EP1206552A2 - Polypeptide ztryp1 de type tryptase - Google Patents

Polypeptide ztryp1 de type tryptase

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Publication number
EP1206552A2
EP1206552A2 EP00954026A EP00954026A EP1206552A2 EP 1206552 A2 EP1206552 A2 EP 1206552A2 EP 00954026 A EP00954026 A EP 00954026A EP 00954026 A EP00954026 A EP 00954026A EP 1206552 A2 EP1206552 A2 EP 1206552A2
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EP
European Patent Office
Prior art keywords
amino acid
seq
acid sequence
acid number
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP00954026A
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German (de)
English (en)
Inventor
Scott R. Presnell
David W. Taft
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Zymogenetics Inc
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Zymogenetics Inc
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Publication date
Application filed by Zymogenetics Inc filed Critical Zymogenetics Inc
Publication of EP1206552A2 publication Critical patent/EP1206552A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Human tryptase is almost exclusively found in the secretory granules of mast cells and is released along with heparin and histamine upon mast cell activation in inflammatory response.
  • Mast cells containing tryptase have been identified in lung, gut mucosa, and skin mast cells.
  • Human tryptase has been established as an important mediator of airway response and is implicated in increasing the magnitude of broncoconstriction in asthma. Tryptase has neuropeptidase activity that may directly induce asthmatic response by degrading broncoactive neuropeptides having broncodilatory activities hence increasing broncoconstriction.
  • Neuropeptides cleaved by tryptase include vasoactive intestinal peptide (VIP), and peptide histidine methionine (PHM).
  • VIP vasoactive intestinal peptide
  • PLM peptide histidine methionine
  • tryptase has been shown to stimulate cytokine production, and exhibit mitogenic effects in a variety of cell types including human and rat lung fibroblasts, human bronchial epithelial cells, and dog airway smooth muscle cells and hence may also contribute to the hyperplasia and fibrotic changes observed in diseases such as asthma.
  • tryptase inhibitors have been found effective in reducing asthmatic response to exposure of antigen in animal models (allergic sheep) and in human trials, and in blocking the mitogenic effects described above.
  • tryptase has been implicated in other activities such as cleaving fibrinogen ⁇ and ⁇ chains, collagen IV, gelatinase and fibronectin. As such it is implicated in inhibition of coagulation and in tissue remodeling in the lung and other tissues.
  • tryptase is shown to cleave calcitonin gene-related peptide (CGRP) which is a potent vasodilator and hence may potentiate gastric ulceration or increase cutaneous neurogenic inflammation, and promote smooth muscle contraction.
  • CGRP calcitonin gene-related peptide
  • tryptases are implicated in matrix degradation, wound healing and tumor metastasis.
  • the present invention provides an isolated polynucleotide encoding a serine protease polypeptide comprising a sequence of amino acid residues that is at least 90% identical to an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ED NO:2 from amino acid number 44 (Val) to amino acid number 276 (De); (b) the amino acid sequence as shown in SEQ ID NO: 15 from amino acid number 43 (Val) to amino acid number 275 (Arg); (c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 24 (Leu) to amino acid number 276 (De); (d) the amino acid sequence as shown in SEQ ID NO: 15 from amino acid number 19 (Arg) to amino acid number 275 (Arg); (e) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 44 (Val) to amino acid number 314 (Leu); (f) the amino acid sequence as shown in SEQ ID NO: 15 from amino acid number 43 (Val) to amino acid
  • the isolated polynucleotide disclosed above encodes a serine protease polypeptide that comprises a sequence of amino acid residues an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 44 (Val) to amino acid number 276 (De); (b) the amino acid sequence as shown in SEQ ID NO: 15 from amino acid number 43 (Val) to amino acid number 275 (Arg); (c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 24 (Leu) to amino acid number 276 (De); (d) the amino acid sequence as shown in SEQ ID NO: 15 from amino acid number 19 (Arg) to amino acid number 275 (Arg); (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 44 (Val) to amino acid number 314 (Leu); (f) the amino acid sequence as shown in SEQ ID NO: 15 from amino acid number 43 (Val) to amino acid number 312 (Leu); (g) the group consisting
  • the isolated polynucleotide disclosed above encodes a serine protease polypeptide that has protease activity.
  • the isolated polynucleotide disclosed above encodes a serine protease polypeptide consisting of a sequence of amino acid residues as shown in SEQ D NO:2 from amino acid number 44 (Val) to amino acid number 276 (De) or of a sequence of amino acid residues as shown in SEQ ID NO: 15 from amino acid number 43 (Val) to amino acid number 275 (Arg).
  • the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a serine protease polypeptide with an amino acid sequence consisting of: (a) the amino acid sequence as shown in SEQ DD NO:2 from amino acid number 44 (Val) to amino acid number 276 (De); (b) the amino acid sequence as shown in SEQ ID NO: 15 from amino acid number 43 (Val) to amino acid number 275 (Arg); (c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 24 (Leu) to amino acid number 314 (Leu); (d) the amino acid sequence as shown in SEQ ID NO: 15 from amino acid number 19 (Arg) to amino acid number 312 (Leu); and (e) the amino acid sequence as shown in SEQ ID NO:24; and a transcription terminator.
  • the expression vector disclosed above further comprises a secretory signal sequence operably linked to the DNA segment.
  • the present invention provides a cultured cell into which has been introduced an expression vector as disclosed above, wherein the cell expresses a polypeptide encoded by the DNA segment.
  • the present invention provides a DNA construct encoding a fusion protein, the DNA construct comprising: a first DNA segment encoding a polypeptide that is selected from the group consisting of: (a) the amino acid sequence of SEQ ED NO: 2 from residue number 1 (Met), to residue number 23 (Ser); (b) the amino acid sequence of SEQ ID NO: 15 from residue number 1 (Met), to residue number 18 (Ala); (c) the amino acid sequence of SEQ ED NO: 2 from residue number 24 (Leu), to residue number 43 (Lys); (d) the amino acid sequence of SEQ ID NO: 15 from residue number 19 (Arg), to residue number 42 (Arg); (e) the amino acid sequence of SEQ ED NO: 2 from residue number 24 (Leu), to residue number 276 (De); (f) the amino acid sequence of SEQ ID NO: 15 from residue number 19 (Arg), to residue number 275 (Arg); (g) the amino acid sequence of SEQ D NO: 2 from residue number 24 (E_eu), to residue
  • the present invention provides a fusion protein produced by a method comprising: culturing a host cell into which has been introduced a vector comprising the following operably linked elements: (a) a transcriptional promoter; (b) a DNA construct encoding a fusion protein as disclosed above; and (c) a transcriptional terminator; and recovering the protein encoded by the DNA segment.
  • the present invention provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 90% identical to an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ DD NO:2 from amino acid number 44 (Val) to amino acid number 276 (De); (b) the amino acid sequence as shown in SEQ DD NO: 15 from amino acid number 43 (Val) to amino acid number 275 (Arg); (c) the amino acid sequence as shown in SEQ DD NO: 2 from amino acid number 24 (Leu) to amino acid number 276 (De); (d) the amino acid sequence as shown in SEQ DD NO: 15 from amino acid number 19 (Arg) to amino acid number 275 (Arg); (e) the amino acid sequence as shown in SEQ DD NO:2 from amino acid number 44 (Val) to amino acid number 314 (Leu); (f) the amino acid sequence as shown in SEQ ED NO: 15 from amino acid number 43 (Val) to amino acid number 312 (Leu); (g) the amino acid sequence as shown
  • the isolated polypeptide disclosed above comprises a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ED NO:2 from amino acid number 44 (Val) to amino acid number 276 (De); (b) the amino acid sequence as shown in SEQ ED NO: 15 from amino acid number 43 (Val) to amino acid number 275 (Arg); (c) the amino acid sequence as shown in SEQ ED NO:2 from amino acid number 24 (Leu) to amino acid number 276 (De); (d) the amino acid sequence as shown in SEQ ED NO: 15 from amino acid number 19 (Arg) to amino acid number 275 (Arg); (e) the amino acid sequence as shown in SEQ DD NO:2 from amino acid number 44 (Val) to amino acid number 314 (E_eu); (f) the amino acid sequence as shown in SEQ ED NO: 15 from amino acid number 43 (Val) to amino acid number 312 (Leu); (g) the amino acid sequence as shown in SEQ ED NO: 2 from
  • the isolated polypeptide disclosed above has protease activity.
  • the isolated polypeptide disclosed above has a sequence of amino acid residues is as shown in SEQ ID NO: 2 from amino acid number 44 (Val) to amino acid number 276 (De), or the sequence of amino acids is as shown in SEQ DD NO: 15 from amino acid number 43 (Val) to amino acid number 275 (Arg).
  • the present invention provides a method of producing a serine protease polypeptide comprising: culturing a cell as disclosed above; and isolating the serine protease polypeptide produced by the cell.
  • the present invention provides a method of producing an antibody to serine protease polypeptide comprising: inoculating an animal with a polypeptide selected from the group consisting of: (a) a polypeptide consisting of 9 to 232 amino acids, wherein the polypeptide is a contiguous sequence of amino acids in SEQ ED NO:2 from amino acid number 44 (Val) to amino acid number 276 (De); (b) a polypeptide consisting of 9 to 233 amino acids, wherein the polypeptide is a contiguous sequence of amino acids in SEQ DD NO: 15 from amino acid number 43 (Val) to amino acid number 275 (Arg), excluding the sequence in SEQ DD NO: 15 from amino acid number 169 to 230; (c) a polypeptide as
  • the present invention provides an antibody produced by the method as disclosed above, which binds to a serine protease polypeptide.
  • the antibody disclosed above is a monoclonal antibody.
  • the present invention provides an antibody that -_, specifically binds to a polypeptide as disclosed above.
  • affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
  • affinity tag any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag.
  • Affinity tags include a poly- histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzvmol. 198:3. 1991), glutathione S transferase (Smith and Johnson, Gene 67:31,
  • allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
  • amino-terminal and “carboxyl-terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
  • complement/anti-complement pair denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions.
  • biotin and avidin are prototypical members of a complement/anti-complement pair.
  • Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of ⁇ 10 ⁇ M " 1 .
  • complements of a polynucleotide molecule denotes a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence.
  • sequence 5' ATGCACGGG 3' is complementary to 5 ' CCCGTGC AT 3 '.
  • degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide).
  • Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).
  • expression vector is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription.
  • additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc.
  • Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • isolated when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
  • isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
  • Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985).
  • an "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue.
  • the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • the term “isolated” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • operably linked when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
  • ortholog denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
  • Parenters are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, ⁇ - globin, ⁇ -globin, and myoglobin are paralogs of each other.
  • polynucleotide is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides
  • nt nt
  • kb kilobases
  • polypeptide is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides”.
  • promoter is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
  • a "protein” is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
  • receptor denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell.
  • a bioactive molecule i.e., a ligand
  • Membrane-bound receptors are characterized by a multi-peptide structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell.
  • Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids.
  • receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).
  • secretory signal sequence denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • the larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • splice variant is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.
  • the present invention provides novel serine protease polypeptides and proteins.
  • the novel serine protease termed ztrypl was discovered and identified to be a novel tryptase by the presence of polypeptide and polynucleotide features characteristic of tryptases (e.g., a- and ⁇ -tryptases).
  • each the human and mouse ztrypl polypeptide was obtained from a clone believed to contain its corresponding polynucleotide sequence.
  • Libraries that might be searched for such sequences include testis, heart, lung, and the like.
  • the nucleotide sequence of a representative mouse ztrypl -encoding DNA is described in SEQ DD NOT, and its deduced 314 amino acid sequence is described in SEQ ID NO:2.
  • the ztrypl polypeptide represents a full- length polypeptide segment (pre-pro-polypeptide) (residue 1 (Met) to residue 314 (Leu) of SEQ ID NO:2).
  • pre-pro-polypeptide pre-pro-polypeptide
  • SEQ DD NO:2 314 amino acids
  • SEQ DD NO:2 a predicted signal peptide of 23 amino acid residues (residue 1 (Met) to residue 23 (Ser) of SEQ DD NO:2), and a pro-polypeptide (or pro-protein) of 291 amino acids (residue 24 (Leu) to residue 314 (Leu) of SEQ ID NO:2).
  • the Ztrypl pro- protein has an unglycosylated molecular weight of approximately 33,005 Daltons (D). Further analysis of SEQ ID NO:2 indicates the presence of the following structural regions and features:
  • a pro-peptide from residue 24 (Leu) to residue 43 (Lys) is present, which when cleaved generates a mature ztrypl polypeptide corresponding to residue 44 (Val) to residue 314 (Leu) of SEQ ED NO:2.
