Isolated human serine protease, nucleic acid molecules encoding human serine protease, and uses thereof

Abstract

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the protease peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the protease peptides, and methods of identifying modulators of the protease peptides.

Description

FIELD OF THE INVENTION

The present invention is in the field of protease proteins that are related to the serine protease subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect protein cleavage/processing/turnover and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION

The proteases may be categorized into families by the different amino acid sequences (generally between 2 and 10 residues) located on either side of the cleavage site of the protease.

The proper functioning of the cell requires careful control of the levels of important structural proteins, enzymes, and regulatory proteins. One of the ways that cells can reduce the steady state level of a particular protein is by proteolytic degradation. Further, one of the ways cells produce functioning proteins is to produce pre or pro-protein precursors that are processed by proteolytic degradation to produce an active moiety. Thus, complex and highly-regulated mechanisms have been evolved to accomplish this degradation.

Proteases regulate many different cell proliferation, differentiation, and signaling processes by regulating protein turnover and processing. Uncontrolled protease activity (either increased or decreased) has been implicated in a variety of disease conditions including inflammation, cancer, arteriosclerosis, and degenerative disorders.

An additional role of intracellular proteolysis is in the stress-response. Cells that are subject to stress such as starvation, heat-shock, chemical insult or mutation respond by increasing the rates of proteolysis. One function of this enhanced proteolysis is to salvage amino acids from non-essential proteins. These amino acids can then be re-utilized in the synthesis of essential proteins or metabolized directly to provide energy. Another function is in the repair of damage caused by the stress. For example, oxidative stress has been shown to damage a variety of proteins and cause them to be rapidly degraded.

The International Union of Biochemistry and Molecular Biology (IUBMB) has recommended to use the term peptidase for the subset of peptide bond hydrolases (Subclass E.C 3.4.). The widely used term protease is synonymous with peptidase. Peptidases comprise two groups of enzymes: the endopeptidases and the exopeptidases, which cleave peptide bonds at points within the protein and remove amino acids sequentially from either N or C-terminus respectively. The term proteinase is also used as a synonym word for endopeptidase and four mechanistic classes of proteinases are recognized by the IUBMB: two of these are described below (also see: Handbook of Proteolytic Enzymes by Barrett, Rawlings, and Woessner AP Press, NY 1998). Also, for a review of the various uses of proteases as drug targets, see: Weber M, Emerging treatments for hypertension: potential role for vasopeptidase inhibition; Am J Hypertens 1999 November;12(11 Pt 2):139S-147S; Kentsch M, Otter W, Novel neurohormonal modulators in cardiovascular disorders. The therapeutic potential of endopeptidase inhibitors, Drugs R D 1999 April;1(4):331-8; Scarborough R M, Coagulation factor Xa: the prothrombinase complex as an emerging therapeutic target for small molecule inhibitors, J Enzym Inhib 1998;14(1):15-25; Skotnicki J S, et al., Design and synthetic considerations of matrix metalloproteinase inhibitors, Ann N Y Acad Sci 1999 June 30;878:61-72; McKerrow J H, Engel J C, Caffrey C R, Cysteine protease inhibitors as chemotherapy for parasitic infections, Bioorg Med Chem 1999 April;7(4):639-44; Rice K D, Tanaka R D, Katz B A, Numerof R P, Moore W R, Inhibitors of tryptase for the treatment of mast cell-mediated diseases, Curr Pharm Des 1998 October;4(5):381-96; Materson B J, Will angiotensin converting enzyme genotype, receptor mutation identification, and other miracles of molecular biology permit reduction of NNT Am J Hypertens 1998 August;11(8 Pt 2):138S-142S.

Serine Proteases

The serine proteases (SP) are a large family of proteolytic enzymes that include the digestive enzymes, trypsin and chymotrypsin, components of the complement cascade and of the blood-clotting cascade, and enzymes that control the degradation and turnover of macromolecules of the extracellular matrix. SP are so named because of the presence of a serine residue in the active catalytic site for protein cleavage. SP have a wide range of substrate specificities and can be subdivided into subfamilies on the basis of these specificities. The main sub-families are trypases (cleavage after arginine or lysine), aspases (cleavage after aspartate), chymases (cleavage after phenylalanine or leucine), metases (cleavage after methionine), and serases (cleavage after serine).

A series of six SP have been identified in murine cytotoxic T-lymphocytes (CTL) and natural killer (NK) cells. These SP are involved with CTL and NK cells in the destruction of virally transformed cells and tumor cells and in organ and tissue transplant rejection (Zunino, S. J. et al. (1990) J. Immunol. 144:2001-9; Sayers, T. J. et al. (1994) J. Immunol. 152:2289-97). Human homologs of most of these enzymes have been identified (Trapani, J. A. et al. (1988) Proc. Natl. Acad. Sci. 85:6924-28; Caputo, A. et al. (1990) J. Immunol. 145:737-44). Like all SP, the CTL-SP share three distinguishing features: 1) the presence of a catalytic triad of histidine, serine, and aspartate residues which comprise the active site; 2) the sequence GDSGGP which contains the active site serine; and 3) an N-terminal IIGG sequence which characterizes the mature SP.

The SP are secretory proteins which contain N-terminal signal peptides that serve to export the immature protein across the endoplasmic reticulum and are then cleaved (von Heijne (1986) Nuc. Acid. Res. 14:5683-90). Differences in these signal sequences provide one means of distinguishing individual SP. Some SP, particularly the digestive enzymes, exist as inactive precursors or preproenzymes, and contain a leader or activation peptide sequence 3′ of the signal peptide. This activation peptide may be 2-12 amino acids in length, and it extends from the cleavage site of the signal peptide to the N-terminal IIGG sequence of the active, mature protein. Cleavage of this sequence activates the enzyme. This sequence varies in different SP according to the biochemical pathway and/or its substrate (Zunino et al, supra; Sayers et al, supra). Other features that distinguish various SP are the presence or absence of N-linked glycosylation sites that provide membrane anchors, the number and distribution of cysteine residues that determine the secondary structure of the SP, and the sequence of a substrate binding sites such as S′. The S′ substrate binding region is defined by residues extending from approximately +17 to +29 relative to the N-terminal I (+1). Differences in this region of the molecule are believed to determine SP substrate specificities (Zunino et al, supra).

Trypsin-like serine proteases have been isolated from patients with chronic airway diseases and may play a role in respiratory diseases and host defense systems on the mucous membranes of the respiratory system (see Yamaoka et al., J. Biol. Chem. 273: 11895-11901, 1998 and Yasuoka et al., Am. J. Resp. Cell Molec. Biol. 16: 300-308, 1997). Therefore, novel human serine protease proteins, and encoding genes, may be useful for screening for, diagnosing, preventing, and/or treating disorders such as respiratory diseases. For example, serine protease genes/proteins may be useful in drug development, such as by serving as novel drug targets for respiratory disease, and SNPs in serine protease genes may be useful markers for diagnostic kits for respiratory diseases.

Trypsinogens

The trypsinogens are serine proteases secreted by exocrine cells of the pancreas (Travis J and Roberts R. Biochemistry 1969; 8: 2884-9; Mallory P and Travis J, Biochemistry 1973; 12: 2847-51). Two major types of trypsinogen isoenzymes have been characterized, trypsinogen-1, also called cationic trypsinogen, and trypsinogen-2 or anionic trypsinogen. The trypsinogen proenzymes are activated to trypsins in the intestine by enterokinase, which removes an activation peptide from the N-terminus of the trypsinogens. The trypsinogens show a high degree of sequence homology, but they can be separated on the basis of charge differences by using electrophoresis or ion exchange chromatography. The major form of trypsinogen in the pancreas and pancreatic juice is trypsinogen-1 (Guy C O et al., Biochem Biophys Res Commun 1984; 125: 516-23). In serum of healthy subjects, trypsinogen-1 is also the major form, whereas in patients with pancreatitis, trypsinogen-2 is more strongly elevated (Itkonen et al., J Lab Clin Med 1990; 115:712-8). Trypsinogens also occur in certain ovarian tumors, in which trypsinogen-2 is the major form (Koivunen et al., Cancer Res 1990; 50: 2375-8). Trypsin-1 in complex with alpha-1-antitrypsin, also called alpha-1-antiprotease, has been found to occur in serum of patients with pancreatitis (Borgstrom A and Ohlsson K, Scand J Clin Lab Invest 1984; 44: 381-6) but determination of this complex has not been found useful for differentiation between pancreatic and other gastrointestinal diseases (Borgstrom et al., Scand J Clin Lab Invest 1989; 49:757-62).

Trypsinogen-1 and -2 are closely related immunologically (Kimland et al., Clin Chim Acta 1989; 184: 31-46; Itkonen et al., 1990), but by using monoclonal antibodies (Itkonen et al., 1990) or by absorbing polyclonal antisera (Kimland et al., 1989) it is possible to obtain reagents enabling specific measurement of each form of trypsinogen.

When active trypsin reaches the blood stream, it is inactivated by the major trypsin inhibitors alpha-2-macroglobulin and alpha-1-antitrypsin (AAT). AAT is a 58 kilodalton serine protease inhibitor synthesized in the liver and is one of the main protease inhibitors in blood. Whereas complexes between trypsin-1 and AAT are detectable in serum (Borgstrom and Ohlsson, 1984) the complexes with alpha-2-macroglobulin are not measurable with antibody-based assays (Ohlsson K, Acta Gastroenterol Belg 1988; 51: 3-12).

Inflammation of the pancreas or pancreatitis may be classified as either acute or chronic by clinical criteria. With treatment, acute pancreatitis can often be cured and normal function restored. Chronic pancreatitis often results in permanent damage. The precise mechanisms which trigger acute inflammation are not understood. However, some causes in the order of their importance are alcohol ingestion, biliary tract disease, post-operative trauma, and hereditary pancreatitis. One theory provides that autodigestion, the premature activation of proteolytic enzymes in the pancreas rather than in the duodenum, causes acute pancreatitis. Any number of other factors including endotoxins, exotoxins, viral infections, ischemia, anoxia, and direct trauma may activate the proenzymes. In addition, any internal or external blockage of pancreatic ducts can also cause an accumulation of pancreatic juices in the pancreas resulting cellular damage.

