WO2007048103A2 - Pkd ligands and polynucleotides encoding pkd ligands - Google Patents

Pkd ligands and polynucleotides encoding pkd ligands Download PDF

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WO2007048103A2
WO2007048103A2 PCT/US2006/060062 US2006060062W WO2007048103A2 WO 2007048103 A2 WO2007048103 A2 WO 2007048103A2 US 2006060062 W US2006060062 W US 2006060062W WO 2007048103 A2 WO2007048103 A2 WO 2007048103A2
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amino acid
seq
peptide
leu
acid residue
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PCT/US2006/060062
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WO2007048103A9 (en
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Thomas Reed
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Intrexon Corporation
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Priority to PCT/US2006/060062 priority Critical patent/WO2007048103A2/en
Priority to US12/090,462 priority patent/US8153598B2/en
Publication of WO2007048103A2 publication Critical patent/WO2007048103A2/en
Publication of WO2007048103A9 publication Critical patent/WO2007048103A9/en
Priority to US13/348,515 priority patent/US8993531B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases

Definitions

  • PKD LIGANDSAND POLYNUCLEOTIDES ENCODING PKD LIGANDS
  • the invention relates to mammalian kinase ligands, substrates and modulators.
  • the invention relates to polypeptides, polypeptide compositions and polynucleotides that encode polypeptides that are ligands, substrates, and/or modulators of PKD.
  • the invention also relates to polyligands that are homopolyligands or heteropolyligands that modulate PKD activity.
  • the invention also relates to ligands and polyligands tethered to a subcellular location.
  • Kinases are enzymes that catalyze the addition of phosphate to a molecule.
  • the addition of phosphate by a kinase is called phosphorylation.
  • the kinase substrate is a protein molecule, the amino acids commonly phosphorylated are serine, threonine and tyrosine.
  • Phosphatases are enzymes that remove phosphate from a molecule. The removal of phosphate is called dephosphorylation.
  • Kinases and phosphatases often represent competing forces within a cell to transmit, attenuate, or otherwise modulate cellular signals and cellular control mechanisms.
  • Kinases and phosphatases have both overlapping and unique natural substrates. Cellular signals and control mechanisms, as regulated by kinases, phosphatases, and their natural substrates are a target of research tool design and drug design.
  • Mammalian Protein Kinase D is also known as PKD.
  • the enzymatic activity, activation and autoregulation of PKD have been studied.
  • Several cellular substrates of PKD have been identified.
  • Substrates and modified substrates are genetically referred to herein as ligands.
  • Natural and synthetic peptide ligands have been studied to examine PKD substrate specificty. While peptide ligands and variants thereof have been studied as individuals PKD ligands, mixed ligands linked together as polyligands have not been demonstrated before this invention.
  • SEQ ID NOS: 1-104 represent examples of monomelic peptide ligand sequences.
  • SEQ ID NOS: 1-52 are fragments of natural substrates of mammlain PKD, wherein each fragment contains at least one amino acid residue phosphorylatable by PKD.
  • SEQ ID NOS:53-104 are fragments of natural PKD substrates, where the location of the PKD phosphorylatable serine or threonine in the natural polypeptide is designated as Xaa.
  • SEQ ID NOS: 105-116 are example polyligands and polynucleotides encoding them.
  • SEQ ID NO:105 is encoded by SEQ ID NO:106, SEQ ID NO:107 and by SEQ ID NO:108, wherein the the codons of SEQ ID NO: 107 and SEQ ID NO: 108 have been optimized for vector insertion.
  • SEQ ID NO: 108 includes flanking restriction sites.
  • SEQ ID NO: 105 is an embodiment of a polyligand of the structure X-S1-Y-S2-Z, wherein X is SEQ ID NO:53, Y is SEQ ID NO:55, Z is SEQ
  • a polyligand of structure A-S1-B-S2-C is called herein a heteropolyligand, shown generically in FIGURE 4B.
  • SEQ ID NO:109 is an embodiment of a polyligand of the structure X-S1-Y-S2-Z, wherein X is SEQ ID NO:61, Y is SEQ ID NO:65, Z is SEQ ID NO:66, wherein Xaa is alanine, wherein Sl is a four amino acid spacer of amino acid sequence AGAG and S2 is a four amino acid spacer of amino acid sequence GAGA.
  • the PKD polyligand of SEQ ID NO:109 is encoded by SEQ ID NO:110, SEQ ID NO:111 and by SEQ ID NO:112, wherein the the codons of SEQ ID NO:111 and SEQ ID NO:112 have been optimized for vector insertion.
  • SEQ ID NO: 112 includes flanking restriction sites.
  • a polyligand of structure X-S1-Y-S2-Z is also called herein a heteropolyligand, shown generically in FIGURE 4B.
  • SEQ ID NO:113 is an embodiment of a polyligand of the structure X-Y-S3-Y-X, wherein X is SEQ ID NO:53, Y is SEQ ID NO:54, wherein Xaa is alanine, and wherein S3 is a five amino acid spacer with the sequence PAGAG.
  • the PKD polyligand of SEQ ID NO:113 is encoded by SEQ ID NO:114, SEQ ID NO:115, and by SEQ ID NO:116, wherein the the codons of SEQ ID NO:115 and SEQ ID NO:116 have been optimized for vector insertion.
  • SEQ ID NO:116 includes flanking restriction sites.
  • a polyligand of structure X-Y-S3-Y-X is also called herein a heteropolyligand, shown generically in FIGURE 4C.
  • SEQ ID NOS: 117-132 are full length PKlD substrates. These sequences have the following public database accession numbers: AAH64840, NP000354, CAA79356, NP001015053, NP009112, NP001531, NP065789, AAH00029, AAB48596, NP_536728, AAH47282, NP004283, NP005219, CAB95729, NP_057541, NP_849231. Each of the protein sequences represented by these accession numbers is incorporated by reference herein.
  • SEQ ID NOS: 117-132 the positions of the amino acid(s) phosphorylatable by PKD are represented by Xaa. In wild-type proteins, Xaa is serine or threonine. In the ligands of the invention, Xaa is any amino acid.
  • Amino acid sequences containing Xaa encompass polypeptides where Xaa is any amino acid.
  • FIGURES 1A-1C show examples of homopolymeric ligands without spacers.
  • FIGURES 2A-2C show examples of homopolymeric ligands with spacers.
  • FIGURES 3A-3C show examples of heteropolymeric ligands without spacers.
  • FIGURES 4A-4C show examples of heteropolymeric ligands with spacers.
  • FIGURES 5A-5G show examples of ligands and polymeric ligands linked to an optional epitope tag.
  • FIGURES 6A-6G show examples of ligands and polymeric ligands linked to an optional reporter.
  • FIGURES 7A-7G show examples of ligands and polymeric ligands linked to an optional localization signal.
  • FIGURES 8A-8G show examples of ligands and polymeric ligands linked to an optional localization signal and an optional epitope tag.
  • FIGURES 9A-9G show examples of gene constructs where ligands and polyligands are linked to an optional localization signal, an optional epitope tag, and an optional reporter.
  • FIGURES 10A- 1OD show examples of vectors containing ligand gene constructs.
  • FIGURE 11 shows an example of a sequential cloning process useful for combinatorial synthesis of polyligands.
  • the invention relates to polypeptide ligands and polyligands for PKD.
  • PKD ligands and polyligands are represented in SEQ ID NOS: 1-132. More specifically, the invention relates to ligands, homopolyligands, and heteropolyligands that comprise any one or more of SEQ ID NOS: 1-
  • the invention relates to ligands and polyligands comprising one or more subsequences of SEQ ID NOS: 117-132 or any portion thereof. Furthermore, the invention relates to polyligands with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% sequence identity to a polyligand comprising one or more of SEQ ID NOS:1-104 or any portion thereof. Furthermore, the invention relates to polyligands with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% sequence identity to a polyligand comprising one or more subsequences of SEQ ID NOS: 117-132 or portions thereof.
  • Polyligands which can be homopolyligands or heteropolyligands, are chimeric ligands composed of two or more monomeric polypeptide ligands.
  • An example of a monomelic ligand is the polypeptide represented by SEQ ID NO:61, wherein Xaa is any amino acid.
  • An example of a homopolyligand is a polypeptide comprising a dimer or multimer of SEQ ID NO:61, wherein Xaa is any amino acid.
  • An example of a heteropolyligand is a polypeptide comprising SEQ ID NO:1 and one or more of SEQ ID NOS:2-104, wherein Xaa is any amino acid.
  • SEQ ID NOS:1-104 into homopolymeric or heteropolymeric ligands. Furthermore, there are numerous ways to combine portions or subsequences of SEQ ID NOS:117-132 with each other and with SEQ ID NOS:1-104 to make polymeric ligands that modulate PKD.
  • the polyligands of the invention optionally comprise spacer amino acids before, after, or between monomers.
  • SEQ ID NO: 105 is an embodiment of a polyligand of the structure X-S1-Y-S2-Z, wherein X is SEQ ID NO:53, Y is SEQ ID NO:55, Z is SEQ ID NO:58, wherein Xaa is alanine, and wherein Sl and S2 are four amino acid spacers.
  • This invention intends to capture all combinations of homopolyligands and heteropolyligands without limitation to the examples given above or below.
  • ligand(s) encompasses monomeric ligands, polymeric ligands, homopolymeric ligands and/or heteropolymeric ligands.
  • a monomeric ligand is a polypeptide where at least a portion of the polypeptide is capable of being recognized by PKD.
  • the portion of the polypeptide capable of recognition is termed the recognition motif.
  • recognition motifs can be natural or synthetic. Examples of recognition motifs are well known in the art and include, but are not limited to, naturally occurring PKD substrates and pseudosubstrate motifs.
  • a polymeric ligand comprises two or more monomeric ligands.
  • a homopolymeric ligand is a polymeric ligand where each of the monomeric ligands is identical in amino acid sequence, except that a phosphorylatable residue may be substituted or modified in one or more of the monomeric ligands.
  • a heteropolymeric ligand is a polymeric ligand where some of the monomeric ligands do not have an identical amino acid sequence.
  • the ligands of the invention are optionally linked to additional molecules or amino acids that provide an epitope tag, a reporter, and/or a cellular localization signal.
  • the cellular localization signal targets the ligands to a region of a cell.
  • the epitope tag and/or reporter and/or localization signal may be the same molecule.
  • the epitope tag and/or reporter and/or localization signal may also be different molecules.
  • the invention also encompasses polynucleotides comprising a nucleotide sequence encoding ligands, homopolyligands, and heteropoly ligands.
  • the nucleic acids of the invention are optionally linked to additional nucleotide sequences encoding polypeptides with additional features, such as an epitope tag, a reporter, and/or a cellular localization signal.
  • the polynucleotides are optionally flanked by nucleotide sequences comprising restriction endonuclease sites and other nucleotides needed for restriction endonuclese activity.
  • the flanking sequences optionally provide unique cloning sites within a vector and optionally provide directionality of subsequence cloning.
  • the nucleic acids of the invention are optionally incorporated into vector polynucleotides.
  • the ligands, polyligands, and polynucleotides of this invention have utility as research tools and/or therapeutics.
  • the present invention relates to ligands and polyligands that are PKD modulators.
  • ligands and polyligands are represented in SEQ ID NOS: 1-132.
  • Polyligands are chimeric ligands comprising two or more monomelic polypeptide ligands.
  • An example of a monomelic ligand is the polypeptide represented by SEQ ID NO:54, wherein Xaa is any amino acid.
  • Another example of a monomelic ligand is the polypeptide represented by SEQ ID NO:1.
  • Each of SEQ ID NOS: 1-104 represents an individual polypeptide ligand in monomeric form, wherein Xaa is any amino acid.
  • monomeric ligands are subsequence portions of SEQ ID NOS:117-132 containing a PKD recognition motif.
  • Monomeric ligand subsequences of SEQ ID NOS:117-132 may be wild-type subsequences. Additionally, monomeric ligand subsequences of SEQ ID NOS: 117-132 may have the PKD phosphorylatable amino acids replaced by other amino acids.
  • monomeric ligands and polyligands may have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a ligand comprising an amino acid sequence in one or more of SEQ ID NOS:1-104. Furthermore, monomeric ligands and polyligands may have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% sequence identity to a subsequence of SEQ ID NOS:117-132.
  • An example of a homopolyligand is a polypeptide comprising a dimer or multimer of SEQ ID NO:66, wherein Xaa is any amino acid.
  • An example of a heteropolyligand is a polypeptide comprising SEQ ID NO: 104 and one or more of SEQ ID NOS: 1-103, wherein Xaa is any amino acid.
  • SEQ ID NOS:1-104 into homopolymeric or heteropolymeric ligands.
  • Polyligands may comprise any two or more of a sequence selected from SEQ ID NOS: 1-104 and recognition motif-containing portions of SEQ ID NOS: 117-132, wherein Xaa is any amino acid.
  • a dimer or multimer of SEQ ID NO:91 is an example of a homopolyligand.
  • An example of a heteropolyligand is a polypeptide comprising SEQ ID NO:5 and one or more of a recognition motif-containing portion of SEQ ID NOS:117-132.
  • SEQ ID NOS:1-104 and portions of SEQ ID NOS:117-132 into homopolymeric or heteropolymeric ligands. The instant invention is directed to all possible combinations of homopolyligands and heteropolyligands without limitation.
  • SEQ ID NOS:53-104 and SEQ ID NOS:117-132 show polypeptides that contain at least one serine or threonine residue phosphorylatable by PKD, the positions of which are represented by Xaa.
  • Xaa is, generally speaking, serine or threonine.
  • Xaa can be any amino acid.
  • Ligands where Xaa is serine or threonine can be used as part of a polyligand, however in one embodiment, at least one phosphorylatable serine or threonine is replaced with another amino acid, such as one of the naturally occurring amino acids including, alanine, aspartate, asparagine, cysteine, glutamate, glutamine, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, arginine, valine, tryptophan, or tyrosine.
  • the Xaa may also be a non-naturally occurring amino acid.
  • the PKD phosphorylatable serine(s) or threonine(s) are replaced by alanine.
  • the ligands and polyligands of the invention are designed to modulate the endogenous effects of PKD.
  • ligand monomers based on natural PKD substrates are built by isolating a putative PKD phosphorylation recognition motif in a PKD substrate. Sometimes it is desirable to modify the phosphorylatable residue to an amino acid other than serine or threonine. Additional monomers include the PKD recognition motif as well as amino acids adjacent and contiguous on either side of the PKD recognition motif. Monomeric ligands may therefore be any length provided the monomer includes the PKD recognition motif.
  • the monomer may comprise a PKD recognition motif and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30-100 or more amino acids adjacent to the recognition motif.
  • the invention comprises an inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 194-201 of SEQ ID NO: 117, wherein the amino acid residue corresponding to amino acid residue 199 of SEQ ID NO : 117 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 191-203 of SEQ ID NO: 117, wherein the amino acid residue corresponding to amino acid residue 199 of SEQ ID NO : 117 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 189-205 of SEQ ID NO: 117, wherein the amino acid residue residues 189-205 of
  • the terms “correspond(s) to” and “corresponding to,” as they relate to sequence alignment, are intended to mean enumerated positions within a reference protein, e.g., HDAC5 (SEQ ID NO:119), and those positions that align with the positions on the reference protein.
  • a reference protein e.g., HDAC5 (SEQ ID NO:119)
  • the amino acids in the subject peptide sequence that "correspond to" certain enumerated positions of the reference peptide sequence are those that align with these positions of the reference peptide sequence, but are not necessarily in these exact numerical positions of the reference sequence.
  • Additional embodiments of the invention include monomers (as described above) based on any putative or real substrate for PKD, such as substrates identified by SEQ ID NOS: 1-104 and SEQ ID NOS: 117- 132. Furthermore, if the substrate has more than one recognition motif, then more than one monomer may be identified therein.
  • Another embodiment of the invention is a nucleic acid molecule comprising a polynucleotide sequence encoding at least one copy of a ligand peptide.
  • Another embodiment of the invention is a nucleic acid molecule wherein the polynucleotide sequence encodes one or more copies of one or more peptide ligands.
  • Another embodiment of the invention is a nucleic acid molecule wherein the polynucleotide sequence encodes at least a number of copies of the peptide selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • Another embodiment of the invention is a vector comprising a nucleic acid molecule encoding at least one copy of a ligand or polyligand.
  • Another embodiment of the invention is a recombinant host cell comprising a vector comprising a nucleic acid molecule encoding at least one copy of a ligand or polyligand.
  • Another embodiment of the invention is a method of inhibiting PKD in a cell comprising transfecting a vector comprising a nucleic acid molecule encoding at least one copy of a ligand or polyligand into a host cell and culturing the transfected host cell under conditions suitable to produce at least one copy of the ligand or polyligand.
  • the invention also relates to modified inhibitors that are at least about 80%, 85%, 90% 95%, 96%, 97%, 98% or 99% identical to a reference inhibitor.
  • a "modified inhibitor” is used to mean a peptide that can be created by addition, deletion or substitution of one or more amino acids in the primary structure (amino acid sequence) of a inhibitor protein or polypeptide.
  • a "modified recognition motif is a naturally occurring PKD recognition motif that has been modified by addition, deletion, or substitution of one or more amino acids in the primary structure (amino acid sequence) of the motif.
  • a modified PKD recognition motif may be a motif where the phosphorylatable amino acid has been modified to a non-phosphorylatable amino acid.
  • the terms "protein” and “polypeptide” are used interchangeably herein.
  • the reference inhibitor is not necessarily a wild-type protein or a portion thereof.
  • the reference inhibitor may be a protein or peptide whose sequence was previously modified over a wild- type protein.
  • the reference inhibitor may or may not be the wild-type protein from a particular organism.
  • a polypeptide having an amino acid sequence at least, for example, about 95% "identical" to a reference an amino acid sequence is understood to mean that the amino acid sequence of the polypeptide is identical to the reference sequence except that the amino acid sequence may include up to about five modifications per each 100 amino acids of the reference amino acid sequence encoding the reference peptide.
  • up to about 5% of the amino acid residues of the reference sequence may be deleted or substituted with another amino acid or a number of amino acids up to about 5% of the total amino acids in the reference sequence may be inserted into the reference sequence.
  • These modifications of the reference sequence may occur at the N-terminus or C-terminus positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • identity is a measure of the identity of nucleotide sequences or amino acid sequences compared to a reference nucleotide or amino acid sequence. In general, the sequences are aligned so that the highest order match is obtained. "Identity” per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g., Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York (1988); Biocomputing: Informatics And Genome Projects, Smith, D.W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H.
  • identity is well known to skilled artisans (Carillo, H. & Lipton, D., Siam J Applied Math 48:1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J.
  • Computer programs may also contain methods and algorithms that calculate identity and similarity. Examples of computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al, Nucleic Acids Research 12(i):387 (1984)), BLASTP, ExPASy, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990)) and FASTDB. Examples of methods to determine identity and similarity are discussed in Michaels, G.
  • the algorithm used to determine identity between two or more polypeptides is BLASTP.
  • the algorithm used to determine identity between two or more polypeptides is FASTDB, which is based upon the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990), incorporated by reference).
  • FASTDB sequence alignment the query and subject sequences are amino sequences. The result of sequence alignment is in percent identity.
  • the percent identity is corrected by calculating the number of amino acids of the query sequence that are N-and C-terminus to the reference sequence that are not matched/aligned, as a percent of the total bases of the query sequence. The results of the FASTDB sequence alignment determine matching/alignment.
  • the alignment percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
  • This corrected score can be used for the purposes of determining how alignments "correspond" to each other, as well as percentage identity. Residues of the query (subject) sequences or the reference sequence that extend past the N-or C-termini of the reference or subject sequence, respectively, may be considered for the purposes of manually adjusting the percent identity score. That is, residues that are not matched/aligned with the N-or C-termini of the comparison sequence may be counted when manually adjusting the percent identity score or alignment numbering.
  • a 90 amino acid residue subject sequence is aligned with a 100 residue reference sequence to determine percent identity.
  • the deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a match/alignment of the first 10 residues at the N- terminus.
  • the 10 unpaired residues represent 10% of the sequence (number of residues at the N-and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%.
  • a 90 residue subject sequence is compared with a 100 reference sequence. This time the deletions are internal deletions so there are no residues at the N-or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected.
  • the polyligands of the invention optionally comprise spacer amino acids before, after, or between monomers.
  • the length and composition of the spacer may vary.
  • An example of a spacer is glycine, alanine, polyglycine, or polyalanine.
  • Specific examples of spacers used between monomers in SEQ ID NO: 105 are the four amino acids AGAG, and the four amino acids GAGA.
  • Spacer amino acids may be any amino acid and are not limited to alanine and glycine.
  • the instant invention is directed to all combinations of homopolyligands and heteropolyligands, with or without spacers, and without limitation to the examples given above or below.
  • the ligands and polyligands of the invention are optionally linked to additional molecules or amino acids that provide an epitope tag, a reporter, and/or localize the ligand to a region of a cell (See FIGURES 5A- 5G, FIGURES 6A-6G, FIGURES 7A-7G, and FIGURES 8A-8G).
  • epitope tags are FLAGTM (Kodak; Rochester, N.Y.), HA (hemagluttinin), c-Myc and His6. Additional examples of epitope tags are given in Jarvik & Telmer 1998 Annual Reviw of Genetics 32:601-18.
  • Non-limiting examples of reporters are alkaline phosphatase, galactosidase, peroxidase, luciferase and green fluorescent protein (GFP).
  • Non-limiting examples of cellular localizations are sarcoplamic reticulum, endoplasmic reticulum, mitochondria, golgi apparatus, nucleus, plasma membrane, apical membrane, and basolateral membrane.
  • the epitopes, reporters and localization signals are given by way of example and without limitation.
  • the epitope tag, reporter and/or localization signal may be the same molecule.
  • the epitope tag, reporter and/or localization signal may also be different molecules.
  • Ligands and polyligands and optional amino acids linked thereto can be synthesized chemically or recombinantly using techniques known in the art.
  • Chemical synthesis techniques include but are not limited to peptide synthesis which is often performed using an automated peptide synthesizer.
  • Pepetides can also be synthesized utilizing non-automated peptide sythesis methods known in the art.
  • Recombinant techniques include insertion of ligand-encoding nucleic acids into expression vectors, wherein nucleic acid expression products are synthesized using cellular factors and processes.
  • Linkage of a cellular localization signal, epitope tag, or reporter to a ligand or polyligand can include covalent or enzymatic linkage to the ligand.
  • the localization signal comprises material other than a polypeptide, such as a lipid or carbohydrate
  • a chemical reaction to link molecules may be utilized.
  • non-standard amino acids and amino acids modified with lipids, carbohydrates, phosphate or other molecules may be used as precursors to peptide synthesis.
  • the ligands of the invention have therapeutic utility with or without localization signals. However, ligands linked to localization signals have utility as subcellular tools or therapeutics.
  • FIGURES 7A-7G and FIGURES 8A-8G represent ligands with utility as subcellular tools or therapeutics.
  • PKD ligand-containing gene constructs are also delivered via gene therapy.
  • FIGURES 1OB and 1OC depict embodiments of gene therapy vectors for delivering and controlling polypeptide expression in vivo.
  • Polynucleotide sequences linked to the gene construct in FIGURES 1OB and 1OC include genome integration domains to facilitate integration of the transgene into a viral genome and/or host genome.
  • FIGURE 1OA shows a vector containing a PKD ligand gene construct, wherein the ligand gene construct is releasable from the vector as a unit useful for generating transgenic animals.
  • the ligand gene construct, or transgene is released from the vector backbone by restriction endonuclease digestion. The released transgene is then injected into pronuclei of fertilized mouse eggs; or the transgene is used to transform embryonic stem cells.
  • the vector containing a ligand gene construct of FIGURE 1OA is also useful for transient transfection of the trangene, wherein the promoter and codons of the transgene are optimized for the host organism.
  • the vector containing a ligand gene construct of FIGURE 1OA is also useful for recombinant expression of polypeptides in fermentable organisms adaptable for small or large scale production, wherein the promoter and codons of the transgene are optimized for the fermentation host organism.
  • FIGURE 1OD shows a vector containing a PKD ligand gene construct useful for generating stable cell lines.
  • the invention also encompasses polynucleotides comprising nucleotide sequences encoding ligands, homopolyligands, and heteropolyligands.
  • the polynucleotides of the invention are optionally linked to additional nucleotide sequences encoding epitopes, reporters and/or localization signals.
  • the nucleic acids of the invention are optionally incorporated into vector polynucleotides.
  • the polynucleotides are optionally flanked by nucleotide sequences comprising restriction endonuclease sites and other nucleotides needed for restriction endonuclese activity. The flanking sequences optionally provide cloning sites within a vector.
  • the restriction sites can include, but are not limited to, any of the commonly used sites in most commercially available cloning vectors. Examples of such sites are those recognized by BamHI, CIaI, EcoRI, EcoRV, Spel, AfIII, Ndel, Nhel, Xbal, Xhol, Sphl, Nael, SexAI, Hindlll, Hpal, and Pstl restriction endonucleases. Sites for cleavage by other restriction enzymes, including homing endonucleases, are also used for this purpose.
  • the polynucleotide flanking sequences also optionally provide directionality of subsequence cloning. It is preferred that 5' and 3' restriction endonuclease sites differ from each other so that double-stranded DNA can be directionally cloned into corresponding complementary sites of a cloning vector.
  • Ligands and polyligands with or without localization signals, epitopes or reporters are alternatively synthesized by recombinant techniques.
  • Polynucleotide expression constructs are made containing desired components and inserted into an expression vector. The expression vector is then transfected into cells and the polypeptide products are expressed and isolated.
  • Ligands made according to recombinant DNA techniques have utility as research tools and/or therapeutics.
  • nucleotide sequences encoding ligands, homopolyligands, and heteropolyligands are produced ia as follows. Complimentary oligonucleotides encoding the ligands and flanking sequences are synthesized and annealled. The resulting double-stranded DNA molecule is inserted into a cloning vector using techniques known in the art. When the ligands and polyligands are placed in-frame adjacent to sequences within a transgenic gene construct that is translated into a protein product, they form part of a fusion protein when expressed in cells or transgenic animals.
  • the promotor portion of the recombinant gene can be a constitutive promotor, a non- constitutive promotor, a tissue-specific promotor (constitutive or non-constitutive) or a selectively controlled promotor.
  • Different selectively controlled promotors are controlled by different mechanisms.
  • RheoSwitch R is an inducible promotor system available from RheoGene. Temperature sensitive promotors can also be used to increase or decrease gene expression.
  • An embodiemt of the invention comprises a ligand or polyligand gene construct whose expression is controlled by an inducible promotor.
  • Polyligands are modular in nature.
  • An aspect of the instant invention is the combinatorial modularity of the disclosed polyligands.
  • Another aspect of the invention are methods of making these modular polyligands easily and conveniently.
  • an embodiment of the invention comprises methods of modular subsequence cloning of genetic expression components.
  • the ligands, homopolyligands, heteropolyligands and optional amino acid expression components are synthesized recombinantly, one can consider each clonable element as a module.
  • One aspect of the invention encompasses module flanking sequences that, at one end of the module, are utilized for restriction enzyme digestion once, and at the other end, utilized for restriction enzyme digestion as many times as desired.
  • a restriction site in one part of the module is utilized and destroyed in order to effect sequential cloning of modular elements.
  • restriction sites flanking a coding region of interest are sequences recognized by the restriction enzymes NgoM IV and CIa I; or Xma I and CIa I.
  • flanking sequences remaining in the third circular DNA still contain intact 5' NgoM IV and 3' CIa I and Xma I sites.
  • This process can be repeated numerous times to achieve directional, sequential, modular cloning events. Restriction sites recognized by NgoM IV, Xma I, and CIa I endonucleases represent a group of sites that permit sequential cloning when used as flanking sequences.
  • restriction sites flanking a coding region module are sequences recognized by the restriction enzymes NgoM IV and CIa I; or Xma I and CIa I.
  • NgoM IV and CIa I the restriction enzymes
  • CIa I and CIa I the restriction enzymes
  • FIGURE 11 a first circular DNA is cut with Xma I and CIa I to yield linear DNA with a 5' CIa I overhang and a 3' Xma I overhang.
  • a second linear double-stranded DNA is generated by PCR amplification followed by digestion, or by synthesizing and annealing complimentary oligonucleotides.
  • the second linear DNA has 5' NgoM IV overhang and a 3' CIa I overhang, which are compatible cohesive ends with the first DNA linearized.
  • first and second DNA fragments are mixed together, annealed, and ligated to form a third circular DNA fragment, the Xma I site that was in the first DNA and the NgoM IV site that was in the second DNA are destroyed in the third circular DNA. Flanking sequences remaining in the third circular DNA still contain intact 5' NgoM IV and 3' CIa I and Xma I sites. This process can be repeated numerous times to achieve directional, sequential, modular cloning events. Restriction sites recognized by NgoM IV, Xma I, and CIa I endonucleases represent a group of sites that permit sequential cloning when used as flanking sequences. This process is depicted in FIGURE 11.
  • restriction site groups can accomplish sequential, directional cloning as described herein.
  • Preferred criteria for restriction endonuclease selection are selecting a pair of endonucleases that generate compatible cohesive ends but whose sites are destroyed upon ligation with each other. Another criteria is to select a third endouclease site that does not generate sticky ends compatible with either of the first two.
  • ligands, polyligands and other coding regions or expression components can be combinatorially assembled as desired. The same sequential process can be utilzed for epitope, reporter, and/or localization signals.
  • Therapeutics include delivery of purified ligand or polyligand with or without a localization signal to a cell.
  • ligands and polyligands with or without a localization signals are delivered via adenovirus, lentivirus, adeno-associated virus, or other viral constructs that express protein product in a cell.
  • a polypeptide comprising a heteropolyligand, an endoplasmic reticulum cellular localization signal, and a His6 epitope is synthesized.
  • Examples of such polypeptides are generically represented by FIGURES 8B, 8D, 8E and 8F.
  • the polypeptide is synthesized on an automated peptide synthesizer or is recombinantly expressed and purified. Purified polypeptide is solubilized in media and added to cells. The polypeptide is endocytosed by the cells, and transported to the endoplasmic reticulum. Verification is performed by immunohistochemical staining using an anti-His6 antibody.
  • a transgene is constructed using a human cytomegalovirus (CMV) promoter to direct expression of a fusion protein comprising SEQ ID NO: 113 (POLYLIGAND), green fluorescent protein (REPORTER), and a nuclear localization signal (LOCALIZATION SIGNAL).
  • CMV human cytomegalovirus
  • POLYLIGAND human cytomegalovirus
  • REPORTER green fluorescent protein
  • LOCALIZATION SIGNAL nuclear localization signal
  • Example 3 A transgene construct is built to produce a protein product with expression driven by a tissue-specific promoter.