  • Within the mature ztrypl polypeptide is a transmembrane domain at the C-terminus (from approximately residue 293 (Phe) to residue 314 (Leu)).
  • ztrypl polypeptide Also within the mature ztrypl polypeptide is a dibasic Lys-Arg cleavage site at residues 277 and 278, which upon cleavage generates a 232 amino acid residue active ztrypl polypeptide corresponding from residue 44 (Val) to residue 276 (De) of SEQ DD NO:2.
  • This ztrypl active polypeptide has an unglycosylated molecular weight of approximately 26,685 Daltons (D). 2)
  • the active ztrypl polypeptide contains critical active site residues typical of serine proteases at His 84 , Asp 130 , and Ser 33 (as shown in SEQ ID NO:2).
  • the active ztrypl polypeptide contains a binding pocket comprising residues 221 (Ser) to 227 (Asp) and residues 254 (Asn) to 267 (Thr) of SEQ ID NO:2. Within the binding pocket the presence of Asp 227 indicates that the ztrypl active polypeptide is trypsin-like in its enzymatic specificity.
  • the active ztrypl polypeptide further contains two N-linked glycosylation sites at residues 107 (Asn) to 109 (Thr) and residues 242 (Asn) to 244 (Thr) of SEQ FD NO:2. Similar N-linked glycosylation sites are seen in elastase and dog tryptase.
  • the nucleotide sequence of a representative human ztrypl -encoding DNA is described in SEQ DD NO: 14, and its deduced 312 amino acid sequence is described in SEQ ID NO: 15.
  • the ztrypl polypeptide (SEQ FD NO: 15) represents a full- length polypeptide segment (pre-pro-polypeptide) (residue 1 (Met) to residue 312 (Leu) of SEQ ED NO: 15).
  • pre-pro-polypeptide pre-pro-polypeptide
  • SEQ ED NO: 15 312 amino acids comprising a predicted signal peptide of 18 amino acid residues (residue 1 (Met) to residue 18 (Ala) of SEQ DD NO: 15), and a pro-polypeptide (or pro-protein) of 294 amino acids (residue 19 (Arg) to residue 312 (Leu) of SEQ DD NO: 15).
  • the Ztrypl pro- protein has an unglycosylated molecular weight of approximately 32-24 kiloDaltons (kD). Further analysis of SEQ DD NO: 15 indicates the presence of the following structural regions and features:
  • a pro-peptide from residue 19 (Arg) to residue 42 (Arg) is present, which when cleaved generates a mature ztrypl polypeptide corresponding to residue 43 (Val) to residue 312 (Leu) of SEQ DD NO: 15.
  • a transmembrane domain at the C-terminus (from approximately residue 291 (Phe) to residue 312 (Leu)).
  • a cleavage site at residues 275, which upon cleavage generates a 233 amino acid residue active ztrypl polypeptide corresponding from residue 43 (Val) to residue 275 (Arg) of SEQ DD NO: 15.
  • This mature ztrypl active polypeptide has an unglycosylated molecular weight of approximately 26-28 kD.
  • the mature ztrypl polypeptide can be made in an active soluble form corresponding from approximately residue 43 (Val) to residue 275 (Arg) of SEQ ID NO: 15.
  • the active ztrypl polypeptide contains critical active site residues typical of serine proteases at His 8 , Asp ⁇ 8 , and Ser 2 1 (as shown in SEQ ID NO: 15).
  • the ztrypl polypeptide contains an Asp at residue 225 +/- 2 (as shown in SEQ DD NO: 15) wherein when the mouse ztrypl and human ztrypl amino acid sequences are optimally aligned, the mouse residue 227 (Asp) corresponds to the Asp in the human sequence.
  • this critical Asp residue is within a binding pocket of about 7 residues comprising residues 219 +/- 2 to 225 +1-1 (Asp) and about 14 residues 252 +1-1 to 265 +1-1 of SEQ ED NO: 15. Within the binding pocket the presence of the critical Asp residue described above indicates that the ztrypl active polypeptide is trypsin-like in its enzymatic specificity.
  • the active ztrypl polypeptide further contains two N-linked glycosylation sites at residues 106 (Asn) to 108 (Thr) of SEQ DD NO: 15. A similar relinked glycosylation site is seen in elastase and dog tryptase.
  • RT-PCR reverse transcription- polymerase chain reaction
  • highly degenerate primers designed from the ztrypl sequences are useful for this purpose.
  • the present invention also provides polynucleotide molecules, including DNA and RNA molecules that encode the ztrypl polypeptides disclosed herein.
  • polynucleotide molecules including DNA and RNA molecules that encode the ztrypl polypeptides disclosed herein.
  • SEQ ED NO: 3 and SEQ DD NO: 16 is a degenerate DNA sequence that encompasses all DNAs that encode the ztrypl polypeptide of SEQ ED NO:2 and SEQ ED NO: 15 respectively.
  • SEQ ED NO:3 and SEQ ED NO: 16 also provides all RNA sequences encoding SEQ ED NO:2 or SEQ ED NO: 15 by substituting U for T.
  • ztrypl polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 942 of SEQ ID NO:3, and ztrypl polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 942 of SEQ ED NO: 16, and their RNA equivalents are contemplated by the present invention.
  • Table 1 sets forth the one-letter codes used within SEQ ED NO:3 and SEQ ED NO: 16 to denote degenerate nucleotide positions.
  • “Resolutions” are the nucleotides denoted by a code letter.
  • “Complement” indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.
  • any X NNN One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid.
  • the degenerate codon for serine can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY).
  • WSN can, in some circumstances, encode arginine
  • MGN degenerate codon for arginine
  • AGY serine
  • polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ED NO:2 or SEQ FD NO: 15. Variant sequences can be readily tested for functionality as described herein.
  • preferential codon usage or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2).
  • the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
  • Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art.
  • the degenerate codon sequence disclosed in SEQ ED NO:3 or SEQ ID NO: 16 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
  • the isolated polynucleotides will hybridize to similar sized regions of SEQ DD NOT or SEQ DD
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Numerous equations for calculating T m are known in the art, and are specific for DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences of varying length (see, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual.
  • Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake, MN) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto, CA), as well as sites on the Internet, are available tools for analyzing a given sequence and calculating T m based on user defined criteria. Such programs can also analyze a given sequence under defined conditions and identify suitable probe sequences.
  • hybridization of longer polynucleotide sequences is performed at temperatures of about 20-25°C below the calculated T m .
  • hybridization is typically carried out at the T m or 5-10°C below. This allows for the maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids. Higher degrees of stringency at lower temperatures can be achieved with the addition of formamide which reduces the T m of the hybrid about 1°C for each 1% formamide in the buffer solution.
  • Suitable stringent hybridization conditions are equivalent to about a 5 h to overnight incubation at about 42°C in a solution comprising: about 40-50% formamide, up to about 6X SSC, about 5X Denhardt's solution, zero up to about 10% dextran sulfate, and about 10-20 ⁇ g/ml denatured commercially-available carrier DNA.
  • stringent conditions include temperatures of 20-70°C and a hybridization buffer containing up to 6x SSC and 0-50% formamide; hybridization is then followed by washing filters in up to about 2X SSC.
  • a suitable wash stringency is equivalent to 0.1X SSC to 2X SSC, 0.1% SDS, at 55°C to 65°C.
  • Different degrees of stringency can be used during hybridization and washing to achieve maximum specific binding to the target sequence.
  • the washes following hybridization are performed at increasing degrees of stringency to remove non- hybridized polynucleotide probes from hybridized complexes.
  • Stringent hybridization and wash conditions depend on the length of the probe, reflected in the Tm, hybridization and wash solutions used, and are routinely determined empirically by one of skill in the art.
  • the isolated polynucleotides of the present invention include DNA and RNA.
  • Methods for preparing DNA and RNA are well known in the art.
  • RNA is isolated from a tissue or cell that produces large amounts of ztrypl RNA.
  • Such tissues and cells are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980), and include testis, including whole testis tissue extracts or testicular cells, such as Sertoli cells, Leydig cells, spermatogonia, or epididymis, cells from vas deferens, and cervical cells, although DNA may also be prepared using RNA from other tissues, such as heart, lung, or gastrointestinal tissues, or isolated as genomic DNA.
  • Total RNA can be prepared using guanidinium isothiocyanate extraction followed by isolation by centrifugation in a CsCI gradient (Chirgwin et al., Biochemistry 18:52-94, 1979).
  • Poly (A)+ RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA
  • Complementary DNA is prepared from poly(A)+ RNA using known methods. In the alternative, genomic DNA can be isolated. Polynucleotides encoding ztrypl polypeptides are then identified and isolated by, for example, hybridization or PCR. A full-length clone encoding ztrypl can be obtained by conventional cloning procedures.
  • Complementary DNA (cDNA) clones are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron.
  • Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library.
  • Expression libraries can be probed with antibodies to ztrypl, receptor fragments, or other specific binding partners.
  • Ztrypl polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a ztrypl gene. In view of the tissue-specific expression observed for ztrypl by Northern blotting, this gene region is expected to provide for testis-specific expression. Promoter elements from a ztrypl gene could thus be used to direct the tissue-specific expression of heterologous genes in, for example, transgenic animals or patients treated with gene therapy. Cloning of 5' flanking sequences also facilitates production of ztrypl proteins by "gene activation" as disclosed in U.S. Patent No. 5,641,670.
  • an endogenous ztrypl gene in a cell is altered by introducing into the ztrypl locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site.
  • the targeting sequence is a ztrypl 5' non-coding sequence that permits homologous recombination of the construct with the endogenous ztrypl locus, whereby the sequences within the construct become operably linked with the endogenous ztrypl coding sequence.
  • an endogenous ztrypl promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.
  • the polynucleotides of the present invention can also be synthesized using DNA synthesis machines.
  • U chemically synthesized double stranded DNA is required for an application such as the synthesis of a DNA or a DNA fragment, then each complementary strand is made separately, for example via the phosphoramidite method known in the art.
  • the production of short polynucleotides 60 to 80 bp is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them.
  • special strategies are usually employed for producing longer polynucleotides (longer than about 300 bp).
  • synthetic DNAs double-stranded
  • One method for building a synthetic DNA involves producing a set of overlapping, complementary oligonucleotides. Each internal section of the DNA has complementary 3' and 5' terminal extensions designed to base pair precisely with an adjacent section. After the DNA is assembled, the process is completed by ligating the nicks along the backbones of the two strands.
  • synthetic DNAs can be designed with terminal sequences that facilitate insertion into a restriction endonuclease site of a cloning vector.
  • Alternative ways to prepare a full-length DNA are also known in the art. See Glick and Pasternak, Molecular Biotechnology. Principles & Applications of Recombinant DNA, (ASM Press, Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-56, 1984 and Climie et al., Proc. Natl. Acad. Sci. USA 87:633-7, 1990.
  • the present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are ztrypl polypeptides from other mammalian species, including human, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of ztrypl can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
  • a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses ztrypl as disclosed herein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue or cell line. A ztrypl -encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences. A cDNA can also be cloned using the polymerase chain reaction (PCR) (Mullis, U.S. Patent No.
  • PCR polymerase chain reaction
  • the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to ztrypl polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
  • SEQ D NOT or SEQ DD NO: 14 represents a single allele of ztrypl and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the DNA sequence shown in SEQ ED NO: 1 or SEQ ED NO: 14, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ D NO:2 or SEQ ED NO: 15.
  • cDNAs generated from alternatively spliced mRNAs, which retain the properties of the ztrypl polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs.
  • Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.
  • the present invention also provides isolated ztrypl polypeptides that are substantially homologous to the polypeptides of SEQ DD NO:2 or SEQ ED NO: 15 and their orthologs.
  • substantially homologous is used herein to denote polypeptides having 70%, preferably 80%, more preferably at least 85%, sequence identity to the sequences shown in SEQ ED NO:2 or SEQ ED NO: 15 or their orthologs.
  • polypeptides will more preferably be at least 90% identical, and most preferably
  • Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table
  • the "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant ztrypl.
  • the FASTA algorithm is described by Pearson and Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63 (1990).
  • the ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score.
  • the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
  • the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allows for amino acid insertions and deletions.
  • FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above.
  • the ktup value can range between one to six, preferably from three to six, most preferably three, with other FASTA program parameters set as default.
  • the BLOSUM62 table (Table 3) is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nafl Acad. Sci. USA 89:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely upon chemical properties (as discussed below), the language "conservative amino acid substitution” preferably refers to a substitution represented by a BLOSUM62 value of greater than -1.
  • an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
  • preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 1 or 3).
  • Variant ztrypl polypeptides or substantially homologous ztrypl polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
  • the present invention thus includes polypeptides of from about 200 to 345 amino acid residues that comprise a sequence that is at least 90%, preferably at least 95%, and more preferably 99% or more identical to the corresponding region of SEQ DD NO:2 or SEQ DD NO: 15.
  • Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the ztrypl polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites. Table 4
  • Aromatic phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine
  • the present invention further provides a variety of other polypeptide fusions and related multimeric proteins comprising one or more polypeptide fusions.
  • a ztrypl polypeptide can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584.
  • Preferred dimerizing proteins in this regard include immunoglobulin constant region domains.
  • Immunoglobulin- ztrypl polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric ztrypl analogs.
  • Auxiliary domains can be fused to ztrypl polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen).
  • a ztrypl polypeptide or protein could be targeted to a predetermined cell type by fusing a ztrypl polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell.
  • polypeptides and proteins can be targeted for therapeutic or diagnostic purposes.
  • a ztrypl polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain.
  • Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34: 1-9, 1996.
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, tran_.-3-methylproline, 2,4-methanoproline, cw-4-hydroxyproline, trans-A- hydroxyproline, N-methylglycine, ⁇ ./othreonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine.
  • coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
  • a natural amino acid that is to be replaced e.g., phenylalanine
  • desired non-naturally occurring amino acid(s) e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine.
  • the non-naturally occurring amino acid is inco ⁇ orated into the protein in place of its natural counterpart.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for ztrypl amino acid residues.
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-502, 1991).
  • site-directed mutagenesis or alanine-scanning mutagenesis Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-502, 1991.
  • single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271:4699-708, 1996.
  • Sites of ligand-receptor or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992.
  • the identities of essential amino acids can also be inferred from analysis of homologies with related polypeptides sucha s human ⁇ -tryptase and ⁇ -tryptase.
  • Determination of amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can determine specific residues that will be more or less tolerant of change and maintain the overall tertiary structure of the molecule.
  • Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity and computer analysis using available software (e.g., the Insight E® viewer and homology modeling tools; MSI, San Diego, CA), secondary structure propensities, binary patterns, complementary packing and buried polar interactions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al., Current Opin. Struct. Biol. 6:3-10, 1996).
  • modifications to molecules or identifying specific fragments determination of structure will be accompanied by evaluating activity of modified molecules.
  • Amino acid sequence changes are made in ztrypl polypeptides so as to minimize disruption of higher order structure essential to biological activity.
  • the ztrypl polypeptide comprises one or more helices
  • changes in amino acid residues will be made so as not to disrupt the helix geometry and other components of the molecule where changes in conformation abate some critical function, for example, binding of the molecule to its binding partners.
  • the effects of amino acid sequence changes can be predicted by, for example, computer modeling as disclosed above or determined by analysis of crystal structure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995).
  • a Hopp/Woods hydrophilicity profile of the ztrypl protein sequence as shown in SEQ DD NO:2 or SEQ ED NO: 15 can be generated (Hopp et al., Proc. Natl. Acad. Sci.78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 and Triquier et al., Protein Engineering JT: 153-169, 1998). The profile is based on a sliding six- residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored.
  • hydrophilic regions include: (1) amino acid number 125 (Asn) to amino acid number 130 (Asp) of SEQ ED NO:2; (2) amino acid number 172 (Glu) to amino acid number 177 (His) of SEQ ED NO:2; (3) amino acid number 256 (Asp) to amino acid number 261 (Gin) of SEQ DD NO:2; (4) amino acid number 277 (Lys) to amino acid number 282 (Asp) of SEQ ED NO:2.
  • corresponding hydrophilic regions include: (1) amino acid number 117 (Gin) to amino acid number 122 (Ser) of SEQ DD NO: 15; (2) amino acid number 57 (Arg) to amino acid number 62 (Lys) of SEQ ED NOT5; (3) amino acid number 116 (His) to amino acid number 121 (Asn) of SEQ ID NO: 15; (4) amino acid number 147 (Lys) to amino acid number 152 (Ser) of SEQ ED NO: 15.
  • hydrophilicity or hydrophobicity will be taken into account when designing modifications in the amino acid sequence of a ztrypl polypeptide, so as not to disrupt the overall structural and biological profile.
  • hydrophobic residues selected from the group consisting of Val, F_eu and De or the group consisting of Met, Gly, Ser, Ala, Tyr and Tip.
  • residues tolerant of substitution could include such as shown in SEQ ED NO:2 or SEQ ED NO: 15. Cysteine residues of SEQ DD NO:2 OR SEQ ED NO: 15, will be relatively intolerant of substitution.
  • the identities of essential amino acids can also be inferred from analysis of sequence similarity between other tryptase family members with ztrypl. Using methods such as "FASTA" analysis described previously, regions of high similarity are identified within a family of proteins and used to analyze amino acid sequence for conserved regions.
  • An alternative approach to identifying a variant ztrypl polynucleotide on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant ztrypl polynucleotide can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ DD NOT or SEQ DD NO: 14, as discussed above.
  • the present invention also includes functional fragments of ztrypl polypeptides and nucleic acid molecules encoding such functional fragments.
  • a "functional" ztrypl or fragment thereof defined herein is characterized by its proliferative or differentiating activity, by its ability to induce or inhibit specialized cell functions, or by its ability to bind specifically to an anti- ztrypl antibody or ztrypl receptor or binding partner (either soluble or immobilized).
  • ztrypl is characterized by a serine protease and tryptase-like structure as shown in SEQ DD NO:2 or SEQ ED NO: 15.
  • the present invention further provides fusion proteins encompassing: (a) polypeptide molecules comprising one or more of the domains, motifs, or residues described herein; and (b) functional fragments comprising one or more of these domains, motifs, or residues.
  • the other polypeptide portion of the fusion protein may be contributed by another tryptase family member, such as human tryptase, trypsin, or the like or by a non-native and/or an unrelated secretory signal peptide that facilitates secretion of the fusion protein.
  • Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a ztrypl polypeptide.
  • DNA molecules having the nucleotide sequence of SEQ DD NOT or SEQ DD NO: 14 or fragments thereof can be digested with ⁇ /31 nuclease to obtain a series of nested deletions. These DNA fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for ztrypl activity, or for the ability to bind anti-ztrypl antibodies or ztrypl receptor.
  • exonuclease digestion is to use oligonucleotide- directed mutagenesis to introduce deletions or stop codons to specify production of a desired ztry l fragment.
  • particular fragments of a ztrypl polynucleotide can be synthesized using the polymerase chain reaction.
  • phage display e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204
  • region- directed mutagenesis e.g., region- directed mutagenesis
  • variants of the disclosed ztrypl DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 9 10747-51, 1994 and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
  • Mutagenesis methods as disclosed herein can be combined with high- throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells.
  • Mutagenized DNA molecules that encode active polypeptides e.g., those with protease activity, that induce signal transduction or bind anti-trypl antibodies, and the like
  • These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
  • polypeptide fragments or variants of SEQ ID NO:2 or SEQ FD NO: 15 can be identified and/or prepare a variety of polypeptide fragments or variants of SEQ ID NO:2 or SEQ FD NO: 15 or that retain, for example, tryptase-like properties, protease activity, mitogenic activity, induce cytokine secretion, binding, cell-cell communication, or signal transduction activity of the wild-type ztrypl protein.
  • polypeptides may also include additional polypeptide segments, such as affinity tags, as generally disclosed herein.
  • any ztrypl polypeptide including variants and fusion proteins
  • one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above.
  • the ztrypl polypeptides of the present invention can be produced in genetically engineered host cells according to conventional techniques.
  • Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred.
  • a DNA sequence encoding a ztrypl polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
  • the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector.
  • the secretory signal sequence may be that of ztrypl, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo.
  • the secretory signal sequence is operably linked to the ztrypl DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).
  • the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway.
  • the present invention provides for such fusion polypeptides.
  • a signal fusion polypeptide can be made wherein a secretory signal sequence derived from residue 1 (Met) to residue 23 (Ser) of SEQ DD NO:2, or from residue 1 (Met) to residue 18 (Ala) of SEQ DD NO: 15 is operably linked to a DNA sequence encoding another polypeptide using methods known in the art and disclosed herein.
  • the secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway.
  • these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein. Such fusions may be used in vivo or in vitro to direct peptides through the secretory pathway.
  • Cultured mammalian cells are suitable hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
  • Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-Kl; ATCC No. CCL 61) cell lines.
  • COS-1 ATCC No. CRL 1650
  • COS-7 ATCC No. CRL 1651
  • BHK ATCC No. CRL 1632
  • BHK 570 ATCC No. CRL 10314
  • 293 ATCC No. CRL 1573
  • Graham et al. J. Gen. Virol. 36:59-72, 1977
  • Chinese hamster ovary e.g. CHO-Kl; ATCC No. CCL 61
  • Suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, VA.
  • strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalo virus. See, e.g., U.S. Patent No. 4,956,288.
  • Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants".
  • stable transfectants Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants.”
  • a preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
  • a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
  • Other drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • Alternative markers that introduce an altered phenotype such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
  • eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells.
  • Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) JT:47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Patent No. 5,162,222 and WIPO publication WO 94/06463.
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa calif ornica nuclear polyhedrosis virus (AcNPV). See, King,
  • This system utilizes a transfer vector, pFastBaclTM (Life Technologies) containing a Tn7 transposon to move the DNA encoding the ztrypl polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid.”
  • the pFastBaclTM transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case ztrypl.
  • pFastBaclTM can be modified to a considerable degree.
  • the polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins.
  • the baculovirus basic protein promoter also known as Pcor, p6.9 or MP promoter
  • Pcor baculovirus basic protein promoter
  • MP promoter baculovirus basic protein promoter
  • transfer vectors can be constructed which replace the native ztrypl secretory signal sequences with secretory signal sequences derived from insect proteins.
  • a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin (Invitrogen, Carlsbad, CA), or baculovirus gp67 (PharMingen, San Diego, CA) can be used in constructs to replace the native ztrypl secretory signal sequence.
  • transfer vectors can include an in- frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed ztrypl polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985).
  • a transfer vector containing ztrypl is transformed into E. Coli, and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus.
  • the bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells. Recombinant virus that expresses ztrypl is subsequently produced. Recombinant viral stocks are made by methods commonly used the art.
  • the recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda. See, in general, Glick and
  • Suitable cell line is the High FiveOTM cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent No.5,300,435).
  • Commercially available serum-free media are used to grow and maintain the cells. Suitable media are Sf900 ETM (Life Technologies) or ESF 921TM (Expression Systems) for the Sf9 cells; and Ex-ceDO405TM (JRH Biosciences, Lenexa, KS) or Express FiveOTM (Life Technologies) for the T. ni cells.
  • the cells are grown up from an inoculation density of approximately 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
  • MOI multiplicity of infection
  • Fungal cells including yeast cells, can also be used within the present invention.
  • Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.
  • Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075.
  • Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).
  • a preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No.
  • Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533.
  • Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565.
  • DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation.
  • the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUG1 or AUG2).
  • DHAS dihydroxyacetone synthase
  • FMD formate dehydrogenase
  • CAT catalase
  • a preferred selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine.
  • host cells For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (AUG1 and AUG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P.
  • methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
  • Prokaryotic host cells including strains of the bacteria Escherichia coli,
  • Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.).
  • the polypeptide When expressing a ztrypl polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea.
  • the denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
  • the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
  • Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells.
  • suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required.
  • the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co- transfected into the host cell.
  • P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25 °C to 35°C.
  • Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors.
  • a preferred culture medium for P. methanolica is Y ⁇ PD (2% D-glucose, 2% BactoTM Peptone (Difco Laboratories, Detroit, MI), 1% BactoTM yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).
  • polypeptides of the present invention it is preferred to purify the polypeptides of the present invention to >80% purity, more preferably to >90% purity, even more preferably >95% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
  • a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • Expressed recombinant ztrypl polypeptides (or chimeric ztrypl polypeptides) can be purified using fractionation and/or conventional purification methods and media. Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
  • Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography.
  • Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.
  • Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross- linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used.
  • These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
  • Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries.
  • These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography:
  • polypeptides of the present invention can be isolated by exploitation of their structural or biochemical properties.
  • immobilized metal ion adsorption (E AC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • polypeptide fusions, or hybrid ztrypl proteins are constructed using regions or domains of the inventive ztrypl in combination with those of other tryptase family proteins (e.g. human ⁇ -tryptase and ⁇ -tryptase, or mouse ztrypl), or heterologous proteins (Sambrook et al., ibid., Altschul et al., ibid., Picard, Cur. Opin. Biology, 5:511-5, 1994, and references therein). These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest. Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure.
  • tryptase family proteins e.g. human ⁇ -tryptase and ⁇ -tryptase, or mouse ztrypl
  • Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them.
  • a polynucleotide encoding both components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein.