Anatomy, physiology, and diseases of the pancreas are reviewed, inter alia, in Guyton A C (1991) Textbook of Medical Physiology, W B Saunders Co, Philadelphia Pa.; Isselbacher K J et al (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, New York City; Johnson K E (1991) Histology and Cell Biology, Harwal Publishing, Media Pa.; and The Merck Manual of Diagnosis and Therapy (1992) Merck Research Laboratories, Rahway N.J.

Metalloprotease

The metalloproteases may be one of the older classes of proteinases and are found in bacteria, fungi as well as in higher organisms. They differ widely in their sequences and their structures but the great majority of enzymes contain a zinc atom which is catalytically active. In some cases, zinc may be replaced by another metal such as cobalt or nickel without loss of the activity. Bacterial thermolysin has been well characterized and its crystallographic structure indicates that zinc is bound by two histidines and one glutamic acid. Many enzymes contain the sequence HEXXH, which provides two histidine ligands for the zinc whereas the third ligand is either a glutamic acid (thermolysin, neprilysin, alanyl aminopeptidase) or a histidine (astacin). Other families exhibit a distinct mode of binding of the Zn atom. The catalytic mechanism leads to the formation of a non covalent tetrahedral intermediate after the attack of a zinc-bound water molecule on the carbonyl group of the scissile bond. This intermediate is further decomposed by transfer of the glutamic acid proton to the leaving group.

Metalloproteases contain a catalytic zinc metal center which participates in the hydrolysis of the peptide backbone (reviewed in Power and Harper, in Protease Inhibitors, A. J. Barrett and G. Salversen (eds.) Elsevier, Amsterdam, 1986, p. 219). The active zinc center differentiates some of these proteases from calpains and trypsins whose activities are dependent upon the presence of calcium. Examples of metalloproteases include carboxypeptidase A, carboxypeptidase B, and thermolysin.

Metalloproteases have been isolated from a number of procaryotic and eucaryotic sources, e.g. Bacillus subtilis (McConn et al., 1964, J. Biol. Chem. 239:3706); Bacillus megaterium ; Serratia (Miyata et al., 1971, Agr. Biol. Chem. 35:460); Clostridium bifermentans (MacFarlane et al., 1992, App. Environ. Microbiol. 58:1195-1200), Legionella pneumophila (Moffat et al., 1994, Infection and Immunity 62:751-3). In particular, acidic metalloproteases have been isolated from broad-banded copperhead venoms (Johnson and Ownby, 1993, Int. J. Biochem. 25:267-278), rattlesnake venoms (Chlou et al., 1992, Biochem. Biophys. Res. Commun. 187:389-396) and articular cartilage (Treadwell et al., 1986, Arch. Biochem. Biophys. 251:715-723). Neutral metalloproteases, specifically those having optimal activity at neutral pH have, for example, been isolated from Aspergillus sojae (Sekine, 1973, Agric. Biol. Chem. 37:1945-1952). Neutral metalloproteases obtained from Aspergillus have been classified into two groups, npI and npII (Sekine, 1972, Agric. Biol. Chem. 36:207-216). So far, success in obtaining amino acid sequence information from these fungal neutral metalloproteases has been limited. An npII metalloprotease isolated from Aspergillus oryzae has been cloned based on amino acid sequence presented in the literature (Tatsumi et al., 1991, Mol. Gen. Genet. 228:97-103). However, to date, no npI fungal metalloprotease has been cloned or sequenced. Alkaline metalloproteases, for example, have been isolated from Pseudomonas aeruginosa (Baumann et al., 1993, EMBO J 12:3357-3364) and the insect pathogen Xenorhabdus luminescens (Schmidt et al., 1998, Appl. Environ. Microbiol. 54:2793-2797).

Metalloproteases have been devided into several distinct families based primarily on activity and sturcture: 1) water nucleophile; water bound by single zinc ion ligated to two His (within the motif HEXXH) and Glu, His or Asp; 2) water nucleophile; water bound by single zinc ion ligated to His, Glu (within the motif HXXE) and His; 3) water nucleophile; water bound by single zinc ion ligated to His, Asp and His; 4) Water nucleophile; water bound by single zinc ion ligated to two His (within the motif HXXEH) and Glu and 5) water nucleophile; water bound by two zinc ions ligated by Lys, Asp, Asp, Asp, Glu.

Examples of members of the metalloproteinase family include, but are not limited to, membrane alanyl aminopeptidase ( Homo sapiens ), germinal peptidyl-dipeptidase A ( Homo sapiens ), thimet oligopeptidase ( Rattus norvegicus ), oligopeptidase F ( Lactococcus lactis ), mycolysin ( Streptomyces cacaoi ), immune inhibitor A ( Bacillus thuringiensis ), snapalysin ( Streptomyces lividans ), leishmanolysin ( Leishmania major ), microbial collagenase ( Vibrio alginolyticus ), microbial collagenase, class I ( Clostridium perfringens ), collagenase 1 ( Homo sapiens ), serralysin ( Serratia marcescens ), fragilysin ( Bacteroides fragilis ), gametolysin ( Chlamydomonas reinhardtii ), astacin ( Astacus fluviatilis ), adamalysin ( Crotalus adamanteus ), ADAM 10 ( Bos taurus ), neprilysin ( Homo sapiens ), carboxypeptidase A ( Homo sapiens ), carboxypeptidase E ( Bos taurus ), gamma-D-glutamyl-(L)-meso-diaminopimelate peptidase I ( Bacillus sphaericus ), vanY D-Ala-D-Ala carboxypeptidase ( Enterococcus faecium ), endolysin (bacteriophage A118), pitrilysin ( Escherichia coli ), mitochondrial processing peptidase ( Saccharomyces cerevisiae ), leucyl aminopeptidase ( Bos taurus ), aminopeptidase I ( Saccharomyces cerevisiae ), membrane dipeptidase ( Homo sapiens ), glutamate carboxypeptidase (Pseudomonas sp.), Gly-X carboxypeptidase ( Saccharomyces cerevisiae ), O-sialoglycoprotein endopeptidase ( Pasteurella haemolytica ), beta-lytic metalloendopeptidase ( Achromobacter lyticus ), methionyl aminopeptidase I ( Escherichia coli ), X-Pro aminopeptidase ( Escherichia coli ), X-His dipeptidase ( Escherichia coli ), IgA1-specific metalloendopeptidase ( Streptococcus sanguis ), tentoxilysin ( Clostridium tetani ), leucyl aminopeptidase ( Vibrio proteolyticus ), aminopeptidase ( Streptomyces griseus ), IAP aminopeptidase ( Escherichia coli ), aminopeptidase T ( Thermus aquaticus ), hyicolysin ( Staphylococcus hyicus ), carboxypeptidase Taq ( Thermus aquaticus ), anthrax lethal factor ( Bacillus anthracis ), penicillolysin ( Penicillium citrinum ), fungalysin ( Aspergillus fumigatus ), lysostaphin ( Staphylococcus simulans ), beta-aspartyl dipeptidase ( Escherichia coli ), carboxypeptidase Ss1 ( Sulfolobus solfataricus ), FtsH endopeptidase ( Escherichia coli ), glutamyl aminopeptidase ( Lactococcus lactis ), cytophagalysin (Cytophaga sp.), metalloendopeptidase (vaccinia virus), VanX D-Ala-D-Ala dipeptidase ( Enterococcus faecium ), Ste24p endopeptidase ( Saccharomyces cerevisiae ), dipeptidyl-peptidase III ( Rattus norvegicus ), S2P protease ( Homo sapiens ), sporulation factor SpoIVFB ( Bacillus subtilis ), and HYBD endopeptidase ( Escherichia coli ).

Metalloproteases have been found to have a number of uses. For example, there is strong evidence that a metalloprotease is involved in the in vivo proteolytic processing of the vasoconstrictor, endothelin-1. Rat metalloprotease has been found to be involved in peptide hormone processing. One important subfamily of the metalloproteases are the matrix metalloproteases.

A number of diseases are thought to be mediated by excess or undesired metalloprotease activity or by an imbalance in the ratio of the various members of the protease family of proteins. These include: a) osteoarthritis (Woessner, et al., J. Biol.Chem. 259(6), 3633, 1984; Phadke, et al., J. Rheumatol. 10, 852, 1983), b) rheumatoid arthritis (Mullins, et al., Biochim. Biophys. Acta 695, 117, 1983; Woolley, et al., Arthritis Rheum. 20, 1231, 1977; Gravallese, et al., Arthritis Rheum. 34, 1076, 1991), c) septic arthritis (Williams, et al., Arthritis Rheum. 33, 533, 1990), d) tumor metastasis (Reich, et al., Cancer Res. 48, 3307, 1988, and Matrisian, et al., Proc. Nat'l. Acad. Sci., USA 83, 9413, 1986), e) periodontal diseases (Overall, et al., J. Periodontal Res. 22, 81, 1987), f) corneal ulceration (Burns, et al., Invest. Opthalmol. Vis. Sci. 30, 1569, 1989), g) proteinuria (Baricos, et al., Biochem. J. 254, 609, 1988), h) coronary thrombosis from atherosclerotic plaque rupture (Henney, et al., Proc. Nat'l. Acad. Sci., USA 88, 8154-8158, 1991), i) aneurysmal aortic disease (Vine, et al., Clin. Sci. 81, 233, 1991), j) birth control (Woessner, et al., Steroids 54, 491, 1989), k) dystrophobic epidermolysis bullosa (Kronberger, et al., J. Invest. Dermatol. 79, 208, 1982), and 1) degenerative cartilage loss following traumatic joint injury, m) conditions leading to inflammatory responses, osteopenias mediated by MMP activity, n) tempero mandibular joint disease, o) demyelating diseases of the nervous system (Chantry, et al., J. Neurochem. 50, 688, 1988).

Aspartic Protease

Aspartic proteases have been divided into several distinct families based primarily on activity and structure. These include 1) water nucleophile; water bound by two Asp from monomer or dimer; all endopeptidases, from eukaryote organisms, viruses or virus-like organisms and 2) endopeptidases that are water nucleophile and are water bound by Asp and Asn.

Most of aspartic proteases belong to the pepsin family. The pepsin family includes digestive enzymes such as pepsin and chymosin as well as lysosomal cathepsins D and processing enzymes such as renin, and certain fungal proteases (penicillopepsin, rhizopuspepsin, endothiapepsin). A second family comprises viral proteases such as the protease from the AIDS virus (HIV) also called retropepsin. Crystallographic studies have shown that these enzymes are bilobed molecules with the active site located between two homologous lobes. Each lobe contributes one aspartate residue of the catalytically active diad of aspartates. These two aspartyl residues are in close geometric proximity in the active molecule and one aspartate is ionized whereas the second one is unionized at the optimum pH range of 2-3. Retropepsins, are monomeric, i.e carry only one catalytic aspartate and then dimerization is required to form an active enzyme.