  • the transgene comprises a synthetic gene expression unit engineered to encode three domains. Each of these three domains is synthesized as a pair of complimentary polynucleotides that are annealed in solution, ligated and inserted into a vector. Starting at the ammo-terminus, the three domains in the expression unit are nucleotide sequences that encode a PKD ligand, a FLAGTM epitope, and an endoplasmic reticulum localization signal.
  • the PKD ligand is a monomelic ligand, homopolymeric ligand or heteropolymeric ligand as described herein.
  • Nucleotide sequences encoding a FLAGTM epitope are placed downstream of nucleotide sequences encoding the PKD ligand. Finally, nucleotide sequences encoding the localization signal are placed downstream of those encoding the FLAGTM epitope.
  • the assembled gene expression unit is subsequently subcloned into an expression vector, such as that shown in FIGURE 1OA, and used to transiently transfect cells. Verification is performed by immunohistochemical staining using an anti-FLAGTM antibody.
  • Example 4 Ligand function and localization is demonstrated in vivo by making a transgene construct used to generate mice expressing a ligand fusion protein targeted to the endoplasmic reticulum.
  • the transgene construct is shown generically in FIGURE 1OB.
  • the expression unit contains nucleotides that encode a tetramer of SEQ ID NO:33, a hemagluttinin epitope, and a mitochondrial localization signal. This expression unit is subsequently subcloned into a pBluscript-based vector (Stratagene; La Jolla, Calif.) between nucleotide sequences including a promoter and an SV40 polyadenylation signal.
  • pBluscript-based vector Stratagene; La Jolla, Calif.
  • transgenic founder mice are bred with wild-type mice.
  • Heterozygous transgenic animals from at least the third generation are used for the following tests, with their non-transgenic littermates serving as controls.
  • Test 1 Southern blotting analysis is performed to determine the copy number. Southern blots are hybridized with a radio-labeled probe generated from a fragment of the transgene. The probe detects bands containing DNA from transgenic mice, but does not detect bands containing DNA from non- transgenic mice. Intensities of the transgenic mice bands are measured and compared with the transgene plasmid control bands to estimate copy number. This demonstrates that mice in Example 4 harbor the transgene in their genomes.
  • Test 2 Tissue homogenates are prepared for Western blot analysis. This experiment demonstrates the transgene is expressed in tissues of transgenic mice because hemagluttinin epitope is detected in transgenic homogenates but not in non-transgenic homogenates. These examples demonstrate delivery of ligands to a localized region of a cell for therapeutic or experimental purposes.
  • the purified polypeptide ligands can be formulated for oral or parenteral administration, topical administration, or in tablet, capsule, or liquid form, intranasal or inhaled aerosol, subcutaneous, intramuscular, intraperitoneal, or other injection; intravenous instillation; or any other routes of administration.
  • nucleotide sequences encoding the ligands permit incorporation into a vector designed to deliver and express a gene product in a cell.
  • vectors include plasmids, cosmids, artificial chromosomes, and modified viruses.
  • Delivery to eukaryotic cells can be accomplished in vivo or ex vivo. Ex vivo delivery methods include isolation of the intended recipient's cells or donor cells and delivery of the vector to those cells, followed by treatment of the recipient with the cells.
  • ligands and polyligands that modulate PKD activity and methods of making and using these ligands.
  • the ligands and polyligands are synthesized chemically or recombinantly and are utilized as research tools or as therapeutics.
  • the invention includes linking the ligands and polyligands to cellular localization signals for subcellular therapeutics.
  • Ala lie GIu Ser Pro Ala VaI Ala Ala Pro Ala Tyr Ser Arg Ala Leu
  • GIy VaI Ser GIu lie Arg His Thr Ala Asp Arg Trp Arg VaI Ser Leu 35 40 45 Asp VaI Asn His Phe Ala Pro Asp GIu Arg Thr VaI Lys Thr 50 55 60
  • ⁇ 400> 34 lie Arg Arg Arg Ser Ser Asn Tyr Arg Ala 1 5 10
  • GIy Ser Ser Asp Ala Ala Arg GIu Pro Arg Pro Ala Pro Ala Pro lie 1 5 10 15 Arg Arg Arg Ser Ser Asn Tyr Arg Ala Tyr Ala Thr GIu Pro His Ala 20 25 30
  • Lys Lys Lys Lys Ser Lys lie Ser Ala Ser 35 40
  • Xaa can be any naturally occurring amino acid ⁇ 400> 53
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid ⁇ 400> 56
  • Ala lie GIu Ser Pro Ala VaI Ala Ala Pro Ala Tyr Ser Arg Ala Leu 1 5 10 15
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid ⁇ 400> 58
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • ⁇ 210> 61 ⁇ 211> 21 ⁇ 212> PRT ⁇ 213> artificial ⁇ 220> ⁇ 223> synthetic ⁇ 220> ⁇ 221> misc_feature ⁇ 222> (11) .. (11) ⁇ 223> Xaa can be any naturally occurring amino acid ⁇ 400> 61
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid ⁇ 220> ⁇ 221> raisc__feature
  • Xaa can be any naturally occurring amino acid
  • ⁇ 210> 64 ⁇ 211> 21 ⁇ 212> PRT ⁇ 213> artificial ⁇ 220> ⁇ 223> synthetic ⁇ 220> ⁇ 221> misc_feature ⁇ 222> (11) .. (H) ⁇ 223> Xaa can be any naturally occurring amino acid ⁇ 400> 64
  • Xaa can be any naturally occurring amino acid
  • ⁇ 210> 66 ⁇ 211> 21 ⁇ 212> PRT ⁇ 213> artificial ⁇ 220> ⁇ 223> synthetic ⁇ 220> ⁇ 221> misc_feature ⁇ 222> (11) -.(ID ⁇ 223> Xaa can be any naturally occurring amino acid ⁇ 400> 66
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid ⁇ 400> 68
  • Xaa can be any naturally occurring amino acid ⁇ 400> 69 GIy GIy His Arg Pro Leu Ser Arg Ala Gin Xaa Ser Pro Ala Ala Pro 1 5 10 15
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid ⁇ 400> 73 GIn Met GIn Arg Thr lie Thr Arg Gin Met Xaa Phe Asp Leu Thr Lys 1 5 10 15
  • Xaa can be any naturally occurring amino acid ⁇ 400> 74
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • GIy Asp Trp Arg Lys Asn lie Asp Ala Leu Xaa GIy Met GIu GIy Arg 1 5 10 15 Lys Lys Lys Phe GIu 20
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • Lys Lys Lys Lys Ser Lys lie Ser Ala Ser 35 40
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid ⁇ 400> 82 Met Gin GIy Leu Ala GIu Arg lie Xaa VaI Leu 1 5 10
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid ⁇ 400> 86 GIy GIy Ala Cys Leu Pro Gin Asp His GIu Met GIn GIy Leu Ala GIu 1 5 10 15
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid ⁇ 400> 91 Leu Lys Arg Thr Ala Xaa Asn Pro Lys VaI 1 5 10
  • ⁇ 210> 92 ⁇ 211> 9 ⁇ 212> PRT ⁇ 213> artificial ⁇ 220> ⁇ 223> synthetic ⁇ 220> ⁇ 221> misc_feature ⁇ 222> (6) .. (6) ⁇ 223> Xaa can be any naturally occurring amino acid ⁇ 400> 92
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid ⁇ 400> 96 Leu Ala GIu Arg lie Xaa lie Leu 1 5
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • ⁇ 400> 98 lie Thr Arg Gin Met Xaa Phe Asp Leu 1 5 ⁇ 210> 99 ⁇ 211> 10 ⁇ 212> PRT ⁇ 213> artificial ⁇ 220> ⁇ 223> synthetic ⁇ 220> ⁇ 221> misc_feature ⁇ 222> (8) .. (8) ⁇ 223> Xaa can be any naturally occurring amino acid ⁇ 400> 99
  • Xaa can be any naturally occurring amino acid ⁇ 400> 100 lie Arg Arg Arg Xaa Xaa Asn Tyr Arg Ala 1 5 10
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid ⁇ 400> 102
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • Asp Phe GIy lie Ser Ala Gin lie GIy Ala Thr Leu Ala Arg Arg Leu 1 5 10 15 Ala Phe lie GIy Thr Pro Tyr Trp Met Ala Pro GIu Ala Ala Leu VaI 20 25 30

Abstract

The invention relates to kinase ligands and polyligands. In particular, the invention relates to ligands, homopolyligands, and heteropolyligands that modulate protein kinase D (PKD) activity. The ligands and polyligands are utilized as research tools or as therapeutics. The invention includes linkage of the ligands and polyligands to cellular localization signals, epitope tags and/or reporters. The invention also includes polynucleotides encoding the ligands and polyligands.

Description

PKD LIGANDSAND POLYNUCLEOTIDES ENCODING PKD LIGANDS
Inventor: Thomas D. Reed
FIELD OF INVENTION
The invention relates to mammalian kinase ligands, substrates and modulators. In particular, the invention relates to polypeptides, polypeptide compositions and polynucleotides that encode polypeptides that are ligands, substrates, and/or modulators of PKD. The invention also relates to polyligands that are homopolyligands or heteropolyligands that modulate PKD activity. The invention also relates to ligands and polyligands tethered to a subcellular location.
This application has subject matter related to application Nos. 10/724,532 (now US 7,071,295), 10/682,764 (US2004/0185556, PCT/US2004/013517, WO2005/040336), 11/233,246, and US20040572011P (WO2005116231). Each of these patents and applications is hereby incorporated by reference.
BACKGROUND AND PRIOR ART
Kinases are enzymes that catalyze the addition of phosphate to a molecule. The addition of phosphate by a kinase is called phosphorylation. When the kinase substrate is a protein molecule, the amino acids commonly phosphorylated are serine, threonine and tyrosine. Phosphatases are enzymes that remove phosphate from a molecule. The removal of phosphate is called dephosphorylation. Kinases and phosphatases often represent competing forces within a cell to transmit, attenuate, or otherwise modulate cellular signals and cellular control mechanisms. Kinases and phosphatases have both overlapping and unique natural substrates. Cellular signals and control mechanisms, as regulated by kinases, phosphatases, and their natural substrates are a target of research tool design and drug design.
Mammalian Protein Kinase D is also known as PKD. The enzymatic activity, activation and autoregulation of PKD have been studied. Several cellular substrates of PKD have been identified. Substrates and modified substrates are genetically referred to herein as ligands. Natural and synthetic peptide ligands have been studied to examine PKD substrate specificty. While peptide ligands and variants thereof have been studied as individuals PKD ligands, mixed ligands linked together as polyligands have not been demonstrated before this invention. Design and synthesis of polypeptide ligands that modulate calcium/calmodulin-dependent protein kinase and that localize to the cardiac sarco(endo)plasmic reticulum was performed by Ji et al. (J Biol Chem (2003) 278:25063-71). Ji et al. accomplished this by generating expression constructs that localized calcium/calmodulin-dependent protein kinase inhibitory polypeptide ligands to the sarcoplasmic reticulum by fusing a sarcoplasmic reticulum localization signal derived from phospholamban to a polypeptide ligand. See also US 7,071,295.
DETAILED DESCRIPTION OF POLYPEPTIDE AND POLYNUCLEOTIDE SEQUENCES
SEQ ID NOS: 1-104 represent examples of monomelic peptide ligand sequences.
Specifically, SEQ ID NOS: 1-52 are fragments of natural substrates of mammlain PKD, wherein each fragment contains at least one amino acid residue phosphorylatable by PKD. SEQ ID NOS:53-104 are fragments of natural PKD substrates, where the location of the PKD phosphorylatable serine or threonine in the natural polypeptide is designated as Xaa.
SEQ ID NOS: 105-116 are example polyligands and polynucleotides encoding them.
Specifically, SEQ ID NO:105 is encoded by SEQ ID NO:106, SEQ ID NO:107 and by SEQ ID NO:108, wherein the the codons of SEQ ID NO: 107 and SEQ ID NO: 108 have been optimized for vector insertion. SEQ ID NO: 108 includes flanking restriction sites. SEQ ID NO: 105 is an embodiment of a polyligand of the structure X-S1-Y-S2-Z, wherein X is SEQ ID NO:53, Y is SEQ ID NO:55, Z is SEQ
ID NO:58, wherein Xaa is alanine, and wherein Sl is a four amino acid spacer of the amino acid sequence AGAG and S2 is a four amino acid spacer of amino acid sequence GAGA. A polyligand of structure A-S1-B-S2-C is called herein a heteropolyligand, shown generically in FIGURE 4B.
SEQ ID NO:109 is an embodiment of a polyligand of the structure X-S1-Y-S2-Z, wherein X is SEQ ID NO:61, Y is SEQ ID NO:65, Z is SEQ ID NO:66, wherein Xaa is alanine, wherein Sl is a four amino acid spacer of amino acid sequence AGAG and S2 is a four amino acid spacer of amino acid sequence GAGA. The PKD polyligand of SEQ ID NO:109 is encoded by SEQ ID NO:110, SEQ ID NO:111 and by SEQ ID NO:112, wherein the the codons of SEQ ID NO:111 and SEQ ID NO:112 have been optimized for vector insertion. SEQ ID NO: 112 includes flanking restriction sites. A polyligand of structure X-S1-Y-S2-Z is also called herein a heteropolyligand, shown generically in FIGURE 4B. SEQ ID NO:113 is an embodiment of a polyligand of the structure X-Y-S3-Y-X, wherein X is SEQ ID NO:53, Y is SEQ ID NO:54, wherein Xaa is alanine, and wherein S3 is a five amino acid spacer with the sequence PAGAG. The PKD polyligand of SEQ ID NO:113 is encoded by SEQ ID NO:114, SEQ ID NO:115, and by SEQ ID NO:116, wherein the the codons of SEQ ID NO:115 and SEQ ID NO:116 have been optimized for vector insertion. SEQ ID NO:116 includes flanking restriction sites. A polyligand of structure X-Y-S3-Y-X is also called herein a heteropolyligand, shown generically in FIGURE 4C.
SEQ ID NOS: 117-132 are full length PKlD substrates. These sequences have the following public database accession numbers: AAH64840, NP000354, CAA79356, NP001015053, NP009112, NP001531, NP065789, AAH00029, AAB48596, NP_536728, AAH47282, NP004283, NP005219, CAB95729, NP_057541, NP_849231. Each of the protein sequences represented by these accession numbers is incorporated by reference herein. In SEQ ID NOS: 117-132, the positions of the amino acid(s) phosphorylatable by PKD are represented by Xaa. In wild-type proteins, Xaa is serine or threonine. In the ligands of the invention, Xaa is any amino acid.
Amino acid sequences containing Xaa encompass polypeptides where Xaa is any amino acid.
DETAILED DESCRIPTION OF DRAWINGS
FIGURES 1A-1C show examples of homopolymeric ligands without spacers.
FIGURES 2A-2C show examples of homopolymeric ligands with spacers.
FIGURES 3A-3C show examples of heteropolymeric ligands without spacers.
FIGURES 4A-4C show examples of heteropolymeric ligands with spacers. FIGURES 5A-5G show examples of ligands and polymeric ligands linked to an optional epitope tag.
FIGURES 6A-6G show examples of ligands and polymeric ligands linked to an optional reporter.
FIGURES 7A-7G show examples of ligands and polymeric ligands linked to an optional localization signal.
FIGURES 8A-8G show examples of ligands and polymeric ligands linked to an optional localization signal and an optional epitope tag.
FIGURES 9A-9G show examples of gene constructs where ligands and polyligands are linked to an optional localization signal, an optional epitope tag, and an optional reporter.
FIGURES 10A- 1OD show examples of vectors containing ligand gene constructs. FIGURE 11 shows an example of a sequential cloning process useful for combinatorial synthesis of polyligands.
BRIEF DESCRIPTION OF THE INVENTION
The invention relates to polypeptide ligands and polyligands for PKD. Various embodiments of the PKD ligands and polyligands are represented in SEQ ID NOS: 1-132. More specifically, the invention relates to ligands, homopolyligands, and heteropolyligands that comprise any one or more of SEQ ID NOS: 1-
104. Additionally, the invention relates to ligands and polyligands comprising one or more subsequences of SEQ ID NOS: 117-132 or any portion thereof. Furthermore, the invention relates to polyligands with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% sequence identity to a polyligand comprising one or more of SEQ ID NOS:1-104 or any portion thereof. Furthermore, the invention relates to polyligands with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% sequence identity to a polyligand comprising one or more subsequences of SEQ ID NOS: 117-132 or portions thereof.
Polyligands, which can be homopolyligands or heteropolyligands, are chimeric ligands composed of two or more monomeric polypeptide ligands. An example of a monomelic ligand is the polypeptide represented by SEQ ID NO:61, wherein Xaa is any amino acid. An example of a homopolyligand is a polypeptide comprising a dimer or multimer of SEQ ID NO:61, wherein Xaa is any amino acid. An example of a heteropolyligand is a polypeptide comprising SEQ ID NO:1 and one or more of SEQ ID NOS:2-104, wherein Xaa is any amino acid. There are numerous ways to combine SEQ ID NOS:1-104 into homopolymeric or heteropolymeric ligands. Furthermore, there are numerous ways to combine portions or subsequences of SEQ ID NOS:117-132 with each other and with SEQ ID NOS:1-104 to make polymeric ligands that modulate PKD.
The polyligands of the invention optionally comprise spacer amino acids before, after, or between monomers. SEQ ID NO: 105 is an embodiment of a polyligand of the structure X-S1-Y-S2-Z, wherein X is SEQ ID NO:53, Y is SEQ ID NO:55, Z is SEQ ID NO:58, wherein Xaa is alanine, and wherein Sl and S2 are four amino acid spacers. This invention intends to capture all combinations of homopolyligands and heteropolyligands without limitation to the examples given above or below. In this description, use of the term "ligand(s)" encompasses monomeric ligands, polymeric ligands, homopolymeric ligands and/or heteropolymeric ligands. A monomeric ligand is a polypeptide where at least a portion of the polypeptide is capable of being recognized by PKD. The portion of the polypeptide capable of recognition is termed the recognition motif. In the present invention, recognition motifs can be natural or synthetic. Examples of recognition motifs are well known in the art and include, but are not limited to, naturally occurring PKD substrates and pseudosubstrate motifs.
A polymeric ligand comprises two or more monomeric ligands.
A homopolymeric ligand is a polymeric ligand where each of the monomeric ligands is identical in amino acid sequence, except that a phosphorylatable residue may be substituted or modified in one or more of the monomeric ligands.
A heteropolymeric ligand is a polymeric ligand where some of the monomeric ligands do not have an identical amino acid sequence.
The ligands of the invention are optionally linked to additional molecules or amino acids that provide an epitope tag, a reporter, and/or a cellular localization signal. The cellular localization signal targets the ligands to a region of a cell. The epitope tag and/or reporter and/or localization signal may be the same molecule. The epitope tag and/or reporter and/or localization signal may also be different molecules.
The invention also encompasses polynucleotides comprising a nucleotide sequence encoding ligands, homopolyligands, and heteropoly ligands. The nucleic acids of the invention are optionally linked to additional nucleotide sequences encoding polypeptides with additional features, such as an epitope tag, a reporter, and/or a cellular localization signal. The polynucleotides are optionally flanked by nucleotide sequences comprising restriction endonuclease sites and other nucleotides needed for restriction endonuclese activity. The flanking sequences optionally provide unique cloning sites within a vector and optionally provide directionality of subsequence cloning. Further, the nucleic acids of the invention are optionally incorporated into vector polynucleotides. The ligands, polyligands, and polynucleotides of this invention have utility as research tools and/or therapeutics.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to ligands and polyligands that are PKD modulators. Various embodiments of ligands and polyligands are represented in SEQ ID NOS: 1-132. Polyligands are chimeric ligands comprising two or more monomelic polypeptide ligands. An example of a monomelic ligand is the polypeptide represented by SEQ ID NO:54, wherein Xaa is any amino acid. Another example of a monomelic ligand is the polypeptide represented by SEQ ID NO:1. Each of SEQ ID NOS: 1-104 represents an individual polypeptide ligand in monomeric form, wherein Xaa is any amino acid. Additional examples of monomeric ligands are subsequence portions of SEQ ID NOS:117-132 containing a PKD recognition motif. Monomeric ligand subsequences of SEQ ID NOS:117-132 may be wild-type subsequences. Additionally, monomeric ligand subsequences of SEQ ID NOS: 117-132 may have the PKD phosphorylatable amino acids replaced by other amino acids. Furthermore, monomeric ligands and polyligands may have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a ligand comprising an amino acid sequence in one or more of SEQ ID NOS:1-104. Furthermore, monomeric ligands and polyligands may have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% sequence identity to a subsequence of SEQ ID NOS:117-132.
An example of a homopolyligand is a polypeptide comprising a dimer or multimer of SEQ ID NO:66, wherein Xaa is any amino acid. An example of a heteropolyligand is a polypeptide comprising SEQ ID NO: 104 and one or more of SEQ ID NOS: 1-103, wherein Xaa is any amino acid. There are numerous ways to combine SEQ ID NOS:1-104 into homopolymeric or heteropolymeric ligands. Furthermore, there are numerous ways to combine PKD recogntition motif-containing portions of SEQ ID NOS :117- 132 with each other and with SEQ ID NOS:1-104 to make polymeric ligands.
Polyligands may comprise any two or more of a sequence selected from SEQ ID NOS: 1-104 and recognition motif-containing portions of SEQ ID NOS: 117-132, wherein Xaa is any amino acid. A dimer or multimer of SEQ ID NO:91 is an example of a homopolyligand. An example of a heteropolyligand is a polypeptide comprising SEQ ID NO:5 and one or more of a recognition motif-containing portion of SEQ ID NOS:117-132. There are numerous ways to combine SEQ ID NOS:1-104 and portions of SEQ ID NOS:117-132 into homopolymeric or heteropolymeric ligands. The instant invention is directed to all possible combinations of homopolyligands and heteropolyligands without limitation.
SEQ ID NOS:53-104 and SEQ ID NOS:117-132 show polypeptides that contain at least one serine or threonine residue phosphorylatable by PKD, the positions of which are represented by Xaa. In nature, Xaa is, generally speaking, serine or threonine. In one embodiment of the instant invention, Xaa can be any amino acid. Ligands where Xaa is serine or threonine can be used as part of a polyligand, however in one embodiment, at least one phosphorylatable serine or threonine is replaced with another amino acid, such as one of the naturally occurring amino acids including, alanine, aspartate, asparagine, cysteine, glutamate, glutamine, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, arginine, valine, tryptophan, or tyrosine. The Xaa may also be a non-naturally occurring amino acid. In another embodiment, the PKD phosphorylatable serine(s) or threonine(s) are replaced by alanine. The ligands and polyligands of the invention are designed to modulate the endogenous effects of PKD.
In general, ligand monomers based on natural PKD substrates are built by isolating a putative PKD phosphorylation recognition motif in a PKD substrate. Sometimes it is desirable to modify the phosphorylatable residue to an amino acid other than serine or threonine. Additional monomers include the PKD recognition motif as well as amino acids adjacent and contiguous on either side of the PKD recognition motif. Monomeric ligands may therefore be any length provided the monomer includes the PKD recognition motif. For example, the monomer may comprise a PKD recognition motif and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30-100 or more amino acids adjacent to the recognition motif.
For example, in one embodiment, the invention comprises an inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 194-201 of SEQ ID NO: 117, wherein the amino acid residue corresponding to amino acid residue 199 of SEQ ID NO : 117 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 191-203 of SEQ ID NO: 117, wherein the amino acid residue corresponding to amino acid residue 199 of SEQ ID NO : 117 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 189-205 of SEQ ID NO: 117, wherein the amino acid residue corresponding to amino acid residue 199 of SEQ ID NO: 117 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 184-207 of SEQ ID NO: 117, wherein the amino acid residue corresponding to amino acid residue 199 of SEQ ID NO:117 is an amino acid residue other than serine or threonine. As used herein, the terms "correspond(s) to" and "corresponding to," as they relate to sequence alignment, are intended to mean enumerated positions within a reference protein, e.g., HDAC5 (SEQ ID NO:119), and those positions that align with the positions on the reference protein. Thus, when the amino acid sequence of a subject peptide is aligned with the amino acid sequence of a reference peptide, e.g., SEQ ID NO: 119, the amino acids in the subject peptide sequence that "correspond to" certain enumerated positions of the reference peptide sequence are those that align with these positions of the reference peptide sequence, but are not necessarily in these exact numerical positions of the reference sequence. Methods for aligning sequences for determining corresponding amino acids between sequences are described below.
Additional embodiments of the invention include monomers (as described above) based on any putative or real substrate for PKD, such as substrates identified by SEQ ID NOS: 1-104 and SEQ ID NOS: 117- 132. Furthermore, if the substrate has more than one recognition motif, then more than one monomer may be identified therein.
Another embodiment of the invention is a nucleic acid molecule comprising a polynucleotide sequence encoding at least one copy of a ligand peptide.
Another embodiment of the invention is a nucleic acid molecule wherein the polynucleotide sequence encodes one or more copies of one or more peptide ligands.
Another embodiment of the invention is a nucleic acid molecule wherein the polynucleotide sequence encodes at least a number of copies of the peptide selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9 or 10.
Another embodiment of the invention is a vector comprising a nucleic acid molecule encoding at least one copy of a ligand or polyligand.
Another embodiment of the invention is a recombinant host cell comprising a vector comprising a nucleic acid molecule encoding at least one copy of a ligand or polyligand.
Another embodiment of the invention is a method of inhibiting PKD in a cell comprising transfecting a vector comprising a nucleic acid molecule encoding at least one copy of a ligand or polyligand into a host cell and culturing the transfected host cell under conditions suitable to produce at least one copy of the ligand or polyligand.
The invention also relates to modified inhibitors that are at least about 80%, 85%, 90% 95%, 96%, 97%, 98% or 99% identical to a reference inhibitor. A "modified inhibitor" is used to mean a peptide that can be created by addition, deletion or substitution of one or more amino acids in the primary structure (amino acid sequence) of a inhibitor protein or polypeptide. A "modified recognition motif is a naturally occurring PKD recognition motif that has been modified by addition, deletion, or substitution of one or more amino acids in the primary structure (amino acid sequence) of the motif. For example, a modified PKD recognition motif may be a motif where the phosphorylatable amino acid has been modified to a non-phosphorylatable amino acid. The terms "protein" and "polypeptide" are used interchangeably herein. The reference inhibitor is not necessarily a wild-type protein or a portion thereof. Thus, the reference inhibitor may be a protein or peptide whose sequence was previously modified over a wild- type protein. The reference inhibitor may or may not be the wild-type protein from a particular organism.
A polypeptide having an amino acid sequence at least, for example, about 95% "identical" to a reference an amino acid sequence is understood to mean that the amino acid sequence of the polypeptide is identical to the reference sequence except that the amino acid sequence may include up to about five modifications per each 100 amino acids of the reference amino acid sequence encoding the reference peptide. In other words, to obtain a peptide having an amino acid sequence at least about 95% identical to a reference amino acid sequence, up to about 5% of the amino acid residues of the reference sequence may be deleted or substituted with another amino acid or a number of amino acids up to about 5% of the total amino acids in the reference sequence may be inserted into the reference sequence. These modifications of the reference sequence may occur at the N-terminus or C-terminus positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
As used herein, "identity" is a measure of the identity of nucleotide sequences or amino acid sequences compared to a reference nucleotide or amino acid sequence. In general, the sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g., Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York (1988); Biocomputing: Informatics And Genome Projects, Smith, D.W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); von Heinje, G., Sequence Analysis In Molecular Biology, Academic Press (1987); and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York (1991)). While there exist several methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo, H. & Lipton, D., Siam J Applied Math 48:1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego (1994) and Carillo, H. & Lipton, D., Siam J Applied Math 48:1073 (1988). Computer programs may also contain methods and algorithms that calculate identity and similarity. Examples of computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al, Nucleic Acids Research 12(i):387 (1984)), BLASTP, ExPASy, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990)) and FASTDB. Examples of methods to determine identity and similarity are discussed in Michaels, G. and Garian, R., Current Protocols in Protein Science, VoI 1, John Wiley & Sons, Inc. (2000), which is incorporated by reference. In one embodiment of the present invention, the algorithm used to determine identity between two or more polypeptides is BLASTP.
In another embodiment of the present invention, the algorithm used to determine identity between two or more polypeptides is FASTDB, which is based upon the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990), incorporated by reference). In a FASTDB sequence alignment, the query and subject sequences are amino sequences. The result of sequence alignment is in percent identity. Parameters that may be used in a FASTDB alignment of amino acid sequences to calculate percent identity include, but are not limited to: Matrix=PAM, k-tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject amino sequence, whichever is shorter.
If the subject sequence is shorter or longer than the query sequence because of N-terminus or C-terminus additions or deletions, not because of internal additions or deletions, a manual correction can be made, because the FASTDB program does not account for N-terminus and C-terminus truncations or additions of the subject sequence when calculating percent identity. For subject sequences truncated at both ends, relative to the query sequence, the percent identity is corrected by calculating the number of amino acids of the query sequence that are N-and C-terminus to the reference sequence that are not matched/aligned, as a percent of the total bases of the query sequence. The results of the FASTDB sequence alignment determine matching/alignment. The alignment percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score can be used for the purposes of determining how alignments "correspond" to each other, as well as percentage identity. Residues of the query (subject) sequences or the reference sequence that extend past the N-or C-termini of the reference or subject sequence, respectively, may be considered for the purposes of manually adjusting the percent identity score. That is, residues that are not matched/aligned with the N-or C-termini of the comparison sequence may be counted when manually adjusting the percent identity score or alignment numbering.
For example, a 90 amino acid residue subject sequence is aligned with a 100 residue reference sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a match/alignment of the first 10 residues at the N- terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N-and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 reference sequence. This time the deletions are internal deletions so there are no residues at the N-or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected.
The polyligands of the invention optionally comprise spacer amino acids before, after, or between monomers. The length and composition of the spacer may vary. An example of a spacer is glycine, alanine, polyglycine, or polyalanine. Specific examples of spacers used between monomers in SEQ ID NO: 105 are the four amino acids AGAG, and the four amino acids GAGA. Spacer amino acids may be any amino acid and are not limited to alanine and glycine. The instant invention is directed to all combinations of homopolyligands and heteropolyligands, with or without spacers, and without limitation to the examples given above or below. The ligands and polyligands of the invention are optionally linked to additional molecules or amino acids that provide an epitope tag, a reporter, and/or localize the ligand to a region of a cell (See FIGURES 5A- 5G, FIGURES 6A-6G, FIGURES 7A-7G, and FIGURES 8A-8G). Non-limiting examples of epitope tags are FLAG™ (Kodak; Rochester, N.Y.), HA (hemagluttinin), c-Myc and His6. Additional examples of epitope tags are given in Jarvik & Telmer 1998 Annual Reviw of Genetics 32:601-18. Non-limiting examples of reporters are alkaline phosphatase, galactosidase, peroxidase, luciferase and green fluorescent protein (GFP). Non-limiting examples of cellular localizations are sarcoplamic reticulum, endoplasmic reticulum, mitochondria, golgi apparatus, nucleus, plasma membrane, apical membrane, and basolateral membrane. The epitopes, reporters and localization signals are given by way of example and without limitation. The epitope tag, reporter and/or localization signal may be the same molecule. The epitope tag, reporter and/or localization signal may also be different molecules.