  • part or all of a domain(s) conferring a biological function may be swapped between ztrypl of the present invention with the functionally equivalent domain(s) from another family member, such as human ⁇ - tryptase, ⁇ -tryptase, mouse ztrypl or trypsin.
  • Such domains include, but are not limited to, the secretory signal sequence, pre-pro peptide, conserved motifs and active site amino acids, binding pocket, active polypeptide, and transmembrane domain.
  • Such fusion proteins would be expected to have a biological functional profile that is the same or similar to polypeptides of the present invention or other known tryptase family proteins, depending on the fusion constructed. Moreover, such fusion proteins may exhibit other properties as disclosed herein.
  • Standard molecular biological and cloning techniques can be used to swap the equivalent domains between the ztrypl polypeptide and those polypeptides to which they are fused.
  • a DNA segment that encodes a domain of interest e.g., a ztrypl active polypeptide or motif described herein, is operably linked in frame to at least one other DNA segment encoding an additional polypeptide and inserted into an appropriate expression vector, as described herein.
  • DNA constructs are made such that the several DNA segments that encode the corresponding regions of a polypeptide are operably linked in frame to make a single construct that encodes the entire fusion protein, or a functional portion thereof.
  • a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising a signal polypeptide followed by a pro-polypeptide (or pro-protein); or a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising a signal polypeptide followed by a pro-peptide, active polypeptide and C-terminal region; or as interchanged with equivalent regions from another protein.
  • a DNA construct would encode a mature form of ztrypl comprising amino acids operably linked in frame comprising amino acids 43 (Val) through 168 (Gly) of SEQ DD NO: 15 followed by amino acids 171 (Leu) to 232 (Thr) of SEQ ED NO:2 followed by 231 (Ser) to 275 (Arg) of SEQ ED NO: 15, as shown in SEQ ED NO:24.
  • the binding pocket of SEQ ED NO:24 comprises the mouse ztrypl binding pocket, as described herein.
  • Such fusion proteins can be expressed, isolated, and assayed for activity as described herein.
  • Ztrypl polypeptides or fragments thereof may also be prepared through chemical synthesis, ztrypl polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.
  • Polypeptides of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. Methods for synthesizing polypeptides are well known in the art. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Kaiser et al., Anal. Biochem. 34:595, 1970.
  • the peptide-resin is with a reagent which cleaves the polypeptide from the resin and removes most of the side-chain protecting groups.
  • the activity of molecules of the present invention can be measured using a variety of assays that measure cleavage of a substrate indicator molecule, synthetic substrate, or substrate polypeptide.
  • assays are well known in the art. For a general reference, see Chan, H. et al., Prot. Express. Purif. 15:251-257, 1999; and Cromlish, J.A. et al., J. Biol. Chem. 262:1363-1373, 1987.
  • Specific assays include, but are not limited to bioassays measuring fluorescence due to hydrolysis of 4- methylumbelliferone, hydrolysis of a substrate such as Tosyl-Gly-Pro-Lys-pNA (where
  • tosyl is a 4-toluenesulfonyl and "pNA” is a p-nitroaniline moiety) and detection of UV, fibrinogen cleavage, and inhibition studies using various general and specific protease inhibitors which are routine in the art.
  • Proteins of the present invention are useful for inducing cell proliferation, inflammatory response, and cytokine production and in treating reproductive, testicular, heart, and other disorders.
  • IL-8 production inflammatory response or cell proliferation can be measured in vitro using cultured cells or in vivo by administering molecules of the claimed invention to the appropriate animal model.
  • host cells expressing a secreted form of ztrypl polypeptide may be embedded in an alginate environment and injected (implanted) into recipient animals.
  • Alginate-poly-L-lysine microencapsulation, permselective membrane encapsulation and diffusion chambers are a means to entrap transfected mammalian cells or primary mammalian cells to permit the diffusion of proteins and other macromolecules secreted or released by the captured cells to the recipient animal. Most importantly, the capsules mask and shield the foreign, embedded cells from the recipient animal's immune response. Such encapsulations can extend the life of the injected cells from a few hours or days (naked cells) to several weeks (embedded cells). Alginate threads provide a simple and quick means for generating embedded cells and testing, in vivo, the proteins secreted therefrom.
  • alginate threads The materials needed to generate the alginate threads are known in the art.
  • 3% alginate is prepared in sterile H2O, and sterile filtered. Just prior to preparation of alginate threads, the alginate solution is again filtered.
  • An approximately 50% cell suspension (containing about 5 x 10 ⁇ to about 5 x
  • viruses for this purpose include adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus (AAV).
  • Adenovirus a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for review, see T.C. Becker et al., Meth. Cell Biol. 43:161-89, 1994; and J.T. Douglas and D.T. Curiel, Science & Medicine 4:44-53, 1997).
  • adenovirus can accommodate relatively large DNA inserts; (ii) can be grown to high- titer; (iii) infect a broad range of mammalian cell types; and (iv) can be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
  • adenovirus vectors where portions of the adenovirus genome are deleted, inserts are incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid.
  • the essential El gene has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary).
  • adenovirus When intravenously administered to intact animals, adenovirus primarily targets the liver. E the adeno viral delivery system has an El gene deletion, the virus cannot replicate in the host cells. However, the host's tissue (e.g., liver) will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein. Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.
  • tissue e.g., liver
  • adenoviral vectors containing various deletions of viral genes can be used in an attempt to reduce or eliminate immune responses to the vector.
  • Such adenoviruses are El deleted, and in addition contain deletions of E2A or E4 (Lusky, M. et al., J. Virol. 72:2022-2032, 1998; Raper, S.E. et al., Human Gene Therapy 9:671- 679, 1998).
  • deletion of E2b is reported to reduce immune responses (Amalfitano, A. et al., J. Virol. 72:926-933, 1998).
  • by deleting the entire adenovirus genome very large inserts of heterologous DNA can be accommodated.
  • the adenovirus system can also be used for protein production in vitro.
  • the cells By culturing adenovirus-infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division.
  • adenovirus vector infected 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (See Garnier et al., Cytotechnol. 15:145-55, 1994).
  • an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant, lysate, or membrane fractions depending on the disposition of the expressed protein in the cell. Within the infected 293 cell production protocol, non-secreted proteins may also be effectively obtained.
  • Ztrypl is highly expressed in a tissue that contracts, and may affect other contractile tissues. Like human tryptase, ztrypl may affect contractile tissues such as lung, gastrointestinal and heart tissues.
  • contractile tissues in which ztrypl is expressed include tissues in testis, e.g., vas deferens; prostate tissues; others where ztrypl may be expressed or active include gastrointestinal tissues, e.g., colon and small intestine; and heart.
  • the effects of ztrypl polypeptide, its antagonists and agonists, on tissue contractility can be measured in vitro using a tensiometer with or without electrical field stimulation.
  • Such assays are known in the art and can be applied to tissue samples, such as aortic rings, vas deferens, ilium, uterine and other contractile tissue samples, as well as to organ systems, such as atria, and can be used to determine whether ztrypl polypeptide, its agonists or antagonists, enhance or depress contractility.
  • Molecules of the present invention are hence useful for treating dysfunction associated with contractile tissues or can be used to suppress or enhance contractility in vivo.
  • molecules of the present invention have utility in treating cardiovascular disease, infertility, in vitro fertilization, birth control, treating impotence or other male reproductive dysfunction, as well as inducing birth.
  • ztrypl polypeptides, antagonists and agonists of the present invention can be measured in a tensiometer that measures contractility and relaxation in tissues. See, Dainty et al., J. Pharmacol. 100:767, 1990; Rhee et al., Neurotox. 16: 179, 1995; Anderson, M.B., Endocrinol. 114:364-368. 1984; and Downing, S.J. and Sherwood, O.D, Endocrinol. JT6: 1206-1214, 1985.
  • measuring vasodilatation of aortic rings is well known in the art. Briefly, aortic rings are taken from 4 month old Sprague Dawley rats and placed in a buffer solution, such as modified Krebs solution (118.5 mM NaCl, 4.6 mM KCl, 1.2 mM
  • the tissues are adjusted to 1 gram resting tension and allowed to stabilize for about one hour before testing.
  • the integrity of the rings can be tested with norepinepherin (Sigma Co., St. Louis, MO) and Carbachol, a muscarinic acetylcholine agonist (Sigma Co.). After integrity is checked, the rings are washed three times with fresh buffer and allowed to rest for about one hour. To test a sample for vasodilatation, or relaxation of the aortic ring tissue, the rings are contracted to two grams tension and allowed to stabilize for fifteen minutes. A ztrypl polypeptide sample is then added to 1, 2 or 3 of the 4 baths, without flushing, and tension on the rings recorded and compared to the control rings containing buffer only. Enhancement or relaxation of contractility by ztrypl polypeptides, their agonists and antagonists is directly measured by this method, and it can be applied to other contractile tissues such as uterus, prostate, gastrointestinal tissues and testis.
  • Gastric motility is generally measured in the clinical setting as the time required for gastric emptying and subsequent transit time through the gastrointestinal tract.
  • Gastric emptying scans are well known to those skilled in the art, and briefly, comprise use of an oral contrast agent, such as barium, or a radiolabeled meal. Solids and liquids can be measured independently.
  • a test food or liquid is radiolabeled with an isotope (e.g. 99m Tc), and after ingestion or administration, transit time through the gastrointestinal tract and gastric emptying are measured by visualization using gamma cameras (Meyer et al.. Am. J. Dig. Pis. 21:296, 1976; Collins et al., Gut 24: 1117, 1983; Maughan et al., Diabet. Med.
  • the activity of ztrypl polypeptide can be measured by a silicon-based biosensor microphysiometer which measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent physiologic cellular responses.
  • An exemplary device is the CytosensorTM Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA.
  • CytosensorTM Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA.
  • a variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method. See, for example, McConnell, H.M. et al., Science 257:1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol. 228:84-108, 1997; Arimilli, S.
  • the microphysiometer can be used for assaying adherent or non- adherent eukaryotic or prokaryotic cells. By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including ztrypl polypeptide, its agonists, or antagonists. Preferably, the microphysiometer is used to measure responses of a ztrypl -responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to ztrypl polypeptide.
  • Ztrypl -responsive eukaryotic cells comprise cells into which a receptor for ztrypl has been transfected creating a cell that is responsive to ztrypl; or cells naturally responsive to ztrypl such as cells derived from testicular tissue. Differences, measured by a change, for example, an increase or diminution in extracellular acidification, in the response of cells exposed to ztrypl polypeptide, relative to a control not exposed to ztrypl, are a direct measurement of ztrypl -modulated cellular responses. Moreover, such ztrypl -modulated responses can be assayed under a variety of stimuli.
  • a method of identifying agonists of ztrypl polypeptide comprising providing cells responsive to a ztrypl polypeptide, culturing a first portion of the cells in the absence of a test compound, culturing a second portion of the cells in the presence of a test compound, and detecting a change, for example, an increase or diminution, in a cellular response of the second portion of the cells as compared to the first portion of the cells.
  • the change in cellular response is shown as a measurable change extracellular acidification rate.
  • culturing a third portion of the cells in the presence of ztrypl polypeptide and the absence of a test compound can be used as a positive control for the ztrypl -responsive cells, and as a control to compare the agonist activity of a test compound with that of the ztrypl polypeptide.
  • a method of identifying antagonists of ztrypl polypeptide comprising providing cells responsive to a ztrypl polypeptide, culturing a first portion of the cells in the presence of ztrypl and the absence of a test compound, culturing a second portion of the cells in the presence of ztrypl and the presence of a test compound, and detecting a change, for example, an increase or a diminution in a cellular response of the second portion of the cells as compared to the first portion of the cells.
  • the change in cellular response is shown as a measurable change extracellular acidification rate.
  • Antagonists and agonists, for ztrypl polypeptide can be rapidly identified using this method.
  • ztrypl can be used to identify cells, tissues, or cell lines which respond to a ztrypl -stimulated pathway.
  • the microphysiometer, described above can be used to rapidly identify ligand-responsive cells, such as cells responsive to ztrypl of the present invention.
  • Cells can be cultured in the presence or absence of ztrypl polypeptide. Those cells which elicit a measurable change in extracellular acidification in the presence of ztrypl are responsive to ztrypl.
  • Such cell lines can be used to identify antagonists and agonists of ztrypl polypeptide as described above.
  • Ztrypl polypeptide can be used to identify inhibitors (antagonists) of its activity.
  • Test compounds are added to the biological or biochemical assays disclosed herein to identify compounds that inhibit the activity of ztrypl.
  • samples can be tested for inhibition of ztrypl activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of ztrypl -dependent cellular responses.
  • ztrypl -responsive cell lines can be transfected with a reporter gene construct that is responsive to a ztrypl -stimulated cellular pathway.