In contrast to serine and cysteine proteases, catalysis by aspartic protease do not involve a covalent intermediate though a tetrahedral intermediate exists. The nucleophilic attack is achieved by two simultaneous proton transfer: one from a water molecule to the diad of the two carboxyl groups and a second one from the diad to the carbonyl oxygen of the substrate with the concurrent CO—NH bond cleavage. This general acid-base catalysis, which may be called a “push-pull” mechanism leads to the formation of a non covalent neutral tetrahedral intermediate.

Examples of the aspartic protease family of proteins include, but are not limited to, pepsin A ( Homo sapiens ), HIV1 retropepsin (human immunodeficiency virus type 1), endopeptidase (cauliflower mosaic virus), bacilliform virus putative protease (rice tungro bacilliform virus), aspergillopepsin II ( Aspergillus niger ), thermopsin ( Sulfolobus acidocaldarius ), nodavirus endopeptidase (flock house virus), pseudomonapepsin (Pseudomonas sp. 101), signal peptidase II ( Escherichia coli ), polyprotein peptidase (human spumaretrovirus), copia transposon ( Drosophila melanogaster ), SIRE-1 peptidase ( Glycine max ), retrotransposon bs1 endopeptidase ( Zea mays ), retrotransposon peptidase ( Drosophila buzzatii ), Tas retrotransposon peptidase ( Ascaris lumbricoides ), Pao retrotransposon peptidase ( Bombyx mori ), putative proteinase of Skippy retrotransposon ( Fusarium oxysporum ), tetravirus endopeptidase ( Nudaurelia capensis omega virus), presenilin 1 ( Homo sapiens ).

Proteases and Cancer

Proteases are critical elements at several stages in the progression of metastatic cancer. In this process, the proteolytic degradation of structural protein in the basal membrane allows for expansion of a tumor in the primary site, evasion from this site as well as homing and invasion in distant, secondary sites. Also, tumor induced angiogenesis is required for tumor growth and is dependent on proteolytic tissue remodeling. Transfection experiments with various types of proteases have shown that the matrix metalloproteases play a dominant role in these processes in particular gelatinases A and B (MMP-2 and MMP-9, respectively). For an overview of this field see Mullins, et al., Biochim. Biophys. Acta 695, 177, 1983; Ray, et al., Eur. Respir. J. 7, 2062, 1994; Birkedal-Hansen, et al., Crit. Rev. Oral Biol. Med. 4, 197, 1993.

Furthermore, it was demonstrated that inhibition of degradation of extracellular matrix by the native matrix metalloprotease inhibitor TIMP-2 (a protein) arrests cancer growth (DeClerck, et al., Cancer Res. 52, 701, 1992) and that TIMP-2 inhibits tumor-induced angiogenesis in experimental systems (Moses, et al. Science 248, 1408, 1990). For a review, see DeClerck, et al., Ann. N. Y. Acad. Sci. 732, 222, 1994. It was further demonstrated that the synthetic matrix metalloprotease inhibitor batimastat when given intraperitoneally inhibits human colon tumor growth and spread in an orthotopic model in nude mice (Wang, et al. Cancer Res. 54, 4726, 1994) and prolongs the survival of mice bearing human ovarian carcinoma xenografts (Davies, et. al., Cancer Res. 53, 2087, 1993). The use of this and related compounds has been described in Brown, et al., WO-9321942 A2.

There are several patents and patent applications claiming the use of metalloproteinase inhibitors for the retardation of metastatic cancer, promoting tumor regression, inhibiting cancer cell proliferation, slowing or preventing cartilage loss associated with osteoarthritis or for treatment of other diseases as noted above (e.g. Levy, et al., WO-9519965 A1; Beckett, et al., WO-9519956 A1; Beckett, et al., WO-9519957 A1; Beckett, et al., WO-9519961 A1; Brown, et al., WO-9321942 A2; Crimmin, et al., WO-9421625 A1; Dickens, et al., U.S. Pat. No. 4,599,361; Hughes, et al., U.S. Pat. No. 5,190,937; Broadhurst, et al., EP 574758 A1; Broadhurst, et al., EP 276436; and Myers, et al., EP 520573 A1.

Protease proteins, particularly members of the serine subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of protease proteins. The present invention advances the state of the art by providing a previously unidentified human protease proteins that have homology to members of the serine subfamily.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of amino acid sequences of human protease peptides and proteins that are related to the serine protease subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate protease activity in cells and tissues that express the protease. Experimental data as provided in FIG. 1 indicates expression in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers.

DESCRIPTION OF THE FIGURE SHEETS

FIG. 1 provides the nucleotide sequence of a cDNA molecule that encodes the protease protein of the present invention. (SEQ ID NO:1) In addition, structure and functional information is provided, such as ATG start, stop and tissue distribution, where available, that allows one to readily determine specific uses of inventions based on this molecular sequence. Experimental data as provided in FIG. 1 indicates expression in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers.

FIG. 2 provides the predicted amino acid sequence of the protease of the present invention. (SEQ ID NO:2) In addition structure and functional information such as protein family, function, and modification sites is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence.

FIG. 3 provides genomic sequences that span the gene encoding the protease protein of the present invention. (SEQ ID NO:3) In addition structure and functional information, such as intron/exon structure, promoter location, etc., is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence. As indicated in FIG. 3 , SNPs, including insertion/deletion polymorphisms (“indels”), were identified at 69 different nucleotide positions in and around the gene encoding the serine protease protein of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

General Description

The present invention is based on the sequencing of the human genome. During the sequencing and assembly of the human genome, analysis of the sequence information revealed previously unidentified fragments of the human genome that encode peptides that share structural and/or sequence homology to protein/peptide/domains identified and characterized within the art as being a protease protein or part of a protease protein and are related to the serine protease subfamily. Utilizing these sequences, additional genomic sequences were assembled and transcript and/or cDNA sequences were isolated and characterized. Based on this analysis, the present invention provides amino acid sequences of human protease peptides and proteins that are related to the serine protease subfamily, nucleic acid sequences in the form of transcript sequences, cDNA sequences and/or genomic sequences that encode these protease peptides and proteins, nucleic acid variation (allelic information), tissue distribution of expression, and information about the closest art known protein/peptide/domain that has structural or sequence homology to the protease of the present invention.

In addition to being previously unknown, the peptides that are provided in the present invention are selected based on their ability to be used for the development of commercially important products and services. Specifically, the present peptides are selected based on homology and/or structural relatedness to known protease proteins of the serine protease subfamily and the expression pattern observed. Experimental data as provided in FIG. 1 indicates expression in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers. The art has clearly established the commercial importance of members of this family of proteins and proteins that have expression patterns similar to that of the present gene. Some of the more specific features of the peptides of the present invention, and the uses thereof, are described herein, particularly in the Background of the Invention and in the annotation provided in the Figures, and/or are known within the art for each of the known serine family or subfamily of protease proteins.

Specific Embodiments

Peptide Molecules

The present invention provides nucleic acid sequences that encode protein molecules that have been identified as being members of the protease family of proteins and are related to the serine protease subfamily (protein sequences are provided in FIG. 2 , transcript/cDNA sequences are provided in FIG. 1 and genomic sequences are provided in FIG. 3 ). The peptide sequences provided in FIG. 2 , as well as the obvious variants described herein, particularly allelic variants as identified herein and using the information in FIG. 3 , will be referred herein as the protease peptides of the present invention, protease peptides, or peptides/proteins of the present invention.

The present invention provides isolated peptide and protein molecules that consist of, consist essentially of, or comprise the amino acid sequences of the protease peptides disclosed in the FIG. 2 , (encoded by the nucleic acid molecule shown in FIG. 1 , transcript/cDNA or FIG. 3 , genomic sequence), as well as all obvious variants of these peptides that are within the art to make and use. Some of these variants are described in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when it is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components (the features of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includes preparations of the peptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins. When the peptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or other chemicals” includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the protease peptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.

The isolated protease peptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. Experimental data as provided in FIG. 1 indicates expression in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers. For example, a nucleic acid molecule encoding the protease peptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell. The protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Many of these techniques are described in detail below.

Accordingly, the present invention provides proteins that consist of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). The amino acid sequence of such a protein is provided in FIG. 2. A protein consists of an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein.

The present invention further provides proteins that comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids. The preferred classes of proteins that are comprised of the protease peptides of the present invention are the naturally occurring mature proteins. A brief description of how various types of these proteins can be made/isolated is provided below.

The protease peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins. Such chimeric and fusion proteins comprise a protease peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the protease peptide. “Operatively linked” indicates that the protease peptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the protease peptide.

In some uses, the fusion protein does not affect the activity of the protease peptide per se. For example, the fusion protein can include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant protease peptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a protein can be increased by using a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al., Current Protocols in Molecular Biology, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A protease peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protease peptide.

As mentioned above, the present invention also provides and enables obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, allelic/sequence variants of the peptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the peptides. Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.

Such variants can readily be identified/made using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the protease peptides of the present invention. The degree of homology/identity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. ( Computational Molecular Biology , Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects , Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology , von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer , Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ( J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. ( J Mol. Biol. 215:403-10 1990)). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. ( Nucleic Acids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the protease peptides of the present invention as well as being encoded by the same genetic locus as the protease peptide provided herein. The gene provided by the present invention is located on a genome component that has been mapped to human chromosome 4 (as indicated in FIG. 3 ), which is supported by multiple lines of evidence, such as STS and BAC map data.

Allelic variants of a protease peptide can readily be identified as being a human protein having a high degree (significant) of sequence homology/identity to at least a portion of the protease peptide as well as being encoded by the same genetic locus as the protease peptide provided herein. Genetic locus can readily be determined based on the genomic information provided in FIG. 3 , such as the genomic sequence mapped to the reference human. The gene provided by the present invention is located on a genome component that has been mapped to human chromosome 4 (as indicated in FIG. 3 ), which is supported by multiple lines of evidence, such as STS and BAC map data. As used herein, two proteins (or a region of the proteins) have significant homology when the amino acid sequences are typically at least about 70-80%, 80-90%, and more typically at least about 90-95% or more homologous. A significantly homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence that will hybridize to a protease peptide encoding nucleic acid molecule under stringent conditions as more fully described below.