Ligands and polyligands and optional amino acids linked thereto can be synthesized chemically or recombinantly using techniques known in the art. Chemical synthesis techniques include but are not limited to peptide synthesis which is often performed using an automated peptide synthesizer. Pepetides can also be synthesized utilizing non-automated peptide sythesis methods known in the art. Recombinant techniques include insertion of ligand-encoding nucleic acids into expression vectors, wherein nucleic acid expression products are synthesized using cellular factors and processes.
Linkage of a cellular localization signal, epitope tag, or reporter to a ligand or polyligand can include covalent or enzymatic linkage to the ligand. When the localization signal comprises material other than a polypeptide, such as a lipid or carbohydrate, a chemical reaction to link molecules may be utilized. Additionally, non-standard amino acids and amino acids modified with lipids, carbohydrates, phosphate or other molecules may be used as precursors to peptide synthesis. The ligands of the invention have therapeutic utility with or without localization signals. However, ligands linked to localization signals have utility as subcellular tools or therapeutics. For example, ligands depicted generically in FIGURES 7A-7G and FIGURES 8A-8G represent ligands with utility as subcellular tools or therapeutics. PKD ligand-containing gene constructs are also delivered via gene therapy. FIGURES 1OB and 1OC depict embodiments of gene therapy vectors for delivering and controlling polypeptide expression in vivo. Polynucleotide sequences linked to the gene construct in FIGURES 1OB and 1OC include genome integration domains to facilitate integration of the transgene into a viral genome and/or host genome. FIGURE 1OA shows a vector containing a PKD ligand gene construct, wherein the ligand gene construct is releasable from the vector as a unit useful for generating transgenic animals. For example, the ligand gene construct, or transgene, is released from the vector backbone by restriction endonuclease digestion. The released transgene is then injected into pronuclei of fertilized mouse eggs; or the transgene is used to transform embryonic stem cells. The vector containing a ligand gene construct of FIGURE 1OA is also useful for transient transfection of the trangene, wherein the promoter and codons of the transgene are optimized for the host organism. The vector containing a ligand gene construct of FIGURE 1OA is also useful for recombinant expression of polypeptides in fermentable organisms adaptable for small or large scale production, wherein the promoter and codons of the transgene are optimized for the fermentation host organism.
FIGURE 1OD shows a vector containing a PKD ligand gene construct useful for generating stable cell lines.
The invention also encompasses polynucleotides comprising nucleotide sequences encoding ligands, homopolyligands, and heteropolyligands. The polynucleotides of the invention are optionally linked to additional nucleotide sequences encoding epitopes, reporters and/or localization signals. Further, the nucleic acids of the invention are optionally incorporated into vector polynucleotides. The polynucleotides are optionally flanked by nucleotide sequences comprising restriction endonuclease sites and other nucleotides needed for restriction endonuclese activity. The flanking sequences optionally provide cloning sites within a vector. The restriction sites can include, but are not limited to, any of the commonly used sites in most commercially available cloning vectors. Examples of such sites are those recognized by BamHI, CIaI, EcoRI, EcoRV, Spel, AfIII, Ndel, Nhel, Xbal, Xhol, Sphl, Nael, SexAI, Hindlll, Hpal, and Pstl restriction endonucleases. Sites for cleavage by other restriction enzymes, including homing endonucleases, are also used for this purpose. The polynucleotide flanking sequences also optionally provide directionality of subsequence cloning. It is preferred that 5' and 3' restriction endonuclease sites differ from each other so that double-stranded DNA can be directionally cloned into corresponding complementary sites of a cloning vector.
Ligands and polyligands with or without localization signals, epitopes or reporters are alternatively synthesized by recombinant techniques. Polynucleotide expression constructs are made containing desired components and inserted into an expression vector. The expression vector is then transfected into cells and the polypeptide products are expressed and isolated. Ligands made according to recombinant DNA techniques have utility as research tools and/or therapeutics.
An example of how nucleotide sequences encoding ligands, homopolyligands, and heteropolyligands are produced ia as follows. Complimentary oligonucleotides encoding the ligands and flanking sequences are synthesized and annealled. The resulting double-stranded DNA molecule is inserted into a cloning vector using techniques known in the art. When the ligands and polyligands are placed in-frame adjacent to sequences within a transgenic gene construct that is translated into a protein product, they form part of a fusion protein when expressed in cells or transgenic animals.
Another embodiment of the invention relates to selective control of transgene expression in a desired cell or organism. The promotor portion of the recombinant gene can be a constitutive promotor, a non- constitutive promotor, a tissue-specific promotor (constitutive or non-constitutive) or a selectively controlled promotor. Different selectively controlled promotors are controlled by different mechanisms. RheoSwitchR is an inducible promotor system available from RheoGene. Temperature sensitive promotors can also be used to increase or decrease gene expression. An embodiemt of the invention comprises a ligand or polyligand gene construct whose expression is controlled by an inducible promotor.
Polyligands are modular in nature. An aspect of the instant invention is the combinatorial modularity of the disclosed polyligands. Another aspect of the invention are methods of making these modular polyligands easily and conveniently. In this regard, an embodiment of the invention comprises methods of modular subsequence cloning of genetic expression components. When the ligands, homopolyligands, heteropolyligands and optional amino acid expression components are synthesized recombinantly, one can consider each clonable element as a module. For speed and convenience of cloning, it is desirable to make modular elements that are compatible at cohesive ends and are easy to insert and clone sequentially. This is accomplished by exploiting the natural properties of restriction endonuclease site recognition and cleavage. One aspect of the invention encompasses module flanking sequences that, at one end of the module, are utilized for restriction enzyme digestion once, and at the other end, utilized for restriction enzyme digestion as many times as desired. In other words, a restriction site in one part of the module is utilized and destroyed in order to effect sequential cloning of modular elements. An example of restriction sites flanking a coding region of interest are sequences recognized by the restriction enzymes NgoM IV and CIa I; or Xma I and CIa I. Cutting a first circular DNA with NgoM IV and CIa I to yield linear DNA with a 5' NgoM IV overhang and a 3' CIa I overhang; and cutting a second circular DNA with Xma I and CIa I to yield linear DNA with a 5' CIa I overhang and a 3' Xma I overhang generates first and second DNA fragments with compatible cohesive ends. When these first and second DNA fragments are mixed together, annealed, and ligated to form a third circular DNA fragment, the NgoM IV site that was in the first DNA and the Xma I site that was in the second DNA are destroyed in the third circular DNA. Now this vestigial region of DNA is protected from further Xma I or NgoM IV digestion, but flanking sequences remaining in the third circular DNA still contain intact 5' NgoM IV and 3' CIa I and Xma I sites. This process can be repeated numerous times to achieve directional, sequential, modular cloning events. Restriction sites recognized by NgoM IV, Xma I, and CIa I endonucleases represent a group of sites that permit sequential cloning when used as flanking sequences.
Another way to assemble coding region modules directionally and sequentially employs linear DNA in addition to circular DNA. For example, like the sequential cloning process described above, restriction sites flanking a coding region module are sequences recognized by the restriction enzymes NgoM IV and CIa I; or Xma I and CIa I. Referring to FIGURE 11, a first circular DNA is cut with Xma I and CIa I to yield linear DNA with a 5' CIa I overhang and a 3' Xma I overhang. A second linear double-stranded DNA is generated by PCR amplification followed by digestion, or by synthesizing and annealing complimentary oligonucleotides. The second linear DNA has 5' NgoM IV overhang and a 3' CIa I overhang, which are compatible cohesive ends with the first DNA linearized. When these first and second DNA fragments are mixed together, annealed, and ligated to form a third circular DNA fragment, the Xma I site that was in the first DNA and the NgoM IV site that was in the second DNA are destroyed in the third circular DNA. Flanking sequences remaining in the third circular DNA still contain intact 5' NgoM IV and 3' CIa I and Xma I sites. This process can be repeated numerous times to achieve directional, sequential, modular cloning events. Restriction sites recognized by NgoM IV, Xma I, and CIa I endonucleases represent a group of sites that permit sequential cloning when used as flanking sequences. This process is depicted in FIGURE 11.
One of ordinary skill in the art recognizes that other restriction site groups can accomplish sequential, directional cloning as described herein. Preferred criteria for restriction endonuclease selection are selecting a pair of endonucleases that generate compatible cohesive ends but whose sites are destroyed upon ligation with each other. Another criteria is to select a third endouclease site that does not generate sticky ends compatible with either of the first two. When such criteria are utilized as a system for sequential, directional cloning, ligands, polyligands and other coding regions or expression components can be combinatorially assembled as desired. The same sequential process can be utilzed for epitope, reporter, and/or localization signals.
Polyligands and methods of making polyligands that modulate PKD activity are disclosed. Therapeutics include delivery of purified ligand or polyligand with or without a localization signal to a cell. Alternatively, ligands and polyligands with or without a localization signals are delivered via adenovirus, lentivirus, adeno-associated virus, or other viral constructs that express protein product in a cell.
Example 1
A polypeptide comprising a heteropolyligand, an endoplasmic reticulum cellular localization signal, and a His6 epitope is synthesized. Examples of such polypeptides are generically represented by FIGURES 8B, 8D, 8E and 8F. The polypeptide is synthesized on an automated peptide synthesizer or is recombinantly expressed and purified. Purified polypeptide is solubilized in media and added to cells. The polypeptide is endocytosed by the cells, and transported to the endoplasmic reticulum. Verification is performed by immunohistochemical staining using an anti-His6 antibody.
Example 2
A transgene is constructed using a human cytomegalovirus (CMV) promoter to direct expression of a fusion protein comprising SEQ ID NO: 113 (POLYLIGAND), green fluorescent protein (REPORTER), and a nuclear localization signal (LOCALIZATION SIGNAL). Such a transgene is generically represented by FIGURE 9A-9D. The transgene is transfected into cells for transient expression. Verification of expression and location is performed by visualization of green fluorescent protein (GFP) by confocal microscopy.
Example 3 A transgene construct is built to produce a protein product with expression driven by a tissue-specific promoter. The transgene comprises a synthetic gene expression unit engineered to encode three domains. Each of these three domains is synthesized as a pair of complimentary polynucleotides that are annealed in solution, ligated and inserted into a vector. Starting at the ammo-terminus, the three domains in the expression unit are nucleotide sequences that encode a PKD ligand, a FLAG™ epitope, and an endoplasmic reticulum localization signal. The PKD ligand is a monomelic ligand, homopolymeric ligand or heteropolymeric ligand as described herein. Nucleotide sequences encoding a FLAG™ epitope are placed downstream of nucleotide sequences encoding the PKD ligand. Finally, nucleotide sequences encoding the localization signal are placed downstream of those encoding the FLAG™ epitope. The assembled gene expression unit is subsequently subcloned into an expression vector, such as that shown in FIGURE 1OA, and used to transiently transfect cells. Verification is performed by immunohistochemical staining using an anti-FLAG™ antibody.
Example 4 Ligand function and localization is demonstrated in vivo by making a transgene construct used to generate mice expressing a ligand fusion protein targeted to the endoplasmic reticulum. The transgene construct is shown generically in FIGURE 1OB. The expression unit contains nucleotides that encode a tetramer of SEQ ID NO:33, a hemagluttinin epitope, and a mitochondrial localization signal. This expression unit is subsequently subcloned into a pBluscript-based vector (Stratagene; La Jolla, Calif.) between nucleotide sequences including a promoter and an SV40 polyadenylation signal. The completed transgene is then injected into pronuclei of fertilized mouse oocytes. The resultant pups are screened for the presence of the transgene by PCR. Transgenic founder mice are bred with wild-type mice. Heterozygous transgenic animals from at least the third generation are used for the following tests, with their non-transgenic littermates serving as controls.
Test 1: Southern blotting analysis is performed to determine the copy number. Southern blots are hybridized with a radio-labeled probe generated from a fragment of the transgene. The probe detects bands containing DNA from transgenic mice, but does not detect bands containing DNA from non- transgenic mice. Intensities of the transgenic mice bands are measured and compared with the transgene plasmid control bands to estimate copy number. This demonstrates that mice in Example 4 harbor the transgene in their genomes.
Test 2: Tissue homogenates are prepared for Western blot analysis. This experiment demonstrates the transgene is expressed in tissues of transgenic mice because hemagluttinin epitope is detected in transgenic homogenates but not in non-transgenic homogenates. These examples demonstrate delivery of ligands to a localized region of a cell for therapeutic or experimental purposes. The purified polypeptide ligands can be formulated for oral or parenteral administration, topical administration, or in tablet, capsule, or liquid form, intranasal or inhaled aerosol, subcutaneous, intramuscular, intraperitoneal, or other injection; intravenous instillation; or any other routes of administration. Furthermore, the nucleotide sequences encoding the ligands permit incorporation into a vector designed to deliver and express a gene product in a cell. Such vectors include plasmids, cosmids, artificial chromosomes, and modified viruses. Delivery to eukaryotic cells can be accomplished in vivo or ex vivo. Ex vivo delivery methods include isolation of the intended recipient's cells or donor cells and delivery of the vector to those cells, followed by treatment of the recipient with the cells.
Disclosed are ligands and polyligands that modulate PKD activity and methods of making and using these ligands. The ligands and polyligands are synthesized chemically or recombinantly and are utilized as research tools or as therapeutics. The invention includes linking the ligands and polyligands to cellular localization signals for subcellular therapeutics.
SEQUENCE LISTING
<110> Intrexon Corporation
Reed, Thomas D .
<120> PKD LIGANDS AND POLYNUCLEOTIDES ENCODING PKD LIGANDS
<130> INX
<150> US 60/728,259 <151> 2005-10-19 <160> 132
<170> Patentln version 3.3
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<210> 21
<211> 41
<212> PRT <213> artificial <220>
<223> synthetic
<400> 21
Leu Arg Tyr Lys Pro Lys Lys Ser Leu GIu Arg Arg Lys Asn Pro Leu 1 5 10 15
Leu Arg Lys GIu Ser Ala Pro Pro Ser Leu Arg Arg Arg Pro Ala GIu 20 25 30
Thr Leu GIy Asp Ser Ser Pro Ser Ser 35 40
<210> 22
<211> 21
<212> PRT
<213> artificial
<220> <223> synthetic
<400> 22 GIy Leu His Trp Pro Leu Ser Arg Thr Arg Ser GIu Pro Leu Pro Pro 1 5 10 15
Ser Ala Thr Ala Pro 20
<210> 23
<211> 41
<212> PRT
<213> artificial <220> <223> synthetic
<400> 23
Leu Met Thr Thr GIu Arg Leu Ser GIy Ser GIy Leu His Trp Pro Leu 1 5 10 15
Ser Arg Thr Arg Ser GIu Pro Leu Pro Pro Ser Ala Thr Ala Pro Pro 20 25 30
Pro Pro GIy Pro Met Gin Pro Arg Leu 35 40
<210> 24
<211> 61 <212> PRT
<213> artificial
<220>
<223> synthetic
<400> 24 GIy Pro Leu Pro Phe His Phe Ala Gin Ser Leu Met Thr Thr GIu Arg 1 5 10 15
Leu Ser GIy Ser GIy Leu His Trp Pro Leu Ser Arg Thr Arg Ser GIu 20 25 30
Pro Leu Pro Pro Ser Ala Thr Ala Pro Pro Pro Pro GIy Pro Met GIn 35 40 45
Pro Arg Leu GIu GIn Leu Lys Thr His VaI GIn VaI lie 50 55 60
<210> 25
<211> 21 <212> PRT
<213> artificial
<220>
<223> synthetic
<400> 25 GIy GIy His Arg Pro Leu Ser Arg Ala Gin Ser Ser Pro Ala Ala Pro 1 5 10 15
Ala Ser Leu Ser Ala 20
<210> 26
<211> 41
<212> PRT <213> artificial <220>
<223> synthetic
<400> 26
GIy Asp Thr VaI Leu Leu Pro Leu Ala GIn GIy GIy His Arg Pro Leu 1 5 10 15
Ser Arg Ala GIn Ser Ser Pro Ala Ala Pro Ala Ser Leu Ser Ala Pro 20 25 30
GIu Pro Ala Ser GIn Ala Arg VaI Leu 35 40
<210> 27
<211> 61
<212> PRT
<213> artificial
<220> <223> synthetic
<400> 27
Leu Ala GIy Arg Leu Pro Arg GIy Ser Thr GIy Asp Thr VaI Leu Leu 1 5 10 15
Pro Leu Ala GIn GIy GIy His Arg Pro Leu Ser Arg Ala Gin Ser Ser 20 25 30
Pro Ala Ala Pro Ala Ser Leu Ser Ala Pro GIu Pro Ala Ser GIn Ala 35 40 45
Arg VaI Leu Ser Ser Ser GIu Thr Pro Ala Arg Thr Leu 50 55 60
<210> 28
<211> 9
<212> PRT
<213> artificial
<220> <223> synthetic
<400> 28 lie Thr Arg GIn Met Ser Phe Asp Leu 1 5
<210> 29 <211> 17
<212> PRT
<213> artificial <220>
<223> synthetic
<400> 29
Arg Gin Met Gin Arg Thr lie Thr Arg GIn Met Ser Phe Asp Leu Thr 1 5 10 15
Lys
<210> 30
<211> 21 <212> PRT
<213> artificial
<220>
<223> synthetic
<400> 30 GIn Met GIn Arg Thr lie Thr Arg GIn Met Ser Phe Asp Leu Thr Lys 1 5 10 15
Leu Leu VaI Thr GIu 20
<210> 31 <211> 41
<212> PRT
<213> artificial
<220>
<223> synthetic <400> 31
Leu Asn Arg Arg Asp Thr Tyr Arg Arg Arg Gin Met Gin Arg Thr lie 1 5 10 15
Thr Arg GIn Met Ser Phe Asp Leu Thr Lys Leu Leu VaI Thr GIu Asp 20 25 30 Trp Phe Ser Asp lie Ser Pro GIn Thr 35 40
<210> 32
<211> 61
<212> PRT <213> artificial <220>
<223> synthetic
<400> 32
GIu Met Thr Lys Leu GIy Ser Lys Thr Ala Leu Asn Arg Arg Asp Thr 1 5 10 15
Tyr Arg Arg Arg Gin Met GIn Arg Thr lie Thr Arg Gin Met Ser Phe 20 25 30 Asp Leu Thr Lys Leu Leu VaI Thr GIu Asp Trp Phe Ser Asp lie Ser 35 40 45
Pro Gin Thr Met Arg Arg Leu Leu Asn lie VaI Ser VaI 50 55 60
<210> 33
<211> 10
<212> PRT
<213> artificial <220> <223> synthetic
<400> 33
Arg Lys Asn lie Asp Ala Leu Ser GIy Met 1 5 10
<210> 34
<211> 10
<212> PRT
<213> artificial
<220>
<223> synthetic
<400> 34 lie Arg Arg Arg Ser Ser Asn Tyr Arg Ala 1 5 10
<210> 35
<211> 21 <212> PRT
<213> artificial
<220>
<223> synthetic
<400> 35 GIy Asp Trp Arg Lys Asn lie Asp Ala Leu Ser GIy Met GIu GIy Arg 1 5 10 15
Lys Lys Lys Phe GIu 20
<210> 36 <211> 32
<212> PRT
<213> artificial
<220>
<223> synthetic <400> 36
GIu Asp Thr GIu Lys GIu Asn Arg GIu VaI GIy Asp Trp Arg Lys Asn 1 5 10 15 lie Asp Ala Leu Ser GIy Met GIu GIy Arg Lys Lys Lys Phe GIu Ser 20 25 30
<210> 37
<211> 42
<212> PRT <213> artificial <220>
<223> synthetic
<400> 37
Leu Arg Ala His Leu Lys Gin VaI Lys Lys GIu Asp Thr GIu Lys GIu 1 5 10 15
Asn Arg GIu VaI GIy Asp Trp Arg Lys Asn lie Asp Ala Leu Ser GIy 20 25 30
Met GIu GIy Arg Lys Lys Lys Phe GIu Ser 35 40
<210> 38
<211> 20
<212> PRT
<213> artificial
<220> <223> synthetic
<400> 38
Ala Pro Ala Pro lie Arg Arg Arg Ser Ser Asn Tyr Arg Ala Tyr Ala 1 5 10 15
Thr GIu Pro His 20
<210> 39
<211> 41
<212> PRT
<213> artificial <220>
<223> synthetic
<400> 39
GIy Ser Ser Asp Ala Ala Arg GIu Pro Arg Pro Ala Pro Ala Pro lie 1 5 10 15 Arg Arg Arg Ser Ser Asn Tyr Arg Ala Tyr Ala Thr GIu Pro His Ala 20 25 30
Lys Lys Lys Ser Lys lie Ser Ala Ser 35 40
<210> 40 <211> 53
<212> PRT
<213> artificial <220>
<223> synthetic
<400> 40
Met Ala Asp GIy Ser Ser Asp Ala Ala Arg GIu Pro Arg Pro Ala Pro 1 5 10 15
Ala Pro lie Arg Arg Arg Ser Ser Asn Tyr Arg Ala Tyr Ala Thr GIu 20 25 30
Pro His Ala Lys Lys Lys Ser Lys lie Ser Ala Ser Arg Lys Leu GIn 35 40 45 Leu Lys Thr Leu Leu 50
<210> 41
<211> 8
<212> PRT <213> artificial <220>
<223> synthetic
<400> 41
Leu Ala GIu Arg lie Ser VaI Leu 1 5
<210> 42
<211> 11
<212> PRT
<213> artificial
<220>
<223> synthetic
<400> 42
Met Gin GIy Leu Ala GIu Arg lie Ser VaI Leu 1 5 10
<210> 43
<211> 21
<212> PRT
<213> artificial
<220> <223> synthetic
<400> 43
GIy GIy Ala Cys Pro Pro Gin Asp His Asp Met GIn GIy Leu Ala GIu 1 5 10 15
Arg He Ser VaI Leu 20
<210> 44 <211> 31 <212> PRT