  • Reporter gene constructs of this type are known in the art, and will generally comprise a ztrypl -DNA response element operably linked to a gene encoding an assay detectable protein, such as luciferase.
  • DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE) insulin response element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87:5273-7, 1990) and serum response elements (SRE) (Shaw et al. Cell 56: 563- 72, 1989). Cyclic AMP response elements are reviewed in Roestler et al., J. Biol. Chem. 263 (19):9063-6; 1988 and Habener, Molec. Endocrinol. 4 (8): 1087-94; 1990.
  • CRE cyclic AMP response elements
  • HRE hormone response elements
  • IRE insulin response element
  • SRE serum response elements
  • test sample to inhibit the activity of ztrypl polypeptide on the target cells as evidenced by a decrease in ztrypl stimulation of reporter gene expression in the presence of a test sample relative to a control which was cultured in the absence of a test sample.
  • Assays of this type will detect compounds that directly block ztrypl binding to cell-surface receptors, e.g., dimerization, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding.
  • compounds or other samples can be tested for direct blocking of ztrypl binding to receptor using ztrypl tagged with a detectable label (e.g., 125 I, biotin, horseradish peroxidase, FITC, and the like).
  • Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors.
  • Antagonists are also useful as research reagents for characterizing sites of ligand-receptor interaction.
  • Inhibitors of ztrypl activity include anti-ztrypl antibodies and soluble proteins which bind ztrypl polypeptide, as well as other peptidic and non-peptidic agents (including ribozymes).
  • the above methodology may be used to identify agonists of ztrypl activity.
  • Candidate compounds serving as test samples including solutions, mixtures or extracts, are tested for the ability to mimic the activity of ztrypl polypeptide on the target cells as evidenced by stimulation of reporter gene expression in the presence of a test sample and the absence of ztrypl, relative to a control (cultured in the absence of a test sample and the absence of ztrypl polypeptide), using assays as described above.
  • Mouse or human ztrypl agonists and antagonists may act on the counterpart human ortholog of ztrypl or in other animal species.
  • agonists including the natural ligand/ substrate/ cofactor/ etc.
  • antagonists have enormous potential in both in vitro and in vivo applications.
  • Compounds identified as ztrypl agonists are useful for stimulating cell growth or signal transduction in vitro and in vivo.
  • Agonists and ztrypl are thus useful in specifically promoting the growth and/or development of cells in culture.
  • ztrypl polypeptides and ztrypl agonists may be particularly useful as research reagents, particularly for the growth of testicular types, animal eggs, construction of transgenic or knockout mice, cells from animal embryos or primary cultures derived from these tissues, and in animal breeding applications.
  • ztrypl polypeptide can be provided as a supplement in cell culture medium.
  • both mouse and human ztrypl serve as a useful research tool in molecular biology and biochemical applications.
  • ztrypl cleavage can be used to characterize and identify proteins and protein families by the cleavage patterns generated upon exposure to ztrypl.
  • cleavage patterns or "cleavage maps" are useful in the art in characterizing proteins generally, as they can be used to identify proteins, and characterize their structure, as well as serve as an educational tool to aid in the characterization of proteins; such uses for ztrypl are similar to proteases known in the art, such as trypsin, chymo-trypsin and the like (See, for example, Ausubel et al, (eds.), Current Protocols in Molecular Biology John Wiley and Sons, Inc. 1987)). Moreover, ztrypl can be used to activate proteins that require ztrypl cleavage.
  • ztrypl can be used in preparations of DNA and RNA, and the like, that require inactivation of proteins, DNAses, RNAses, and the like.
  • a protease, such as ztrypl serves as a useful reagent to dissociate adherent cells from tissue culture plates.
  • Such applications for the novel ztrypl protease are well known by one of skill in the art. See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Press 1989); Ausubel et al, (eds.), supra.; Berger and Kimmel (eds.), Guide to Molecular Cloning Techniques, (Academic Press, Inc.
  • ztrypl can have further industrial applicability in industries that utilize proteases, for example, as a detergent enzyme, food processing enzyme, and the like. Such applications for the novel ztrypl protease are well known by one of skill in the art.
  • ztrypl expression suggests a role in spermatogenesis, a process that is remarkably similar to the development of blood cells (hematopoiesis). Briefly, spermatogonia undergo a maturation process similar to the differentiation of hematopoietic stem cells.
  • agonists and antagonists have enormous potential in both in vitro and in vivo applications.
  • Ztrypl polypeptides, agonists and antagonists may also prove useful in modulating spermatogenesis and thus aid in overcoming infertility.
  • Antagonists are useful as research reagents for characterizing sites of ligand-receptor interaction.
  • ztrypl polypeptides, agonists or antagonists may find application in the treatment of male infertility or as a male contraceptive agent, or in animal breeding.
  • the ztrypl polypeptides, antagonists of agonists, of the present invention can also modulate sperm capacitation.
  • the sperm Before reaching the oocyte or egg and initiating an egg-sperm interaction, the sperm must be activated. The sperm undergo a gradual capacitation, lasting up to 3 or 4 hours in vitro, during which the plasma membrane of the sperm head and the outer acrosomal membrane fuse to form vesicles that facilitate the release of acrosomal enzymes.
  • the acrosomal membrane surrounds the acrosome or acrosomal cap which is located at the anterior end of the nucleus in the sperm head. In order for the sperm to fertilize egg the sperm must penetrate the oocyte.
  • sperm must undergo acrosomal exocytosis, also known as the acrosomal reaction, and release the acrosomal enzymes in the vicinity of the oocyte. These enzymes enable the sperm to penetrate the various oocyte layers, (the cumulus oophorus, the corona radiata and the zona pellucida).
  • the released acrosomal enzymes include hyaluronidase and proacrosin, in addition to other enzymes such as proteases.
  • proacrosin is converted to acrosin, the active form of the enzyme, which is required for and must occur before binding and penetration of the zona pellucida is possible.
  • a combination of the acrosomal lytic enzymes and sperm tail movements allow the sperm to penetrate the oocyte layers. Numerous sperm must reach the egg and release acrosomal enzymes before the egg can finally be fertilized.
  • ztrypl As ztrypl has structural similarity to proteases and has protease activity, it may cleave or process hormones and other proteases such as peptide prohormones, and the like to their active forms. Moreover, tissue-specific localization of Ztrypl to the testis, suggests that the ztrypl polypeptides described herein play a role in the function or reproductive processes in this tissue.
  • proteins of the present invention can have applications in enhancing fertilization during assisted reproduction in humans and in animals.
  • assisted reproduction methods are known in the art and include artificial insemination, in vitro fertilization, embryo transfer and gamete intrafallopian transfer. Such methods are useful for assisting men and women who have physiological or metabolic disorders preventing natural conception or can be used to enhance in vitro fertilization.
  • Such methods are also used in animal breeding programs, such as for livestock breeding and can be used as methods for the creation of transgenic animals.
  • Proteins of the present invention can be combined with sperm, an egg or an egg-sperm mixture prior to fertilization of the egg. In some species, sperm capacitate spontaneously during in vitro fertilization procedures, but normally sperm capacitate over an extended period of time both in vivo and in vitro.
  • sperm activation during such procedures to enhance the likelihood of successful fertilization.
  • the washed sperm or sperm removed from the seminal plasma used in such assisted reproduction methods has been shown to have altered reproductive functions, in particular, reduced motility and zona interaction.
  • sperm is capacitated using exogenously added compounds.
  • ztrypl polypeptide or polypeptide fragments may function as germ-cell-specific antigens for use as components in "immunocontraceptive" or "anti-fertility” vaccines to induce formation of antibodies and/or cell mediated immunity to selectively inhibit a process, or processes, critical to successful reproduction in humans and animals.
  • sperm and testis antigens in the development of immunocontraceptives have been described (O'Hern et al., Biol Reprod. 52:311-39, 1995; Diekman and Herr, Am. J. Reprod. Immunol. 37:111-17, 1997: Zhu and Naz, Proc. Natl. Acad. Sci. USA 94:4704-9,1997).
  • HCG human chorionic gonadotrophin
  • tetanus a vaccine based on human chorionic gonadotrophin (HCG) linked to a diphtheria or tetanus carrier was in clinical trials (Talwar et al., Proc. Natl. Acad. Sci. USA 91:8532- 36, 1994).
  • a single injection resulted in production of high titer antibodies that persisted for nearly a year in rabbits (Stevens, Am. J. Reprod. Immunol. 29:176-88, 1993).
  • Such methods of immunocontraception using vaccines would include a ztrypl testes-specific protein or fragment thereof.
  • the Ztrypl protein or fragments can be conjugated to a carrier protein or peptide, such as tetanus or diphtheria toxoid.
  • an adjuvant as described above, can be included and the protein or fragment can be non- covalently associated with other molecules to enhance intrinsic immunoreactivity.
  • Methods for administration and methods for determining the number of administrations are known in the art. Such a method might include a number of primary injections over several weeks followed by booster injections as needed to maintain a suitable antibody titer.
  • the anti-ztrypl antibodies of the present invention can also be used as immunocontraceptives.
  • mouse ztrypl transgenic and knockout mice and mice can be used as a fertility or contraception animal model, and can be used to test the effects of test compounds that influence ztrypl activity on fertility.
  • the polypeptides, nucleic acid and/or antibodies of the present invention can be used as a model to study or be applied in treatment of disorders associated with asthma, the immune system, gastrointestinal system, heart, inflammation, lymph system, and testis.
  • the polypeptides, agonists, antagonists, nucleic acid and/or antibodies of the present invention can be used in treatment of disorders associated with asthma, vascular function such as stroke, inflammation and testicular function.
  • the molecules of the present invention may used to modulate or to treat or prevent development of pathological conditions. In particular, certain syndromes or diseases can be amenable to such diagnosis, treatment or prevention.
  • polypeptides of the present invention can be used for their ability to modify inflammation.
  • Methods to assess proinflammatory or antiinflammatory qualities of ztrypl are known in the art. For example, suppression of cAMP production is an indication of anti-inflammatory effects (Nihei, Y., et al., Arch. Dermatol. Res., 287:546-552, 1995).
  • Ztrypl likewise can exhibit similar anti- inflammatory effects, and may exert these effects in tissues in which it is expressed. For example, ztrypl is expressed in testis and can be useful in treatment of inflammation in this tissue.
  • ztrypl may be expressed in the small intestine, and can be useful in treatment of inflammatory bowel disease, diverticulitis, inflammation during and after intestinal surgery, and the like.
  • ztrypl if expressed in PBLs and bone marrow, can have other antiinflammatory actions in heart, pelvic inflammatory disease, (PDD), psoriasis, arthritis, and other inflammatory diseases.
  • PPD pelvic inflammatory disease
  • psoriasis psoriasis
  • arthritis and other inflammatory diseases.
  • mouse ztrypl transgenic and knockout mice and mice can be used as an animal model for inflammation, and can be used to test the effects of test compounds, agonists and antagonists that influence ztrypl activity on inflammation.
  • ztrypl polypeptide As such, ztrypl polypeptide, or its agonists or antagonists, have potential uses in inflammatory diseases such as asthma and arthritis.
  • ztrypl like known tryptases, can be inflammatory; thus, antagonists would be valuable in asthma therapy or other anti-inflammatory therapies where migration of lymphocytes is damaging.
  • ztrypl can have an inhibitory or competitive effect on inflammatory agents and may serve directly as an asthma therapeutic or anti- inflammatory.
  • ztrypl can serve other important roles in lung function, for instance, bronchodilation or broncoconstriction, tissue elasticity, recruitment of lymphocytes in lung infection and damage. Assays to assess the activity of ztrypl in lung cells are discussed in Laberge, S.
  • proinflammatory effects of ztrypl polypeptide can be directly tested in assays using a TranswellTM (Costar), wherein endothelial cells are plated on a semi-permeable membrane and ztrypl polypeptide is present in the lower chamber of the transwell and Cr 51 or fluorescentiy-labeled neutrophils (PMNs), lymphocytes, HL60 cells, K562 cells, or the like are added on to the upper chamber of the transwell.
  • TranswellTM Costar
  • PMNs fluorescentiy-labeled neutrophils
  • ztrypl polypeptide in inducing these cells to secrete factors that enhance migration of PMNs, i.e., inflammation, can be measured.
  • the bFGF can be used as a positive control for indirect assay.
  • Anti-inflammatory effects of ztrypl polypeptide can also be measured when added on the upper chamber in the presence of PMN's using a similar transwell assay known in the art.
  • a ztrypl polypeptide can be expressed as a fusion with an immunoglobulin heavy chain constant region, typically an F c fragment, which contains two constant region domains and lacks the variable region.
  • an immunoglobulin heavy chain constant region typically an F c fragment
  • Methods for preparing such fusions are disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584.