FIG. 3 provides information on SNPs that have been identified in the gene encoding the protease protein of the present invention. SNPs, including indels (indicated by a “-”), were identified at 69 different nucleotide positions. Non-synonymous cSNPs were identified at position 30496. The changes in the amino acid sequence caused by these SNPs is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. SNPs outside the ORF and in introns may affect control/regulatory elements.

Paralogs of a protease peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the protease peptide, as being encoded by a gene from humans, and as having similar activity or function. Two proteins will typically be considered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70% or greater homology through a given region or domain. Such paralogs will be encoded by a nucleic acid sequence that will hybridize to a protease peptide encoding nucleic acid molecule under moderate to stringent conditions as more fully described below.

Orthologs of a protease peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the protease peptide as well as being encoded by a gene from another organism. Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents. Such orthologs will be encoded by a nucleic acid sequence that will hybridize to a protease peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins. The gene provided by the present invention is located on a genome component that has been mapped to human chromosome 4 (as indicated in FIG. 3 ), which is supported by multiple lines of evidence, such as STS and BAC map data.

FIG. 3 provides information on SNPs that have been identified in the gene encoding the protease protein of the present invention. SNPs, including indels (indicated by a “-”), were identified at 69 different nucleotide positions. Non-synonymous cSNPs were identified at position 30496. The changes in the amino acid sequence caused by these SNPs is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. SNPs outside the ORF and in introns may affect control/regulatory elements.

Non-naturally occurring variants of the protease peptides of the present invention can readily be generated using recombinant techniques. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the protease peptide. For example, one class of substitutions are conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a protease peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).

Variant protease peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind substrate, ability to cleave substrate, ability to participate in a signaling pathway, etc. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. FIG. 2 provides the result of protein analysis and can be used to identify critical domains/regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.

Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085 (1989)), particularly using the results provided in FIG. 2 . The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as protease activity or in assays such as an in vitro proliferative activity. Sites that are critical for binding partner/substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).

The present invention further provides fragments of the protease peptides, in addition to proteins and peptides that comprise and consist of such fragments, particularly those comprising the residues identified in FIG. 2 . The fragments to which the invention pertains, however, are not to be construed as encompassing fragments that may be disclosed publicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or more contiguous amino acid residues from a protease peptide. Such fragments can be chosen based on the ability to retain one or more of the biological activities of the protease peptide or could be chosen for the ability to perform a function, e.g. bind a substrate or act as an immunogen. Particularly important fragments are biologically active fragments, peptides that are, for example, about 8 or more amino acids in length. Such fragments will typically comprise a domain or motif of the protease peptide, e.g., active site, a transmembrane domain or a substrate-binding domain. Further, possible fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments, and fragments containing immunogenic structures. Predicted domains and functional sites are readily identifiable by computer programs well known and readily available to those of skill in the art (e.g., PROSITE analysis). The results of one such analysis are provided in FIG. 2 .

Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in protease peptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art (some of these features are identified in FIG. 2 ).

Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins , B. C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. ( Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. ( Ann. N.Y. Acad Sci. 663:48-62 (1992)).

Accordingly, the protease peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature protease peptide is fused with another compound, such as a compound to increase the half-life of the protease peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature protease peptide, such as a leader or secretory sequence or a sequence for purification of the mature protease peptide or a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial and specific assays related to the functional information provided in the Figures; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or ligand) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Where the protein binds or potentially binds to another protein or ligand (such as, for example, in a protease-effector protein interaction or protease-ligand interaction), the protein can be used to identify the binding partner/ligand so as to develop a system to identify inhibitors of the binding interaction. Any or all of these uses are capable of being developed into reagent grade or kit format for commercialization as commercial products.

Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are based primarily on the source of the protein as well as the class/action of the protein. For example, proteases isolated from humans and their human/mammalian orthologs serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the protease. Experimental data as provided in FIG. 1 indicates that protease proteins of the present invention are expressed in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers. Specifically, a virtual northern blot shows expression in cancers. In addition, PCR-based tissue screening panels indicate expression in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, and bone marrow. A large percentage of pharmaceutical agents are being developed that modulate the activity of protease proteins, particularly members of the serine subfamily (see Background of the Invention). The structural and functional information provided in the Background and Figures provide specific and substantial uses for the molecules of the present invention, particularly in combination with the expression information provided in FIG. 1 . Experimental data as provided in FIG. 1 indicates expression in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers. Such uses can readily be determined using the information provided herein, that which is known in the art, and routine experimentation.

The proteins of the present invention (including variants and fragments that may have been disclosed prior to the present invention) are useful for biological assays related to proteases that are related to members of the serine subfamily. Such assays involve any of the known protease functions or activities or properties useful for diagnosis and treatment of protease-related conditions that are specific for the subfamily of proteases that the one of the present invention belongs to, particularly in cells and tissues that express the protease. Experimental data as provided in FIG. 1 indicates that protease proteins of the present invention are expressed in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers. Specifically, a virtual northern blot shows expression in cancers. In addition, PCR-based tissue screening panels indicate expression in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, and bone marrow.

The proteins of the present invention are also useful in drug screening assays, in cell-based or cell-free systems. Cell-based systems can be native, i.e., cells that normally express the protease, as a biopsy or expanded in cell culture. Experimental data as provided in FIG. 1 indicates expression in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers. In an alternate embodiment, cell-based assays involve recombinant host cells expressing the protease protein.

The polypeptides can be used to identify compounds that modulate protease activity of the protein in its natural state or an altered form that causes a specific disease or pathology associated with the protease. Both the proteases of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the protease. These compounds can be further screened against a functional protease to determine the effect of the compound on the protease activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the protease to a desired degree.

Further, the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the protease protein and a molecule that normally interacts with the protease protein, e.g. a substrate or a component of the signal pathway that the protease protein normally interacts (for example, a protease). Such assays typically include the steps of combining the protease protein with a candidate compound under conditions that allow the protease protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the protease protein and the target, such as any of the associated effects of signal transduction such as protein cleavage, cAMP turnover, and adenylate cyclase activation, etc.

Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab′) 2 , Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries).

One candidate compound is a soluble fragment of the receptor that competes for substrate binding. Other candidate compounds include mutant proteases or appropriate fragments containing mutations that affect protease function and thus compete for substrate. Accordingly, a fragment that competes for substrate, for example with a higher affinity, or a fragment that binds substrate but does not allow release, is encompassed by the invention.

The invention further includes other end point assays to identify compounds that modulate (stimulate or inhibit) protease activity. The assays typically involve an assay of events in the signal transduction pathway that indicate protease activity. Thus, the cleavage of a substrate, inactivation/activation of a protein, a change in the expression of genes that are up- or down-regulated in response to the protease protein dependent signal cascade can be assayed.

Any of the biological or biochemical functions mediated by the protease can be used as an endpoint assay. These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art or that can be readily identified using the information provided in the Figures, particularly FIG. 2 . Specifically, a biological function of a cell or tissues that expresses the protease can be assayed. Experimental data as provided in FIG. 1 indicates that protease proteins of the present invention are expressed in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers. Specifically, a virtual northern blot shows expression in cancers. In addition, PCR-based tissue screening panels indicate expression in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, and bone marrow.

Binding and/or activating compounds can also be screened by using chimeric protease proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions. For example, a substrate-binding region can be used that interacts with a different substrate then that which is recognized by the native protease. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. This allows for assays to be performed in other than the specific host cell from which the protease is derived.

The proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the protease (e.g. binding partners and/or ligands). Thus, a compound is exposed to a protease polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. Soluble protease polypeptide is also added to the mixture. If the test compound interacts with the soluble protease polypeptide, it decreases the amount of complex formed or activity from the protease target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the protease. Thus, the soluble polypeptide that competes with the target protease region is designed to contain peptide sequences corresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable to immobilize either the protease protein, or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.

Techniques for immobilizing proteins on matrices can be used in the drug screening assays. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., 35 S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of protease-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques. For example, either the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art. Alternatively, antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation. Preparations of a protease-binding protein and a candidate compound are incubated in the protease protein-presenting wells and the amount of complex trapped in the well can be quantitated. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the protease protein target molecule, or which are reactive with protease protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.

Agents that modulate one of the proteases of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.

Modulators of protease protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the protease pathway, by treating cells or tissues that express the protease. Experimental data as provided in FIG. 1 indicates expression in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers. These methods of treatment include the steps of administering a modulator of protease activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein.

In yet another aspect of the invention, the protease proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with the protease and are involved in protease activity. Such protease-binding proteins are also likely to be involved in the propagation of signals by the protease proteins or protease targets as, for example, downstream elements of a protease-mediated signaling pathway. Alternatively, such protease-binding proteins are likely to be protease inhibitors.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a protease protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a protease-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the protease protein.

This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a protease-modulating agent, an antisense protease nucleic acid molecule, a protease-specific antibody, or a protease-binding partner) can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

The protease proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. Experimental data as provided in FIG. 1 indicates expression in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers. The method involves contacting a biological sample with a compound capable of interacting with the protease protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable of selectively binding to protein. A biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.

The peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are known for other members of the family of proteins to which the present one belongs. Thus, the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant peptide. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification. Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered protease activity in cell-based or cell-free assay, alteration in substrate or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

In vitro techniques for detection of peptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent. Alternatively, the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample.

The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. ( Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. ( Clin. Chem. 43(2):254-266 (1997)). The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism. Thus, the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes effects both the intensity and duration of drug action. Thus, the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype. The discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages. Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymorphism may lead to allelic protein variants of the protease protein in which one or more of the protease functions in one population is different from those in another population. The peptides thus allow a target to ascertain a genetic predisposition that can affect treatment modality. Thus, in a ligand-based treatment, polymorphism may give rise to amino terminal extracellular domains and/or other substrate-binding regions that are more or less active in substrate binding, and protease activation. Accordingly, substrate dosage would necessarily be modified to maximize the therapeutic effect within a given population containing a polymorphism. As an alternative to genotyping, specific polymorphic peptides could be identified.

The peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Experimental data as provided in FIG. 1 indicates expression in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers. Accordingly, methods for treatment include the use of the protease protein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one of the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof. As used herein, an antibody selectively binds a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins. An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge. The antibodies of the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab′) 2 , and Fv fragments.

Many methods are known for generating and/or identifying antibodies to a given target peptide. Several such methods are described by Hallows, Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse. The full-length protein, an antigenic peptide fragment or a fusion protein can be used. Particularly important fragments are those covering functional domains, such as the domains identified in FIG. 2 , and domain of sequence homology or divergence amongst the family, such as those that can readily be identified using protein alignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments of the protease proteins. Antibodies can be prepared from any region of the peptide as described herein. However, preferred regions will include those involved in function/activity and/or protease/binding partner interaction. FIG. 2 can be used to identify particularly important regions while sequence alignment can be used to identify conserved and unique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguous amino acid residues. The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more amino acid residues. Such fragments can be selected on a physical property, such as fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions or can be selected based on sequence uniqueness (see FIG. 2 ).

Detection on an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitations. The antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells. In addition, such antibodies are useful to detect the presence of one of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development. Experimental data as provided in FIG. 1 indicates that protease proteins of the present invention are expressed in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers. Specifically, a virtual northern blot shows expression in cancers. In addition, PCR-based tissue screening panels indicate expression in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, and bone marrow. Further, such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression. Also, such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the full length protein can be used to identify turnover.

Further, the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed form, the antibody can be prepared against the normal protein. Experimental data as provided in FIG. 1 indicates expression in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant protein.

The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism. Experimental data as provided in FIG. 1 indicates expression in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers. The diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared against polymorphic proteins can be used to identify individuals that require modified treatment modalities. The antibodies are also useful as diagnostic tools as an immunological marker for aberrant protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.

The antibodies are also useful for tissue typing. Experimental data as provided in FIG. 1 indicates expression in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers. Thus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type.

The antibodies are also useful for inhibiting protein function, for example, blocking the binding of the protease peptide to a binding partner such as a substrate. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function. An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity. Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane. See FIG. 2 for structural information relating to the proteins of the present invention.

The invention also encompasses kits for using antibodies to detect the presence of a protein in a biological sample. The kit can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use. Such a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail below for nucleic acid arrays and similar methods have been developed for antibody arrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid molecules that encode a protease peptide or protein of the present invention (cDNA, transcript and genomic sequence). Such nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the protease peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof.

As used herein, an “isolated” nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. However, there can be some flanking nucleotide sequences, for example up to about 5KB, 4KB, 3KB, 2KB, or 1KB or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the genomic sequence. The important point is that the nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector are considered isolated. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules that consist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2 , SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule.

The present invention further provides nucleic acid molecules that consist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2 , SEQ ID NO:2. A nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule.

The present invention further provides nucleic acid molecules that comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2 , SEQ ID NO:2. A nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at least part of the final nucleotide sequence of the nucleic acid molecule. In such a fashion, the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences. Such a nucleic acid molecule can have a few additional nucleotides or can comprises several hundred or more additional nucleotides. A brief description of how various types of these nucleic acid molecules can be readily made/isolated is provided below.

In FIGS. 1 and 3 , both coding and non-coding sequences are provided. Because of the source of the present invention, humans genomic sequence ( FIG. 3 ) and cDNA/transcript sequences (FIG. 1 ), the nucleic acid molecules in the Figures will contain genomic intronic sequences, 5′ and 3′ non-coding sequences, gene regulatory regions and non-coding intergenic sequences. In general such sequence features are either noted in FIGS. 1 and 3 or can readily be identified using computational tools known in the art. As discussed below, some of the non-coding regions, particularly gene regulatory elements such as promoters, are useful for a variety of purposes, e.g. control of heterologous gene expression, target for identifying gene activity modulating compounds, and are particularly claimed as fragments of the genomic sequence provided herein.

The isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the protease peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5′ and 3′ sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encode obvious variants of the protease proteins of the present invention that are described above. Such nucleic acid molecules may be naturally occurring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.

The present invention further provides non-coding fragments of the nucleic acid molecules provided in FIGS. 1 and 3 . Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents. A promoter can readily be identified as being 5′ to the ATG start site in the genomic sequence provided in FIG. 3 .

A fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30,40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described in the Peptide Section, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence shown in the Figure sheets or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence shown in the Figure sheets or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene.

As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology , John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45 C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65 C. Examples of moderate to low stringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays. The nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptide described in FIG. 2 and to isolate cDNA and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides shown in FIG. 2 . As indicated in FIG. 3 , SNPs, including insertion/deletion polymorphisms (“indels”), were identified at 69 different nucleotide positions in and around the gene encoding the transporter protein of the present invention.

The probe can correspond to any sequence along the entire length of the nucleic acid molecules provided in the Figures. Accordingly, it could be derived from 5′ noncoding regions, the coding region, and 3′ noncoding regions. However, as discussed, fragments are not to be construed as encompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplify any given region of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all of, the peptide sequences. Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence, such as into the cellular genome, to alter in situ expression of a gene and/or gene product. For example, an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenic portions of the proteins.

The nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods. The gene provided by the present invention is located on a genome component that has been mapped to human chromosome 4 (as indicated in FIG. 3 ), which is supported by multiple lines of evidence, such as STS and BAC map data.

The nucleic acid molecules are also useful in making vectors containing the gene regulatory regions of the nucleic acid molecules of the present invention.

The nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.

The nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful as hybridization probes for determining the presence, level, form and distribution of nucleic acid expression. Experimental data as provided in FIG. 1 indicates that protease proteins of the present invention are expressed in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers. Specifically, a virtual northern blot shows expression in cancers. In addition, PCR-based tissue screening panels indicate expression in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, and bone marrow. Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms. The nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to assess expression and/or gene copy number in a given cell, tissue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in protease protein expression relative to normal results.

In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting DNA includes Southern hybridizations and in situ hybridization.

Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a protease protein, such as by measuring a level of a protease-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a protease gene has been mutated. Experimental data as provided in FIG. 1 indicates that protease proteins of the present invention are expressed in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers. Specifically, a virtual northern blot shows expression in cancers. In addition, PCR-based tissue screening panels indicate expression in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, and bone marrow.

Nucleic acid expression assays are useful for drug screening to identify compounds that modulate protease nucleic acid expression.

The invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the protease gene, particularly biological and pathological processes that are mediated by the protease in cells and tissues that express it. Experimental data as provided in FIG. 1 indicates expression in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers. The method typically includes assaying the ability of the compound to modulate the expression of the protease nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired protease nucleic acid expression. The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the protease nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.

The assay for protease nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the protease protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.

Thus, modulators of protease gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined. The level of expression of protease mRNA in the presence of the candidate compound is compared to the level of expression of protease mRNA in the absence of the candidate compound. The candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression. When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression. When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate protease nucleic acid expression in cells and tissues that express the protease. Experimental data as provided in FIG. 1 indicates that protease proteins of the present invention are expressed in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers. Specifically, a virtual northern blot shows expression in cancers. In addition, PCR-based tissue screening panels indicate expression in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, and bone marrow. Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.

Alternatively, a modulator for protease nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the protease nucleic acid expression in the cells and tissues that express the protein. Experimental data as provided in FIG. 1 indicates expression in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers.

The nucleic acid molecules are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the protease gene in clinical trials or in a treatment regimen. Thus, the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance. The gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays for qualitative changes in protease nucleic acid expression, and particularly in qualitative changes that lead to pathology. The nucleic acid molecules can be used to detect mutations in protease genes and gene expression products such as mRNA. The nucleic acid molecules can be used as hybridization probes to detect naturally occurring genetic mutations in the protease gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the protease gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a protease protein.

Individuals carrying mutations in the protease gene can be detected at the nucleic acid level by a variety of techniques. FIG. 3 provides information on SNPs that have been identified in the gene encoding the protease protein of the present invention. SNPs, including indels (indicated by a “-”), were identified at 69 different nucleotide positions. Non-synonymous cSNPs were identified at position 30496. The changes in the amino acid sequence caused by these SNPs is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. SNPs outside the ORF and in introns may affect control/regulatory elements. The gene provided by the present invention is located on a genome component that has been mapped to human chromosome 4 (as indicated in FIG. 3 ), which is supported by multiple lines of evidence, such as STS and BAC map data. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.

Alternatively, mutations in a protease gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and S1 protection or the chemical cleavage method. Furthermore, sequence differences between a mutant protease gene and a wild-type gene can be determined by direct DNA sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C. W., (1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (Myers et al., Nature 313:495 (1985)). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship). Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the protease gene in an individual in order to select an appropriate compound or dosage regimen for treatment.

Thus nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs to control protease gene expression in cells, tissues, and organisms. A DNA antisense nucleic acid molecule is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of protease protein. An antisense RNA or DNA nucleic acid molecule would hybridize to the mRNA and thus block translation of mRNA into protease protein. FIG. 3 provides information on SNPs that have been identified in the gene encoding the protease protein of the present invention. SNPs, including indels (indicated by a “-”), were identified at 69 different nucleotide positions. Non-synonymous cSNPs were identified at position 30496. The changes in the amino acid sequence caused by these SNPs is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. SNPs outside the ORF and in introns may affect control/regulatory elements.

Alternatively, a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of protease nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired protease nucleic acid expression. This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the protease protein, such as substrate binding.

The nucleic acid molecules also provide vectors for gene therapy in patients containing cells that are aberrant in protease gene expression. Thus, recombinant cells, which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired protease protein to treat the individual.

The invention also encompasses kits for detecting the presence of a protease nucleic acid in a biological sample. Experimental data as provided in FIG. 1 indicates that protease proteins of the present invention are expressed in humans in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in cancers. Specifically, a virtual northern blot shows expression in cancers. In addition, PCR-based tissue screening panels indicate expression in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal brain, and bone marrow. For example, the kit can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting protease nucleic acid in a biological sample; means for determining the amount of protease nucleic acid in the sample; and means for comparing the amount of protease nucleic acid in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect protease protein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

As used herein “Arrays” or “Microarrays” refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support. In one embodiment, the microarray is prepared and used according to the methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93; 10614-10619), all of which are incorporated herein in their entirety by reference. In other embodiments, such arrays are produced by the methods described by Brown et al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 5′, or 3′, sequence, sequential oligonucleotides which cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for a microarray or detection kit, the gene(s) of interest (or an ORF identified from the contigs of the present invention) is typically examined using a computer algorithm which starts at the 5′ or at the 3′ end of the nucleotide sequence. Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit. The “pairs” will be identical, except for one nucleotide that preferably is located in the center of the sequence. The second oligonucleotide in the pair (mismatched by one) serves as a control. The number of oligonucleotide pairs may range from two to one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a “gridded” array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit, the RNA or DNA from a biological sample is made into hybridization probes. The mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the microarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit. The biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large-scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples.