<213> artificial
<220>
<223> synthetic
<400> 44
GIy Ser GIy Leu Pro Thr Asp Arg Asp Leu GIy GIy Ala Cys Pro Pro 1 5 10 15
GIn Asp His Asp Met Gin GIy Leu Ala GIu Arg lie Ser VaI Leu 20 25 30
<210> 45
<211> 8
<212> PRT
<213> artificial
<220> <223> synthetic
<400> 45
Leu Ala GIu Arg lie Ser lie Leu 1 5
<210> 46 <211> 11
<212> PRT
<213> artificial
<220>
<223> synthetic <400> 46
Met Gin GIy Leu Ala GIu Arg lie Ser lie Leu 1 5 10
<210> 47
<211> 21
<212> PRT
<213> artificial
<220>
<223> synthetic
<400> 47 GIy GIy Ala Cys Leu Pro GIn Asp His GIu Met GIn GIy Leu Ala GIu 1 5 10 15
Arg lie Ser lie Leu 20
<210> 48 <211> 29
<212> PRT
<213> artificial <220>
<223> synthetic <400> 48
GIy Thr Pro Ala GIu GIy Asp Leu GIy GIy Ala Cys Leu Pro GIn Asp 1 5 10 15
His GIu Met GIn GIy Leu Ala GIu Arg lie Ser lie Leu 20 25
<210> 49
<211> 10
<212> PRT
<213> artificial <220>
<223> synthetic
<400> 49
Leu Leu Arg Ser Met Ser Ala Ala Phe Cys 1 5 10
<210> 50
<211> 22
<212> PRT
<213> artificial
<220> <223> synthetic
<400> 50
Leu GIy Arg Arg Arg Pro Leu Leu Arg Ser Met Ser Ala Ala Phe Cys 1 5 10 15
Ser Leu Leu Ala Pro GIu 20
<210> 51
<211> 39
<212> PRT
<213> artificial <220>
<223> synthetic
<400> 51
Arg GIy Ser Pro Ala Thr Ser Pro His Leu GIy Arg Arg Arg Pro Leu 1 5 10 15 Leu Arg Ser Met Ser Ala Ala Phe Cys Ser Leu Leu Ala Pro GIu Arg 20 25 30
GIn VaI GIy Arg Ala Ala Ala 35
<210> 52 <211> 60
<212> PRT
<213> artificial <220>
<223> synthetic
<400> 52
Ser Ser GIu GIu GIu GIy VaI Pro GIy Ser Arg GIy Ser Pro Ala Thr 1 5 10 15
Ser Pro His Leu GIy Arg Arg Arg Pro Leu Leu Arg Ser Met Ser Ala 20 25 30
Ala Phe Cys Ser Leu Leu Ala Pro GIu Arg GIn VaI GIy Arg Ala Ala 35 40 45 Ala Ala Leu Met Gin Asp Arg His Thr Ala Ala GIy 50 55 60
<210> 53
<211> 28
<212> PRT
<213> artificial
<220>
<223> synthetic
<220>
<221> misc feature
<222> (17) .. (17)
<223> Xaa can be any naturally occurring amino acid <400> 53
Asp Phe GIy lie Ser Ala GIn lie GIy Ala Thr Leu Ala Arg Arg Leu 1 5 10 15
Xaa Phe lie GIy Thr Pro Tyr Trp Met Ala Pro GIu 20 25
<210> 54
<211> 14
<212> PRT <213> artificial
<220>
<223> synthetic
<220>
<221> misc_feature <222> (8) .. (8)
<223> Xaa can be any naturally occurring amino acid
<400> 54
Ala Ala Leu VaI Arg GIn Met Xaa VaI Ala Phe Phe Phe Lys 1 5 10
<210> 55
<211> 22
<212> PRT
<213> artificial
<220> <223> synthetic <220>
<221> misc_feature
<222> (11) .. (12)
<223> Xaa can be any naturally occurring amino acid
<400> 55
Ala Tyr Ser Arg Ala Leu Ser Arg GIn Leu Xaa Xaa GIy VaI Ser GIu 1 5 10 15 lie Arg His Thr Ala Asp 20
<210> 56
<211> 42
<212> PRT
<213> artificial
<220> <223> synthetic
<220>
<221> misc_feature
<222> (21) .. (22)
<223> Xaa can be any naturally occurring amino acid <400> 56
Ala lie GIu Ser Pro Ala VaI Ala Ala Pro Ala Tyr Ser Arg Ala Leu 1 5 10 15
Ser Arg GIn Leu Xaa Xaa GIy VaI Ser GIu lie Arg His Thr Ala Asp 20 . 25 30 Arg Trp Arg VaI Ser Leu Asp VaI Asn His 35 40
<210> 57
<211> 62
<212> PRT <213> artificial
<220>
<223> synthetic
<220>
<221> misc_feature <222> (31).. (32)
<223> Xaa can be any naturally occurring amino acid
<400> 57
Pro GIy Tyr VaI Arg Pro Leu Pro Pro Ala Ala lie GIu Ser Pro Ala 1 5 10 15 VaI Ala Ala Pro Ala Tyr Ser Arg Ala Leu Ser Arg Gin Leu Xaa Xaa 20 25 30
GIy VaI Ser GIu lie Arg His Thr Ala Asp Arg Trp Arg VaI Ser Leu 35 40 45
Asp VaI Asn His Phe Ala Pro Asp GIu Arg Thr VaI Lys Thr 50 55 60 <210> 58 <211> 21 <212> PRT <213> artificial <220> <223> synthetic <220> <221> misc_feature <222> (11) .. (H) <223> Xaa can be any naturally occurring amino acid <400> 58
Ser Leu Ser Ser Asn Leu Lys Arg Thr Ala Xaa Asn Pro Lys VaI GIu 1 5 10 15
Asn GIu Asp GIu Pro 20
<210> 59
<211> 41
<212> PRT
<213> artificial <220>
<223> synthetic
<220>
<221> misc__feature
<222> (21) .. (21) <223> Xaa can be any naturally occurring amino acid
<400> 59
Arg Ser Lys Ser Asp Ala Thr Ala Ser lie Ser Leu Ser Ser Asn Leu 1 5 10 15
Lys Arg Thr Ala Xaa Asn Pro Lys VaI GIu Asn GIu Asp GIu Pro VaI 20 25 30
Arg Leu Ala Pro GIu Arg GIu Phe lie 35 40
<210> 60
<211> 61 <212> PRT
<213> artificial
<220>
<223> synthetic
<220> <221> misc_feature
<222> (31) .. (31)
<223> Xaa can be any naturally occurring amino acid
<400> 60
GIy Leu Ser Pro Ser Lys Arg Thr His Gin Arg Ser Lys Ser Asp Ala 1 5 10 15 Thr Ala Ser lie Ser Leu Ser Ser Asn Leu Lys Arg Thr Ala Xaa Asn 20 25 30
Pro Lys VaI GIu Asn GIu Asp GIu Pro VaI Arg Leu Ala Pro GIu Arg 35 40 45
GIu Phe lie Lys Ser Leu Met Ala lie GIy Lys Arg Leu 50 55 60
<210> 61 <211> 21 <212> PRT <213> artificial <220> <223> synthetic <220> <221> misc_feature <222> (11) .. (11) <223> Xaa can be any naturally occurring amino acid <400> 61
Pro GIu His Phe Pro Leu Arg Lys Thr VaI Xaa GIu Pro Asn Leu Lys 1 5 10 15 Leu Arg Tyr Lys Pro 20
<210> 62
<211> 41
<212> PRT <213> artificial
<220>
<223> synthetic
<220>
<221> misc_feature <222> (21) .. (21)
<223> Xaa can be any naturally occurring amino acid
<400> 62
Pro Pro VaI Pro Ser Leu Pro Ser Asp Pro Pro GIu His Phe Pro Leu 1 5 10 15 Arg Lys Thr VaI Xaa GIu Pro Asn Leu Lys Leu Arg Tyr -Lys Pro Lys 20 25 30
Lys Ser Leu GIu Arg Arg Lys Asn Pro 35 40
<210> 63 <211> 87
<212> PRT
<213> artificial
<220>
<223> synthetic <220> <221> misc_feature
<222> (31) .. (31)
<223> Xaa can be any naturally occurring amino acid <220> <221> raisc__feature
<222> (57) .. (57)
<223> Xaa can be any naturally occurring amino acid
<400> 63
Ala Thr Arg Ser Met Leu Ser Ser Phe Leu Pro Pro VaI Pro Ser Leu 1 5 10 15
Pro Ser Asp Pro Pro GIu His Phe Pro Leu Arg Lys Thr VaI Xaa GIu 20 25 30
Pro Asn Leu Lys Leu Arg Tyr Lys Pro Lys Lys Ser Leu GIu Arg Arg 35 40 45 Lys Asn Pro Leu Leu Arg Lys GIu Xaa Ala Pro Pro Ser Leu Arg Arg 50 55 60
Arg Pro Ala GIu Thr Leu GIy Asp Ser Ser Pro Ser Ser Ser Ser Thr 65 70 75 80
Pro Ala Ser GIy Cys Ser Ser 85
<210> 64 <211> 21 <212> PRT <213> artificial <220> <223> synthetic <220> <221> misc_feature <222> (11) .. (H) <223> Xaa can be any naturally occurring amino acid <400> 64
Arg Arg Lys Asn Pro Leu Leu Arg Lys GIu Xaa Ala Pro Pro Ser Leu 1 5 10 15
Arg Arg Arg Pro Ala 20
<210> 65
<211> 41
<212> PRT
<213> artificial <220>
<223> synthetic
<220>
<221> misc_feature
<222> (21) .. (21) <223> Xaa can be any naturally occurring amino acid
<400> 65 Leu Arg Tyr Lys Pro Lys Lys Ser Leu GIu Arg Arg Lys Asn Pro Leu 1 5 10 15
Leu Arg Lys GIu Xaa Ala Pro Pro Ser Leu Arg Arg Arg Pro Ala GIu 20 25 30
Thr Leu GIy Asp Ser Ser Pro Ser Ser 35 40
<210> 66 <211> 21 <212> PRT <213> artificial <220> <223> synthetic <220> <221> misc_feature <222> (11) -.(ID <223> Xaa can be any naturally occurring amino acid <400> 66
GIy Leu His Trp Pro Leu Ser Arg Thr Arg Xaa GIu Pro Leu Pro Pro 1 5 10 15 Ser Ala Thr Ala Pro 20
<210> 67
<211> 41
<212> PRT <213> artificial
<220>
<223> synthetic
<220>
<221> misc_feature <222> (21) .. (21)
<223> Xaa can be any naturally occurring amino acid
<400> 67
Leu Met Thr Thr GIu Arg Leu Ser GIy Ser GIy Leu His Trp Pro Leu 1 5 10 15 Ser Arg Thr Arg Xaa GIu Pro Leu Pro Pro Ser Ala Thr Ala Pro Pro 20 25 30
Pro Pro GIy Pro Met GIn Pro Arg Leu 35 40
<210> 68 <211> 61
<212> PRT
<213> artificial <220>
<223> synthetic <220>
<221> misc_feature
<222> (31) .. (31)
<223> Xaa can be any naturally occurring amino acid <400> 68
GIy Pro Leu Pro Phe His Phe Ala Gin Ser Leu Met Thr Thr GIu Arg 1 5 10 15
Leu Ser GIy Ser GIy Leu His Trp Pro Leu Ser Arg Thr Arg Xaa GIu 20 25 30 Pro Leu Pro Pro Ser Ala Thr Ala Pro Pro Pro Pro GIy Pro Met Gin 35 40 45
Pro Arg Leu GIu Gin Leu Lys Thr His VaI GIn VaI lie 50 55 60
<210> 69 <211> 21 <212> PRT <213> artificial <220> <223> synthetic <220> <221> misc_feature <222> (11) .. (H) <223> Xaa can be any naturally occurring amino acid <400> 69 GIy GIy His Arg Pro Leu Ser Arg Ala Gin Xaa Ser Pro Ala Ala Pro 1 5 10 15
Ala Ser Leu Ser Ala
20
<210> 70 <211> 41
<212> PRT
<213> artificial
<220>
<223> synthetic <220>
<221> misc_feature
<222> (21) .. (21)
<223> Xaa can be any naturally occurring amino acid
<400> 70 GIy Asp Thr VaI Leu Leu Pro Leu Ala GIn GIy GIy His Arg Pro Leu 1 5 10 15
Ser Arg Ala Gin Xaa Ser Pro Ala Ala Pro Ala Ser Leu Ser Ala Pro 20 25 30
GIu Pro Ala Ser Gin Ala Arg VaI Leu 35 40 <210> 71
<211> 61
<212> PRT
<213> artificial <220>
<223> synthetic <220>
<221> misc_feature
<222> (31) .. (31) <223> Xaa can be any naturally occurring amino acid
<400> 71
Leu Ala GIy Arg Leu Pro Arg GIy Ser Thr GIy Asp Thr VaI Leu Leu 1 5 10 15
Pro Leu Ala GIn GIy GIy His Arg Pro Leu Ser Arg Ala Gin Xaa Ser 20 25 30
Pro Ala Ala Pro Ala Ser Leu Ser Ala Pro GIu Pro Ala Ser GIn Ala 35 40 45
Arg VaI Leu Ser Ser Ser GIu Thr Pro Ala Arg Thr Leu 50 55 60
<210> 72
<211> 17
<212> PRT
<213> artificial
<220>
<223> synthetic
<220>
<221> misc featu
<222> (12) .. (12)
<223> X Xaaaa ccaann bbee any naturally occurring amino acid
<400> 72
Arg Gin Met Gin Arg Thr lie Thr Arg Gin Met Xaa Phe Asp Leu Thr 1 5 10 15
Lys
<210> 73
<211> 21
<212> PRT
<213> artificial
<220> <223> synthetic
<220>
<221> misc_feature
<222> (11) .. (11)
<223> Xaa can be any naturally occurring amino acid <400> 73 GIn Met GIn Arg Thr lie Thr Arg Gin Met Xaa Phe Asp Leu Thr Lys 1 5 10 15
Leu Leu VaI Thr GIu 20
<210> 74
<211> 41
<212> PRT
<213> artificial
<220> <223> synthetic
<220>
<221> misc_feature
<222> (21) .. (21)
<223> Xaa can be any naturally occurring amino acid <400> 74
Leu Asn Arg Arg Asp Thr Tyr Arg Arg Arg GIn Met GIn Arg Thr lie 1 5 10 15
Thr Arg GIn Met Xaa Phe Asp Leu Thr Lys Leu Leu VaI Thr GIu Asp 20 25 30 Trp Phe Ser Asp lie Ser Pro Gin Thr 35 40
<210> 75
<211> 61
<212> PRT <213> artificial
<220>
<223> synthetic
<220>
<221> misc_feature <222> (31) .. (31)
<223> Xaa can be any naturally occurring amino acid
<400> 75
GIu Met Thr Lys Leu GIy Ser Lys Thr Ala Leu Asn Arg Arg Asp Thr 1 5 10 15 - Tyr Arg Arg Arg GIn Met -Gin Arg Thr lie Thr Arg GIn Met Xaa Phe 20 25 30
Asp Leu Thr Lys Leu Leu VaI Thr GIu Asp Trp Phe Ser Asp lie Ser 35 40 45
Pro GIn Thr Met Arg Arg Leu Leu Asn lie VaI Ser VaI 50 55 60
<210> 76
<211> 21
<212> PRT
<213> artificial <220>
<223> synthetic <220>
<221> misc_feature <222> (11) .. (11)
<223> Xaa can be any naturally occurring amino acid
<400> 76
GIy Asp Trp Arg Lys Asn lie Asp Ala Leu Xaa GIy Met GIu GIy Arg 1 5 10 15 Lys Lys Lys Phe GIu 20
<210> 77
<211> 32
<212> PRT <213> artificial
<220>
<223> synthetic
<220>
<221> misc_feature <222> (21) .. (21)
<223> Xaa can be any naturally occurring amino acid
<400> 77
GIu Asp Thr GIu Lys GIu Asn Arg GIu VaI GIy Asp Trp Arg Lys Asn 1 5 10 15 He Asp Ala Leu Xaa GIy Met GIu GIy Arg Lys Lys Lys Phe GIu Ser 20 25 30
<210> 78
<211> 42
<212> PRT <213> artificial
<220>
<223> synthetic
<220>
<221> misc_feature <222> (31) .. (31)
<223> Xaa can be any naturally occurring amino acid
<400> 78
Leu Arg Ala His Leu Lys GIn VaI Lys Lys GIu Asp Thr GIu Lys GIu 1 5 10 15 Asn Arg GIu VaI GIy Asp Trp Arg Lys Asn He Asp Ala Leu Xaa GIy 20 25 30
Met GIu GIy Arg Lys Lys Lys Phe GIu Ser 35 40
<210> 79 <211> 20 <212> PRT
<213> artificial
<220>
<223> synthetic
<220>
<221> misc feature
<222> (9) .. (10)
<223> Xaa can be any naturally occurring amino acid
<400> 79 Ala Pro Ala Pro lie Arg Arg Arg Xaa Xaa Asn Tyr Arg Ala Tyr Ala 1 5 10 15
Thr GIu Pro His 20
<210> 80 <211> 41
<212> PRT
<213> artificial
<220>
<223> synthetic <220>
<221> misc_feature
<222> (20) .. (21)
<223> Xaa can be any naturally occurring amino acid
<400> 80 GIy Ser Ser Asp Ala Ala Arg GIu Pro Arg Pro Ala Pro Ala Pro lie 1 5 10 15
Arg Arg Arg Xaa Xaa Asn Tyr Arg Ala Tyr Ala Thr GIu Pro His Ala 20 25 30
Lys Lys Lys Ser Lys lie Ser Ala Ser 35 40
<210> 81
<211> 53
<212> PRT
<213> artificial <220>
<223> synthetic
<220>
<221> misc_feature
<222> (23) .. (24) <223> Xaa can be any naturally occurring amino acid
<400> 81
Met Ala Asp GIy Ser Ser Asp Ala Ala Arg GIu Pro Arg Pro Ala Pro 1 5 10 15
Ala Pro lie Arg Arg Arg Xaa Xaa Asn Tyr Arg Ala Tyr Ala Thr GIu 20 25 30
Pro His Ala Lys Lys Lys Ser Lys lie Ser Ala Ser Arg Lys Leu Gin 35 40 45
Leu Lys Thr Leu Leu 50
<210> 82 <211> 11 <212> PRT <213> artificial <220> <223> synthetic <220> <221> misc_feature <222> (9) .. O) <223> Xaa can be any naturally occurring amino acid <400> 82 Met Gin GIy Leu Ala GIu Arg lie Xaa VaI Leu 1 5 10
<210> 83
<211> 21
<212> PRT <213> artificial
<220>
<223> synthetic
<220>
<221> misc__feature <222> (19) .. (19)
<223> Xaa can be any naturally occurring amino acid
<400> 83
GIy GIy Ala Cys Pro Pro GIn Asp His Asp Met GIn GIy Leu Ala GIu 1 5 10 15 Arg lie Xaa VaI Leu 20
<210> 84
<211> 31
<212> PRT <213> artificial
<220>
<223> synthetic
<220>
<221> misc_feature <222> (29).. (29)
<223> Xaa can be any naturally occurring amino acid
<400> 84
GIy Ser GIy Leu Pro Thr Asp Arg Asp Leu GIy GIy Ala Cys Pro Pro 1 5 10 15 Gin Asp His Asp Met GIn GIy Leu Ala GIu Arg lie Xaa VaI Leu 20 25 30 <210> 85
<211> 11
<212> PRT
<213> artificial
<220>
<223> synthetic
<220>
<221> misc featu
<222> (9) .. (9)
<223> X Xaaaa ccaann bbee any naturally occurring amino acid
<400> 85
Met GIn GIy Leu Ala GIu Arg lie Xaa lie Leu 1 5 10
<210> 86
<211> 21
<212> PRT
<213> artificial
<220>
<223> synthetic
<220>
<221> misc feature
<222> (19) .. (19)
<223> Xaa can be any naturally occurring amino acid <400> 86 GIy GIy Ala Cys Leu Pro Gin Asp His GIu Met GIn GIy Leu Ala GIu 1 5 10 15
Arg lie Xaa lie Leu 20
<210> 87 <211> 29
<212> PRT
<213> artificial
<220>
<223> synthetic <220>
<221> misc_feature
<222> (27) .. (27)
<223> Xaa can be any naturally occurring amino acid
<400> 87 GIy Thr Pro Ala GIu GIy Asp Leu GIy GIy Ala Cys Leu Pro GIn Asp 1 5 10 15
His GIu Met GIn GIy Leu Ala GIu Arg lie Xaa lie Leu 20 25
<210> 88 <211> 9
<212> PRT
<213> artificial
<220>
<223> synthetic
<220>
<221> misc feature
<222> (6) .. (6)
<223> Xaa can be any naturally occurring amino acid
<400> 88
Leu Ala Arg Arg Leu Xaa Phe lie GIy 1 5
<210> 89
<211> 8
<212> PRT
<213> artificial
<220>
<223> synthetic
<220>
<221> misc feature
<222> (6) .. (6)
<223> Xaa can be any naturally occurring amino acid
<400> 89
Leu VaI Arg GIn Met Xaa VaI Ala 1 5
<210> 90
<211> 9
<212> PRT
<213> artificial
<220>
<223> synthetic
<220>
<221> misc feature
<222> (6) .. (6)
<223> Xaa can be any naturally occurring amino acid
<400> 90
Leu Ser Arg Gin Leu Xaa Ser GIy VaI 1 5
<210> 91 <211> 10 <212> PRT <213> artificial <220> <223> synthetic <220> <221> misc_feature <222> (6) .. (6) <223> Xaa can be any naturally occurring amino acid <400> 91 Leu Lys Arg Thr Ala Xaa Asn Pro Lys VaI 1 5 10
<210> 92 <211> 9 <212> PRT <213> artificial <220> <223> synthetic <220> <221> misc_feature <222> (6) .. (6) <223> Xaa can be any naturally occurring amino acid <400> 92
Leu Arg Lys Thr VaI Xaa GIu Pro Asn 1 5
<210> 93
<211> 8
<212> PRT
<213> artificial
<220>
<223> synthetic
<220>
<221> misc feature
<222> (6) .. (6)
<223> Xaa can be any naturally occurring amino acid
<400> 93
Leu Leu Arg Lys GIu Xaa Ala Pro 1 5
<210> 94
<211> 9
<212> PRT
<213> artificial
<220>
<223> synthetic
<220>
<221> misc feature
<222> (6) .. (6)
<223> Xaa can be any naturally occurring amino acid
<400> 94 Leu Ser Arg Ala GIn Xaa Ser Pro Ala 1 5
<210> 95
<211> 9
<212> PRT <213> artificial <220> <223> synthetic <220> <221> misc_feature <222> (6) .. (6) <223> Xaa can be any naturally occurring amino acid <400> 95
Leu Ser Arg Thr Arg Xaa GIu Pro Leu 1 5
<210> 96 <211> 8 <212> PRT <213> artificial <220> <223> synthetic <220> <221> misc_feature <222> (6) .. (6) <223> Xaa can be any naturally occurring amino acid <400> 96 Leu Ala GIu Arg lie Xaa lie Leu 1 5
<210> 97
<211> 8
<212> PRT
<213> artificial
<220>
<223> synthetic
<220>
<221> misc feature
<222> (6) .. (6)
<223> Xaa can be any naturally occurring amino acid
<400> 97
Leu Ala GIu Arg lie Xaa VaI Leu 1 5
<210> 98
<211> 9
<212> PRT
<213> artificial
<220>
<223> synthetic
<220>
<221> raise feature
<222> (6) .. (6)
<223> Xaa can be any naturally occurring amino acid
<400> 98 lie Thr Arg Gin Met Xaa Phe Asp Leu 1 5 <210> 99 <211> 10 <212> PRT <213> artificial <220> <223> synthetic <220> <221> misc_feature <222> (8) .. (8) <223> Xaa can be any naturally occurring amino acid <400> 99
Arg Lys Asn lie Asp Ala Leu Xaa GIy Met 1 5 10
<210> 100 <211> 10 <212> PRT <213> artificial <220> <223> synthetic <220> <221> misc_feature <222> (5) .. (6) <223> Xaa can be any naturally occurring amino acid <400> 100 lie Arg Arg Arg Xaa Xaa Asn Tyr Arg Ala 1 5 10
<210> 101
<211> 10
<212> PRT
<213> artificial
<220>
<223> synthetic
<220>
<221> misc feature
<222> (6) .. (6)
<223> Xaa can be any naturally occurring amino acid
<400> 101
Leu Leu Arg Ser Met Xaa Ala Ala Phe Cys 1 5 10
<210> 102
<211> 22
<212> PRT
<213> artificial
<220>
<223> synthetic
<220>
<221> misc feature
<222> (12) .. (12) <223> Xaa can be any naturally occurring amino acid <400> 102
Leu GIy Arg Arg Arg Pro Leu Leu Arg Ser Met Xaa Ala Ala Phe Cys 1 5 10 15 Ser Leu Leu Ala Pro GIu 20
<210> 103
<211> 39
<212> PRT <213> artificial
<220>
<223> synthetic
<220>
<221> misc_feature <222> (21) .. (21)
<223> Xaa can be any naturally occurring amino acid
<400> 103
Arg GIy Ser Pro Ala Thr Ser Pro His Leu GIy Arg Arg Arg Pro Leu 1 5 10 15 Leu Arg Ser Met Xaa Ala Ala Phe Cys Ser Leu Leu Ala Pro GIu Arg 20 25 30
GIn VaI GIy Arg Ala Ala Ala 35
<210> 104 <211> 60
<212> PRT
<213> artificial
<220>
<223> synthetic <220>
<221> misc_feature
<222> (31) .. (31)
<223> Xaa can be any naturally occurring amino acid
<400> 104 Ser Ser GIu GIu GIu GIy VaI Pro GIy Ser Arg GIy Ser Pro Ala Thr 1 5 10 15
Ser Pro His Leu GIy Arg Arg Arg Pro Leu Leu Arg Ser Met Xaa Ala 20 25 30
Ala Phe Cys Ser Leu Leu Ala Pro GIu Arg GIn VaI GIy Arg Ala Ala 35 40 45
Ala Ala Leu Met GIn Asp Arg His Thr Ala Ala GIy 50 55 60
<210> 105 <211> 79
<212> PRT
<213> artificial <220> <223> synthetic
<400> 105
Asp Phe GIy lie Ser Ala Gin lie GIy Ala Thr Leu Ala Arg Arg Leu 1 5 10 15
Ala Phe lie GIy Thr Pro Tyr Trp Met Ala Pro GIu Ala GIy Ala GIy 20 25 30
Ala Tyr Ser Arg Ala Leu Ser Arg GIn Leu Ala Ser GIy VaI Ser GIu 35 40 45 lie Arg His Thr Ala Asp GIy Ala GIy Ala Ser Leu Ser Ser Asn Leu 50 55 60 Lys Arg Thr Ala Ala Asn Pro Lys VaI GIu Asn GIu Asp GIu Pro 65 70 75
<210> 106
<211> 237
<212> DNA <213> artificial <220>
<223> synthetic
<400> 106 gacttcggca tcagcgccca gatcggcgcc accctggcca ggaggctggc cttcatcggc 60 accccctact ggatggcccc cgaggccggc gccggcgcct acagcagggc cctgagcagg 120 cagctggcca gcggcgtgag cgagatcagg cacaccgccg acggcgccgg cgccagcctg 180 agcagcaacc tgaagaggac cgccgccaac cccaaggtgg agaacgagga cgagccc 237
<210> 107
<211> 237 <212> DNA
<213> artificial
<220>
<223> synthetic
<400> 107 gacttcggca tcagcgccca gatcggagcc accctggcta ggaggctggc cttcatcggc 60 accccctact ggatggctcc cgaggctggc gctggagcct acagcagagc cctgagcagg 120 cagctcgcca gcggcgtgag cgagatcagg cacaccgccg acggcgctgg agccagcctg 180 agcagcaacc tgaagaggac cgccgccaac cccaaggtgg agaacgagga cgagccc 237
<210> 108 <211> 265
<212> DNA <213> artificial <220>
<223> synthetic <400> 108 gccggcgact tcggcatcag cgcccagatc ggagccaccc tggctaggag gctggccttc 60 atcggcaccc cctactggat ggctcccgag gctggcgctg gagcctacag cagagccctg 120 agcaggcagc tcgccagcgg cgtgagcgag atcaggcaca ccgccgacgg cgctggagcc 180 agcctgagca gcaacctgaa gaggaccgcc gccaacccca aggtggagaa cgaggacgag 240 ccccccgggg gaggcggaat cgatt 265
<210> 109
<211> 91
<212> PRT
<213> artificial
<220> <223> synthetic
<400> 109
Pro GIu His Phe Pro Leu Arg Lys Thr VaI Ala GIu Pro Asn Leu Lys 1 5 10 15
Leu Arg Tyr Lys Pro Ala GIy Ala GIy Leu Arg Tyr Lys Pro Lys Lys 20 25 30
Ser Leu GIu Arg Arg Lys Asn Pro Leu Leu Arg Lys GIu Ala Ala Pro 35 40 45
Pro Ser Leu Arg Arg Arg Pro Ala GIu Thr Leu GIy Asp Ser Ser Pro 50 55 60 Ser Ser GIy Ala GIy Ala GIy Leu His Trp Pro Leu Ser Arg Thr Arg 65 70 75 80
Ala GIu Pro Leu Pro Pro Ser Ala Thr Ala Pro 85 90
<210> 110 <211> 273
<212> DNA
<213> artificial
<220>
<223> synthetic <400> 110 cccgagcact tccccctgag gaagaccgtg gccgagccca acctgaagct gaggtacaag 60 cccgccggcg ccggcctgag gtacaagccc aagaagagcc tggagaggag gaagaacccc 120 ctgctgagga aggaggccgc cccccccagc ctgaggagga ggcccgccga gaccctgggc 180 gacagcagcc ccagcagcgg cgccggcgcc ggcctgcact ggcccctgag caggaccagg 240 gccgagcccc tgccccccag cgccaccgcc ccc 273 <210> 111
<211> 273
<212> DNA
<213> artificial <220>
<223> synthetic
<400> 111 cccgagcact tccccctgag gaagaccgtg gccgagccca acctgaagct gaggtacaag 60 cccgccggag ctggcctgag gtacaagccc aagaaaagcc tggagaggag gaagaacccc 120 ctgctgagga aggaggccgc cccccctagc ctgaggagga ggcccgccga gaccctgggc 180 gacagcagcc ctagcagcgg agctggcgct ggcctgcact ggcccctgag caggaccagg 240 gccgagcccc tgccccctag cgccaccgcc ccc 273
<210> 112
<211> 301 <212> DNA
<213> artificial
<220>
<223> synthetic
<400> 112 gccggccccg agcacttccc cctgaggaag accgtggccg agcccaacct gaagctgagg 60 tacaagcccg ccggagctgg cctgaggtac aagcccaaga aaagcctgga gaggaggaag 120 aaccccctgc tgaggaagga ggccgccccc cctagcctga ggaggaggcc cgccgagacc 180 ctgggcgaca gcagccctag cagcggagct ggcgctggcc tgcactggcc cctgagcagg 240 accagggccg agcccctgcc ccctagcgcc accgcccccc ccgggggagg cggaatcgat 300 t 301
<210> 113
<211> 89
<212> PRT
<213> artificial <220>
<223> synthetic
<400> 113
Asp Phe GIy lie Ser Ala Gin lie GIy Ala Thr Leu Ala Arg Arg Leu 1 5 10 15 Ala Phe lie GIy Thr Pro Tyr Trp Met Ala Pro GIu Ala Ala Leu VaI 20 25 30
Arg GIn Met Ala VaI Ala Phe Phe Phe Lys Pro Ala GIy Ala GIy Ala 35 40 45
Ala Leu VaI Arg GIn Met Ala VaI Ala Phe Phe Phe Lys Asp Phe GIy 50 55 60 lie Ser Ala GIn lie GIy Ala Thr Leu Ala Arg Arg Leu Ala Phe lie 65 70 75 80
GIy Thr Pro Tyr Trp Met Ala Pro GIu 85
<210> 114
<211> 267
<212> DNA
<213> artificial <220>
<223> synthetic
<400> 114 gacttcggca tcagcgccca gatcggcgcc accctggcca ggaggctggc cttcatcggc 60 accccctact ggatggcccc cgaggccgcc ctggtgaggc agatggccgt ggccttcttc 120 ttcaagcccg ccggcgccgg cgctgctctc gtcagacaaa tggctgtcgc tttttttttt 180 aaagattttg gaatttccgc tcaaattgga gctacactcg ctagaagact cgcttttatt 240 ggaacacctt attggatggc tcctgaa 267