  • Such fusions are typically secreted as multimeric molecules wherein the Fc portions are disulfide bonded to each other and two non-Ig polypeptides are arrayed in closed proximity to each other. Fusions of this type can be used to affinity purify complementary polypeptides, as an in vitro assay tool, or zrtypl antagonist.
  • the chimeras are bound to a support via the F c region and used in an ELISA format.
  • a ztrypl receptor-binding polypeptide can also be used for purification of receptor.
  • the polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
  • a solid support such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
  • Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation.
  • the resulting medium will generally be configured in the form of a column, and fluids, cell lysates or membrane fractions containing receptors are passed through the column one or more times to allow ligand to bind to the receptor polypeptide.
  • the receptor is then eluted using changes in salt concentration, chaotropic agents (guanidine HCl), or pH to disrupt ligand-receptor binding.
  • Molecules of the present invention can be used to identify and isolate receptors involved in testicular function.
  • proteins and peptides of the present invention can be immobilized on a column and membrane preparations run over the column (Immobilized Affinity Ligand Techniques, Hermanson et al., eds., Academic Press, San Diego, CA, 1992, pp.195-202).
  • Proteins and peptides can also be radiolabeled (Methods in Enzymol., vol. 182, "Guide to Protein Purification", M. Deutscher, ed., Acad. Press, San Diego, 1990, 721-37) or photoaffmity labeled (Brunner et al., Ann. Rev. Biochem. 62:483-514, 1993 and Fedan et al., Biochem. Pharmacol. 33:1167-80, 1984) and specific cell-surface proteins can be identified.
  • An assay system that uses a ligand-binding receptor (or an antibody, one member of a complement/ anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BEAcore, Pharmacia Biosensor, Piscataway, NJ) may be advantageously employed.
  • a ligand-binding receptor or an antibody, one member of a complement/ anti-complement pair
  • a commercially available biosensor instrument BEAcore, Pharmacia Biosensor, Piscataway, NJ
  • Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip.
  • Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145:229-40, 1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993.
  • a receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell.
  • a test sample is passed through the cell. If a ligand, epitope, or opposite member of the complement/anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film.
  • This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.
  • Ligand-binding receptor polypeptides can also be used within other assay systems known in the art. Such systems include Scatchard analysis for determination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949) and calorimetric assays (Cunningham et al., Science 253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).
  • Ztrypl polypeptides can also be used to prepare antibodies that bind to ztrypl epitopes, peptides or polypeptides.
  • the ztrypl polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response.
  • antigenic, epitope-bearing polypeptides contain a sequence of at least 6, preferably at least 9, and more preferably at least 15 to about 30 contiguous amino acid residues of a ztrypl polypeptide (e.g., SEQ ED NO:2 or SEQ ED NO: 15).
  • Polypeptides comprising a larger portion of a ztrypl polypeptide, i.e., from 30 to 10 residues up to the entire length of the amino acid sequence are included.
  • Antigens or immunogenic epitopes can also include attached tags, adjuvants and carriers, as described herein. Suitable antigens include the ztrypl polypeptide encoded by SEQ DD NO:2 from amino acid number 44 (Val) to amino acid number 276 (De) (i.e., the active polypeptide), or a contiguous 9 to 232 amino acid fragment thereof.
  • Suitable antigens include the ztrypl polypeptide encoded by SEQ DD NO: 15 from amino acid number 43 (Val) to amino acid number 275 (Arg) (i.e., the mature active polypeptide), or a contiguous 9 to 233 amino acid fragment thereof.
  • Other suitable antigens include the pre-pro peptide, binding pocket, hydrophilic domains, extracellular domains, motifs, domains, regions, epitopes, as disclosed herein.
  • Preferred peptides to use as antigens are hydrophilic peptides such as those predicted by one of skill in the art from a hydrophobicity plot such as a Hopp/Woods hydrophilicity profile based on a sliding six -residue window, with buried G, S, and T residues and exposed H, Y, and W residues ignored.
  • Such Ztrypl hydrophilic peptides include peptides comprising amino acid sequences selected from the group consisting of: (1) amino acid number 125 (Asn) to amino acid number 130 (Asp) of SEQ DD NO:2; (2) amino acid number 172 (Glu) to amino acid number 177 (His) of SEQ DD NO:2; (3) amino acid number 256 (Asp) to amino acid number 261 (Gin) of SEQ DD NO:2; (4) amino acid number 277 (Lys) to amino acid number 282 (Asp) of SEQ D NO:2; and (5) amino acid number 117 (Gin) to amino acid number 122 (Ser) of SEQ ED NO: 15; (6) amino acid number 57 (Arg) to amino acid number 62 (Lys) of SEQ ED NO: 15; (7) amino acid number 116 (His) to amino acid number 121 (Asn) of SEQ ED NO: 15; (8) amino acid number 147 (Lys)
  • Antigenic epitopes as predicted from a Jameson -Wolf plot of SEQ ED NO:2 or SEQ ED NO: 15, for example, using a DNA*STAR program, are also included as antigens for use in generating anti-ztrypl antibodies.
  • Antibodies from an immune response generated by inoculation of an animal with these antigens can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in Immunology, Cooligan, et al.
  • polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a ztrypl polypeptide or a fragment thereof.
  • the immunogenicity of a ztrypl polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of ztrypl or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
  • the polypeptide immunogen may be a full-length molecule or a portion thereof.
  • E the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid
  • antibodies includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2 and Fab proteolytic fragments.
  • Non-human antibodies may be humanized by grafting non-human
  • humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics.
  • biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
  • human antibodies can be produced in transgenic, non-human animals that have been engineered to contain human immunoglobulin genes as disclosed in WIPO Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination.
  • Antibodies are considered to be specifically binding if: 1) they exhibit a threshold level of binding activity, and 2) they do not significantly cross-react with related polypeptide molecules.
  • a threshold level of binding is determined if anti-ztrypl antibodies herein bind to a ztrypl polypeptide, peptide or epitope with an affinity at least 10-fold greater than the binding affinity to control (non-ztrypl) polypeptide. It is preferred that the antibodies exhibit a binding affinity (K a ) of 10 M " or greater,
  • binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51 . : 660-672, 1949).
  • anti-ztrypl antibodies do not significantly cross-react with related polypeptide molecules is shown, for example, by the antibody detecting ztrypl polypeptide but not known related polypeptides using a standard Western blot analysis (Ausubel et al., ibid.).
  • known related polypeptides are those disclosed in the prior art, such as known orthologs, and paralogs, and similar known members of a protein family (e.g., known human tryptases). Screening can also be done using known non- mouse or human ztrypl, and ztrypl mutant polypeptides.
  • antibodies can be "screened against" known related polypeptides, to isolate a population that specifically binds to the ztrypl polypeptides. For example, antibodies raised to ztrypl are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to ztrypl will flow through the matrix under the proper buffer conditions. Screening allows isolation of polyclonal and monoclonal antibodies non-crossreactive to known closely related polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995).
  • assays known to those skilled in the art can be utilized to detect antibodies which bind to ztrypl proteins or polypeptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant ztrypl protein or polypeptide.
  • Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to ztrypl protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled ztrypl protein or peptide).
  • Genes encoding polypeptides having potential ztrypl polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli.
  • Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
  • random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., US Patent NO. 5,223,409; Ladner et al., US Patent NO. 4,946,778; Ladner et al., US Patent NO. 5,403,484 and Ladner et al., US Patent NO. 5,571,698) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from Clontech (Palo Alto, CA), Invitrogen Inc.
  • Random peptide display libraries can be screened using the ztrypl sequences disclosed herein to identify proteins which bind to ztrypl.
  • These "binding polypeptides" which interact with ztrypl polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like.
  • binding polypeptides can also be used in analytical methods such as for screening expression libraries and neutralizing activity, e.g., for blocking interaction between ligand and receptor, or viral binding to a receptor.
  • binding polypeptides can also be used for diagnostic assays for determining circulating levels of ztrypl polypeptides; for detecting or quantitating soluble ztrypl polypeptides as marker of underlying pathology or disease. These binding polypeptides can also act as ztrypl "antagonists" to block ztrypl binding and signal transduction in vitro and in vivo. These anti-ztrypl binding polypeptides would be useful for inhibiting ztrypl activity or protein-binding.
  • Antibodies to ztrypl may be used for tagging cells that express ztrypl; for isolating ztrypl by affinity purification; for diagnostic assays for determining circulating levels of ztrypl polypeptides; for detecting or quantitating soluble ztrypl as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block ztrypl activity in vitro and in vivo.
  • Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti- complement pairs as intermediates.
  • Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • antibodies to ztrypl or fragments thereof may be used in vitro to detect denatured ztrypl or fragments thereof in assays, for example, Western Blots or other assays known in the art.
  • Antibodies or polypeptides herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance).
  • ztrypl polypeptides or anti-ztrypl antibodies, or bioactive fragments or portions thereof can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti- complementary molecule.
  • Suitable detectable molecules may be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like.
  • Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium- 188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance).
  • Polypeptides or antibodies may also be conjugated to cytotoxic drugs, such as adriamycin.
  • cytotoxic drugs such as adriamycin.
  • the detectable or cytotoxic molecule can be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the polypeptide or antibody portion.
  • biotin/streptavidin is an exemplary complementary/ anticomplementary pair.
  • polypeptide-toxin fusion proteins or antibody- toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues).
  • a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest.
  • the anti-complementary molecule can be conjugated to a detectable or cytotoxic molecule.
  • Such domain-complementary molecule fusion proteins thus represent a generic targeting vehicle for cell/tissue- specific delivery of generic anti-complementary-detectable/ cytotoxic molecule conjugates.
  • ztrypl -cytokine fusion proteins or antibody- cytokine fusion proteins can be used for enhancing in vivo killing of target tissues (for example, blood and bone marrow cancers), if the ztrypl polypeptide or anti-ztrypl antibody targets the hyperproliferative blood or bone marrow cell (See, generally, Hornick et al., Blood 89:4437-47, 1997).
  • target tissues for example, blood and bone marrow cancers
  • ztrypl polypeptide or anti-ztrypl antibody targets the hyperproliferative blood or bone marrow cell.
  • fusion proteins enable targeting of a cytokine to a desired site of action, thereby providing an elevated local concentration of cytokine.
  • Suitable ztrypl polypeptides or anti-ztrypl antibodies target an undesirable cell or tissue (i.e., a tumor or a leukemia), and the fused cytokine mediated improved target cell lysis by effector cells.
  • Suitable cytokines for this purpose include interleukin 2 and granulocyte-macrophage colony-stimulating factor (GM- CSF), for instance.
  • the ztrypl polypeptide or anti-ztrypl antibody targets vascular cells or tissues
  • such polypeptide or antibody may be conjugated with a radionuclide, and particularly with a beta-emitting radionuclide, to reduce restenosis.
  • a radionuclide and particularly with a beta-emitting radionuclide
  • Such therapeutic approach poses less danger to clinicians who administer the radioactive therapy.
  • iridium-192 impregnated ribbons placed into stented vessels of patients until the required radiation dose was delivered showed decreased tissue growth in the vessel and greater luminal diameter than the control group, which received placebo ribbons. Further, revascularisation and stent thrombosis were significantly lower in the treatment group. Similar results are predicted with targeting of a bioactive conjugate containing a radionuclide, as described herein.
  • the bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intraarterially or intraductally, or may be introduced locally at the intended site of action
  • Differentiation is a progressive and dynamic process, beginning with pluripotent stem cells and ending with terminally differentiated cells.
  • Pluripotent stem cells that can regenerate without commitment to a lineage express a set of differentiation markers that are lost when commitment to a cell lineage is made.
  • Progenitor cells express a set of differentiation markers that may or may not continue to be expressed as the cells progress down the cell lineage pathway toward maturation.
  • Differentiation markers that are expressed exclusively by mature cells are usually functional properties such as cell products, enzymes to produce cell products, and receptors. The stage of a cell population's differentiation is monitored by identification of markers present in the cell population.
  • Myocytes, osteoblasts, adipocytes, chrondrocytes, fibroblasts and reticular cells are believed to originate from a common mesenchymal stem cell (Owen et al., Ciba Fdn. Syrnp. 136:42-46, 1988). Markers for mesenchymal stem cells have not been well identified (Owen et al., J. of Cell Sci. 87:731-738, 1987), so identification is usually made at the progenitor and mature cell stages.
  • the novel polypeptides of the present invention may be useful for studies to isolate mesenchymal stem cells and myocyte or other progenitor cells, both in vivo and ex vivo.
  • the present invention includes a polypeptide that may be indirectly involved (e.g., via processing a prohormone or other regulatory peptide to an active polypeptide) in stimulating or inhibiting the proliferation of lymphoid cells, hematopoietic cells, endothelial cells, testicular cells, or cancer cells.