Using such arrays, the present invention provides methods to identify the expression of the protease proteins/peptides of the present invention. In detail, such methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample. Such assays will typically involve arrays comprising many genes, at least one of which is a gene of the present invention and or alleles of the protease gene of the present invention. FIG. 3 provides information on SNPs that have been identified in the gene encoding the protease protein of the present invention. SNPs, including indels (indicated by a “-”), were identified at 69 different nucleotide positions. Non-synonymous cSNPs were identified at position 30496. The changes in the amino acid sequence caused by these SNPs is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. SNPs outside the ORF and in introns may affect control/regulatory elements.

Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the novel fragments of the Human genome disclosed herein. Examples of such assays can be found in Chard, T, An Introduction to Radioimmunoassay and Related Techniques , Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry , Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology , Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein or membrane extracts of cells. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts or of cells are well known in the art and can be readily be adapted in order to obtain a sample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.

Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the Human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.

In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe. One skilled in the art will readily recognize that the previously unidentified protease gene of the present invention can be routinely identified using the sequence information disclosed herein can be readily incorporated into one of the established kit formats which are well known in the art, particularly expression arrays.

Vectors/host Cells

The invention also provides vectors containing the nucleic acid molecules described herein. The term “vector” refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules. When the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules. Alternatively, the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules. The vectors can function in prokaryotic or eukaryotic cells or in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell. The nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription. Thus, the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector. Alternatively, a trans-acting factor may be supplied by the host cell. Finally, a trans-acting factor can be produced from the vector itself. It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.

The regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli , the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.

In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 nd. ed ., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

A variety of expression vectors can be used to express a nucleic acid molecule. Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 nd. ed ., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, E. coli , Streptomyces, and Salmonella typhimurium . Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.

As described herein, it may be desirable to express the peptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production of the peptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enteroprotease. Typical fusion expression vectors include pGEX (Smith et al, Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)119-128). Alternatively, the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli . (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al, EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187-195 (1987)).

The expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the nucleic acid molecules. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2 nd, ed, Cold Spring Harbor Laboratory , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA. Thus, an antisense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).

The invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, et al. ( Molecular Cloning: A Laboratory Manual. 2 nd, ed., Cold Spring Harbor Laboratory , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, the nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing trans-acting factors for expression vectors. When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced or joined to the nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.

Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell-free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.

Where secretion of the peptide is desired, which is difficult to achieve with multi-transmembrane domain containing proteins such as proteases, appropriate secretion signals are incorporated into the vector. The signal sequence can be endogenous to the peptides or heterologous to these peptides.

Where the peptide is not secreted into the medium, which is typically the case with proteases, the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like. The peptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinant production of the peptides described herein, the peptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the peptides may include an initial modified methionine in some cases as a result of a host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein have a variety of uses. First, the cells are useful for producing a protease protein or peptide that can be further purified to produce desired amounts of protease protein or fragments. Thus, host cells containing expression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involving the protease protein or protease protein fragments, such as those described above as well as other formats known in the art. Thus, a recombinant host cell expressing a native protease protein is useful for assaying compounds that stimulate or inhibit protease protein function.

Host cells are also useful for identifying protease protein mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant protease protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native protease protein.

Genetically engineered host cells can be further used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a protease protein and identifying and evaluating modulators of protease protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any of the protease protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the protease protein to particular cells.

Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo , (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.

In another embodiment, transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992). Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein is required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. Nature 385:810-813 (1997) and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G 0 phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring born of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

Transgenic animals containing recombinant cells that express the peptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect substrate binding, protease protein activity/activation, and signal transduction, may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo protease protein function, including substrate interaction, the effect of specific mutant protease proteins on protease protein function and substrate interaction, and the effect of chimeric protease proteins. It is also possible to assess the effect of null mutations, that is mutations that substantially or completely eliminate one or more protease protein functions.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.