<210> 115
<211> 267 <212> DNA
<213> artificial
<220>
<223> synthetic
<400> 115 gacttcggca tcagcgccca gatcggagcc accctggcta ggaggctggc cttcatcggc 60 accccctact ggatggcccc cgaggctgcc ctggtgaggc agatggccgt ggccttcttc 120 ttcaagcccg ccggagccgg agctgctctc gtcagacaaa tggctgtcgc tttttttttt 180 aaagattttg gaatttccgc tcaaattgga gctacactcg ctagaagact cgcttttatt 240 ggaacacctt attggatggc tcctgaa 267
<210> 116
<211> 295
<212> DNA
<213> artificial
<220> <223> synthetic
<400> 116 gccggcgact tcggcatcag cgcccagatc ggagccaccc tggctaggag gctggccttc 60 atcggcaccc cctactggat ggcccccgag gctgccctgg tgaggcagat ggccgtggcc 120 ttcttcttca agcccgccgg agccggagct gctctcgtca gacaaatggc tgtcgctttt 180 ttttttaaag attttggaat ttccgctcaa attggagcta cactcgctag aagactcgct 240 tttattggaa caccttattg gatggctcct gaacccgggg gaggcggaat cgatt 295
<210> 117
<211> 210 <212> PRT
<213> human
<220>
<221> misc__feature
<222> (23) .. (24) <223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (199) .. (199)
<223> Xaa can be any naturally occurring amino acid <400> 117
Met Ala Asp GIy Ser Ser Asp Ala Ala Arg GIu Pro Arg Pro Ala Pro 1 5 10 15
Ala Pro lie Arg Arg Arg Xaa Xaa Asn Tyr Arg Ala Tyr Ala Thr GIu 20 25 30 Pro His Ala Lys Lys Lys Ser Lys lie Ser Ala Ser Arg Lys Leu Gin 35 40 45
Leu Lys Thr Leu Leu Leu GIn lie Ala Lys Gin GIu Leu GIu Arg GIu 50 55 60
Ala GIu GIu Arg Arg GIy GIu Lys GIy Arg Ala Leu Ser Thr Arg Cys 65 70 75 80
Gin Pro Leu GIu Leu Ala GIy Leu GIy Phe Ala GIu Leu GIn Asp Leu 85 90 95
Cys Arg GIn Leu His Ala Arg VaI Asp Lys VaI Asp GIu GIu Arg Tyr 100 105 110 Asp lie GIu Ala Lys VaI Thr Lys Asn He Thr GIu lie Ala Asp Leu 115 120 125
Thr GIn Lys He Phe Asp Leu Arg GIy Lys Phe Lys Arg Pro Thr Leu 130 - 135 140
Arg Arg VaI Arg He Ser Ala Asp Ala Met Met GIn Ala Leu Leu GIy 145 150 155 160
Ala Arg Ala Lys GIu Ser Leu Asp Leu Arg Ala His Leu Lys Gin VaI 165 170 175
Lys Lys GIu Asp Thr GIu Lys GIu Asn Arg GIu VaI GIy Asp Trp Arg 180 185 190 Lys Asn He Asp Ala Leu Xaa GIy Met GIu GIy Arg Lys Lys Lys Phe 195 200 205 GIu Ser 210
<210> 118
<211> 882 <212> PRT
<213> human
<220>
<221> misc_feature
<222> (850).. (850) <223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (853) .. (853)
<223> Xaa can be any naturally occurring amino acid <400> 118
Met GIy Pro Trp Ser Arg Ser Leu Ser Ala Leu Leu Leu Leu Leu GIn 1 5 10 15
VaI Ser Ser Trp Leu Cys GIn GIu Pro GIu Pro Cys His Pro GIy Phe 20 25 30 Asp Ala GIu Ser Tyr Thr Phe Thr VaI Pro Arg Arg His Leu GIu Arg 35 40 45
GIy Arg VaI Leu GIy Arg VaI Asn Phe GIu Asp Cys Thr GIy Arg GIn 50 55 60
Arg Thr Ala Tyr Phe Ser Leu Asp Thr Arg Phe Lys VaI GIy Thr Asp 65 70 75 80
GIy VaI lie Thr VaI Lys Arg Pro Leu Arg Phe His Asn Pro GIn lie 85 90 95
His Phe Leu VaI Tyr Ala Trp Asp Ser Thr Tyr Arg Lys Phe Ser Thr 100 105 110 Lys VaI Thr Leu Asn Thr VaI GIy His His His Arg Pro Pro Pro His 115 120 125
Gin Ala Ser VaI Ser GIy He GIn Ala GIu Leu Leu Thr Phe Pro Asn 130 135 140
Ser Ser Pro GIy Leu Arg Arg Gin Lys Arg Asp Trp VaI He Pro Pro 145 150 155 160
He Ser Cys Pro GIu Asn GIu Lys GIy Pro Phe Pro Lys Asn Leu VaI 165 170 175
Gin He Lys Ser Asn Lys Asp Lys GIu GIy Lys VaI Phe Tyr Ser He 180 185 190 Thr GIy GIn GIy Ala Asp Thr Pro Pro VaI GIy VaI Phe He He GIu 195 200 205
Arg GIu Thr GIy Trp Leu Lys VaI Thr GIu Pro Leu Asp Arg GIu Arg 210 215 220 lie Ala Thr Tyr Thr Leu Phe Ser His Ala VaI Ser Ser Asn GIy Asn 225 230 235 240
Ala VaI GIu Asp Pro Met GIu lie Leu lie Thr VaI Thr Asp GIn Asn 245 250 255
Asp Asn Lys Pro GIu Phe Thr GIn GIu VaI Phe Lys GIy Ser VaI Met 260 265 270
GIu GIy Ala Leu Pro GIy Thr Ser VaI Met GIu VaI Thr Ala Thr Asp 275 280 285 Ala Asp Asp Asp VaI Asn Thr Tyr Asn Ala Ala lie Ala Tyr Thr lie 290 295 300
Leu Ser GIn Asp Pro GIu Leu Pro Asp Lys Asn Met Phe Thr lie Asn 305 310 315 320
Arg Asn Thr GIy VaI lie Ser VaI VaI Thr Thr GIy Leu Asp Arg GIu 325 330 335
Ser Phe Pro Thr Tyr Thr Leu VaI VaI GIn Ala Ala Asp Leu GIn GIy 340 345 350
GIu GIy Leu Ser Thr Thr Ala Thr Ala VaI lie Thr VaI Thr Asp Thr 355 360 365 Asn Asp Asn Pro Pro lie Phe Asn Pro Thr Thr Tyr Lys GIy Gin VaI 370 375 380
Pro GIu Asn GIu Ala Asn VaI VaI lie Thr Thr Leu Lys VaI Thr Asp 385 390 395 400
Ala Asp Ala Pro Asn Thr Pro Ala Trp GIu Ala VaI Tyr Thr He Leu 405 410 415
Asn Asp Asp GIy GIy Gin Phe VaI VaI Thr Thr Asn Pro VaI Asn Asn 420 425 430
Asp GIy He Leu Lys Thr Ala Lys GIy Leu Asp Phe GIu Ala Lys Gin 435 440 445 GIn Tyr He Leu His VaI Ala VaI Thr Asn VaI VaI Pro Phe GIu VaI 450 455 460
Ser Leu Thr Thr Ser Thr Ala Thr VaI Thr VaI Asp VaI Leu Asp VaI 465 470 475 480
Asn GIu Ala Pro He Phe VaI Pro Pro GIu Lys Arg VaI GIu VaI Ser 485 490 495
GIu Asp Phe GIy VaI GIy Gin GIu He Thr Ser Tyr Thr Ala Gin GIu 500 505 510
Pro Asp Thr Phe Met GIu Gin Lys He Thr Tyr Arg He Trp Arg Asp 515 520 525 Thr Ala Asn Trp Leu GIu lie Asn Pro Asp Thr GIy Ala lie Phe Thr 530 535 540
Arg Ala GIu Leu Asp Arg GIu Asp Phe GIu His VaI Lys Asn Ser Thr 545 550 555 560 Tyr Thr Ala Leu lie lie Ala Thr Asp Asn GIy Ser Pro VaI Ala Thr 565 570 575 GIy Thr GIy Thr Leu Leu Leu lie Leu Ser Asp VaI Asn Asp Asn Ala 580 585 590
Pro lie Pro GIu Pro Arg Thr lie Phe Phe Cys GIu Arg Asn Pro Lys 595 600 605
Pro Gin VaI lie Asn lie lie Asp Ala Asp Leu Pro Pro Asn Thr Ser 610 615 620
Pro Phe Thr Ala GIu Leu Thr His GIy Ala Ser Ala Asn Trp Thr lie 625 630 635 640 GIn Tyr Asn Asp Pro Thr GIn GIu Ser lie lie Leu Lys Pro Lys Met
645 650 655
Ala Leu GIu VaI GIy Asp Tyr Lys lie Asn Leu Lys Leu Met Asp Asn
660 665 670
GIn Asn Lys Asp GIn VaI Thr Thr Leu GIu VaI Ser VaI Cys Asp Cys 675 680 685
GIu Gly Ala Ala GIy VaI Cys Arg Lys Ala GIn Pro VaI GIu Ala Gly 690 695 700
Leu Gin He Pro Ala He Leu Gly He Leu Gly Gly He Leu Ala Leu 705 710 715 720 Leu He Leu He Leu Leu Leu Leu Leu Phe Leu Arg Arg Arg Ala VaI 725 730 735
VaI Lys GIu Pro Leu Leu Pro Pro GIu Asp Asp Thr Arg Asp Asn VaI 740 745 750
Tyr Tyr Tyr Asp GIu GIu Gly Gly Gly GIu GIu Asp Gin Asp Phe Asp 755 760 765
Leu Ser GIn Leu His Arg Gly Leu Asp Ala Arg Pro GIu VaI Thr Arg 770 775 780
Asn Asp VaI Ala Pro Thr Leu Met Ser VaI Pro Arg Tyr Leu Pro Arg 785 790 795 800
Pro Ala Asn Pro Asp GIu He Gly Asn Phe He Asp GIu Asn Leu Lys 805 810 815
Ala Ala Asp Thr Asp Pro Thr Ala Pro Pro Tyr Asp Ser Leu Leu VaI 830 Phe Asp Tyr GIu GIy Ser GIy Ser GIu Ala Ala Ser Leu Ser Ser Leu 835 840 845
Asn Xaa Ser GIu Xaa Asp Lys Asp Gin Asp Tyr Asp Tyr Leu Asn GIu 850 855 860 Trp GIy Asn Arg Phe Lys Lys Leu Ala Asp Met Tyr GIy GIy GIy GIu 865 870 875 880
Asp Asp
<210> 119 <211> 1123
<212> PRT
<213> human
<220>
<221> misc_feature <222> (260) .. (260)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (499) .. (499) <223> Xaa can be any naturally occurring amino acid
<400> 119
Met Asn Ser Pro Asn GIu Ser Ala Asp GIy Met Ser GIy Arg GIu Pro 1 5 10 15
Ser Leu GIu lie Leu Pro Arg Thr Ser Leu His Ser lie Pro VaI Thr 20 25 30
VaI GIu VaI Lys Pro VaI Leu Pro Arg Ala Met Pro Ser Ser Met GIy 35 40 45
GIy GIy GIy GIy GIy Ser Pro Ser Pro VaI GIu Leu Arg GIy Ala Leu 50 55 60 VaI GIy Ser VaI Asp Pro Thr Leu Arg GIu GIn GIn Leu Gin Gin GIu 65 70 75 80
Leu Leu Ala Leu Lys GIn Gin Gin GIn Leu GIn Lys GIn Leu Leu Phe 85 90 95
Ala GIu Phe GIn Lys GIn His Asp His Leu Thr Arg GIn His GIu VaI 100 105 110
Gin Leu Gin Lys His Leu Lys GIn Gin Gin GIu Met Leu Ala Ala Lys 115 120 125
Gin GIn GIn GIu Met Leu Ala Ala Lys Arg Gin Gin GIu Leu GIu GIn 130 135 140 GIn Arg GIn Arg GIu Gin Gin Arg Gin GIu GIu Leu GIu Lys Gin Arg 145 150 155 160
Leu GIu Gin Gin Leu Leu lie Leu Arg Asn Lys GIu Lys Ser Lys GIu 165 170 175
Ser Ala lie Ala Ser Thr GIu VaI Lys Leu Arg Leu Gin GIu Phe Leu 180 185 190
Leu Ser Lys Ser Lys GIu Pro Thr Pro GIy GIy Leu Asn His Ser Leu 195 200 205
Pro GIn His Pro Lys Cys Trp GIy Ala His His Ala Ser Leu Asp Gin 210 215 220
Ser Ser Pro Pro GIn Ser GIy Pro Pro GIy Thr Pro Pro Ser Tyr Lys 225 230 235 240 Leu Pro Leu Pro GIy Pro Tyr Asp Ser Arg Asp Asp Phe Pro Leu Arg
245 250 255
Lys Thr Ala Xaa GIu Pro Asn Leu Lys VaI Arg Ser Arg Leu Lys Gin 260 265 270
Lys VaI Ala GIu Arg Arg Ser Ser Pro Leu Leu Arg Arg Lys Asp GIy 275 280 285
Thr VaI lie Ser Thr Phe Lys Lys Arg Ala VaI GIu lie Thr GIy Ala 290 295 300
GIy Pro GIy Ala Ser Ser VaI Cys Asn Ser Ala Pro GIy Ser GIy Pro 305 310 315 320 Ser Ser Pro Asn Ser Ser His Ser Thr lie Ala GIu Asn GIy Phe Thr
325 330 335
GIy Ser VaI Pro Asn lie Pro Thr GIu Met Leu Pro GIn His Arg Ala 340 345 350
Leu Pro Leu Asp Ser Ser Pro Asn Gin Phe Ser Leu Tyr Thr Ser Pro 355 360 365
Ser Leu Pro Asn lie Ser Leu GIy Leu Gin Ala Thr VaI Thr VaI Thr 370 375 380
Asn Ser His Leu Thr Ala Ser Pro Lys Leu Ser Thr GIn Gin GIu Ala 385 390 395 400 GIu Arg GIn Ala Leu Gin Ser Leu Arg Gin GIy GIy Thr Leu Thr GIy
405 410 415
Lys Phe Met Ser Thr Ser Ser lie Pro GIy Cys Leu Leu GIy VaI Ala 420 425 430
Leu GIu GIy Asp GIy Ser Pro His GIy His Ala Ser Leu Leu Gin His 435 440 445
VaI Leu Leu Leu GIu GIn Ala Arg GIn GIn Ser Thr Leu He Ala VaI 450 455 460
Pro Leu His GIy GIn Ser Pro Leu VaI Thr GIy GIu Arg VaI Ala Thr 465 470 475 480 Ser Met Arg Thr VaI GIy Lys Leu Pro Arg His Arg Pro Leu Ser Arg 485 490 495
Thr Gin Xaa Ser Pro Leu Pro GIn Ser Pro GIn Ala Leu GIn Gin Leu 500 505 510 VaI Met GIn GIn GIn His GIn GIn Phe Leu GIu Lys Gin Lys Gin GIn 515 520 525
GIn Leu Gin Leu GIy Lys lie Leu Thr Lys Thr GIy GIu Leu Pro Arg 530 535 540
GIn Pro Thr Thr His Pro GIu GIu Thr GIu GIu GIu Leu Thr GIu Gin 545 550 555 560
GIn GIu VaI Leu Leu GIy GIu GIy Ala Leu Thr Met Pro Arg GIu GIy 565 570 575
Ser Thr GIu Ser GIu Ser Thr GIn GIu Asp Leu GIu GIu GIu Asp GIu 580 585 590 GIu Asp Asp GIy GIu GIu GIu GIu Asp Cys lie Gin VaI Lys Asp GIu 595 600 605
GIu GIy GIu Ser GIy Ala GIu GIu GIy Pro Asp Leu GIu GIu Pro GIy 610 615 620
Ala GIy Tyr Lys Lys Leu Phe Ser Asp Ala Gin Pro Leu GIn Pro Leu 625 630 635 640
GIn VaI Tyr GIn Ala Pro Leu Ser Leu Ala Thr VaI Pro His GIn Ala 645 650 655
Leu GIy Arg Thr Gin Ser Ser Pro Ala Ala Pro GIy GIy Met Lys Ser 660 665 670 Pro Pro Asp GIn Pro VaI Lys His Leu Phe Thr Thr GIy VaI VaI Tyr 675 680 685
Asp Thr Phe Met Leu Lys His Gin Cys Met Cys GIy Asn Thr His VaI 690 695 700
His Pro GIu His Ala GIy Arg lie Gin Ser lie Trp Ser Arg Leu GIn 705 710 715 720
GIu Thr GIy Leu Leu Ser Lys Cys GIu Arg lie Arg GIy Arg Lys Ala 725 730 735
Thr Leu Asp GIu lie Gin Thr VaI His Ser GIu Tyr His Thr Leu Leu 740 745 750 Tyr GIy Thr Ser Pro Leu Asn Arg Gin Lys Leu Asp Ser Lys Lys Leu 755 760 765
Leu GIy Pro lie Ser Gin Lys Met Tyr Ala VaI Leu Pro Cys GIy GIy 770 775 780 lie GIy VaI Asp Ser Asp Thr VaI Trp Asn GIu Met His Ser Ser Ser 785 790 795 800
Ala VaI Arg Met Ala VaI GIy Cys Leu Leu GIu Leu Ala Phe Lys VaI 805 810 815 Ala Ala GIy GIu Leu Lys Asn GIy Phe Ala lie lie Arg Pro Pro GIy 820 825 830
His His Ala GIu GIu Ser Thr Ala Met GIy Phe Cys Phe Phe Asn Ser 835 840 845
VaI Ala lie Thr Ala Lys Leu Leu GIn GIn Lys Leu Asn VaI GIy Lys 850 855 860
VaI Leu lie VaI Asp Trp Asp lie His His GIy Asn GIy Thr GIn GIn 865 870 875 880
Ala Phe Tyr Asn Asp Pro Ser VaI Leu Tyr lie Ser Leu His Arg Tyr 885 890 895 Asp Asn GIy Asn Phe Phe Pro GIy Ser GIy Ala Pro GIu GIu VaI GIy 900 905 910
GIy GIy Pro GIy VaI GIy Tyr Asn VaI Asn VaI Ala Trp Thr GIy GIy 915 920 925
VaI Asp Pro Pro lie GIy Asp VaI GIu Tyr Leu Thr Ala Phe Arg Thr 930 935 940
VaI VaI Met Pro lie Ala His GIu Phe Ser Pro Asp VaI VaI Leu VaI 945 950 955 960
Ser Ala GIy Phe Asp Ala VaI GIu GIy His Leu Ser Pro Leu GIy GIy 965 970 975 Tyr Ser VaI Thr Ala Arg Cys Phe GIy His Leu Thr Arg Gin Leu Met 980 985 990
Thr Leu Ala GIy GIy Arg VaI VaI Leu Ala Leu GIu GIy GIy His Asp 995 1000 1005
Leu Thr Ala lie Cys Asp Ala Ser GIu Ala Cys VaI Ser Ala Leu 1010 1015 1020
Leu Ser VaI GIu Leu GIn Pro Leu Asp GIu Ala VaI Leu GIn GIn 1025 1030 1035
Lys Pro Asn lie Asn Ala VaI Ala Thr Leu GIu Lys VaI lie GIu 1040 1045 1050 lie GIn Ser Lys His Trp Ser Cys VaI GIn Lys Phe Ala Ala GIy 1055 1060 1065
Leu GIy Arg Ser Leu Arg GIu Ala GIn Ala GIy GIu Thr GIu GIu 1070 1075 1080
Ala GIu Thr VaI Ser Ala Met Ala Leu Leu Ser VaI GIy Ala GIu 1085 1090 1095
GIn Ala GIn Ala Ala Ala Ala Arg GIu His Ser Pro Arg Pro Ala 1100 1105 1110
GIu GIu Pro Met GIu GIn GIu Pro Ala Leu 1115 1120
<210> 120
<211> 954
<212> PRT
<213> human <220>
<221> misc_feature
<222> (194) .. (194)
<223> Xaa can be any naturally occurring amino acid
<220> <221> misc_feature
<222> (220) .. (220)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature <222> (360) .. (360)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (488) .. (488) <223> Xaa can be any naturally occurring amino acid
<400> 120
Met His Ser Pro GIy Ala Asp GIy Thr Gin VaI Ser Pro GIy Ala His 1 5 10 15
Tyr Cys Ser Pro Thr GIy Ala GIy Cys Pro Arg Pro Cys Ala Asp Thr 20 25 30
Pro GIy Pro GIn Pro GIn Pro Met Asp Leu Arg VaI GIy Gin Arg Pro 35 40 45
Pro VaI GIu Pro Pro Pro GIu Pro Thr Leu Leu Ala Leu Gin Arg Pro 50 55 60 Gin Arg Leu His His His Leu Phe Leu Ala GIy Leu Gin GIn GIn Arg 65 70 75 80
Ser VaI GIu Pro Met Arg Leu Ser Met Asp Thr Pro Met Pro GIu Leu 85 90 95
Gin VaI GIy Pro GIn GIu GIn GIu Leu Arg GIn Leu Leu His Lys Asp 100 105 110
Lys Ser Lys Arg Ser Ala VaI Ala Ser Ser VaI VaI Lys GIn Lys Leu 115 120 125
Ala GIu VaI lie Leu Lys Lys GIn Gin Ala Ala Leu GIu Arg Thr VaI 130 135 140 His Pro Asn Ser Pro GIy lie Pro Tyr Arg Thr Leu GIu Pro Leu GIu 145 150 155 160
Thr GIu GIy Ala Thr Arg Ser Met Leu Ser Ser Phe Leu Pro Pro VaI 165 170 175 Pro Ser Leu Pro Ser Asp Pro Pro GIu His Phe Pro Leu Arg Lys Thr 180 185 190
VaI Xaa GIu Pro Asn Leu Lys Leu Arg Tyr Lys Pro Lys Lys Ser Leu 195 200 205
GIu Arg Arg Lys Asn Pro Leu Leu Arg Lys GIu Xaa Ala Pro Pro Ser 210 215 220
Leu Arg Arg Arg Pro Ala GIu Thr Leu GIy Asp Ser Ser Pro Ser Ser 225 230 235 240
Ser Ser Thr Pro Ala Ser GIy Cys Ser Ser Pro Asn Asp Ser GIu His 245 250 255 GIy Pro Asn Pro lie Leu GIy Ser GIu Ala Asp Ser Asp Arg Arg Thr 260 265 270
His Pro Thr Leu GIy Pro Arg GIy Pro lie Leu GIy Ser Pro His Thr 275 280 285
Pro Leu Phe Leu Pro His GIy Leu GIu Pro GIu Ala GIy GIy Thr Leu 290 295 300
Pro Ser Arg Leu Gin Pro lie Leu Leu Leu Asp Pro Ser GIy Ser His 305 310 315 320
Ala Pro Leu Leu Thr VaI Pro GIy Leu GIy Pro Leu Pro Phe His Phe 325 330 335 Ala GIn Ser Leu Met Thr Thr GIu Arg Leu Ser GIy Ser GIy Leu His 340 345 350
Trp Pro Leu Ser Arg Thr Arg Xaa GIu Pro Leu Pro Pro Ser Ala Thr 355 360 365
Ala Pro Pro Pro Pro GIy Pro Met GIn Pro Arg Leu GIu GIn Leu Lys 370 375 380
Thr His VaI GIn VaI lie Lys Arg Ser Ala Lys Pro Ser GIu Lys Pro 385 390 395 400
Arg Leu Arg GIn lie Pro Ser Ala GIu Asp Leu GIu Thr Asp GIy GIy 405 410 415 GIy Pro GIy Gin VaI VaI Asp Asp GIy Leu GIu His Arg GIu Leu GIy 420 425 430
His GIy Gin Pro GIu Ala Arg GIy Pro Ala Pro Leu GIn Gin His Pro 435 440 445
Gin VaI Leu Leu Trp GIu Gin GIn Arg Leu Ala GIy Arg Leu Pro Arg 450 455 460
GIy Ser Thr GIy Asp Thr VaI Leu Leu Pro Leu Ala GIn GIy GIy His 465 470 475 480
Arg Pro Leu Ser Arg Ala Gin Xaa Ser Pro Ala Ala Pro Ala Ser Leu 485 490 495
Ser Ala Pro GIu Pro Ala Ser GIn Ala Arg VaI Leu Ser Ser Ser GIu 500 505 510
Thr Pro Ala Arg Thr Leu Pro Phe Thr Thr GIy Leu lie Tyr Asp Ser 515 520 525 VaI Met Leu Lys His GIn Cys Ser Cys GIy Asp Asn Ser Arg His Pro 530 535 540
GIu His Ala GIy Arg He GIn Ser lie Trp Ser Arg Leu GIn GIu Arg 545 550 555 560
GIy Leu Arg Ser Gin Cys GIu Cys Leu Arg GIy Arg Lys Ala Ser Leu 565 570 575
GIu GIu Leu GIn Ser VaI His Ser GIu Arg His VaI Leu Leu Tyr GIy 580 585 590
Thr Asn Pro Leu Ser Arg Leu Lys Leu Asp Asn GIy Lys Leu Ala GIy 595 600 605 Leu Leu Ala GIn Arg Met Phe VaI Met Leu Pro Cys GIy GIy VaI GIy 610 615 620
VaI Asp Thr Asp Thr He Trp Asn GIu Leu His Ser Ser Asn Ala Ala 625 630 635 640
Arg Trp Ala Ala GIy Ser VaI Thr Asp Leu Ala Phe Lys VaI Ala Ser 645 650 655
Arg GIu Leu Lys Asn GIy Phe Ala VaI VaI Arg Pro Pro GIy His His 660 665 670
Ala Asp His Ser Thr Ala Met GIy Phe Cys Phe Phe Asn Ser VaI Ala 675 680 685 He Ala Cys Arg GIn Leu Gin Gin GIn Ser Lys Ala Ser Lys He Leu 690 695 700
He VaI Asp Trp Asp VaI His His GIy Asn GIy Thr Gin GIn Thr Phe 705 710 715 720
Tyr GIn Asp Pro Ser VaI Leu Tyr He Ser Leu His Arg His Asp Asp 725 730 735
GIy Asn Phe Phe Pro GIy Ser GIy Ala VaI Asp GIu VaI GIy Ala GIy 740 745 750
Ser GIy GIu GIy Phe Asn VaI Asn VaI Ala Trp Ala GIy GIy Leu Asp 755 760 765 Pro Pro Met GIy Asp Pro GIu Tyr Leu Ala Ala Phe Arg lie VaI VaI 770 775 780
Met Pro lie Ala Arg GIu Phe Ser Pro Asp Leu VaI Leu VaI Ser Ala 785 790 795 800 GIy Phe Asp Ala Ala GIu GIy His Pro Ala Pro Leu Gly GIy Tyr His
805 810 815
VaI Ser Ala Lys Cys Phe GIy Tyr Met Thr Gin GIn Leu Met Asn Leu 820 825 830
Ala Gly GIy Ala VaI VaI Leu Ala Leu GIu Gly GIy His Asp Leu Thr 835 840 845
Ala lie Cys Asp Ala Ser GIu Ala Cys VaI Ala Ala Leu Leu Gly Asn 850 855 860
Arg VaI Asp Pro Leu Ser GIu GIu Gly Trp Lys GIn Lys Pro Asn Leu 865 870 875 880 Asn Ala lie Arg Ser Leu GIu Ala VaI lie Arg VaI His Ser Lys Tyr
885 890 895
Trp Gly Cys Met Gin Arg Leu Ala Ser Cys Pro Asp Ser Trp VaI Pro 900 905 910
Arg VaI Pro Gly Ala Asp Lys GIu GIu VaI GIu Ala VaI Thr Ala Leu 915 920 925
Ala Ser Leu Ser VaI Gly lie Leu Ala GIu Asp Arg Pro Ser GIu GIn 930 935 940
Leu VaI GIu GIu GIu GIu Pro Met Asn Leu 945 950
<210> 121
<211> 833
<212> PRT
<213> human
<220> <221> misc_feature
<222> (171) .. (171)
<223> Xaa can be any naturally occurring amino acid
<400> 121
Met Asp VaI VaI Asp Pro Asp lie Phe Asn Arg Asp Pro Arg Asp His 1 5 10 15
Tyr Asp Leu Leu GIn Arg Leu Gly Gly Gly Thr Tyr Gly GIu VaI Phe 20 25 30
Lys Ala Arg Asp Lys VaI Ser Gly Asp Leu VaI Ala Leu Lys Met VaI 35 40 45 Lys Met GIu Pro Asp Asp Asp VaI Ser Thr Leu Gin Lys GIu lie Leu 50 55 60 lie Leu Lys Thr Cys Arg His Ala Asn lie VaI Ala Tyr His GIy Ser 65 70 75 80
Tyr Leu Trp Leu GIn Lys Leu Trp lie Cys Met GIu Phe Cys GIy Ala 85 90 95
GIy Ser Leu GIn Asp lie Tyr GIn VaI Thr GIy Ser Leu Ser GIu Leu 100 105 110
GIn lie Ser Tyr VaI Cys Arg GIu VaI Leu GIn GIy Leu Ala Tyr Leu 115 120 125 His Ser Gin Lys Lys lie His Arg Asp lie Lys GIy Ala Asn lie Leu 130 135 140 lie Asn Asp Ala GIy GIu VaI Arg Leu Ala Asp Phe GIy lie Ser Ala 145 150 155 160
GIn lie GIy Ala Thr Leu Ala Arg Arg Leu Xaa Phe lie GIy Thr Pro 165 170 175
Tyr Trp Met Ala Pro GIu VaI Ala Ala VaI Ala Leu Lys GIy GIy Tyr 180 185 190
Asn GIu Leu Cys Asp lie Trp Ser Leu GIy lie Thr Ala lie GIu Leu 195 200 205 Ala GIu Leu Gin Pro Pro Leu Phe Asp VaI His Pro Leu Arg VaI Leu 210 215 220
Phe Leu Met Thr Lys Ser GIy Tyr GIn Pro Pro Arg Leu Lys GIu Lys 225 230 235 240
GIy Lys Trp Ser Ala Ala Phe His Asn Phe lie Lys VaI Thr Leu Thr 245 250 255
Lys Ser Pro Lys Lys Arg Pro Ser Ala Thr Lys Met Leu Ser His Gin 260 265 270
Leu VaI Ser Gin Pro GIy Leu Asn Arg GIy Leu lie Leu Asp Leu Leu 275 280 285 Asp Lys Leu Lys Asn Pro GIy Lys GIy Pro Ser lie GIy Asp lie GIu 290 295 300
Asp GIu GIu Pro GIu Leu Pro Pro Ala lie Pro Arg Arg lie Arg Ser 305 310 315 320
Thr His Arg Ser Ser Ser Leu GIy lie Pro Asp Ala Asp Cys Cys Arg 325 330 335
Arg His Met GIu Phe Arg Lys Leu Arg GIy Met GIu Thr Arg Pro Pro 340 345 350
Ala Asn Thr Ala Arg Leu Gin Pro Pro Arg Asp Leu Arg Ser Ser Ser 355 360 365 Pro Arg Lys GIn Leu Ser GIu Ser Ser Asp Asp Asp Tyr Asp Asp VaI 370 375 380
Asp He Pro Thr Pro Ala GIu Asp Thr Pro Pro Pro Leu Pro Pro Lys 385 390 395 400 Pro Lys Phe Arg Ser Pro Ser Asp GIu GIy Pro GIy Ser Met GIy Asp
405 410 415
Asp GIy Gin Leu Ser Pro GIy VaI Leu VaI Arg Cys Ala Ser GIy Pro 420 425 430
Pro Pro Asn Ser Pro Arg Pro GIy Pro Pro Pro Ser Thr Ser Ser Pro 435 440 445
His Leu Thr Ala His Ser GIu Pro Ser Leu Trp Asn Pro Pro Ser Arg 450 455 460
GIu Leu Asp Lys Pro Pro Leu Leu Pro Pro Lys Lys GIu Lys Met Lys 465 470 475 480 Arg Lys GIy Cys Ala Leu Leu VaI Lys Leu Phe Asn GIy Cys Pro Leu
485 490 495
Arg He His Ser Thr Ala Ala Trp Thr His Pro Ser Thr Lys Asp GIn 500 505 510
His Leu Leu Leu GIy Ala GIu GIu GIy He Phe He Leu Asn Arg Asn 515 520 525
Asp GIn GIu Ala Thr Leu GIu Met Leu Phe Pro Ser Arg Thr Thr Trp 530 535 540
VaI Tyr Ser He Asn Asn VaI Leu Met Ser Leu Ser GIy Lys Thr Pro 545 550 555 560 His Leu Tyr Ser His Ser He Leu GIy Leu Leu GIu Arg Lys GIu Thr
565 570 575
Arg Ala GIy Asn Pro He Ala His He Ser Pro His Arg Leu Leu Ala 580 585 590
Arg Lys Asn Met VaI Ser Thr Lys He GIn Asp Thr Lys GIy Cys Arg 595 600 605
Ala Cys Cys VaI Ala GIu GIy Ala Ser Ser GIy GIy Pro Phe Leu Cys 610 615 620
GIy Ala Leu GIu Thr Ser VaI VaI Leu Leu GIn Trp Tyr GIn Pro Met 625 630 635 640 Asn Lys Phe Leu Leu VaI Arg GIn VaI Leu Phe Pro Leu Pro Thr Pro
645 650 655
Leu Ser VaI Phe Ala Leu Leu Thr GIy Pro GIy Ser GIu Leu Pro Ala 660 665 670 VaI Cys lie GIy VaI Ser Pro GIy Arg Pro GIy Lys Ser VaI Leu Phe 675 680 685
His Thr VaI Arg Phe GIy Ala Leu Ser Cys Trp Leu GIy GIu Met Ser 690 695 700 Thr GIu His Arg GIy Pro VaI Gin VaI Thr Gin VaI GIu GIu Asp Met 705 710 715 720
VaI Met VaI Leu Met Asp GIy Ser VaI Lys Leu VaI Thr Pro GIu GIy 725 730 735
Ser Pro VaI Arg GIy Leu Arg Thr Pro GIu lie Pro Met Thr GIu Ala 740 745 750
VaI GIu Ala VaI Ala Met VaI GIy GIy GIn Leu GIn Ala Phe Trp Lys 755 760 765
His GIy VaI GIn VaI Trp Ala Leu GIy Ser Asp GIn Leu Leu GIn GIu 770 775 780 Leu Arg Asp Pro Thr Leu Thr Phe Arg Leu Leu GIy Ser Pro Arg Leu 785 790 795 800
GIu Cys Ser GIy Thr lie Ser Pro His Cys Asn Leu Leu Leu Pro GIy 805 810 815
Ser Ser Asn Ser Pro Ala Ser Ala Ser Arg VaI Ala GIy lie Thr GIy 820 825 830
Leu
<210> 122
<211> 205 <212> PRT
<213> human
<220>
<221> misc_feature
<222> (82) .. (82) <223> Xaa can be any naturally occurring amino acid
<400> 122
Met Thr GIu Arg Arg VaI Pro Phe Ser Leu Leu Arg GIy Pro Ser Trp 1 5 10 15
Asp Pro Phe Arg Asp Trp Tyr Pro His Ser Arg Leu Phe Asp GIn Ala 20 25 30
Phe GIy Leu Pro Arg Leu Pro GIu GIu Trp Ser GIn Trp Leu GIy GIy 35 40 45