  • polypeptide and antibody molecules of the present invention may have use in inhibiting tumor cells, and particularly testicular tumor cells.
  • Assays measuring differentiation include, for example, measuring cell markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis,
  • ztrypl polypeptide itself can serve as an additional cell-surface or secreted marker associated with stage- specific expression of a tissue.
  • direct measurement of ztrypl polypeptide, or its loss of expression in a tissue as it differentiates can serve as a marker for differentiation of tissues.
  • direct measurement of ztrypl polypeptide, or its loss of expression in a tissue can be determined in a tissue or cells as they undergo tumor progression.
  • ztrypl increases in invasiveness and motility of cells, or the gain or loss of expression of ztrypl in a pre-cancerous or cancerous condition, in comparison to normal tissue, can serve as a diagnostic for transformation, invasion and metastasis in tumor progression.
  • ztrypl is highly expressed in testis, and appears to have very little expression in other tissues, therefore increased expression of ztrypl in non-testis tissues can be indicative of transformation, cancer, or metastasis in the non- testicular tissue.
  • knowledge of a tumor's stage of progression or metastasis will aid the physician in choosing the most proper therapy, or aggressiveness of treatment, for a given individual cancer patient.
  • ztrypl gain or loss of expression of either mRNA or protein
  • appearance or disappearance of polypeptides that regulate cell motility can be used to aid diagnosis and prognosis of prostate cancer (Banyard, J. and Zetter, B.R., Cancer and Metast. Rev. 17:449-458, 1999).
  • ztrypl gain or loss of expression of mRNA or polypeptide may serve as a diagnostic for testicular and other cancers.
  • polynucleotide probes, anti- ztrypl antibodies, and detection the presence of ztrypl polypeptides in tissue can be used to assess whether testicular tissue is present, for example, after surgery involving the excision of testis.
  • the polynucleotides, polypeptides, and antibodies of the present invention can be used as an aid to determine whether all testicular tissue is excised after surgery, for example, after surgery for testicular cancer. In such instances, it is especially important to remove all potentially diseased tissue to maximize recovery from the cancer, and to minimize recurrence.
  • Preferred embodiments include fluorescent, radiolabeled, or calorimetrically labeled antibodies, that can be used in situ.
  • tumor cells passaged in culture are implanted into mice of the same strain as the tumor donor.
  • the cells will develop into tumors having similar characteristics in the recipient mice, and metastasis will also occur in some of the models.
  • Appropriate tumor models for our studies include the Lewis lung carcinoma (ATCC No. CRL- 1642) and B 16 melanoma (ATCC No. CRL-6323), amongst others. These are both commonly used tumor lines, syngeneic to the C57BL6 mouse that are readily cultured and manipulated in vitro.
  • Tumors resulting from implantation of either of these cell lines are capable of metastasis to the lung in C57BL6 mice.
  • the Lewis lung carcinoma model has recently been used in mice to identify an inhibitor of angiogenesis (O'Reilly MS, et al. Cell 79: 315-328,1994).
  • C57BL6/J mice are treated with an experimental agent either through daily injection of recombinant protein, agonist or antagonist or a one-time injection of recombinant adenovirus. Three days following this treatment, 10 to 10 cells are implanted under the dorsal skin.
  • the cells themselves may be infected with recombinant adenovirus, such as one expressing ztrypl, before implantation so that the protein is synthesized at the tumor site or intracellularly, rather than systemically.
  • adenovirus such as one expressing ztrypl
  • the mice normally develop visible tumors within 5 days. The tumors are allowed to grow for a
  • the implanted cells can be transiently transfected with ztrypl.
  • Use of stable ztrypl transfectants as well as use of induceable promoters to activate ztrypl expression in vivo are known in the art and can be used in this system to assess ztrypl induction of metastasis.
  • purified ztrypl or ztrypl conditioned media can be directly injected in to this mouse model, and hence be used in this system.
  • O'Reilly MS et al. Cell 79:315-328, 1994
  • Rusciano D et al. Murine Models of Liver Metastasis. Invasion Metastasis 14:349-361, 1995.
  • ztrypl and its derivatives (conjugates) on growth and dissemination of tumor cells derived from human hematologic malignancies can also be measured in vivo in a mouse xenograft model.
  • Several mouse models have been developed in which human tumor cells are implanted into immunodeficient mice, collectively referred to as xenograft models. See Cattan, AR and Douglas, E Leuk. Res. 18:513-22, 1994; and Flavell, DJ, Hematological Oncology 14:67-82, 1996.
  • the characteristics of the disease model vary with the type and quantity of cells delivered to the mouse. Typically, the tumor cells will proliferate rapidly and can be found circulating in the blood and populating numerous organ systems.
  • Therapeutic strategies appropriate for testing in such a model include antibody induced toxicity, ligand-toxin conjugates or cell-based therapies.
  • the latter method commonly referred to adoptive immunotherapy, involves treatment of the animal with components of the human immune system (i.e. lymphocytes, NK cells) and may include ex vivo incubation of cells with ztrypl or other immunomodulatory agents.
  • Polynucleotides encoding ztrypl polypeptides are useful within gene therapy applications where it is desired to increase or inhibit ztrypl activity.
  • E a mammal has a mutated or absent ztrypl gene, the ztrypl gene can be introduced into the cells of the mammal.
  • a gene encoding a ztrypl polypeptide is introduced in vivo in a viral vector.
  • viral vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like.
  • HSV herpes simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred.
  • a defective virus is not infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • Examples of particular vectors include, but are not limited to, a defective herpes simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-30, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest.
  • HSV1 herpes simplex virus 1
  • a ztrypl gene can be introduced in a retroviral vector, e.g., as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., J. Virol. 62: 1120, 1988; Temin et al., U.S. Patent No. 5,124,263; International Patent Publication No.
  • the vector can be introduced by lipofection in vivo using liposomes.
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027-31, 1988).
  • the use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages.
  • Molecular targeting of liposomes to specific cells represents one area of benefit. More particularly, directing transfection to particular cells represents one area of benefit. For instance, directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting.
  • Targeted peptides e.g., hormones or neurotransmitters
  • proteins such as antibodies, or non- peptide molecules can be coupled to liposomes chemically.
  • DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem. 267:963-7, 1992; Wu et al., J. Biol. Chem. 263: 14621-4, 1988.
  • Antisense methodology can be used to inhibit ztrypl gene transcription, such as to inhibit cell proliferation in vivo.
  • Polynucleotides that are complementary to a segment of a ztrypl -encoding polynucleotide e.g., a polynucleotide as set forth in
  • SEQ ED NOT or SEQ D NO: 14 are designed to bind to ztrypl -encoding mRNA and to inhibit translation of such mRNA.
  • antisense polynucleotides are used to inhibit expression of ztrypl polypeptide-encoding genes in cell culture or in a subject.
  • the present invention also provides reagents which will find use in diagnostic applications.
  • the ztrypl gene a probe comprising ztrypl DNA or RNA or a subsequence thereof can be used to determine if the ztrypl gene is present on a chromosome or if a mutation has occurred.
  • Detectable chromosomal aberrations at the ztrypl gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements.
  • Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid. ; Ausubel et. al., ibjd.; Marian, Chest 108:255-65, 1995).
  • molecular genetic techniques such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid. ; Ausubel et. al., ibjd.; Marian, Chest 108:255-65, 1995).
  • Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high-resolution, contiguous maps of mammalian chromosomes (Cox et al., Science 250:245-50, 1990). Partial or full knowledge of a gene's sequence allows one to design PCR primers suitable for use with chromosomal radiation hybrid mapping panels. Radiation hybrid mapping panels are commercially available which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL). These panels enable rapid, PCR-based chromosomal localizations and ordering of genes, sequence-tagged sites (STSs), and other nonpolymorphic and polymorphic markers within a region of interest.
  • STSs sequence-tagged sites
  • the precise knowledge of a gene's position can be useful for a number of purposes, including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region or corresponding human chromosomal region; and 3) cross- referencing model organisms, such as between mouse and human, which may aid in determining what function a particular gene might have.
  • Sequence tagged sites STSs
  • STSs can also be used independently for chromosomal localization.
  • An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome.
  • An STS is defined by a pair of oligonucleotide primers that are used in a polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences.
  • STSs are based solely on DNA sequence they can be completely described within an electronic database, for example, Database of Sequence Tagged Sites (dbSTS), GenBank, (National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov), and can be searched with a gene sequence of interest for the mapping data contained within these short genomic landmark STS sequences.
  • dbSTS Database of Sequence Tagged Sites
  • GenBank National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov
  • the present invention also provides reagents that will find use in diagnostic applications.
  • the ztrypl gene a probe comprising ztrypl DNA or RNA or a subsequence thereof can be used to determine if the ztrypl gene is present on human chromosome 11 or if a mutation has occurred.
  • human Ztrypl is located at the l lq23-24 region of human chromosome 11.
  • Detectable chromosomal aberrations at the ztrypl gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements.
  • Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, fluorescence in situ hybridization methods, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).
  • molecular genetic techniques such as restriction fragment length polymorphism (RFLP) analysis, fluorescence in situ hybridization methods, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).
  • the precise knowledge of a gene's position can be useful for a number of purposes, including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing model organisms, such as mouse, which may aid in determining what function a particular gene might have.
  • the ztrypl gene is located at the l lq23-24 region of chromosome 11.
  • chromosome 11 Several genes of known function or correlated with human disease map to this region. For example, many gross chromosomal abnormalities such as chromosomal deletions, or loss of heterogeneity (LOH), and translocations in the l lq23 region are associated with various human tumors, including lymphoid cancers such as acute lymphoid and myeloid leukemias (acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); mixed lineage leukemias; MLL), breast and lung cancers, carotid body tumors and tumor suppressor gene on 1 lq is suspected to be involved (E.g., see Rowley, JD et al., Proc.
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • MLL mixed lineage leukemias
  • breast and lung cancers carotid body
  • ztrypl polynucleotide probes can be used to detect abnormalities or genotypes associated with cancers and other diseases associated with chromosomal translocations in Ilq23-q24.
  • ztrypl polynucleotide probes can be used to detect abnormalities or genotypes associated with some genetically linked forms of tuberous sclerosis and Ewing sarcoma, wherein variants of the disease are linked to chromosomal translocation breakpoints in the 1 Iq region between I lqll-IA, such as reciprocal translocations involving l lq23-24 and chromosomes 22.
  • translocations between chromosomes 4, 9, 10 and 19 with the l lq23 locus are involved in many MLL and some ALL.
  • the most frequent translocation seen hereditary in breast cancer is between l lq23 and 22ql l (Iselius, L. et al., Hum.
  • LOH of zrtypl may be involved in progression of human cancers and metastasis.
  • deletions, rearrangements, or other defects in the ztrypl gene itself can increase susceptibility to cancers, or other immune or testicular dysfunction.
  • defects in the ztrypl gene may fully or in part cause known human diseases for which all the genetic defects are not known, for example, those diseases associated with Jacobsen's Syndrome (l lq23-25 translocations), and hydrolethalus syndrome (I lq23-q25).
  • Molecules of the present invention such as the polypeptides, antagonists, agonists, polynucleotides and antibodies of the present invention would aid in the detection, diagnosis prevention, and treatment associated with a ztrypl genetic defect.
  • a diagnostic can assist physicians in determining the type of disease and appropriate associated therapy, or assistance in genetic counseling.
  • inventive anti-ztrypl antibodies, polynucleotides, and polypeptides can be used for the detection of ztrypl polypeptide, mRNA or anti-ztrypl antibodies, thus serving as markers and be directly used for detecting or genetic diseases or cancers, as described herein, using methods known in the art and described herein.
  • ztrypl polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome l lq23-24 deletions, LOH, and translocations associated with human diseases, other translocations involved with malignant progression of tumors or other l lq23-24 mutations, which are expected to be involved in chromosome rearrangements in malignancy; or in other cancers.
  • ztrypl polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome l lq23-24 trisomy and chromosome loss (e.g. LOH) associated with human diseases or spontaneous abortion.
  • chromosome loss e.g. LOH
  • ztrypl polynucleotide probes can be used to detect allelic differences between diseased or non-diseased individuals at the ztrypl chromosomal locus. As such, the ztrypl sequences can be used as diagnostics in forensic DNA profiling.
  • Analytical probes will be generally at least 20 nt in length, although somewhat shorter probes can be used (e.g., 14-17 nt).
  • PCR primers are at least 5 nt in length, preferably 15 or more, more preferably 20-30 nt.
  • a ztrypl polynucleotide probe may comprise an entire exon or more. Exons are readily determined by one of skill in the art by comparing ztrypl sequences (SEQ DD NOT or SEQ DD NO: 14) with the genomic DNA for ztrypl.