4 1 1225 DNA Human 1cgcccttatg ctgaagccat ggatgattgc cgttctcatt gtgttgtccc tgacagtggt 60ggcagtgacc ataggtctcc tggttcactt cctagtattt gaccaaaaaa aggagtacta 120tcatggctcc tttaaaattt tagatccaca aatcaatttc aatttcggac aaagcaacac 180atatcaactt aaggacttac gagagacgac cgaaaatttg gtggatgaga tatttataga 240ttcagcctgg aagaaaaatt atatcaagaa ccaagtagtc agactgactc cagaggaaga 300tggtgtgaaa gtagatgtca ttatggtgtt ccagttcccc tctactgaac aaagggcagt 360aagagagaag aaaatccaaa gcatcttaaa tcagaagata aggaatttaa gagccttgcc 420aataaatgcc tcatcagttc aagttaatgc aatgagctca tcaacagggg agttaactgt 480ccaagcaagt tgtggtaaac gagttgttcc attaaacgtc aacagaatag catctggagt 540cattgcaccc aaggcggcct ggccttggca agcttccctt cagtatgata acatccatca 600gtgtggggcc accttgatta gtaacacatg gcttgtcact gcagcacact gcttccagaa 660gtataaaaat ccacatcaat ggactgttag ttttggaaca aaaatcaacc ctcccttaat 720gaaaagaaat gtcagaagat ttattatcca tgagaagtac cgctctgcag caagagagta 780cgacattgct gttgtgcagg tctcttccag agtcaccttt tcggatgaca tacgccggat 840ttgtttgcca gaagcctctg catccttcca accaaatttg actgtccaca tcacaggatt 900tggagcactt tactatggtg gggaatccca aaatgatctc cgagaagcca gagtgaaaat 960cataagtgac gatgtctgca agcaaccaca ggtgtatggc aatgatataa aacctggaat 1020gttctgtgcc ggatatatgg aaggaattta tgatgcctgc aggggtgatt ctgggggacc 1080tttagtcaca agggatctga aagatacgtg gtatctcatt ggaattgtaa gctggggaga 1140taactgtggt caaaaggaca agcctggagt ctacacacaa gtgacttatt accgaaactg 1200gattgcttca aaaacaggca tctaa 1225 2 405 PRT Human 2Met Leu Lys Pro Trp Met Ile Ala Val Leu Ile Val Leu Ser Leu Thr 1 5 10 15Val Val Ala Val Thr Ile Gly Leu Leu Val His Phe Leu Val Phe Asp 20 25 30Gln Lys Lys Glu Tyr Tyr His Gly Ser Phe Lys Ile Leu Asp Pro Gln 35 40 45Ile Asn Phe Asn Phe Gly Gln Ser Asn Thr Tyr Gln Leu Lys Asp Leu 50 55 60Arg Glu Thr Thr Glu Asn Leu Val Asp Glu Ile Phe Ile Asp Ser Ala65 70 75 80Trp Lys Lys Asn Tyr Ile Lys Asn Gln Val Val Arg Leu Thr Pro Glu 85 90 95Glu Asp Gly Val Lys Val Asp Val Ile Met Val Phe Gln Phe Pro Ser 100 105 110Thr Glu Gln Arg Ala Val Arg Glu Lys Lys Ile Gln Ser Ile Leu Asn 115 120 125Gln Lys Ile Arg Asn Leu Arg Ala Leu Pro Ile Asn Ala Ser Ser Val 130 135 140Gln Val Asn Ala Met Ser Ser Ser Thr Gly Glu Leu Thr Val Gln Ala145 150 155 160Ser Cys Gly Lys Arg Val Val Pro Leu Asn Val Asn Arg Ile Ala Ser 165 170 175Gly Val Ile Ala Pro Lys Ala Ala Trp Pro Trp Gln Ala Ser Leu Gln 180 185 190Tyr Asp Asn Ile His Gln Cys Gly Ala Thr Leu Ile Ser Asn Thr Trp 195 200 205Leu Val Thr Ala Ala His Cys Phe Gln Lys Tyr Lys Asn Pro His Gln 210 215 220Trp Thr Val Ser Phe Gly Thr Lys Ile Asn Pro Pro Leu Met Lys Arg225 230 235 240Asn Val Arg Arg Phe Ile Ile His Glu Lys Tyr Arg Ser Ala Ala Arg 245 250 255Glu Tyr Asp Ile Ala Val Val Gln Val Ser Ser Arg Val Thr Phe Ser 260 265 270Asp Asp Ile Arg Arg Ile Cys Leu Pro Glu Ala Ser Ala Ser Phe Gln 275 280 285Pro Asn Leu Thr Val His Ile Thr Gly Phe Gly Ala Leu Tyr Tyr Gly 290 295 300Gly Glu Ser Gln Asn Asp Leu Arg Glu Ala Arg Val Lys Ile Ile Ser305 310 315 320Asp Asp Val Cys Lys Gln Pro Gln Val Tyr Gly Asn Asp Ile Lys Pro 325 330 335Gly Met Phe Cys Ala Gly Tyr Met Glu Gly Ile Tyr Asp Ala Cys Arg 340 345 350Gly Asp Ser Gly Gly Pro Leu Val Thr Arg Asp Leu Lys Asp Thr Trp 355 360 365Tyr Leu Ile Gly Ile Val Ser Trp Gly Asp Asn Cys Gly Gln Lys Asp 370 375 380Lys Pro Gly Val Tyr Thr Gln Val Thr Tyr Tyr Arg Asn Trp Ile Ala385 390 395 400Ser Lys Thr Gly Ile 405 3 38844 DNA Human 3ttatattcat aaaagtaggc agtaagttga agatttattc atataggatt tagtagctgc 60agctttaacc tgtggcttct gtagcttttg taatctggca gtgcgcatct gctatattat 120ctaaatgttt cctcaaaagg agaaacactc taacaactta tcaccctagt ctgctggcca 180ccattttccc tcagatgctc acagcttctt ccgtgggatt tgaagatatg acttccatga 240cacttgatca gtatgtcaat gggtattgaa ccactcttca gctctgatcc cacggttcag 300ttcctttcag tgtgactatg tgtcttggtg gtgggagatg tgattctttt atctactttc 360tccatttatc ttactcagag gaactgtgct ctaataggga aatagattga aagcttataa 420atttccttga gttttaactt ttctcctttg gtcttttttt cttttcaaat gacttgaaga 480cacattgata agattctatg agaaaatgaa gagttgaaca aattgaatat gtatgagtga 540atgaatagat taatacataa atgataaatt tattaaataa tttgaacgaa atcaatcgag 600aggcaccgag aataaatttg tgtcctagaa gtaagaagac ctgagtttga gataactagt 660agttctatta tactggagaa attacttaat catcactgga cttcattttt ctcatatgga 720aagtaattca atcacactaa acaatcttta aggtctcctt cacttataaa tgtatgtttt 780aagccattta ggaggttaaa taatgtcatg tcccatggga cttctgtttg ttgttctatt 840caagcatgtt agcttgtttc tatcacagga cctgctgcct ttccgcagcc agttctctag 900attattttta atcagtcggt gcacacatgg tcaatattta ctcaatagaa ttcaggtttc 960ccaaattcca tgaggattct tgattaattt tattacttat gccaaaacta ttatcttctt 1020aactatttta ggtccaaaca gttttaactt ttatcctggc atttatatat aaaaaacttt 1080tgtaagaccg ggtgcagtgg ctcatgcctg taatcccagc actttgggag gccgaggtgg 1140gtggatcacc aggtcaggag atggagacca tcctggctaa caccatgaaa ccctgtttct 1200actaaaaata caaaaaatta gccgggcgtg gtggtggacg cctttagtcc cagctattca 1260ggaggctgag gcaggagaat ggcgtgaacc tgggaggcag agcttgcagt gagcagagat 1320cacaccactg cactccagcc tggcagcctg gatgacacag cgagactccg tctcaaaaaa 1380aaaaaaaaaa aaaagaaaaa aactgtttta tagtcaaaag aaaaactttc tataaatcaa 1440ccaatcctgt gaagaaaata tgaaaaatat cctctgtttc caaaaaaatt taggctatca 1500atatatacac ataaagagat aaactctgat aaattggata aataaaattc actataatag 1560caagttttag agaacaagca cgggagttag tcgacctggg cccttaaaca gatatcctct 1620ctctcatcct gtgttatttc ctgtgtaatg ttggtatcat tcctgcctga ctctcataga 1680tttatatgat tcctactctg tccaggtgcc ttattgggtc ttagcggtaa aaagatgaac 1740aaggctaatg cagcccattg agaagctatc tgtaagtgaa catacatgca aactaatact 1800tgattcaatg tgagaagcac tgttgctgat cataggtgcc agaagaacag caaagagtta 1860ttttttcctc caaaattgtg gaaaaatttt tatccccggt gtgatgcaat ataaaataca 1920cagcaccacc tttgaagtat tcttgccaaa tgaatttaac caaaatctaa tcaagacttc 1980agagctaaag aaaatctaaa ggtaatccaa tttataggaa atgagggata taaaagaaca 2040agttaaataa taccacagga aagcattcag acaagtccag aaagtaagat attctaaagg 2100atgtttagct tgatctcttc aacagtcaat gtcattaaaa actaaaaaag aagcaggact 2160cttttagatt aaaagagatt aaaaaggcat aacaaacaag tgcactgcat ggtcctcgat 2220tatgtcttgg cttttacaaa tcatgtgtaa ttataatgaa accatggagg gaacttgaag 2280atggactggg tattagatga tatggcagaa atatcattaa ttttttagga gtgttaagag 2340tatcatggtt atgttggata tatcctaatt gtctataata atgatttggt aaaaagtcac 2400gatgttttat ttcacattaa aatatagcag cagaaaaaat aaatgagcca aatacagtaa 2460aattttcaac aattgatata ataatgtgat atatatatgg atgttcaatt atactattct 2520tagtaatttt ttatgtctga acattttcat aatacttaaa aataaaagat aaaagataaa 2580aataaatgag ataatagatt taaaatcact ttgtaaactc taaaaggata gacagataaa 2640agagataaca aagtgctgga gaaaggagga atggtccctt ttcaagcatg tatgccacct 2700tggaccatgc tgctaagaga aaccattcct gaccaccaca aagaggccac caaatgcctc 2760taaaatagaa agcaggagca acattaggat tcccagatcc tgatattttt tttttaacac 2820atcttctcag accaagatga cattgaacaa aattaaagac ctttttgcag ggaaaggtag 2880gctacagcaa cttgaacttg tctaaggaga gctggaaaac ctgcaagcat tgctatctga 2940gagtaaccag tgggcccttc cttttctcag gacagtggga tttggcaccc gaagcagaaa 3000tgctgaagcc atggatgatt gccgttctca ttgtgttgtc cctgacagtg gtggcagtga 3060ccataggtct cctggttcac ttcctagtat ttggtaggta aaattaaaga tttcactcta 3120tttgatttta tttttctgca aagctccatt tacatatatg taaatgtaac ttcatctaaa 3180aaattgcaca tttaccttca aatttccaca gagtatattt aactgtttca gtcatttcat 3240caacaaacaa gtactaaatt cttattatat gtgagtactt ttctggatat tcaagataca 3300gctttaagca aagtagacag atttctaatt tccttagagc tctcaaccca gaattctttt 3360gagaatctac acaaaaagat caaaaattgt aattgtctga aacttactag taattataat 3420aaacaactca tcacttatta tatattaaaa tgaaaagcta tgataaatta gttattaaaa 3480ttggctcttt tactcatgaa ccatcatttt ctgtccaaca tttctaaggc aaaagaaaaa 3540cacttgtcta ataaaataag gaatttcaaa atgattgaaa acctatacgt atgacacaat 3600attatcattt atttttagag aaaaaaaatt ttactctttc caaaacaata ttcagggatt 3660atatttttat caactaatat atttgtaatt acacaaataa tgcacttcaa gattctcttt 3720ttacattcag tctctttctg gggagaatgc aagccattta cattttttca caaatctcta 3780caatgtgact ctcacatgga tgtatgtgat aaaacaaata actcaggctg ctcactttaa 3840cgctcttatc tgctgtcacc ttcacagagt caatggggga gcaaagactc tacttggagc 3900cttaaagggc ttaagatcat agtcctaggc cttatatgat aaccccagct gtagtttata 3960ccattggcaa aagattctca ggtcacttta tttggttgca taaaagtctc tttacaatga 4020gagtaaggtt tgttaacagt atggattata tgggtaagta atcaggatgt ccaaaaatgt 4080attacaaggt ccagagattt cccacttaag acatatgcct tcctgatatc cctgtttctt 4140tccttggttt gtagtctcga aacccactcc ctcttccctg agccaggctt ctcaaggatt 4200gaggttgttt tgtatttttc ccattctcta tctttaactc tgtatctttc ttactccctc 4260tgggccttac tcctcagatt accaaattcc ttaggagtct caactgcttt cctttcttac 4320atttcctaat agatttatcc ctgtttcatg ctcgtcttgt cttcaatctc agacagctct 4380tctctacact ttcttttcag gtttttctta gtgtgcctgg ctctcttgtt aaaaatcaaa 4440attcacaagg acattcactt atctctactt ccactagagt gtatgatggt acacatttca 4500actcagcaag gagcaatgta gcaatgaaat gttcaagctc tacagctaga ctggatttaa 4560aacttggaca ggccacctac tagttacaga acaatttact taatgcctct gtgccttaat 