Ser 50 55 60
Asp Val Gly Thr Ser Val Gly Phe Asp Ser Trp Gly Pro Gly Thr Leu 65
70 75 80 Val Thr Val Ser Leu
85 8092PRTOryctolagus cuniculus 80Asp Ser His His His
Cys Gln Ala Ser Glu Ser Val Tyr Ser Asn Asn 1 5
10 15 Arg Leu Ser Trp Phe Gln Gln Lys Pro Gly
Gln Pro Pro Lys Leu Leu 20 25
30 Ile Tyr Leu Val Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe
Lys 35 40 45 Gly
Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val Val 50
55 60 Cys Asp Asp Ala Ala Thr
Tyr Tyr Cys Val Gly Tyr Lys Ser Ser Thr 65 70
75 80 Thr Asp Gly Leu Ala Phe Gly Gly Gly Thr Asp
Gly 85 90 8190PRTOryctolagus
cuniculus 81Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Ser Val Tyr Ser Asn
Asn 1 5 10 15 Arg
Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu
20 25 30 Ile Tyr Leu Val Ser
Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys 35
40 45 Gly Ser Gly Ser Gly Thr Gln Phe Thr
Leu Thr Ile Ser Asp Val Val 50 55
60 Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Val Gly Tyr Lys
Ser Ser Thr 65 70 75
80 Thr Asp Gly Leu Ala Phe Gly Gly Gly Thr 85
90 8259PRTOryctolagus cuniculus 82Gly Thr Leu Val Thr Val Ser Ser
Thr Ile Thr Arg Asn Thr Asn Leu 1 5 10
15 Asn Thr Val Thr Leu Lys Met Thr Ser Leu Thr Ala Ala
Asp Thr Ala 20 25 30
Thr Tyr Phe Cys Ala Arg Arg Ser Asp Val Gly Thr Ser Val Gly Phe
35 40 45 Asp Ser Trp Gly
Pro Gly Thr Leu Ala Pro Ser 50 55
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