  • Most diagnostic methods comprise the steps of (a) obtaining a genetic sample from a potentially diseased patient, diseased patient or potential non-diseased carrier of a recessive disease allele; (b) producing a first reaction product by incubating the genetic sample with a ztrypl polynucleotide probe wherein the polynucleotide will hybridize to complementary polynucleotide sequence, such as in RFLP analysis or by incubating the genetic sample with sense and antisense primers in a PCR reaction under appropriate PCR reaction conditions; (iii) Visualizing the first reaction product by gel electrophoresis and/or other known method such as visualizing the first reaction product with a ztrypl polynucleotide probe wherein the polynucleotide will hybridize to the complementary polynucleotide sequence of the first reaction; and (iv) comparing the visualized first reaction product to a second control reaction product of a genetic sample from wild type patient.
  • a difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the diseased or potentially diseased patient, or the presence of a heterozygous recessive carrier phenotype for a non-diseased patient, or the presence of a genetic defect in a tumor from a diseased patient, or the presence of a genetic abnormality in a fetus or pre -implantation embryo.
  • a difference in restriction fragment pattern, length of PCR products, length of repetitive sequences at the ztrypl genetic locus, and the like are indicative of a genetic abnormality, genetic aberration, or allelic difference in comparison to the normal wild type control. Controls can be from unaffected family members, or unrelated individuals, depending on the test and availability of samples.
  • Genetic samples for use within the present invention include genomic DNA, mRNA, and cDNA isolated form any tissue or other biological sample from a patient, such as but not limited to, blood, saliva, semen, embryonic cells, amniotic fluid, and the like.
  • the polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NOT or SEQ DD NO: 14, the complement of SEQ ED NOT or SEQ ED NO: 14, or an RNA equivalent thereof.
  • Such methods of showing genetic linkage analysis to human disease phenotypes are well known in the art. For reference to PCR based methods in diagnostics see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc.
  • Aberrations associated with the ztrypl locus can be detected using nucleic acid molecules of the present invention by employing standard methods for direct mutation analysis, such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted mismatch analysis, and other genetic analysis techniques known in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995), Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc.
  • standard methods for direct mutation analysis such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-a
  • mice engineered to express the ztrypl gene referred to as "transgenic mice,” and mice that exhibit a complete absence of ztrypl gene function, referred to as “knockout mice,” may also be generated (Snouwaert et al., Science 257:1083, 1992; Lowell et al., Nature 366:740-42, 1993; Capecchi, M.R., Science 244: 1288-1292, 1989; Palmiter, R.D. et al. Annu Rev Genet. 20: 465-499, 1986).
  • transgenic mice that over-express ztrypl, either ubiquitously or under a tissue-specific or tissue-restricted promoter can be used to ask whether over-expression causes a phenotype.
  • over-expression of a wild-type ztrypl polypeptide, polypeptide fragment or a mutant thereof may alter normal cellular processes, resulting in a phenotype that identifies a tissue in which ztrypl expression is functionally relevant and may indicate a therapeutic target for the ztrypl, its agonists or antagonists.
  • a preferred transgenic mouse to engineer is one that over-expresses the ztrypl active polypeptide (approximately amino acids 44 (Val) to 276 (De) of SEQ DD NO:2; approximately amino acids 43 (Val) to 275 (Arg) of SEQ DD NO: 15).
  • over-expression may result in a phenotype that shows similarity with human diseases.
  • knockout ztrypl mice can be used to determine where ztrypl is absolutely required in vivo. The phenotype of knockout mice is predictive of the in vivo effects of that a ztrypl antagonist, such as those described herein, may have.
  • the mouse ztrypl cDNA can be used to generate knockout mice or used to isolate murine ztrypl mRNA, and genomic DNA, which are subsequently used to generate knockout mice. These mice may be employed to study the ztrypl gene and the protein encoded thereby in an in vivo system, and can be used as in vivo models for corresponding human diseases. Moreover, transgenic mice expression of ztrypl antisense polynucleotides or ribozymes directed against ztrypl, described herein, can be used analogously to transgenic mice described above.
  • Polynucleotides and polypeptides of the present invention will additionally find use as educational tools as a laboratory practicum kits for courses related to genetics and molecular biology, protein chemistry and antibody production and analysis. Due to its unique polynucleotide and polypeptide sequence molecules of Ztrypl can be used as standards or as "unknowns" for testing purposes.
  • Ztrypl polynucleotides can be used as an aid, such as, for example, to teach a student how to prepare expression constructs for bacterial, viral, and/or mammalian expression, including fusion constructs, wherein Ztrypl is the gene to be expressed; for determining the restriction endonuclease cleavage sites of the polynucleotides; determining mRNA and DNA localization of Ztrypl polynucleotides in tissues (i.e., by Northern and Southern blotting as well as polymerase chain reaction); and for identifying related polynucleotides and polypeptides by nucleic acid hybridization.
  • Ztrypl polypeptides can be used educationally as an aid to teach preparation of antibodies; identifying proteins by Western blotting; protein purification; determining the weight of expressed Ztrypl polypeptides as a ratio to total protein expressed; identifying peptide cleavage sites; coupling amino and carboxyl terminal tags; amino acid sequence analysis, as well as, but not limited to monitoring biological activities of both the native and tagged protein (i.e., receptor binding, signal transduction, proliferation, and differentiation) in vitro and in vivo.
  • native and tagged protein i.e., receptor binding, signal transduction, proliferation, and differentiation
  • Ztrypl polypeptides can also be used to teach analytical skills such as mass spectrometry, circular dichroism to determine conformation, especially of the four alpha helices, x-ray crystallography to determine the three-dimensional structure in atomic detail, nuclear magnetic resonance spectroscopy to reveal the structure of proteins in solution.
  • analytical skills such as mass spectrometry, circular dichroism to determine conformation, especially of the four alpha helices, x-ray crystallography to determine the three-dimensional structure in atomic detail, nuclear magnetic resonance spectroscopy to reveal the structure of proteins in solution.
  • a kit containing the Ztrypl can be given to the student to analyze. Since the amino acid sequence would be known by the professor, the protein can be given to the student as a test to determine the skills or develop the skills of the student, the teacher would then know whether or not the student has correctly analyzed the polypeptide. Since every polypeptide is unique, the educational utility of Ztrypl would be unique unto itself.
  • the antibodies which bind specifically to Ztrypl can be used as a teaching aid to instruct students how to prepare affinity chromatography columns to purify Ztrypl, cloning and sequencing the polynucleotide that encodes an antibody and thus as a practicum for teaching a student how to design humanized antibodies.
  • the Ztrypl gene, polypeptide or antibody would then be packaged by reagent companies and sold to universities so that the students gain skill in art of molecular biology. Because each gene and protein is unique, each gene and protein creates unique challenges and learning experiences for students in a lab practicum. Such educational kits containing the Ztrypl gene, polypeptide or antibody are considered within the scope of the present invention.
  • the PCR product was run on a 2% Agarose gel to identify the 203 bp band expected for positive clones (those containing the OST).
  • the library was de-convoluted down to a positive pool of 250 clones.
  • E.coli DH10B cells (Gibco BRL) were transformed with this 250 clone pool by electroporation following manufacturer's protocol. The transformed culture was titered and then diluted and arrayed into 96 wells at approximately 20 cells/well. The cells were grown up in LB+amp overnight at 37°C.
  • 5' RACE was carried out with oligonucleotides ZC9,739 (SEQ ED NO:6) (API) and ZC 18,364 (SEQ ED NO:4) using cDNA prepared from mouse testis RNA (Clontech) using a Marathon cDNA kit (Clontech).
  • PCR conditions were as follows: one cycle at 94°C for 2 minutes; 5 cycles at 94°C for 30 seconds, 72°C for 1.5 minutes; 30 cycles at 94°C for 30 seconds, 64°C for 20 seconds, 72°C for 1 min. 15 sec; one cycle at 72°C for 5 minutes; followed by 4°C hold.
  • nested RACE was carried out with oligonucleotides ZC9,719 (SEQ DD NO:7) (AP2) and ZC20,900 (SEQ ED NO:8).
  • PCR conditions were as follows: one cycle at 94°C for 3 minutes; 22 cycles at 94°C for 30 sec, 62°C for 20 sec, 72°C for 40 sec; one cycle at 72°C for 5 min.; followed by 4°C hold.
  • the nested PCR reaction was electrophoresed on a 2% agarose gel and a 0.5 kb band was excised and gel purified using QiaexE reagents (Qiagen) according to the manufacturer's protocol. Sequence analysis of the isolated RACE product extended the 5' end of ztrypl to the initiating MET. The full length sequence of mouse ztrypl is shown in SEQ DD NOT and its corresponding amino acid sequence shown in SEQ ED NO:2.
  • Sequencing primers include: ZC 14,063 (SEQ DD NO:9), ZC5,020 (SEQ DD NO: 10), ZC18,364 (SEQ ED NO:4), ZC18,365 (SEQ ED NO:5), ZC20 71 (SEQ ED NOT 1), ZC20,900 (SEQ ED NO:8) and ZC9/719 (SEQ ED NO:7).
  • Example 2
  • the probe was purified using a NUCTRAP push column (Stratagene Cloning Systems, La Jolla, CA).
  • EXPRESSHYB (Clontech, Palo Alto, CA) solution was used for prehybridization and as a hybridizing solution for the Northern blots. Hybridization took place overnight at 55°C. using 1.5 x 10 6 cpm/ml labeled probe. The blots were then washed in 2X SSC,
  • Murine Ztrypl was mapped in mouse to chromosome 9 using the commercially available mouse T31 whole genome radiation hybrid (WGRH) panel (Research Genetics, Inc., Huntsville, AL) and Map Manager QT linkage analysis program.
  • WGRH whole genome radiation hybrid
  • the T31 WGRH panel contains DNAs from each of 100 radiation hybrid clones, plus two control DNAs (the 129aa donor and the A23 recipient).
  • 20 ⁇ l reactions were set up in 96- well microtiter plates (Stratagene, La Jolla, CA) and used in a "RoboCycler Gradient 96" thermal cycler (Stratagene).
  • Each of the 102 PCR reactions consisted of 2 ⁇ l 10X KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, CA), 1.6 ⁇ l dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, CA), 1 ⁇ l sense primer, ZC22,855, (SEQ FD NO: 12), 1 ⁇ l antisense primer, ZC22,856 (SEQ ED NO: 13), 2 ⁇ l "RediLoad” (Research Genetics, Inc., Huntsville, AL), 0.4 ⁇ l 50X Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and ddH 2 O for a total volume of 20 ⁇ l.
  • the reactions were overlaid with an equal amount of mineral oil and sealed.
  • the PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 94°C, 35 cycles of a 45 seconds denaturation at 94°C, 45 seconds annealing at 64°C and 1 minute and 15 seconds extension at 72°C, followed by a final 1 cycle extension of 7 minutes at 72°C.
  • the reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD).
  • a 2% agarose gel Life Technologies, Gaithersburg, MD.
  • a panel of cDNAs from human tissues was screened for human ztrypl expression using PCR.
  • the panel was made in-house and contained 94 marathon cDNA and cDNA samples from various normal and cancerous human tissues and cell lines and is shown in Table 5, below.
  • the cDNAs came from in-house libraries or marathon cDNAs from in-house RNA preps, Clontech RNA, or Invitrogen RNA.
  • the marathon cDNAs were made using the marathon-ReadyTM kit (Clontech, Palo Alto, CA) and QC tested with clathrin primers ZC21,195 (SEQ ED NOT7) and ZC21,196 (SEQ DD NO: 18) and then diluted based on the intensity of the clathrin band.
  • the panel was set up in a 96-well format that included a human genomic DNA (Clontech, Palo Alto, CA) positive control sample. Each well contained approximately 0.2-100 pg/ ⁇ l of cDNA.
  • the PCR reactions for human ztrypl were set up using oligos ZC28,936 (SEQ ED NO:22) and ZC28,937 (SEQ ED NO:23). The amplification was carried out as follows: 1 cycle at 94°C for 2 minutes, 5 cycles of 94°C for 15 seconds, 70°C for 30 seconds, followed by 35 cycles at 94°C for 15 seconds, 62°C for 20 seconds and 72°C for 30 seconds, followed by 1 cycle at 72°C for 5 minutes.

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Abstract

La présente invention concerne des molécules polynucléotidiques et polypeptidiques destinées à un ztryp1 de souris, un nouveau membre de la famille des protéases de sérine de protéines. Ces polynucléotides codant pour ztryp1 de souris peuvent être utilisés pour identifier un orthologue humain ou pour créer un modèle de souris associé à des stades de maladie humaine. Cette invention concerne aussi des techniques de production de cette protéine, des utilisations de celle-ci et des anticorps dirigés contre celle-ci.
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