Ser Ser Trp Pro GIy Tyr VaI Arg Pro Leu Pro Pro Ala Ala lie GIu 50 55 60 Ser Pro Ala VaI Ala Ala Pro Ala Tyr Ser Arg Ala Leu Ser Arg Gin 65 70 75 80 Leu Xaa Ser GIy VaI Ser GIu lie Arg His Thr Ala Asp Arg Trp Arg 85 90 95
VaI Ser Leu Asp VaI Asn His Phe Ala Pro Asp GIu Leu Thr VaI Lys 100 105 110 Thr Lys Asp GIy VaI VaI GIu lie Thr GIy Lys His GIu GIu Arg Gin 115 120 125
Asp GIu His GIy Tyr lie Ser Arg Cys Phe Thr Arg Lys Tyr Thr Leu 130 135 140
Pro Pro GIy VaI Asp Pro Thr Gin VaI Ser Ser Ser Leu Ser Pro GIu 145 150 155 160
GIy Thr Leu Thr VaI GIu Ala Pro Met Pro Lys Leu Ala Thr Gin Ser 165 170 175
Asn GIu lie Thr lie Pro VaI Thr Phe GIu Ser Arg Ala Gin Leu GIy 180 185 190 GIy Pro GIu Ala Ala Lys Ser Asp GIu Thr Ala Ala Lys 195 200 205
<210> 123
<211> 1771
<212> PRT <213> human
<220>
<221> misc_feature
<222> (918).. (918)
<223> Xaa can be any naturally occurring amino acid <400> 123
Met Ser VaI Leu lie Ser GIn Ser VaI lie Asn Tyr VaI GIu GIu GIu 1 5 10 15
Asn lie Pro Ala Leu Lys Ala Leu Leu GIu Lys Cys Lys Asp VaI Asp 20 25 30 GIu Arg Asn GIu Cys GIy GIn Thr Pro Leu Met lie Ala Ala GIu GIn 35 40 45
GIy Asn Leu GIu lie VaI Lys GIu Leu lie Lys Asn GIy Ala Asn Cys 50 55 60
Asn Leu GIu Asp Leu Asp Asn Trp Thr Ala Leu lie Ser Ala Ser Lys 65 70 75 80
GIu GIy His VaI His lie VaI GIu GIu Leu Leu Lys Cys GIy VaI Asn 85 90 95
Leu GIu His Arg Asp Met GIy GIy Trp Thr Ala Leu Met Trp Ala Cys 100 105 110 Tyr Lys GIy Arg Thr Asp VaI VaI GIu Leu Leu Leu Ser His GIy Ala 115 120 125
Asn Pro Ser VaI Thr GIy Leu Tyr Ser VaI Tyr Pro lie lie Trp Ala 130 135 140
Ala GIy Arg GIy His Ala Asp He VaI His Leu Leu Leu Gin Asn GIy 145 150 155 160
Ala Lys VaI Asn Cys Ser Asp Lys Tyr GIy Thr Thr Pro Leu VaI Trp 165 170 175
Ala Ala Arg Lys GIy His Leu GIu Cys VaI Lys His Leu Leu Ala Met 180 185 190 GIy Ala Asp VaI Asp GIn GIu GIy Ala Asn Ser Met Thr Ala Leu He 195 200 205
VaI Ala VaI Lys GIy GIy Tyr Thr GIn Ser VaI Lys GIu He Leu Lys 210 215 220
Arg Asn Pro Asn VaI Asn Leu Thr Asp Lys Asp GIy Asn Thr Ala Leu 225 230 235 240
Met He Ala Ser Lys GIu GIy His Thr GIu He VaI Gin Asp Leu Leu 245 250 255
Asp Ala GIy Thr Tyr VaI Asn He Pro Asp Arg Ser GIy Asp Thr VaI 260 265 270 Leu He GIy Ala VaI Arg GIy GIy His VaI GIu He VaI Arg Ala Leu 275 280 285
Leu GIn Lys Tyr Ala Asp He Asp He Arg GIy GIn Asp Asn Lys Thr 290 295 300
Ala Leu Tyr Trp Ala VaI GIu Lys GIy Asn Ala Thr Met VaI Arg Asp 305 310 315 320
He Leu GIn Cys Asn Pro Asp Thr GIu He Cys Thr Lys Asp GIy GIu 325 330 335
Thr Pro Leu He Lys Ala Thr Lys Met Arg Asn He GIu VaI VaI GIu 340 345 350 Leu Leu Leu Asp Lys GIy Ala Lys VaI Ser Ala VaI Asp- Lys Lys GIy 355 360 365
Asp Thr Pro Leu His He Ala He Arg GIy Arg Ser Arg Lys Leu Ala 370 375 380
GIu Leu Leu Leu Arg Asn Pro Lys Asp GIy Arg Leu Leu Tyr Arg Pro 385 390 395 400
Asn Lys Ala GIy GIu Thr Pro Tyr Asn He Asp Cys Ser His Gin Lys 405 410 415
Ser He Leu Thr Gin He Phe GIy Ala Arg His Leu Ser Pro Thr GIu 420 425 430 Thr Asp GIy Asp Met Leu GIy Tyr Asp Leu Tyr Ser Ser Ala Leu Ala 435 440 445
Asp lie Leu Ser GIu Pro Thr Met GIn Pro Pro lie Cys VaI GIy Leu 450 455 460 Tyr Ala GIn Trp GIy Ser GIy Lys Ser Phe Leu Leu Lys Lys Leu GIu 465 470 475 480
Asp GIu Met Lys Thr Phe Ala GIy GIn GIn lie GIu Pro Leu Phe GIn 485 490 495
Phe Ser Trp Leu lie VaI Phe Leu Thr Leu Leu Leu Cys GIy GIy Leu 500 505 510
GIy Leu Leu Phe Ala Phe Thr VaI His Pro Asn Leu GIy lie Ala VaI 515 520 525
Ser Leu Ser Phe Leu Ala Leu Leu Tyr lie Phe Phe lie VaI lie Tyr 530 535 540 Phe GIy GIy Arg Arg GIu GIy GIu Ser Trp Asn Trp Ala Trp VaI Leu 545 550 555 560
Ser Thr Arg Leu Ala Arg His lie GIy Tyr Leu GIu Leu Leu Leu Lys 565 570 575
Leu Met Phe VaI Asn Pro Pro GIu Leu Pro GIu Gin Thr Thr Lys Ala 580 585 590
Leu Pro VaI Arg Phe Leu Phe Thr Asp Tyr Asn Arg Leu Ser Ser VaI 595 600 605
GIy GIy GIu Thr Ser Leu Ala GIu Met lie Ala Thr Leu Ser Asp Ala 610 615 620 Cys GIu Arg GIu Phe GIy Phe Leu Ala Thr Arg Leu Phe Arg VaI Phe 625 630 635 640
Lys Thr GIu Asp Thr GIn GIy Lys Lys Lys Trp Lys Lys Thr Cys Cys 645 650 655
Leu Pro Ser Phe VaI He Phe Leu Phe He He GIy Cys He He Ser 660 - 665 670
GIy He Thr Leu Leu Ala He Phe Arg VaI Asp Pro Lys His Leu Thr 675 680 685
VaI Asn Ala VaI Leu He Ser He Ala Ser VaI VaI GIy Leu Ala Phe 690 695 700 VaI Leu Asn Cys Arg Thr Trp Trp Gin VaI Leu Asp Ser Leu Leu Asn 705 710 715 720
Ser GIn Arg Lys Arg Leu His Asn Ala Ala Ser Lys Leu His Lys Leu 725 730 735 Lys Ser GIu GIy Phe Met Lys VaI Leu Lys Cys GIu VaI GIu Leu Met 740 745 750
Ala Arg Met Ala Lys Thr lie Asp Ser Phe Thr GIn Asn Gin Thr Arg 755 760 765 Leu VaI VaI lie He Asp GIy Leu Asp Ala Cys GIu GIn Asp Lys VaI 770 775 780
Leu GIn Met Leu Asp Thr VaI Arg VaI Leu Phe Ser Lys GIy Pro Phe 785 790 795 800 lie Ala He Phe Ala Ser Asp Pro His He He He Lys Ala He Asn 805 810 815
GIn Asn Leu Asn Ser VaI Leu Arg Asp Ser Asn He Asn GIy His Asp 820 825 830
Tyr Met Arg Asn He VaI His Leu Pro VaI Phe Leu Asn Ser Arg GIy 835 840 845 Leu Ser Asn Ala Arg Lys Phe Leu VaI Thr Ser Ala Thr Asn GIy Asp 850 855 860
VaI Pro Cys Ser Asp Thr Thr GIy He Gin GIu Asp Ala Asp Arg Arg 865 870 875 880
VaI Ser GIn Asn Ser Leu GIy GIu Met Thr Lys Leu GIy Ser Lys Thr 885 890 895
Ala Leu Asn Arg Arg Asp Thr Tyr Arg Arg Arg GIn Met GIn Arg Thr 900 905 910
He Thr Arg GIn Met Xaa Phe Asp Leu Thr Lys Leu Leu VaI Thr GIu 915 920 925 Asp Trp Phe Ser Asp He Ser Pro GIn Thr Met Arg Arg Leu Leu Asn 930 935 940
He VaI Ser VaI Thr GIy Arg Leu Leu Arg Ala Asn GIn He Ser Phe 945 950 955 960
Asn Trp Asp Arg Leu Ala Ser Trp He Asn Leu Thr GIu Gin Trp Pro 965 970 975
Tyr Arg Thr Ser Trp Leu He Leu Tyr Leu GIu GIu Thr GIu GIy He 980 985 990
Pro Asp Gin Met Thr Leu Lys Thr He Tyr GIu Arg He Ser Lys Asn 995 1000 1005 He Pro Thr Thr Lys Asp VaI Glu Pro Leu Leu GIu He Asp GIy 1010 1015 1020
Asp He Arg Asn Phe Glu VaI Phe Leu Ser Ser Arg Thr Pro VaI 1025 1030 1035
Leu VaI Ala Arg Asp VaI Lys VaI Phe Leu Pro Cys Thr VaI Asn 1040 1045 1050
Leu Asp Pro Lys Leu Arg GIu lie lie Ala Asp VaI Arg Ala Ala 1055 1060 1065
Arg GIu GIn lie Ser He GIy GIy Leu Ala Tyr Pro Pro Leu Pro 1070 1075 1080
Leu His GIu GIy Pro Pro Arg Ala Pro Ser GIy Tyr Ser Gin Pro 1085 1090 1095
Pro Ser VaI Cys Ser Ser Thr Ser Phe Asn GIy Pro Phe Ala GIy 1100 1105 1110 GIy VaI VaI Ser Pro GIn Pro His Ser Ser Tyr Tyr Ser GIy Met 1115 1120 1125
Thr GIy Pro Gin His Pro Phe Tyr Asn Arg Pro Phe Phe Ala Pro 1130 1135 1140
Tyr Leu Tyr Thr Pro Arg Tyr Tyr Pro GIy Gly Ser GIn His Leu 1145 1150 1155
He Ser Arg Pro Ser VaI Lys Thr Ser Leu Pro Arg Asp Gin Asn 1160 1165 1170
Asn GIy Leu GIu VaI He Lys GIu Asp Ala Ala GIu GIy Leu Ser 1175 1180 1185 Ser Pro Thr Asp Ser Ser Arg GIy Ser Gly Pro Ala Pro GIy Pro 1190 1195 1200
VaI VaI Leu Leu Asn Ser Leu Asn VaI Asp Ala VaI Cys GIu Lys 1205 1210 1215
Leu Lys GIn He GIu Gly Leu Asp Gin Ser Met Leu Pro Gin Tyr 1220 1225 1230
Cys Thr Thr He Lys Lys Ala Asn He Asn Gly Arg VaI Leu Ala 1235 1240 1245
GIn Cys Asn He Asp GIu Leu Lys Lys GIu Met Asn Met Asn Phe 1250 1255 1260 Gly Asp Trp His Leu Phe Arg Ser Thr VaI Leu GIu Met Arg Asn 1265 1270 1275
Ala GIu Ser His VaI VaI Pro GIu Asp Pro Arg Phe Leu Ser GIu 1280 1285 1290
Ser Ser Ser Gly Pro Ala Pro His Gly GIu Pro Ala Arg Arg Ala 1295 1300 1305
Ser His Asn GIu Leu Pro His Thr GIu Leu Ser Ser GIn Thr Pro 1310 1315 1320
Tyr Thr Leu Asn Phe Ser Phe GIu GIu Leu Asn Thr Leu Gly Leu 1325 1330 1335 Asp GIu GIy Ala Pro Arg His Ser Asn Leu Ser Trp GIn Ser GIn 1340 1345 1350
Thr Arg Arg Thr Pro Ser Leu Ser Ser Leu Asn Ser Gin Asp Ser 1355 1360 1365 Ser lie GIu lie Ser Lys Leu Thr Asp Lys VaI GIn Ala GIu Tyr 1370 1375 1380
Arg Asp Ala Tyr Arg GIu Tyr lie Ala Gin Met Ser GIn Leu GIu 1385 1390 1395
GIy GIy Pro GIy Ser Thr Thr lie Ser GIy Arg Ser Ser Pro His 1400 1405 1410
Ser Thr Tyr Tyr Met GIy GIn Ser Ser Ser GIy GIy Ser lie His 1415 1420 1425
Ser Asn Leu GIu GIn GIu Lys GIy Lys Asp Ser GIu Pro Lys Pro 1430 1435 1440 Asp Asp GIy Arg Lys Ser Phe Leu Met Lys Arg GIy Asp VaI lie 1445 1450 1455
Asp Tyr Ser Ser Ser GIy VaI Ser Thr Asn Asp Ala Ser Pro Leu 1460 1465 1470
Asp Pro lie Thr GIu GIu Asp GIu Lys Ser Asp GIn Ser GIy Ser 1475 1480 1485
Lys Leu Leu Pro GIy Lys Lys Ser Ser GIu Arg Ser Ser Leu Phe 1490 1495 1500
GIn Thr Asp Leu Lys Leu Lys GIy Ser GIy Leu Arg Tyr GIn Lys 1505 1510 1515 Leu Pro Ser Asp GIu Asp GIu Ser GIy Thr GIu GIu Ser Asp Asn 1520 1525 1530
Thr Pro Leu Leu Lys Asp Asp Lys Asp Arg Lys Ala GIu GIy Lys 1535 1540 1545
VaI GIu Arg VaI Pro Lys Ser Pro GIu His Ser Ala GIu Pro lie - 1550 1555 1560
Arg Thr Phe lie Lys Ala Lys GIu Tyr Leu Ser Asp Ala Leu Leu 1565 1570 1575
Asp Lys Lys Asp Ser Ser Asp Ser GIy VaI Arg Ser Ser GIu Ser 1580 1585 1590 Ser Pro Asn His Ser Leu His Asn GIu VaI Ala Asp Asp Ser GIn 1595 1600 1605
Leu GIu Lys Ala Asn Leu lie GIu Leu GIu Asp Asp Ser His Ser 1610 1615 1620 GIy Lys Arg GIy lie Pro His Ser Leu Ser GIy Leu GIn Asp Pro
1625 1630 1635 lie lie Ala Arg Met Ser lie Cys Ser GIu Asp Lys Lys Ser Pro
1640 1645 1650 Ser GIu Cys Ser Leu lie Ala Ser Ser Pro GIu GIu Asn Trp Pro
1655 1660 1665
Ala Cys Gin Lys Ala Tyr Asn Leu Asn Arg Thr Pro Ser Thr VaI
1670 1675 1680
Thr Leu Asn Asn Asn Ser Ala Pro Ala Asn Arg Ala Asn Gin Asn 1685 1690 1695
Phe Asp GIu Met GIu GIy lie Arg GIu Thr Ser GIn VaI lie Leu
1700 1705 1710
Arg Pro Ser Ser Ser Pro Asn Pro Thr Thr lie GIn Asn GIu Asn
1715 1720 1725 Leu Lys Ser Met Thr His Lys Arg Ser Gin Arg Ser Ser Tyr Thr
1730 1735 1740
Arg Leu Ser Lys Asp Pro Pro GIu Leu His Ala Ala Ala Ser Ser
1745 1750 1755
GIu Ser Thr GIy Phe GIy GIu GIu Arg GIu Ser lie Leu 1760 1765 1770
<210> 124
<211> 801
<212> PRT
<213> mouse <220>
<221> misc_feature
<222> (294) .. (294)
<223> Xaa can be any naturally occurring amino acid
<400> 124 Met GIy Asp Thr VaI VaI GIu Pro Ala Pro Leu Lys Pro Thr Ser GIu 1 5 10 15
Pro Thr Ser GIy Pro Pro GIy Asn Asn GIy GIy Ser Leu Leu Ser VaI 20 25 30 lie Thr GIu GIy VaI GIy GIu Leu Ser VaI lie Asp Pro GIu VaI Ala 35 40 45
GIn Lys Ala Cys Gin GIu VaI Leu GIu Lys VaI Lys Leu Leu His GIy 50 55 60
GIy VaI Ala VaI Ser Ser Arg GIy Thr Pro Leu GIu Leu VaI Asn GIy 65 70 75 80 Asp GIy VaI Asp Ser GIu He Arg Cys Leu Asp Asp Pro Pro Ala Gin
85 90 95 lie Arg GIu GIu GIu Asp GIu Met GIy Ala Ala VaI Ala Ser GIy Thr 100 105 110
Ala Lys GIy Ala Arg Arg Arg Arg GIn Asn Asn Ser Ala Lys Gin Ser 115 120 125 Trp Leu Leu Arg Leu Phe GIu Ser Lys Leu Phe Asp lie Ser Met Ala 130 135 140 lie Ser Tyr Leu Tyr Asn Ser Lys GIu Pro GIy VaI GIn Ala Tyr lie 145 150 155 160
GIy Asn Arg Leu Phe Cys Phe Arg Asn GIu Asp VaI Asp Phe Tyr Leu 165 170 175
Pro Gin Leu Leu Asn Met Tyr lie His Met Asp GIu Asp VaI GIy Asp 180 185 190
Ala lie Lys Pro Tyr lie VaI His Arg Cys Arg Gin Ser lie Asn Phe 195 200 205 Ser Leu Gin Cys Ala Leu Leu Leu GIy Ala Tyr Ser Ser Asp Met His 210 215 220 lie Ser Thr Gin Arg His Ser Arg GIy Thr Lys Leu Arg Lys Leu lie 225 230 235 240
Leu Ser Asp GIu Leu Lys Pro Ala His Arg Lys Arg GIu Leu Pro Ser 245 250 255
Leu Ser Pro Ala Pro Asp Thr GIy Leu Ser Pro Ser Lys Arg Thr His 260 265 270
Gin Arg Ser Lys Ser Asp Ala Thr Ala Ser lie Ser Leu Ser Ser Asn 275 280 285 Leu Lys Arg Thr Ala Xaa Asn Pro Lys VaI GIu Asn GIu Asp GIu Pro 290 295 300
VaI Arg Leu Ala Pro GIu Arg GIu Phe lie Lys Ser Leu Met Ala lie 305 310 315 320
GIy Lys Arg Leu Ala Thr Leu Pro Thr Lys GIu Gin Lys Thr GIn Arg 325 330 335
Leu lie Ser GIu Leu Ser Leu Leu Asn His Lys Leu Pro Ala Arg VaI 340 345 350
Trp Leu Pro Thr Ala GIy Phe Asp His His VaI VaI Arg VaI Pro His 355 360 365 Thr GIn Ala VaI VaI Leu Asn Ser Lys Asp Lys Ala Pro Tyr Leu lie 370 375 380
Tyr VaI GIu VaI Leu GIu Cys GIu Asn Phe Asp Thr Thr Ser VaI Pro 385 390 395 400 Ala Arg He Pro GIu Asn Arg He Arg Ser Thr Arg Ser VaI GIu Asn 405 410 415
Leu Pro GIu Cys GIy He Thr His GIu GIn Arg Ala GIy Ser Phe Ser 420 425 430
Thr VaI Pro Asn Tyr Asp Asn Asp Asp GIu Ala Trp Ser VaI Asp Asp 435 440 445
He GIy GIu Leu GIn VaI GIu Leu Pro GIu VaI His Thr Asn Ser Cys 450 455 460
Asp Asn He Ser Gin Phe Ser VaI Asp Ser He Thr Ser Gin GIu Ser 465 470 475 480
Lys GIu Pro VaI Phe He Ala Ala GIy Asp He Arg Arg Arg Leu Ser 485 490 495
GIu Gin Leu Ala His Thr Pro Thr Ala Phe Lys Arg Asp Pro GIu Asp 500 505 510 Pro Ser Ala VaI Ala Leu Lys GIu Pro Trp GIn GIu Lys VaI Arg Arg 515 520 525
He Arg GIu GIy Ser Pro Tyr GIy His Leu Pro Asn Trp Arg Leu Leu 530 535 540
Ser VaI He VaI Lys Cys GIy Asp Asp Leu Arg GIn GIu Leu Leu Ala 545 550 555 560
Phe Gin VaI Leu Lys GIn Leu GIn Ser He Trp GIu GIn GIu Arg VaI 565 570 575
Pro Leu Trp He Lys Pro Tyr Lys He Leu VaI He Ser Ala Asp Ser 580 585 590
GIy Met He GIu Pro VaI VaI Asn Ala VaI Ser He His GIn VaI Lys 595 600 605
Lys Gin Ser Gin Leu Ser Leu Leu Asp Tyr Phe Leu GIn GIu His GIy 610 615 620
Ser Tyr Thr Thr GIu Ala Phe Leu Ser Ala Gin Arg Asn Phe VaI Gin 625 630 635 640
Ser Cys Ala GIy Tyr Cys Leu VaI Cys Tyr Leu Leu GIn VaI Lys Asp 645 650 655
Arg His Asn GIy Asn He Leu Leu Asp Ala GIu GIy His He He His 660 665 670
He Asp Phe GIy Phe He Leu Ser Ser Ser Pro Arg Asn Leu GIy Phe 675 680 685
GIu Thr Ser Ala Phe Lys Leu Thr Thr GIu Phe VaI Asp VaI Met GIy 690 695 700
GIy Leu Asp GIy Asp Met Phe Asn Tyr Tyr Lys Met Leu Met Leu GIn 705 710 715 720
GIy Leu He Ala Ala Arg Lys His Met Asp Lys VaI VaI GIn He VaI 725 730 735
GIu He Met GIn GIn GIy Ser GIn Leu Pro Cys Phe His GIy Ser Ser 740 745 750
Thr He Arg Asn Leu Lys GIu Arg Phe His Met Ser Met Thr GIu GIu 755 760 765
GIn Leu GIn Leu Leu VaI GIu GIn Met VaI Asp GIy Ser Met Arg Ser 770 775 780 He Thr Thr Lys Leu Tyr Asp GIy Phe Gin Tyr Leu Thr Asn GIy He 785 790 795 800
Met
<210> 125 <211> 415
<212> PRT
<213> human
<220>
<221> misc_feature <222> (386).. (386)
<223> Xaa can be any naturally occurring amino acid
<400> 125
Met Ser Ser Asn Cys Thr Ser Thr Thr Ala VaI Ala VaI Ala Pro Leu 1 5 10 15 Ser Ala Ser Lys Thr Lys Thr Lys Lys Lys His Phe VaI Cys GIn Lys 20 25 30
VaI Lys Leu Phe Arg Ala Ser GIu Pro He Leu Ser VaI Leu Met Trp 35 40 45
GIy VaI Asn His Thr He Asn GIu Leu Ser Asn VaI Pro VaI Pro VaI 50 55 60
Met Leu Met Pro Asp Asp Phe Lys Ala Tyr Ser Lys He Lys VaI Asp 65 70 75 80
Asn His Leu Phe Asn Lys GIu Asn Leu Pro Ser Arg Phe Lys Phe Lys 85 90 95 GIu Tyr Cys Pro Met VaI Phe Arg Asn Leu Arg GIu Arg Phe GIy He 100 105 HO
Asp Asp GIn Asp Tyr GIn Asn Ser VaI Thr Arg Ser Ala Pro He Asn 115 120 125
Ser Asp Ser GIn GIy Arg Cys GIy Thr Arg Phe Leu Thr Thr Tyr Asp 130 135 140 Arg Arg Phe VaI lie Lys Thr VaI Ser Ser GIu Asp VaI Ala GIu Met 145 150 155 160
His Asn lie Leu Lys Lys Tyr His GIn Phe lie VaI GIu Cys His GIy 165 170 175 Asn Thr Leu Leu Pro GIn Phe Leu GIy Met Tyr Arg Leu Thr VaI Asp 180 185 190
GIy VaI GIu Thr Tyr Met VaI VaI Thr Arg Asn VaI Phe Ser His Arg 195 200 205
Leu Thr VaI His Arg Lys Tyr Asp Leu Lys GIy Ser Thr VaI Ala Arg 210 215 220
GIu Ala Ser Asp Lys GIu Lys Ala Lys Asp Leu Pro Thr Phe Lys Asp 225 230 235 240
Asn Asp Phe Leu Asn GIu GIy Gin Lys Leu His VaI GIy GIu GIu Ser 245 250 255 Lys Lys Asn Phe Leu GIu Lys Leu Lys Arg Asp VaI GIu Phe Leu Ala 260 265 270
Gin Leu Lys lie Met Asp Tyr Ser Leu Leu VaI GIy lie His Asp VaI 275 280 285
Asp Arg Ala GIu Gin GIu GIu Met GIu VaI GIu GIu Arg Ala GIu Asp 290 295 300
GIu GIu Cys GIu Asn Asp GIy VaI GIy GIy Asn Leu Leu Cys Ser Tyr 305 310 315 320
GIy Thr Pro Pro Asp Ser Pro GIy Asn Leu Leu Ser Phe Pro Arg Phe 325 330 335 Phe GIy Pro GIy GIu Phe Asp Pro Ser VaI Asp VaI Tyr Ala Met Lys 340 345 350
Ser His GIu Ser Ser Pro Lys Lys GIu VaI Tyr Phe Met Ala lie lie 355 360 365
Asp lie Leu Thr Pro Tyr Asp Thr Lys Lys Lys Ala Ala His Ala Ala 370 375 380
Lys Xaa VaI Lys His GIy Ala GIy Ala GIu lie Ser Thr VaI Asn Pro 385 390 395 400
GIu Gin Tyr Ser Lys Arg Phe Asn GIu Phe Met Ser Asn lie Leu 405 410 415
<210> 126
<211> 416
<212> PRT
<213> mouse
<220> <221> misc_feature <222> (386) .. (386)
<223> Xaa can be any naturally occurring amino acid <400> 126
Met Ser Ser Asn Cys Thr Ser Thr Thr Ala VaI Ala VaI Ala Pro Leu 1 5 10 15
Ser Ala Ser Lys Thr Lys Thr Lys Lys Lys His Phe VaI Cys GIn Lys 20 25 30
VaI Lys Leu Phe Arg Ala Ser GIu Pro lie Leu Ser VaI Leu Met Trp 35 40 45 GIy VaI Asn His Thr lie Asn GIu Leu Ser Asn VaI Pro VaI Pro VaI 50 55 60
Met Leu Met Pro Asp Asp Phe Lys Ala Tyr Ser Lys lie Lys VaI Asp 65 70 75 80
Asn His Leu Phe Asn Lys GIu Asn Leu Pro Ser Arg Phe Lys Phe Lys 85 90 95
GIu Tyr Cys Pro Met VaI Phe Arg Asn Leu Arg GIu Arg Phe GIy lie 100 105 110
Asp Asp GIn Asp Tyr GIn Asn Ser VaI Thr Arg Ser Ala Pro lie Asn 115 120 125 Ser Asp Ser Gin GIy Arg Cys GIy Thr Arg Phe Leu Thr Thr Tyr Asp 130 135 140
Arg Arg Phe VaI lie Lys Thr VaI Ser Ser GIu Asp VaI Ala GIu Met 145 150 155 160
His Asn lie Leu Lys Lys Tyr His GIn Phe lie VaI GIu Cys His GIy 165 170 175
Asn Thr Leu Leu Pro GIn Phe Leu GIy Met Tyr Arg Leu Thr VaI Asp 180 185 190
GIy VaI GIu Thr Tyr Met VaI VaI Thr Arg Asn VaI Phe Ser His Arg 195 200 205 Leu Thr VaI His Arg Lys Tyr Asp Leu Lys GIy Ser Thr VaI Ala Arg 210 . 215 220
GIu Ala Ser Asp Lys GIu Lys Ala Lys Asp Leu Pro Thr Phe Lys Asp 225 230 235 240
Asn Asp Phe Leu Asn GIu GIy Gin Lys Leu His VaI GIy GIu GIu Ser 245 250 255
Lys Lys Asn Phe Leu GIu Lys Leu Lys Arg Asp VaI GIu Phe Leu Ala 260 265 270
Gin Leu Lys lie Met Asp Tyr Ser Leu Leu VaI GIy lie His Asp VaI 275 280 285 Asp Arg Ala GIu Gin GIu GIu Met GIu VaI GIu GIu Arg Ala GIu GIu 290 295 300
GIu GIu Cys GIu Asn Asp GIy VaI GIy GIy Ser Leu Leu Cys Ser Tyr 305 310 315 320 GIy Thr Pro Pro Asp Ser Pro GIy Asn Leu Leu Ser Phe Pro Arg Phe
325 330 335
Phe GIy Pro GIy GIu Phe Asp Pro Ser VaI Asp VaI Tyr Ala Met Lys 340 345 350
Ser His GIu Ser Ala Pro Lys Lys GIu VaI Tyr Phe Met Ala lie lie 355 360 365
Asp lie Leu Thr Pro Tyr Asp Ala Lys Lys Lys Ala Ala His Ala Ala 370 375 380
Lys Xaa VaI Lys His GIy Ala GIy Ala GIu lie Ser Thr VaI Asn Pro 385 390 395 400 GIu GIn Tyr Ser Lys Arg Phe Asn GIu Phe Met Ser Asn lie Leu Thr
405 410 415
<210> 127
<211> 420
<212> PRT <213> rat
<220>
<221> misc_feature
<222> (390).. (390)
<223> Xaa can be any naturally occurring amino acid <400> 127
Met Ala Ser Ser Ser VaI Pro Pro Ala Thr Ala Pro Ala Ala Ala GIy 1 5 10 15
GIy Pro GIy Pro GIy Phe GIy Phe Ala Ser Lys Thr Lys Lys Lys His 20 25 30 Phe VaI Gin GIn Lys VaI Lys VaI Phe Arg Ala Ala Asp Pro Leu VaI 35 40 45
GIy VaI Phe Leu Trp GIy VaI Ala His Ser lie Asn GIu Leu Ser Gin 50 55 60
VaI Pro Pro Pro VaI Met Leu Leu Pro Asp Asp Phe Lys Ala Ser Ser 65 70 75 80
Lys lie Lys VaI Asn Asn His Leu Phe His Arg GIu Asn Leu Pro Ser 85 90 95
His Phe Lys Phe Lys GIu Tyr Cys Pro GIn VaI Phe Arg Asn Leu Arg 100 105 110 Asp Arg Phe Ala lie Asp Asp His Asp Tyr Leu VaI Ser Leu Thr Arg 115 120 125 Ser Pro Pro Ser Glu Thr GIu GIy Ser Asp GIy Arg Phe Leu lie Ser 130 135 140
Tyr Asp Arg Thr Leu VaI lie Lys GIu VaI Ser Ser GIu Asp He Ala 145 150 155 160 Asp Met His Ser Asn Leu Ser Asn Tyr His Gin Tyr He VaI Lys Cys
165 170 175
His GIy Asn Thr Leu Leu Pro GIn Phe Leu GIy Met Tyr Arg VaI Ser 180 185 190
VaI GIu Asn GIu Asp Ser Tyr Met Leu VaI Met Arg Asn Met Phe Ser 195 200 205
His Arg Leu Pro VaI His Arg Lys Tyr Asp Leu Lys GIy Ser Leu VaI 210 215 220
Ser Arg GIu Ala Ser Asp Lys GIu Lys VaI Lys GIu Leu Pro Thr Leu 225 230 235 240 Lys Asp Met Asp Phe Leu Asn Lys Asn GIn Lys VaI Tyr He GIy GIu
245 250 255
GIu GIu Lys Lys VaI Phe Leu GIu Lys Leu Lys Arg Asp VaI GIu Phe 260 265 270
Leu VaI Gin Leu Lys He Met Asp Tyr Ser Leu Leu Leu GIy He His 275 280 285
Asp He He Arg Gly Ser GIu Pro GIu Glu GIu GIy Pro VaI Arg GIu 290 295 300
Glu Glu Ser Glu Trp Asp GIy Asp Cys Asn Leu Thr GIy Pro Pro Ala 305 310 315 320 Leu VaI GIy Ser Tyr GIy Thr Ser Pro Glu GIy He GIy Gly Tyr He
325 330 335
His Ser His Arg Pro Leu Gly Pro Gly Glu Phe Glu Ser Phe He Asp 340 345 350
VaI Tyr Ala He Arg Ser Ala Glu Gly Ala Pro Glu Gly Gly VaI Phe 355 360 - 365
His Gly Leu He Asp He Leu Thr Gin Tyr Asp Ala Lys Lys Lys Ala 370 375 380
Ala His Ala Ala Lys Xaa VaI Lys His Gly Ala Gly Ala Glu He Ser 385 390 395 400 Thr VaI His Pro Glu GIn Tyr Ala Lys Arg Phe Leu Asp Phe He Ser
405 410 415
Asn He Phe Ala 420 <210> 128
<211> 783
<212> PRT
<213> human <220>
<221> misc_feature
<222> (351) .. (351)
<223> Xaa can be any naturally occurring amino acid
<400> 128 Met GIu Ser Pro GIy GIu Ser GIy Ala GIy Ser Pro GIy Ala Pro Ser 1 5 10 15
Pro Ser Ser Phe Thr Thr GIy His Leu Ala Arg GIu Lys Pro Ala GIn 20 25 30
Asp Pro Leu Tyr Asp VaI Pro Asn Ala Ser GIy GIy Gin Ala GIy GIy 35 40 45
Pro GIn Arg Pro GIy Arg VaI VaI Ser Leu Arg GIu Arg Leu Leu Leu 50 55 60
Thr Arg Pro VaI Trp Leu Gin Leu Gin Ala Asn Ala Ala Ala Ala Leu 65 70 75 80 His Met Leu Arg Thr GIu Pro Pro GIy Thr Phe Leu VaI Arg Lys Ser
85 90 95
Asn Thr Arg Gin Cys Gin Ala Leu Cys Met Arg Leu Pro GIu Ala Ser 100 105 110
GIy Pro Ser Phe VaI Ser Ser His Tyr lie Leu GIu Ser Pro GIy GIy 115 120 125
VaI Ser Leu GIu GIy Ser GIu Leu Met Phe Pro Asp Leu VaI GIn Leu 130 135 140 lie Cys Ala Tyr Cys His Thr Arg Asp lie Leu Leu Leu Pro Leu GIn 145 150 155 160 Leu Pro Arg Ala lie His His Ala Ala Thr His Lys GIu Leu GIu Ala
165 170 175
-lie Ser His Leu GIy lie GIu Phe Trp Ser Ser Ser Leu Asn lie Lys 180 185 190
Ala GIn Arg GIy Pro Ala GIy GIy Pro VaI Leu Pro Gin Leu Lys Ala 195 200 205
Arg Ser Pro GIn GIu Leu Asp GIn GIy Thr GIy Ala Ala Leu Cys Phe 210 215 220
Phe Asn Pro Leu Phe Pro GIy Asp Leu GIy Pro Thr Lys Arg GIu Lys 225 230 235 240 Phe Lys Arg Ser Phe Lys VaI Arg VaI Ser Thr GIu Thr Ser Ser Pro
245 250 255 Leu Ser Pro Pro Ala VaI Pro Pro Pro Pro VaI Pro VaI Leu Pro GIy 260 265 270
Ala VaI Pro Ser Gin Thr GIu Arg Leu Pro Pro Cys GIn Leu Leu Arg 275 280 285 Arg GIu Ser Ser VaI GIy Tyr Arg VaI Pro Ala GIy Ser GIy Pro Ser 290 295 300
Leu Pro Pro Met Pro Ser Leu Gin GIu VaI Asp Cys GIy Ser Pro Ser 305 310 315 320
Ser Ser GIu GIu GIu GIy VaI Pro GIy Ser Arg GIy Ser Pro Ala Thr 325 330 335
Ser Pro His Leu GIy Arg Arg Arg Pro Leu Leu Arg Ser Met Xaa Ala 340 345 350
Ala Phe Cys Ser Leu Leu Ala Pro GIu Arg Gin VaI GIy Arg Ala Ala 355 360 365 Ala Ala Leu Met GIn Asp Arg His Thr Ala Ala GIy Gin Leu VaI GIn 370 375 380
Asp Leu Leu Thr Gin VaI Arg Ala GIy Pro GIu Pro Gin GIu Leu GIn 385 390 395 400
GIy lie Arg Gin Ala Leu Ser Arg Ala Arg Ala Met Leu Ser Ala GIu 405 410 415
Leu GIy Pro GIu Lys Leu Leu Ser Pro Lys Arg Leu GIu His VaI Leu 420 425 430
GIu Lys Ser Leu His Cys Ser VaI Leu Lys Pro Leu Arg Pro lie Leu 435 440 445 Ala Ala Arg Leu Arg Arg Arg Leu Ala Ala Asp GIy Ser Leu GIy Arg 450 455 460
Leu Ala GIu GIy Leu Arg Leu Ala Arg Ala GIn GIy Pro GIy Ala Phe 465 470 475 480
GIy Ser His Leu Ser Leu Pro Ser Pro VaI GIu Leu GIu GIn VaI Arg 485 490 495
GIn Lys Leu Leu Gin Leu Leu Arg Thr Tyr Ser Pro Ser Ala Gin VaI 500 505 510
Lys Arg Leu Leu GIn Ala Cys Lys Leu Leu Tyr Met Ala Leu Arg Thr 515 520 525 Gin GIu GIy GIu GIy Ala GIy Ala Asp GIu Phe Leu Pro Leu Leu Ser 530 535 540
Leu VaI Leu Ala His Cys Asp Leu Pro GIu Leu Leu Leu GIu Ala GIu 545 550 555 560 Tyr Met Ser GIu Leu Leu GIu Pro Ser Leu Leu Thr GIy GIu GIy GIy 565 570 575
Tyr Tyr Leu Thr Ser Leu Ser Ala Ser Leu Ala Leu Leu Ser GIy Leu 580 585 590 GIy Gin Ala His Thr Leu Pro Leu Ser Pro VaI GIn GIu Leu Arg Arg 595 600 605
Ser Leu Ser Leu Trp GIu Gin Arg Arg Leu Pro Ala Thr His Cys Phe 610 615 620
GIn His Leu Leu Arg VaI Ala Tyr Gin Asp Pro Ser Ser GIy Cys Thr 625 630 635 640
Ser Lys Thr Leu Ala VaI Pro Pro GIu Ala Ser lie Ala Thr Leu Asn 645 650 655
GIn Leu Cys Ala Thr Lys Phe Arg VaI Thr Gin Pro Asn Thr Phe GIy 660 665 670 Leu Phe Leu Tyr Lys GIu GIn GIy Tyr His Arg Leu Pro Pro GIy Ala 675 680 685
Leu Ala His Arg Leu Pro Thr Thr GIy Tyr Leu VaI Tyr Arg Arg Ala 690 695 700
GIu Trp Pro GIu Thr GIn GIy Ala VaI Thr GIu GIu GIu GIy Ser GIy 705 710 715 720
GIn Ser GIu Ala Arg Ser Arg GIy GIu GIu GIn GIy Cys GIn GIy Asp 725 730 735
GIy Asp Ala GIy VaI Lys Ala Ser Pro Arg Asp lie Arg GIu GIn Ser 740 745 750 GIu Thr Thr Ala GIu GIy GIy GIn GIy Gin Ala GIn GIu GIy Pro Ala 755 760 765
Gin Pro GIy GIu Pro GIu Ala GIu GIy Ser Arg Ala Ala GIu GIu 770 775 780
<210> 129 <211> 1210
<212> PRT
<213> human
<220>
<221> misc_feature <222> (678).. (678)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (693).. (693) <223> Xaa can be any naturally occurring amino acid
<400> 129
Met Arg Pro Ser GIy Thr Ala GIy Ala Ala Leu Leu Ala Leu Leu Ala 10 15
Ala Leu Cys Pro Ala Ser Arg Ala Leu GIu GIu Lys Lys VaI Cys Gin 20 25 30
GIy Thr Ser Asn Lys Leu Thr GIn Leu GIy Thr Phe GIu Asp His Phe 35 40 45
Leu Ser Leu Gin Arg Met Phe Asn Asn Cys GIu VaI VaI Leu GIy Asn 50 55 60
Leu GIu lie Thr Tyr VaI Gin Arg Asn Tyr Asp Leu Ser Phe Leu Lys 65 70 75 80 Thr lie GIn GIu VaI Ala GIy Tyr VaI Leu lie Ala Leu Asn Thr VaI
85 90 95
GIu Arg lie Pro Leu GIu Asn Leu GIn lie lie Arg GIy Asn Met Tyr 100 105 110
Tyr GIu Asn Ser Tyr Ala Leu Ala VaI Leu Ser Asn Tyr Asp Ala Asn 115 120 125
Lys Thr GIy Leu Lys GIu Leu Pro Met Arg Asn Leu Gin GIu lie Leu 130 135 140
His GIy Ala VaI Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn VaI GIu 145 150 155 160 Ser lie GIn Trp Arg Asp lie VaI Ser Ser Asp Phe Leu Ser Asn Met
165 170 175
Ser Met Asp Phe GIn Asn His Leu GIy Ser Cys GIn Lys Cys Asp Pro 180 185 190
Ser Cys Pro Asn GIy Ser Cys Trp GIy Ala GIy GIu GIu Asn Cys GIn 195 200 205
Lys Leu Thr Lys lie lie Cys Ala GIn GIn Cys Ser GIy Arg Cys Arg 210 215 220
GIy Lys Ser Pro Ser Asp Cys Cys His Asn Gin Cys Ala Ala GIy Cys 225 230 235 240 Thr GIy Pro Arg GIu Ser Asp Cys Leu VaI Cys Arg Lys Phe Arg Asp
245 250 255
GIu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265 270
Thr Thr Tyr GIn Met Asp VaI Asn Pro GIu GIy Lys Tyr Ser Phe GIy 275 280 285
Ala Thr Cys VaI Lys Lys Cys Pro Arg Asn Tyr VaI VaI Thr Asp His 290 295 300
GIy Ser Cys VaI Arg Ala Cys GIy Ala Asp Ser Tyr GIu Met GIu GIu 305 310 315 320 Asp GIy VaI Arg Lys Cys Lys Lys Cys GIu GIy Pro Cys Arg Lys VaI 325 330 335
Cys Asn GIy lie GIy lie GIy GIu Phe Lys Asp Ser Leu Ser lie Asn 340 345 350 Ala Thr Asn lie Lys His Phe Lys Asn Cys Thr Ser lie Ser GIy Asp 355 360 365
Leu His lie Leu Pro VaI Ala Phe Arg GIy Asp Ser Phe Thr His Thr 370 375 380
Pro Pro Leu Asp Pro GIn GIu Leu Asp lie Leu Lys Thr VaI Lys GIu 385 390 395 400 lie Thr GIy Phe Leu Leu lie GIn Ala Trp Pro GIu Asn Arg Thr Asp 405 410 415
Leu His Ala Phe GIu Asn Leu GIu lie lie Arg GIy Arg Thr Lys GIn 420 425 430 His GIy Gin Phe Ser Leu Ala VaI VaI Ser Leu Asn He Thr Ser Leu 435 440 445
GIy Leu Arg Ser Leu Lys GIu He Ser Asp GIy Asp VaI He He Ser 450 455 460
GIy Asn Lys Asn Leu Cys Tyr Ala Asn Thr He Asn Trp Lys Lys Leu 465 470 475 480
Phe GIy Thr Ser GIy GIn Lys Thr Lys He He Ser Asn Arg GIy GIu 485 490 495
Asn Ser Cys Lys Ala Thr GIy GIn VaI Cys His Ala Leu Cys Ser Pro 500 505 510 GIu GIy Cys Trp GIy Pro GIu Pro Arg Asp Cys VaI Ser Cys Arg Asn 515 520 525
VaI Ser Arg GIy Arg GIu Cys VaI Asp Lys Cys Asn Leu Leu GIu GIy 530 535 540
GIu Pro Arg GIu Phe VaI GIu Asn Ser GIu Cys He GIn Cys His Pro 545- - 550 - - 555 - 560
GIu Cys Leu Pro Gin Ala Met Asn He Thr Cys Thr GIy Arg GIy Pro 565 570 575
Asp Asn Cys He Gin Cys Ala His Tyr He Asp GIy Pro His Cys VaI 580 585 590 Lys Thr Cys Pro Ala GIy VaI Met GIy GIu Asn Asn Thr Leu VaI Trp 595 600 605
Lys Tyr Ala Asp Ala GIy His VaI Cys His Leu Cys His Pro Asn Cys 610 615 620 Thr Tyr GIy Cys Thr GIy Pro GIy Leu GIu GIy Cys Pro Thr Asn GIy 625 630 635 640
Pro Lys lie Pro Ser lie Ala Thr GIy Met VaI GIy Ala Leu Leu Leu 645 650 655 Leu Leu VaI VaI Ala Leu GIy lie GIy Leu Phe Met Arg Arg Arg His 660 665 670 lie VaI Arg Lys Arg Xaa Leu Arg Arg Leu Leu Gin GIu Arg GIu Leu 675 680 685
VaI GIu Pro Leu Xaa Pro Ser GIy GIu Ala Pro Asn GIn Ala Leu Leu 690 695 700
Arg lie Leu Lys Glu Thr GIu Phe Lys Lys lie Lys VaI Leu GIy Ser 705 710 715 720
GIy Ala Phe GIy Thr VaI Tyr Lys GIy Leu Trp lie Pro Glu GIy Glu 725 730 735 Lys VaI Lys He Pro VaI Ala He Lys Glu Leu Arg Glu Ala Thr Ser 740 745 750
Pro Lys Ala Asn Lys Glu He Leu Asp Glu Ala Tyr VaI Met Ala Ser 755 760 765
VaI Asp Asn Pro His VaI Cys Arg Leu Leu GIy He Cys Leu Thr Ser 770 775 780
Thr VaI GIn Leu He Thr GIn Leu Met Pro Phe GIy Cys Leu Leu Asp 785 790 795 800
Tyr VaI Arg Glu His Lys Asp Asn He GIy Ser Gin Tyr Leu Leu Asn 805 810 815 Trp Cys VaI Gin He Ala Lys GIy Met Asn Tyr Leu Glu Asp Arg Arg 820 825 830
Leu VaI His Arg Asp Leu Ala Ala Arg Asn VaI Leu VaI Lys Thr Pro 835 840 845
Gin His VaI Lys He Thr Asp Phe GIy Leu Ala Lys Leu Leu GIy Ala 850 855 860
Glu Glu Lys Glu Tyr His Ala Glu GIy GIy Lys VaI Pro He Lys Trp 865 870 875 880
Met Ala Leu Glu Ser He Leu His Arg He Tyr Thr His Gin Ser Asp 885 890 895 VaI Trp Ser Tyr GIy VaI Thr VaI Trp Glu Leu Met Thr Phe GIy Ser 900 905 910
Lys Pro Tyr Asp GIy He Pro Ala Ser Glu He Ser Ser He Leu Glu 915 920 925
Lys GIy Glu Arg Leu Pro Gin Pro Pro He Cys Thr He Asp VaI Tyr 930 935 940
Met lie Met VaI Lys Cys Trp Met lie Asp Ala Asp Ser Arg Pro Lys 945 950 955 960
Phe Arg GIu Leu lie lie GIu Phe Ser Lys Met Ala Arg Asp Pro Gin 965 970 975
Arg Tyr Leu VaI lie GIn GIy Asp GIu Arg Met His Leu Pro Ser Pro 980 985 990
Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp GIu GIu Asp Met Asp 995 1000 1005 Asp VaI VaI Asp Ala Asp GIu Tyr Leu lie Pro GIn GIn GIy Phe 1010 1015 1020
Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu 1025 1030 1035
Ser Ala Thr Ser Asn Asn Ser Thr VaI Ala Cys lie Asp Arg Asn 1040 1045 1050
GIy Leu Gin Ser Cys Pro lie Lys GIu Asp Ser Phe Leu Gin Arg 1055 1060 1065
Tyr Ser Ser Asp Pro Thr GIy Ala Leu Thr GIu Asp Ser lie Asp 1070 1075 1080 Asp Thr Phe Leu Pro VaI Pro GIu Tyr lie Asn Gin Ser VaI Pro 1085 1090 1095
Lys Arg Pro Ala GIy Ser VaI GIn Asn Pro VaI Tyr His Asn Gin 1100 1105 1110
Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gin Asp Pro 1115 1120 1125
His Ser Thr Ala VaI GIy Asn Pro GIu Tyr Leu Asn Thr VaI GIn 1130 1135 1140
Pro Thr Cys VaI Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala 1145 1150 1155 Gin Lys GIy Ser His Gin lie Ser Leu Asp Asn Pro Asp Tyr Gin 1160 1165 1170
GIn Asp Phe Phe Pro Lys GIu Ala Lys Pro Asn GIy lie Phe Lys 1175 1180 1185
GIy Ser Thr Ala GIu Asn Ala GIu Tyr Leu Arg VaI Ala Pro Gin 1190 1195 1200
Ser Ser GIu Phe lie GIy Ala 1205 1210
<210> 130 <211> 839
<212> PRT
<213> human <220> <221> misc_feature
<222> (117) .. (117)
<223> Xaa can be any naturally occurring amino acid
<400> 130
Met Lys Lys Trp Ser Ser Thr Asp Leu GIy Ala Ala Ala Asp Pro Leu 1 5 10 15
Gin Lys Asp Thr Cys Pro Asp Pro Leu Asp GIy Asp Pro Asn Ser Arg 20 25 30
Pro Pro Pro Ala Lys Pro Gin Leu Ser Thr Ala Lys Ser Arg Thr Arg 35 40 45 Leu Phe GIy Lys GIy Asp Ser GIu GIu Ala Phe Pro VaI Asp Cys Pro 50 55 60
His GIu GIu GIy GIu Leu Asp Ser Cys Pro Thr lie Thr VaI Ser Pro 65 70 75 80
VaI lie Thr lie Gin Arg Pro GIy Asp GIy Pro Thr GIy Ala Arg Leu 85 90 95
Leu Ser Gin Asp Ser VaI Ala Ala Ser Thr GIu Lys Thr Leu Arg Leu 100 105 110
Tyr Asp Arg Arg Xaa lie Phe GIu Ala VaI Ala GIn Asn Asn Cys GIn 115 120 125 Asp Leu GIu Ser Leu Leu Leu Phe Leu Gin Lys Ser Lys Lys His Leu 130 135 140
Thr Asp Asn GIu Phe Lys Asp Pro GIu Thr GIy Lys Thr Cys Leu Leu 145 150 155 160
Lys Ala Met Leu Asn Leu His Asp GIy GIn Asn Thr Thr lie Pro Leu 165 170 175
Leu Leu GIu lie Ala Arg GIn Thr Asp Ser Leu Lys GIu Leu VaI Asn 180 185 190
Ala Ser Tyr Thr Asp Ser Tyr Tyr Lys GIy GIn Thr Ala Leu His lie 195 200 205 Ala lie GIu Arg Arg Asn Met Ala Leu VaI Thr Leu Leu VaI GIu Asn 210 215 220
GIy Ala Asp VaI GIn Ala Ala Ala His GIy Asp Phe Phe Lys Lys Thr 225 230 235 240
Lys GIy Arg Pro GIy Phe Tyr Phe GIy GIu Leu Pro Leu Ser Leu Ala 245 250 255
Ala Cys Thr Asn Gin Leu GIy lie VaI Lys Phe Leu Leu GIn Asn Ser 260 265 270
Trp Gin Thr Ala Asp lie Ser Ala Arg Asp Ser VaI GIy Asn Thr VaI 275 280 285
Leu His Ala Leu VaI GIu VaI Ala Asp Asn Thr Ala Asp Asn Thr Lys 290 295 300
Phe VaI Thr Ser Met Tyr Asn GIu lie Leu Met Leu GIy Ala Lys Leu 305 310 315 320
His Pro Thr Leu Lys Leu GIu GIu Leu Thr Asn Lys Lys GIy Met Thr 325 330 335 Pro Leu Ala Leu Ala Ala GIy Thr GIy Lys lie GIy VaI Leu Ala Tyr 340 345 350 lie Leu Gin Arg GIu lie Gin GIu Pro GIu Cys Arg His Leu Ser Arg 355 360 365
Lys Phe Thr GIu Trp Ala Tyr GIy Pro VaI His Ser Ser Leu Tyr Asp 370 375 380
Leu Ser Cys lie Asp Thr Cys GIu Lys Asn Ser VaI Leu GIu VaI lie 385 390 395 400
Ala Tyr Ser Ser Ser GIu Thr Pro Asn Arg His Asp Met Leu Leu VaI 405 410 415 GIu Pro Leu Asn Arg Leu Leu Gin Asp Lys Trp Asp Arg Phe VaI Lys 420 425 430
Arg lie Phe Tyr Phe Asn Phe Leu VaI Tyr Cys Leu Tyr Met lie lie 435 440 445
Phe Thr Met Ala Ala Tyr Tyr Arg Pro VaI Asp GIy Leu Pro Pro Phe 450 455 460
Lys Met GIu Lys Thr GIy Asp Tyr Phe Arg VaI Thr GIy GIu lie Leu 465 470 475 480
Ser VaI Leu GIy GIy VaI Tyr Phe Phe Phe Arg GIy lie GIn Tyr Phe 485 490 495 Leu GIn Arg Arg Pro Ser Met Lys Thr Leu Phe VaI Asp Ser Tyr Ser 500 505 510
GIu Met Leu Phe Phe Leu Gin Ser Leu Phe Met Leu Ala Thr VaI VaI 515 520 525
Leu Tyr Phe Ser His Leu Lys GIu Tyr VaI Ala Ser Met VaI Phe Ser 530 535 540
Leu Ala Leu GIy Trp Thr Asn Met Leu Tyr Tyr Thr Arg GIy Phe GIn 545 550 555 560
Gin Met GIy He Tyr Ala VaI Met He GIu Lys Met He Leu Arg Asp 565 570 575 Leu Cys Arg Phe Met Phe VaI Tyr He VaI Phe Leu Phe GIy Phe Ser 580 585 590
Thr Ala VaI VaI Thr Leu lie GIu Asp GIy Lys Asn Asp Ser Leu Pro 595 600 605 Ser GIu Ser Thr Ser His Arg Trp Arg GIy Pro Ala Cys Arg Pro Pro 610 615 620
Asp Ser Ser Tyr Asn Ser Leu Tyr Ser Thr Cys Leu GIu Leu Phe Lys 625 630 635 640
Phe Thr He GIy Met GIy Asp Leu GIu Phe Thr GIu Asn Tyr Asp Phe 645 650 655
Lys Ala VaI Phe He He Leu Leu Leu Ala Tyr VaI He Leu Thr Tyr 660 665 670
He Leu Leu Leu Asn Met Leu He Ala Leu Met GIy GIu Thr VaI Asn 675 680 685 Lys He Ala GIn GIu Ser Lys Asn He Trp Lys Leu GIn Arg Ala He 690 695 700
Thr He Leu Asp Thr GIu Lys Ser Phe Leu Lys Cys Met Arg Lys Ala 705 710 715 720
Phe Arg Ser GIy Lys Leu Leu Gin VaI GIy Tyr Thr Pro Asp GIy Lys 725 730 735
Asp Asp Tyr Arg Trp Cys Phe Arg VaI Asp GIu VaI Asn Trp Thr Thr 740 745 750
Trp Asn Thr Asn VaI GIy He He Asn GIu Asp Pro GIy Asn Cys GIu 755 760 765 GIy VaI Lys Arg Thr Leu Ser Phe Ser Leu Arg Ser Ser Arg VaI Ser 770 775 780
GIy Arg His Trp Lys Asn Phe Ala Leu VaI Pro Leu Leu Arg GIu Ala 785 790 795 800
Ser Ala Arg Asp Arg GIn Ser Ala GIn Pro GIu GIu VaI Tyr Leu Arg 805 810 815
GIn Phe Ser GIy Ser Leu Lys Pro GIu Asp Ala GIu VaI Phe Lys Ser 820 825 830
Pro Ala Ala Ser GIy GIu Lys 835
<210> 131
<211> 878
<212> PRT
<213> human <220> <221> misc_feature
<222> (876) .. (876)
<223> Xaa can be any naturally occurring amino acid
<400> 131 Met Ala Thr Ala Pro Ser Tyr Pro Ala GIy Leu Pro GIy Ser Pro GIy 1 5 10 15
Pro GIy Ser Pro Pro Pro Pro GIy GIy Leu GIu Leu GIn Ser Pro Pro 20 25 30
Pro Leu Leu Pro GIn lie Pro Ala Pro Gly Ser GIy VaI Ser Phe His 35 40 45 lie Gin lie GIy Leu Thr Arg GIu Phe VaI Leu Leu Pro Ala Ala Ser 50 55 60
GIu Leu Ala His VaI Lys GIn Leu Ala Cys Ser lie VaI Asp GIn Lys 65 70 75 80 Phe Pro GIu Cys GIy Phe Tyr GIy Leu Tyr Asp Lys lie Leu Leu Phe
85 90 95
Lys His Asp Pro Thr Ser Ala Asn Leu Leu GIn Leu VaI Arg Ser Ser 100 105 110
GIy Asp lie Gin GIu Gly Asp Leu VaI GIu VaI VaI Leu Ser Ala Ser 115 120 125
Ala Thr Phe GIu Asp Phe GIn lie Arg Pro His Ala Leu Thr VaI His 130 135 140
Ser Tyr Arg Ala Pro Ala Phe Cys Asp His Cys Gly GIu Met Leu Phe 145 150 155 160 Gly Leu VaI Arg GIn Gly Leu Lys Cys Asp Gly Cys Gly Leu Asn Tyr
165 170 175
His Lys Arg Cys Ala Phe Ser lie Pro Asn Asn Cys Ser Gly Ala Arg 180 185 190
Lys Arg Arg Leu Ser Ser Thr Ser Leu Ala Ser Gly His Ser VaI Arg 195 200 205
Leu Gly Thr -Ser GIu Ser Leu Pro Cys Thr Ala GIu GIu Leu Ser Arg 210 215 220
Ser Thr Thr GIu Leu Leu Pro Arg Arg Pro Pro Ser Ser Ser Ser Ser 225 230 235 240 Ser Ser Ala Ser Ser Tyr Thr Gly Arg Pro lie GIu Leu Asp Lys Met
245 250 255
Leu Leu Ser Lys VaI Lys VaI Pro His Thr Phe Leu lie His Ser Tyr 260 265 270
Thr Arg Pro Thr VaI Cys GIn Ala Cys Lys Lys Leu Leu Lys Gly Leu 275 280 285 Phe Arg Gin GIy Leu GIn Cys Lys Asp Cys Lys Phe Asn Cys His Lys 290 295 300
Arg Cys Ala Thr Arg VaI Pro Asn Asp Cys Leu GIy GIu Ala Leu lie 305 310 315 320 Asn GIy Asp VaI Pro Met GIu GIu Ala Thr Asp Phe Ser GIu Ala Asp
325 330 335
Lys Ser Ala Leu Met Asp GIu Ser GIu Asp Ser GIy VaI lie Pro GIy 340 345 350
Ser His Ser GIu Asn Ala Leu His Ala Ser GIu GIu GIu GIu GIy GIu 355 360 365
GIy GIy Lys Ala GIn Ser Ser Leu GIy Tyr lie Pro Leu Met Arg VaI 370 375 380
VaI GIn Ser VaI Arg His Thr Thr Arg Lys Ser Ser Thr Thr Leu Arg 385 390 395 400 GIu GIy Trp VaI VaI His Tyr Ser Asn Lys Asp Thr Leu Arg Lys Arg
405 410 415
His Tyr Trp Arg Leu Asp Cys Lys Cys lie Thr Leu Phe GIn Asn Asn 420 425 430
Thr Thr Asn Arg Tyr Tyr Lys GIu lie Pro Leu Ser GIu lie Leu Thr 435 440 445
VaI GIu Ser Ala GIn Asn Phe Ser Leu VaI Pro Pro GIy Thr Asn Pro 450 455 460
His Cys Phe GIu lie VaI Thr Ala Asn Ala Thr Tyr Phe VaI GIy GIu 465 470 475 480 Met Pro GIy GIy Thr Pro GIy GIy Pro Ser GIy GIn GIy Ala GIu Ala
485 490 495
Ala Arg GIy Trp GIu Thr Ala He Arg GIn Ala Leu Met Pro VaI He 500 505 510
Leu GIn Asp Ala Pro Ser Ala Pro GIy His Ala Pro His Arg Gin Ala 515 520 525
Ser Leu Ser He Ser VaI Ser Asn Ser GIn He GIn GIu Asn VaI Asp 530 535 540
He Ala Thr VaI Tyr GIn He Phe Pro Asp GIu VaI Leu GIy Ser GIy 545 550 555 560 Gin Phe GIy VaI VaI Tyr GIy GIy Lys His Arg Lys Thr GIy Arg Asp
565 570 575
VaI Ala VaI Lys VaI He Asp Lys Leu Arg Phe Pro Thr Lys Gin GIu 580 585 590 Ser Gin Leu Arg Asn GIu VaI Ala lie Leu GIn Ser Leu Arg His Pro 595 600 605
GIy lie VaI Asn Leu GIu Cys Met Phe GIu Thr Pro GIu Lys VaI Phe 610 615 620 VaI VaI Met GIu Lys Leu His GIy Asp Met Leu GIu Met lie Leu Ser 625 630 635 640
Ser GIu Lys GIy Arg Leu Pro GIu Arg Leu Thr Lys Phe Leu lie Thr 645 650 655
GIn He Leu VaI Ala Leu Arg His Leu His Phe Lys Asn He VaI His 660 665 670
Cys Asp Leu Lys Pro GIu Asn VaI Leu Leu Ala Ser Ala Asp Pro Phe 675 680 685
Pro GIn VaI Lys Leu Cys Asp Phe GIy Phe Ala Arg He He GIy GIu 690 695 700
Lys Ser Phe Arg Arg Ser VaI VaI GIy Thr Pro Ala Tyr Leu Ala Pro 705 710 715 720
GIu VaI Leu Leu Asn Gin GIy Tyr Asn Arg Ser Leu Asp Met Trp Ser 725 730 735
VaI GIy VaI He Met Tyr VaI Ser Leu Ser GIy Thr Phe Pro Phe Asn 740 745 750
GIu Asp GIu Asp He Asn Asp Gin He Gin Asn Ala Ala Phe Met Tyr 755 760 765
Pro Ala Ser Pro Trp Ser His He Ser Ala GIy Ala He Asp Leu He 770 775 780
Asn Asn Leu Leu Gin VaI Lys Met Arg Lys Arg Tyr Ser VaI Asp Lys 785 790 795 800
Ser Leu Ser His Pro Trp Leu Gin GIu Tyr GIn Thr Trp Leu Asp Leu 805 810 815
Arg GIu Leu GIu GIy Lys Met GIy GIu Arg Tyr He Thr His GIu Ser 820 825 830
Asp Asp Ala Arg Trp GIu GIn Phe Ala Ala GIu His Pro Leu Pro GIy 835 840 845
Ser GIy Leu Pro Thr Asp Arg Asp Leu GIy GIy Ala Cys Pro Pro GIn 850 855 860
Asp His Asp Met GIn GIy Leu Ala GIu Arg He Xaa VaI Leu 865 870 875
<210> 132 <211> 875 <212> PRT <213> mouse <220>
<221> misc_feature
<222> (873) .. (873) <223> Xaa can be any naturally occurring amino acid
<400> 132
Met Ala Ala Ala Pro Ser His Pro Ala GIy Leu Pro GIy Ser Pro GIy 1 5 10 15
Pro GIy Ser Pro Pro Pro Pro GIy GIy Leu Asp Leu GIn Ser Pro Pro 20 25 30
Pro Leu Leu Pro GIn lie Pro Ala Pro GIy Ser GIy VaI Ser Phe His 35 40 45 lie Gin lie GIy Leu Thr Arg GIu Phe VaI Leu Leu Pro Ala Ala Ser 50 55 60 GIu Leu Ala His VaI Lys GIn Leu Ala Cys Ser lie VaI Asp GIn Lys 65 70 75 80
Phe Pro GIu Cys GIy Phe Tyr GIy Leu Tyr Asp Lys lie Leu Leu Phe 85 90 95
Lys His Asp Pro Thr Ser Ala Asn Leu Leu Gin Leu VaI Arg Ser Ala 100 105 110
Ala Asp lie GIn GIu GIy Asp Leu VaI GIu VaI VaI Leu Ser Ala Ser 115 120 125
Ala Thr Phe GIu Asp Phe GIn lie Arg Pro His Ala Leu Thr VaI His 130 135 140 Ser Tyr Arg Ala Pro Ala Phe Cys Asp His Cys GIy GIu Met Leu Phe 145 150 155 160
GIy Leu VaI Arg GIn GIy Leu Lys Cys Asp GIy Cys GIy Leu Asn Tyr 165 170 175
His Lys Arg Cys Ala Phe Ser lie Pro Asn Asn Cys Ser GIy Ala Arg 180 185 190
Lys Arg Arg Leu Ser Ser Thr Ser Leu Ala Ser GIy His Ser VaI Arg 195 200 205
Leu Gly Ser Ser GIu Ser Leu Pro Cys Thr Ala GIu GIu Leu Ser Arg 210 215 220 Ser Thr Thr Asp Leu Leu Pro Arg Arg Pro Pro Ser Ser Ser Ser Ser 225 230 235 240
Ser Ser Ser Ser Ser Phe Tyr Thr GIy Arg Pro lie GIu Leu Asp Lys 245 250 255
Met Leu Met Ser Lys VaI Lys VaI Pro His Thr Phe Leu lie His Ser 260 265 270 Tyr Thr Arg Pro Thr VaI Cys GIn Ala Cys Lys Lys Leu Leu Lys GIy 275 280 285
Leu Phe Arg GIn GIy Leu GIn Cys Lys Asp Cys Lys Phe Asn Cys His 290 295 300 Lys Arg Cys Ala Thr Arg VaI Pro Asn Asp Cys Leu GIy GIu Ala Leu 305 310 315 320
He Asn GIy Asp VaI Pro Met GIu GIu Ala Ala Asp Tyr Ser GIu Ala 325 330 335
Asp Lys Ser Ser He Ser Asp GIu Leu GIu Asp Ser GIy VaI He Pro 340 345 350
GIy Ser His Ser GIu Ser Ala Leu His Ala Ser GIu GIu GIu GIu GIy 355 360 365
GIu GIy His Lys Ala Gin Ser Ser Leu GIy Tyr He Pro Leu Met Arg 370 375 380 VaI VaI GIn Ser VaI Arg His Thr Thr Arg Lys Ser Ser Thr Thr Leu 385 390 395 400
Arg GIu GIy Trp VaI VaI His Tyr Ser Asn Lys Asp Thr Leu Arg Lys 405 410 415
Arg His Tyr Trp Arg Leu Asp Cys Lys Cys He Thr Leu Phe GIn Asn 420 425 430
Asn Thr Thr Asn Arg Tyr Tyr Lys GIu He Pro Leu Ser GIu He Leu 435 440 445
Ala VaI GIu Pro Ala GIn Asn Phe Ser Leu VaI Pro Pro GIy Thr Asn 450 455 460 Pro His Cys Phe GIu He He Thr Ala Asn VaI Thr Tyr Phe VaI GIy 465 470 475 480
GIu Thr Pro GIy GIy Ala Pro GIy GIy Pro Ser GIy Gin GIy Thr GIu 485 490 495
Ala VaI Arg GIy Trp GIu Thr Ala He Arg GIn Ala Leu Met Pro VaI 500 505 510
He Leu Gin Asp Ala Pro Ser Ala Pro GIy His Thr Pro His Arg GIn 515 520 525
Ala Ser Leu Ser He Ser VaI Ser Asn Ser GIn He Gin GIu Asn VaI 530 535 540 Asp He Ala Thr VaI Tyr GIn He Phe Pro Asp GIu VaI Leu GIy Ser 545 550 555 560
GIy Gin Phe GIy VaI VaI Tyr GIy GIy Lys His Arg Lys Thr GIy Arg 565 570 575
Asp VaI Ala VaI Lys VaI He Asp Lys Leu Arg Phe Pro Thr Lys GIn 580 585 590
GIu Ser Gin Leu Arg Asn GIu VaI Ala lie Leu GIn Ser Leu Arg His 595 600 605
Pro GIy lie VaI Asn Leu GIu Cys Met Phe GIu Thr Pro GIu Lys VaI 610 615 620
Phe VaI VaI Met GIu Lys Leu His GIy Asp Met Leu GIu Met lie Leu 625 630 635 640
Ser Ser GIu Lys GIy Arg Leu Pro GIu Arg Leu Thr Lys Phe Leu lie 645 650 655 Thr Gin lie Leu VaI Ala Leu Arg His Leu His Phe Lys Asn lie VaI 660 665 670
His Cys Asp Leu Lys Pro GIu Asn VaI Leu Leu Ala Ser Ala Asp Pro 675 680 685
Phe Pro GIn VaI Lys Leu Cys Asp Phe GIy Phe Ala Arg lie lie GIy 690 695 700
GIu Lys Ser Phe Arg Arg Ser VaI VaI GIy Thr Pro Ala Tyr Leu Ala 705 710 715 720
Pro GIu VaI Leu Leu Asn Gin GIy Tyr Asn Arg Ser Leu Asp Met Trp 725 730 735 Ser VaI GIy VaI lie Met Tyr VaI Ser Leu Ser GIy Thr Phe Pro Phe 740 745 750
Asn GIu Asp GIu Asp lie Asn Asp Gin lie GIn Asn Ala Ala Phe Met 755 760 765
Tyr Pro Ala Ser Pro Trp Ser His lie Ser Ser GIy Ala lie Asp Leu 770 775 780 lie Asn Asn Leu Leu Gin VaI Lys Met Arg Lys Arg Tyr Ser VaI Asp 785 790 795 800
Lys Ser Leu Ser His Pro Trp Leu Gin GIu Tyr GIn Thr Trp Leu Asp 805 810 815 Leu Arg GIu Leu GIu GIy Lys Met GIy GIu Arg Tyr lie Thr His GIu 820 825 830
Ser Asp Asp Ala Arg Trp Asp Gin Phe VaI Ala GIu Arg His GIy Thr 835 840 845
Pro Ala GIu GIy Asp Leu GIy GIy Ala Cys Leu Pro Gin Asp His GIu 850 855 860
Met GIn GIy Leu Ala GIu Arg lie Xaa lie Leu 865 870 875

Claims

WHAT IS CLAIMED IS:
1. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 19-29 of SEQ ID NO: 117, wherein the amino acid residue corresponding to amino acid residue 23 of SEQ ID NO: 117 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 18-30 of SEQ ID NO:117, wherein the amino acid residue corresponding to amino acid residue 23 of SEQ ID NO:117 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 15-32 of SEQ ID NO: 117, wherein the amino acid residue corresponding to amino acid residue 23 of SEQ ID NO: 117 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 9-35 of SEQ ID NO: 117, wherein the amino acid residue corresponding to amino acid residue 23 of SEQ ID NO : 117 is an amino acid residue other than serine or threonine.
2. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 19-29 of SEQ ID NO: 117, wherein the amino acid residue corresponding to amino acid residue 24 of SEQ ID NO : 117 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 18-30 of SEQ ID NO:117, wherein the amino acid residue corresponding to amino acid residue 24 of SEQ ID NO:117 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 15-32 of SEQ ID NO: 117, wherein the amino acid residue corresponding to amino acid residue 24 of SEQ ID NO:117 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 9-35 of SEQ ID NO: 117, wherein the amino acid residue corresponding to amino acid residue 24 of SEQ ID NO : 117 is an amino acid residue other than serine or threonine.
3. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 194-201 of SEQ ID NO: 117, wherein the amino acid residue corresponding to amino acid residue 199 of SEQ ID NO : 117 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 191-203 of SEQ ID NO: 117, wherein the amino acid residue corresponding to amino acid residue 199 of SEQ ID NO:117 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 189-205 of SEQ ID NO: 117, wherein the amino acid residue corresponding to amino acid residue 199 of SEQ ID NO : 117 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 184-207 of SEQ ID NO: 117, wherein the amino acid residue corresponding to amino acid residue 199 of SEQ ID NO:117 is an amino acid residue other than serine or threonine.
4. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 847-855 of the amino acid sequence of SEQ ID NO: 118, wherein the amino acid residue corresponding to amino acid residue 850 of SEQ ID NO:118 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 843-852 of the amino acid sequence of SEQ ID NO: 118, wherein the amino acid residue corresponding to amino acid residue 850 of SEQ ID NO:118 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 842-859 of the amino acid sequence of SEQ ID NO: 118, wherein the amino acid residue corresponding to amino acid residue 850 of SEQ ID NO:118 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 839-862 of the amino acid sequence of SEQ ID NO: 118, wherein the amino acid residue corresponding to amino acid residue 850 of SEQ ID NO:118 is an amino acid residue other than serine or threonine.
5. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 848-857 of the amino acid sequence of SEQ ID NO: 118, wherein the amino acid residue corresponding to amino acid residue 853 of SEQ ID NO:118 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 843-859 of the amino acid sequence of SEQ ID NO: 118, wherein the amino acid residue corresponding to amino acid residue 853 of SEQ ID NO:118 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 841-861 of the amino acid sequence of SEQ ID NO:118, wherein the amino acid residue corresponding to amino acid residue 853 of SEQ ID NO:118 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 851-862 of the amino acid sequence of SEQ ID NO:118, wherein the amino acid residue corresponding to amino acid residue 853 of SEQ ID NO:118 is an amino acid residue other than serine or threonine.
6. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 255-264 of the amino acid sequence of SEQ ID NO:119, wherein the amino acid residue corresponding to amino acid residue 260 of SEQ ID NO:119 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 253-266 of the amino acid sequence of SEQ ID NO:119, wherein the amino acid residue corresponding to amino acid residue 260 of SEQ ID NO:119 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 250-270 of the amino acid sequence of SEQ ID NO: 119, wherein the amino acid residue corresponding to amino acid residue 260 of SEQ ID NO:119 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 245-275 of the amino acid sequence of SEQ ID NO: 119 wherein the amino acid residue corresponding to amino acid residue 260 of SEQ ID NO:119 is an amino acid residue other than serine or threonine.
7. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 494-502 of the amino acid sequence of SEQ ID NO:119, wherein the amino acid residue corresponding to amino acid residue 499 of SEQ ID NO:119 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 491-506 of the amino acid sequence of SEQ ID NO:119, wherein the amino acid residue corresponding to amino acid residue 499 of SEQ ID NO:119 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 488-509 of the amino acid sequence of SEQ ID NO: 119, wherein the amino acid residue corresponding to amino acid residue 499 of SEQ ID NO:119 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 485-514 of the amino acid sequence of SEQ ID NO:119 wherein the amino acid residue corresponding to amino acid residue 499 of SEQ ID NO:119 is an amino acid residue other than serine or threonine.
8. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 189-198 of the amino acid sequence of SEQ ID NO: 120, wherein the amino acid residue corresponding to amino acid residue 194 of SEQ ID NO: 120 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 185-202 of the amino acid sequence of SEQ ID NO: 120, wherein the amino acid residue corresponding to amino acid residue 194 of SEQ ID NO: 120 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 182-206 of the amino acid sequence of SEQ ID NO: 120, wherein the amino acid residue corresponding to amino acid residue 194 of SEQ ID NO: 120 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 179-209 of the amino acid sequence of SEQ ID NO: 120, wherein the amino acid residue corresponding to amino acid residue 194 of SEQ ID NO: 120 is an amino acid residue other than serine or threonine.
9. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 216-225 of the amino acid sequence of SEQ ID NO: 120, wherein the amino acid residue corresponding to amino acid residue 220 of SEQ ID NO: 120 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 212-230 of the amino acid sequence of SEQ ID NO: 120, wherein the amino acid residue corresponding to amino acid residue 220 of SEQ ID NO: 120 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 209-234 of the amino acid sequence of SEQ ID NO: 120, wherein the amino acid residue corresponding to amino acid residue 220 of SEQ ID NO: 120 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 204-238 of the amino acid sequence of SEQ ID NO: 120, wherein the amino acid residue corresponding to amino acid residue 220 of SEQ ID NO: 120 is an amino acid residue other than serine or threonine.
10. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 483-492 of the amino acid sequence of SEQ ID NO: 120, wherein the amino acid residue corresponding to amino acid residue 488 of SEQ ID NO:120 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 479-496 of the amino acid sequence of SEQ ID NO: 120, wherein the amino acid residue corresponding to amino acid residue 488 of SEQ ID NO: 120 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 476-502 of the amino acid sequence of SEQ ID NO: 120, wherein the amino acid residue corresponding to amino acid residue 488 of SEQ ID NO:120 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 472-508 of the amino acid sequence of SEQ ID NO: 120, wherein the amino acid residue corresponding to amino acid residue 488 of SEQ ID NO: 120 is an amino acid residue other than serine or threonine.
11. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 355-364 of the amino acid sequence of SEQ ID NO: 120, wherein the amino acid residue corresponding to amino acid residue 360 of SEQ ID NO: 120 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 351-367 of the amino acid sequence of SEQ ID NO:120, wherein the amino acid residue corresponding to amino acid residue 360 of SEQ ID NO:120 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 348-376 of the amino acid sequence of SEQ ID NO: 120, wherein the amino acid residue corresponding to amino acid residue 360 of SEQ ID NO:120 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 344-380 of the amino acid sequence of SEQ ID NO: 120, wherein the amino acid residue corresponding to amino acid residue 360 of SEQ ID NO: 120 is an amino acid residue other than serine or threonine.
12. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 166-175 of the amino acid sequence of SEQ ID NO: 121, wherein the amino acid residue corresponding to amino acid residue 171 of SEQ ID NO: 121 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 164-180 of the amino acid sequence of SEQ ID NO: 121, wherein the amino acid residue corresponding to amino acid residue 171 of SEQ ID NO: 121 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 160-184 of the amino acid sequence of SEQ ID NO: 121, wherein the amino acid residue corresponding to amino acid residue 171 of SEQ ID NO: 121 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 157-188 of the amino acid sequence of SEQ ID NO:121, wherein the amino acid residue corresponding to amino acid residue 171 of SEQ ID NO: 121 is an amino acid residue other than serine or threonine.
13. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 77-85 of the amino acid sequence of SEQ ID NO: 122, wherein the amino acid residue corresponding to amino acid residue 82 of SEQ ID NO: 122 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 73-88 of the amino acid sequence of SEQ ID NO: 122, wherein the amino acid residue corresponding to amino acid residue 82 of SEQ ID NO: 122 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 70-92 of the amino acid sequence of SEQ ID NO: 122, wherein the amino acid residue corresponding to amino acid residue 82 of SEQ ID NO: 122 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 66-97 of the amino acid sequence of SEQ ID NO: 122, wherein the amino acid residue corresponding to amino acid residue 82 of SEQ ID NO: 122 is an amino acid residue other than serine or threonine.
14. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 913-922 of the amino acid sequence of SEQ ID NO: 123, wherein the amino acid residue corresponding to amino acid residue 918 of SEQ ID NO: 123 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 909-926 of the amino acid sequence of SEQ ID NO: 123, wherein the amino acid residue corresponding to amino acid residue 918 of SEQ ID NO: 123 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 904-931 of the amino acid sequence of SEQ ID NO: 123, wherein the amino acid residue corresponding to amino acid residue 918 of SEQ ID NO: 123 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 902-936 of the amino acid sequence of SEQ ID NO: 123, wherein the amino acid residue corresponding to amino acid residue 918 of SEQ ID NO: 123 is an amino acid residue other than serine or threonine.
15. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 289-298 of the amino acid sequence of SEQ ID NO: 124, wherein the amino acid residue corresponding to amino acid residue 294 of SEQ ID NO: 124 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 287-302 of the amino acid sequence of SEQ ID NO: 124, wherein the amino acid residue corresponding to amino acid residue 294 of SEQ ID NO: 124 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 283-308 of the amino acid sequence of SEQ ID NO: 124, wherein the amino acid residue corresponding to amino acid residue 294 of SEQ ID NO: 124 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 279-313 of the amino acid sequence of SEQ ID NO: 124, wherein the amino acid residue corresponding to amino acid residue 294 of SEQ ID NO: 124 is an amino acid residue other than serine or threonine.
16. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 381-390 of the amino acid sequence of SEQ ID NO:125, wherein the amino acid residue corresponding to amino acid residue 386 of SEQ ID NO: 125 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 379-393 of the amino acid sequence of SEQ ID NO: 125, wherein the amino acid residue corresponding to amino acid residue 386 of SEQ ID NO:125 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 374-398 of the amino acid sequence of SEQ ID NO: 125, wherein the amino acid residue corresponding to amino acid residue 386 of SEQ ID NO:125 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 371-402 of the amino acid sequence of SEQ ID NO: 125, wherein the amino acid residue corresponding to amino acid residue 386 of SEQ ID NO: 125 is an amino acid residue other than serine or threonine.
17. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 381-390 of the amino acid sequence of SEQ ID NO:126, wherein the amino acid residue corresponding to amino acid residue 386 of SEQ ID NO:126 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 376-391 of the amino acid sequence of SEQ ID NO:126, wherein the amino acid residue corresponding to amino acid residue 386 of SEQ ID NO:126 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 365-395 of the amino acid sequence of SEQ ID NO: 126, wherein the amino acid residue corresponding to amino acid residue 386 of SEQ ID NO: 126 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 362-398 of the amino acid sequence of SEQ ID NO: 126, wherein the amino acid residue corresponding to amino acid residue 386 of SEQ ID NO: 126 is an amino acid residue other than serine or threonine.
18. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 386-394 of the amino acid sequence of SEQ ID NO:127, wherein the amino acid residue corresponding to amino acid residue 390 of SEQ ID NO:127 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 384-397 of the amino acid sequence of SEQ ID NO:127, wherein the amino acid residue corresponding to amino acid residue 390 of SEQ ID NO: 127 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 381-399 of the amino acid sequence of SEQ ID NO: 127, wherein the amino acid residue corresponding to amino acid residue 390 of SEQ ID NO:127 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 374-395 of the amino acid sequence of SEQ ID NO: 127, wherein the amino acid residue corresponding to amino acid residue 390 of SEQ ID NO: 127 is an amino acid residue other than serine or threonine.
19. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 346-355 of the amino acid sequence of SEQ ID NO: 128, wherein the amino acid residue corresponding to amino acid residue 351 of SEQ ID NO:128 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 344-359 of the amino acid sequence of SEQ ID NO: 128, wherein the amino acid residue corresponding to amino acid residue 351 of SEQ ID NO:128 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 341-364 of the amino acid sequence of SEQ ID NO:128, wherein the amino acid residue corresponding to amino acid residue 351 of SEQ ID NO:128 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues337-367 of the amino acid sequence of SEQ ID NO: 128, wherein the amino acid residue corresponding to amino acid residue 351 of SEQ ID NO:128 is an amino acid residue other than serine or threonine.
20. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 674-682 of the amino acid sequence of SEQ ID NO: 129, wherein the amino acid residue corresponding to amino acid residue 678 of SEQ ID NO: 129 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 670-687 of the amino acid sequence of SEQ ID NO: 129, wherein the amino acid residue corresponding to amino acid residue 678 of SEQ ID NO: 129 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 668-690 of the amino acid sequence of SEQ ID NO: 129, wherein the amino acid residue corresponding to amino acid residue 678 of SEQ ID NO: 129 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 665-692 of the amino acid sequence of SEQ ID NO: 129, wherein the amino acid residue corresponding to amino acid residue 678 of SEQ ID NO: 129 is an amino acid residue other than serine or threonine.
21. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 689-696 of the amino acid sequence of SEQ ID NO: 129, wherein the amino acid residue corresponding to amino acid residue 693 of SEQ ID NO: 129 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 687-698 of the amino acid sequence of SEQ ID NO: 129, wherein the amino acid residue corresponding to amino acid residue 693 of SEQ ID NO: 129 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 683-702 of the amino acid sequence of SEQ ID NO: 129, wherein the amino acid residue corresponding to amino acid residue 693 of SEQ ID NO: 129 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 679-706 of the amino acid sequence of SEQ ID NO: 129, wherein the amino acid residue corresponding to amino acid residue 693 of SEQ ID NO: 129 is an amino acid residue other than serine or threonine.
22. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 111-121 of the amino acid sequence of SEQ ID NO:130, wherein the amino acid residue corresponding to amino acid residue 117 of SEQ ID NO: 130 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino no acid residues 107-125 of the amino acid sequence of SEQ ID NO: 130, wherein the amino acid residue corresponding to amino acid residue 117 of SEQ ID NO: 130 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 104-130 of the amino acid sequence of SEQ ID NO: 130, wherein the amino acid residue corresponding to amino acid residue 117 of SEQ ID NO: 130 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 100-133 of the amino acid sequence of SEQ ID NO:130, wherein the amino acid residue corresponding to amino acid residue 117 of SEQ ID NO: 130 is an amino acid residue other than serine or threonine.
23. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 870-877 of the amino acid sequence of SEQ ID NO: 131, wherein the amino acid residue corresponding to amino acid residue 876 of SEQ ID NO: 131 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 868-878 of the amino acid sequence of SEQ ID NO:131, wherein the amino acid residue corresponding to amino acid residue 876 of SEQ ID NO:131 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 865-878 of the amino acid sequence of SEQ ID NO: 131, wherein the amino acid residue corresponding to amino acid residue 876 of SEQ ID NO:131 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 860-878 of the amino acid sequence of SEQ ID NO:131, wherein the amino acid residue corresponding to amino acid residue 876 of SEQ ID NO: 131 is an amino acid residue other than serine or threonine.
24. An inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of: a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 867-874 of the amino acid sequence of SEQ ID NO: 132, wherein the amino acid residue corresponding to amino acid residue 873 of SEQ ID NO: 132 is an amino acid residue other than serine or threonine; b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 865-875 of the amino acid sequence of SEQ ID NO:132, wherein the amino acid residue corresponding to amino acid residue 873 of SEQ ID NO: 132 is an amino acid residue other than serine or threonine; c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 861-875 of the amino acid sequence of SEQ ID NO: 132, wherein the amino acid residue corresponding to amino acid residue 873 of SEQ ID NO: 132 is an amino acid residue other than serine or threonine; and d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 857-875 of the amino acid sequence of SEQ ID NO:132, wherein the amino acid residue corresponding to amino acid residue 873 of SEQ ID NO:132 is an amino acid residue other than serine or threonine.
25. A nucleic acid molecule comprising a polynucleotide sequence encoding at least one copy of the polypeptide of any one of claims 1-24
26. The nucleic acid molecule of claim 25 wherein the polynucleotide sequence encodes at least a number of copies of the peptide selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9 or 10.
27. A vector comprising the nucleic acid molecule of claim 25.
28. A recombinant host cell comprising the vector of claim 27.
29. A method of inhibiting PKD in a cell comprising transfecting the vector of claim 27 into a host cell and culturing the transfected host cell under conditions suitable to produce at least one copy of the polypeptide.
30. A composition comprising at least two monomelic ligands, wherein at least a portion of each of said ligands is capable of being recognized by PKD and wherein at least one of said ligands does not contain a phosphorylatable amino acid residue.
31. The composition of claim 30, wherein said composition is a homopolymeric ligand.
32. The composition of claim 31, further comprising spacer amino acids between at least two of said monomeric ligands.
33. The composition of claim 31, further comprising a localization signal.
34. The composition of claim 31, further comprising a epitopic peptide.
35. The composition of claim 30, wherein said composition is a heteropolymeric ligand.
36. The composition of claim 35, further comprising spacer amino acids between at least two of said monomeric ligands.
37. The composition of claim 35, further comprising a localization signal.
38. The composition of claim 35, further comprising an epitopic peptide.
39. An isolated polypeptide comprising an amino acid sequence at least about 80% identical to SEQ ID NO:105.
40. An isolated polypeptide comprising an amino acid sequence at least about 80% identical to SEQ ID NO:109.
41. An isolated polypeptide comprising an amino acid sequence at least about 80% identical to SEQ ID NO:113.
42. An isolated polynucleotide encoding a polypeptide of claims 39, 40, or 41.
43. An isolated polynucleotide comprising SEQ ID NO:106 or SEQ ID NO:107 or SEQ ID NO:108 or SEQ ID NO:110 or SEQ ID NO:111 or SEQ ID NO:112 or SEQ ID NO:114 or SEQ ID NO:115 or SEQ ID NO: 116.
44. An isolated fusion polypeptide comprising two or more polypeptides selected from SEQ ID NOS:1- 104, wherein at least one amino acid designated as Xaa is an amino acid other than serine or threonine.
45. An isolated fusion polypeptide comprising two or more polypeptides selected from SEQ ID NOS :53- 104, wherein at least one amino acid designated as Xaa is an amino acid other than serine or threonine.
46. An isolated polypeptide homopolyligand whose monomers are selected from the group consisting of SEQ ID NOS:1-104, wherein Xaa is any amino acid.
47. An isolated polypeptide heteropolyligand whose monomers are selected from the group consisting of SEQ ID NOS: 1-104, wherein at least one amino acid designated as Xaa is an amino acid other than serine or threonine.
48. An isolated polypeptide homopolyligand whose monomers are selected from the group consisting of peptides comprising an amino acid sequence at least 80% identical to any one of SEQ ID NOS: 1-104.
49. An isolated polypeptide heterpolyligand whose monomers are selected from the group consisting of peptides comprising an amino acid sequence at least 80% identical to any one of SEQ ID NOS: 1-104.
50. An isolated polypeptide homopolyligand whose monomers are selected from the group consisting of peptides comprising an amino acid sequence at least 85% identical to any one of SEQ ID NOS:1-104.
51. An isolated polypeptide heteropolyligand whose monomers are selected from the group consisting of peptides comprising an amino acid sequence at least 85% identical to any one of SEQ ID NOS: 1-104.
52. An isolated polypeptide homopolyligand whose monomers are selected from the group consisting of peptides comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOS: 1-104.
53. An isolated polypeptide heteropolyligand whose monomers are selected from the group consisting of peptides comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOS:1-104.
54. An isolated polypeptide homopolyligand whose monomers are selected from the group consisting of peptides comprising an amino acid sequence at least 95% identical to any one of SEQ ID NOS: 1-104.
55. An isolated polypeptide heteropolyligand whose monomers are selected from the group consisting of peptides comprising an amino acid sequence at least 95% identical to any one of SEQ ID NOS:1-104.
56. An isolated polypeptide homopolyligand whose monomers are selected from the group consisting of peptides comprising an amino acid sequence at least 96% identical to any one of SEQ ID NOS: 1-104.
57. An isolated polypeptide heteropolyligand whose monomers are selected from the group consisting of peptides comprising an amino acid sequence at least 96% identical to any one of SEQ ID NOS: 1-104.
58. An isolated polypeptide homopolyligand whose monomers are selected from the group consisting of peptides comprising an amino acid sequence at least 97% identical to any one of SEQ ID NOS:1-104.
59. An isolated polypeptide heteropolyligand whose monomers are selected from the group consisting of peptides comprising an amino acid sequence at least 97% identical to any one of SEQ ID NOS: 1-104.
60. An isolated polypeptide homopolyligand whose monomers are selected from the group consisting of peptides comprising an amino acid sequence at least 98% identical to any one of SEQ ID NOS: 1-104.
61. An isolated polypeptide heteropolyligand whose monomers are selected from the group consisting of peptides comprising an amino acid sequence at least 98% identical to any one of SEQ ID NOS:1-104.
62. An isolated polypeptide homopolyligand whose monomers are selected from the group consisting of peptides comprising an amino acid sequence at least 99% identical to any one of SEQ ID NOS: 1-104.
63. An isolated polypeptide heteropolyligand whose monomers are selected from the group consisting of peptides comprising an amino acid sequence at least 99% identical to any one of SEQ ID NOS:1-104.
64. The polyligand of each of claims 44-63 linked to a subcellular localization signal.
65. The polyligand of each of claims 44-63 linked to an epitope.
66. The polyligand of each of claims 44-63 linked to a reporter.
67. An isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of each of claims 44-66.
68. A vector comprising a polynucleotide of claim 67.
69. A host cell comprising a vector of claim 68.
70. A method of inhibiting PKD in a cell comprising transfecting a vector of claim 68 into a host cell and culturing the transfected host cell under conditions suitable to produce at least one copy of the polypeptide.
71. An isolated polypeptide homopolyligand, wherein the homopolyligand modulates PKD activity.
72. An isolated polypeptide heteropolyligand, wherein the heteropolyligand modulates PKD activity.
73. An isolated polynucleotide encoding each of the polypeptides of claims 44-63, wherein the polynucleotide is flanked on one end by a sequence cleavable by a first restriction endonuclease, and wherein the polynucleotide is flanked on the other end by a sequence cleavable by a second restriction endonuclease, and wherein the first and second restriction endonucleases generate noncompatible cohesive ends.
74. An isolated polynucleotide encoding each of the polypeptides of claims 44-63, wherein the polynucleotide is flanked on one end by a sequence cleavable by NgoM IV, and wherein the polynucleotide is flanked on the other end by sequences cleavable by Xma I and CIa I.
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