4620ttccttatct gtaaaatgaa ggtgatacca atcttagaga gctggtgtgg ggattaaatg 4680ggctaataca taaaaagtgc acaggacagt gcctgccata ttgtagaaac tcaataaatg 4740gcagctatta taattgatat aaaacattaa ctgttatttt ttaaataaaa ctcaattatg 4800aagaggctca gggacatatt caagatttat attggcccca ttgtaattga gttctgaaat 4860ctttgtccaa accatttagt ttcctatttt tcatttccat tgcagaccaa aaaaaggagt 4920actatcatgg ctcctttaaa attttagatc cacaaatcaa taacaatttc ggacaaagca 4980acacatatca acttaaggac ttacgagaga cgaccgaaaa tttggtgagt caggtaaact 5040tctttttatc atagaataat gcaagtggaa gggattttgt ggatcatttc tccatttcta 5100aaaacatgat tttcagaccg ccaacattag aatcatcttg cagattgcta ggccccatcc 5160cagacctgct taatcagagt atgatgagat gggtaggtgg ggagaggaga gtaagggaat 5220ctgcatgtct aacaaatggg tgattctaat aagcctctct ttctaactca gctaccttat 5280ttaaaggtaa gagaattgag gccaagatat cctagcccgt ttcttcccca attccaccac 5340gtttcccctg tagaaaagcc taatcatacc aaaactagtt tttataagtc cacacacttg 5400tttgtaagac cacattttaa gattttgagt attttcagaa tttacgttca tcttgtaagt 5460atattgataa agacaaaaaa ccagacttat tttgtagtaa tcaagtcaaa tgctaataat 5520tttgttaaag ctaaagtgca agactgctcc caaaaagaaa aaaagcacac tcagttgtat 5580aatcattcca ctcagaatgc ccatgaactc tcactcaaaa actaggttca aattaatttt 5640tctaacaagg aagcacagaa gcagagactt attttaaaaa gaaagaaatg acaaatgtat 5700tggtttgttt taatcaaaga accattttta agacactttc tttcccaaat catctaccat 5760tttttcctgt catcatttgc tctttgtcca tagtatacct aatggcatca tatttacaat 5820aatattgtag agtttataat ctctattttc agttaacatt aaatcattca caatttctta 5880attttgtggt ttcatctttc ccaaccaata attaatgtct acagattgat atagattctg 5940cattctttca catgcagagc atcttataaa agagcatttg caatcagttc ttaagttatg 6000ctaggatgaa cggggagcct gcaccaatac acccaaatac cttctctact cctccagtcc 6060taagtgactc cacataacct cctcgatgca aaaagagaaa actcttaact tgccttagtt 6120aaaaagataa acacaccttt gaatgatgga aaatgttaca atttactggg aaattttgaa 6180atttgtttca tttatatttt atggccaaca ttactgctac tgttgttgtt gtaagttaac 6240taggcaattc tgtctttact gaagtaaacg gacaagaatg caataggtct taaaagaagt 6300gagagaaatg cagaggtgca tgttgaacag aaactctatt taaaagtgga gttttaagtt 6360tcacctaagc atgtgttcct tcaaaggcta aggctaagtt aagtaaggac acattatcat 6420catgggtacc tgcaaggccc ttctctggtt gtcattattt atttatcctc ctttatcacc 6480atagcataag cccttaccct ccccccttgc aggaaatcat tctatgtttc atgtggtatt 6540cttttgtttg tattcattct tacaaaaata tgttttgcta ttttgcgtac acttgctttt 6600aacttacatt ttgtgttata aatcactttt gtttcatctc tttttactga gaacttttta 6660aaagatatat gttactaaat atacctttag tttattgctg ttagctgcta attcatagtg 6720tgtatcttcc atatttacct gcctgtcatg ccaagaaatg ccacactaaa cagactccta 6780cttaccccct tatagaccta tgcaagtact tctggaagca gaattactag gtcattgaat 6840gtacatatac ttaacttgac caattggtgc aggtttgctc ttcaaaatgg ctgactcagt 6900gtgcacgccc atctacaatg catgaggatt tctatgtccc cacatctaac caacacttag 6960tgtcttagta tgtttaggct actacaacaa aaaataccat aggctgggta tcttaaacaa 7020caaacaatta tttctcatag ttctggaggc tgaagattcc aagatgaaga tgatcaaggc 7080tctagcagat gtctggtgag agcctgcttc ctggttcata gaataccatc ttgctgtgtc 7140cctcatggca gaagccataa gagaactttc ttttgtaagg acactaatga ctttcatgag 7200aactccaccc tcatgaccta actatcctcc aaaggcccca tctcctctat catcggtttg 7260ggagttaagg tctcaaaata taaatttcag gggaacacaa acattcagtc cacagcactt 7320ggtattattt ggctttctaa atttgccacc ctaatatgta taaagtagta ttttatttgt 7380gatttaattt gcatgtttct aattactaat gagtttgtgc attgttacgt ataattatta 7440actttttgga ctttcatttc tataaattgc ctgtacatat tatttgccta tttttctgtt 7500aaacttgctt tttcacctta tttgtattgc tttgcagaag ttctttacat tttctggata 7560ttgatagtgt gttggttgtg gacactgcgc ttatccattc tgtcttctac taatatggac 7620cgtgttgttc tttatgaaac cgaaatctgt aactgaagta atcatttttt cactgttttg 7680ccttatgatt gtattttgaa gcttttcttt aagaagtcct tcttcccttc taagacataa 7740aaatatttta ctatgttact tattaacctt atagttttat cttttacatt aggtctccaa 7800tacatgtgga atccaccttt ggatgtgtta ggtagattca gttttttaat tcatatagtg 7860agccagtttt tgaatataac tagttaaaat atcttggctt ttcctaatat atggtattat 7920tattgagttc attgcatgca tttcttggca cctgggtctt gcagaaaagg aaacatgaat 7980ctgtctcctc aaattgcttc caatcttttt ggaaagatgt gagtaacaca catggaattg 8040aatatcatga catgatataa ttaagggcta aattacatgt tgaggacagt aagtacagaa 8100aaacttcaaa accaaacaag ggttcccatg gtcagaaaag gctttatatt attttacctt 8160tgtttaaatg agacaggtgt ttttctcctc ccatcccgca ccaggttagc tttagaagaa 8220ttacaggaag agtttatgcc tcatcctgag ccacacctgt ttgttgttgc taaatcccaa 8280tgaatacaac cagattcttc tctctgtcct atatgggtgc taattagaca accaaggaag 8340aacaggttgc acgtcctgtt cttcctcaca ttgggcttta ctgatttgaa tgcaaattga 8400gatgcaaaag taaaaatgag ttcatattta gatattgcta taatccgccc ctgttccctg 8460agatagtgga gcagacatat ctcatctctc atatcattct tcagagaagg gtccattaat 8520cagacattac tgatgtctga ttactgccgg ctggccatcc tgcaggtgga gaagcatggc 8580atccagcaga aactgacagc atgcactttg agggagggaa ggataagcca ggaatttatg 8640ctgaataagc tgcctaagta tacatgttca ataagttcta ggggaagtca caaatactta 8700tgaaaggaga aacataacta tgtgcaattg agctttatgt ctcttcatgt gttgcatgtt 8760caaaaaatgg tggcattagc atgatccaag ggtggagttt tcagccattt gatgttcaaa 8820ggtgaagcag aggacacaaa acccttacta tgcatcctct gtgagtcagc caaaaccagt 8880ctggactgct agctagatta acaaagaaaa aaagagaaag aagatacaaa taagcacgat 8940cagaaatgat agaggtaaca ttacaaccaa tcccacagaa atacaaaaga tcgtctgaga 9000ctcttatgaa cacttctatg tagataaact agaaaatcta gaggaaatgg gtaaattcct 9060ggaaaaacac aatcttccaa gattgaatca gaaagaaatt gaaaccctga acagaccaat 9120attgagttca tacttaaatc agtaatttaa aaaacttacc agccaaaagg aaaaaaaaag 9180gcccaaacta gatggattca cagccaaatt ctaccagacg tacaagaaat agctaggacc 9240aattctagtg aaactattcc aaagaattga gaagagactt cttcttaaat cattctatga 9300agtcagcatt accctaacgc caaaacctca caaagacaga atgaaaaaag aaaattacag 9360gccaatatcc ctgatgaaca tagatataaa aatcctcaac caaataccag caaaccaaat 9420ccagcagcac atcaaaaagt taattttcca aaatcaagta ggctttattt ctgtgatgca 9480agactggttc aacatatgta aatcaataaa tgcgatttac cacataaacc gaattaaaaa 9540caaaaatcat acaattagcc aggcatggtg gctcacactt gtaatcccag cactttggga 9600gaccatggtg ggcaaattac ctgaggtcag aagttcgaga ccaacctggc caacatggtg 9660aaaccccatc tgtattaaaa atacgaaaat tagccgggca tggtggcagg tgcctgtaat 9720cccagctact cggagggctg aggcaggaga atcacttgaa cccaggaggc agaggttgca 9780gtgagccgag atcgtgccat tgcactccag cctgggtgac agagcaaaaa tccatctcaa 9840aaaaattaaa aatttaagaa aattaaaatc atacaatcat ctcaatatat gtagaaaaag 9900cttttgataa aattaaacat cccttcataa taaaaacact tagactaggc atcgaagaaa 9960catacttcaa aataataaga gccatctgtg acaaacccac agccatcatc acactgaatg 10020ggcaaaagct ggaggcacta tccttaagaa cagggaaaaa gacaagaatg ttcactctca 10080ctactcctat tcaacatagt actagaagtt ctagaaagag caatcgagca ggagaaagaa 10140ggaaaatgca tccaaatacg aaaagaggaa gtcaaattat ctctctttac tgacaatatg 10200attatatgcc tagaaaaccc taaagacttt acaaaaagtt tccaaaactg ataaacaact 10260tcagtaaagt ttcaggatac aaaatcaatg tacaaaattc agtagcattt ctaaacaata 10320atgtccaagc tgagaaccaa atcaagaaca caatcccatt ttcaatagcg acacacacac 10380acaaatgaaa tacctaggaa tacatctaac caaggaggta aaagatctct ataaggagaa 10440taaaaaaaca ctattgaaag aaatcggaga tgacacaaat gaatgcaaaa acattccatg 10500ctcatggatt ggaagaatca atattgttaa aatgtcccta ctgcccagag caatctacag 10560attcaatgct attcctatca aactaccaac ataattttcc acacaaagtt agaaaaagct 10620tttgtaaatt tcatatggta caaaaaaaaa aagccccaat agccaaagga ctcctaataa 10680aaaagaacag agccagaggc ctcacattat ctgacttcaa actatacttt aaggctacag 10740taatcaaaac agaatggcat tggtcaaaaa cagacatata aaccaataga acagaataga 10800gaacccagaa ataaagccac acatctacag ccatcagata ttcaataaaa ttaacaaaaa 10860taagcaatgg ggagagaact ttctattcaa taaatggtgc tggaatagct agctagtcag 10920aagcagaaaa atgaaattgg actcctatca ctaaatacaa aaactaactc aagatgcagt 10980aaagaattaa atgtaagacc acaaacaatt aatacaagaa ccctagaaga aaacctagga 11040aatactgttg tagacatcag tcttggcaca gaatttagga ctaagtcctc aaaagcaact 11100gcaacaaaaa caaaaattga taagttggac ctaattaaac taaagaactt ctgcacaata 11160aaagaaacta tcaacagagt aaacaaacaa cctacagact gggagaaaat atttgcaaac 11220tatgcatctg aaaaggtcta atgtccagaa tctgtaaaga acttaaacaa ctcaacaagc 11280aaaagaaacc aagtaacgcc attaaaaagt aggcaaagaa catgaacaga tgcttcacaa 11340aagaagacat acaacgcagt caagaaacat atgaacaaat gctccacatc actaattatc 11400caagtaatgc aaatcaaaac tacagtgaga taatatctca taccagttac aatggctatt 11460attaaagatt aaaaaaataa catgctgatg agactgcgga ggaaagagaa tgcttaaata 11520ctgttggaaa cgtaaatggg ttcagccact gtggaaagca gtttggagac ttctcaaagt 11580acttaaaatg gaactactat tcaacctagc aatcctactt actgggtgta tacccaaagg 11640agtataaact tttttcccag aaagacagct gcactctcac attaattacc acagtattca 11700caatagcaaa gatgtggaat caacctagat atccatcaat ggtggattgg acaaagaaac 11760tgtgagatat atatgtatat atatctatat ataccatgga atactatgta gccataaaaa 11820aggatgaaat catgtccttt gcagcaacat ggatgtaaca ccacaaggaa ggcactttta 11880tctcctcttt acaggtaaga gaaccaagct tctgaaatta aggtccatag ctggaaaatg 11940atggagggga gatttgaagt catctaggca actccacaca tgtgctcttt ccactaaatt 12000gttctactgt caggaaggga ctcagctaag acagaagata aaattattaa aatctaaatc 12060aattcttctc tcatttcatt ttttaaatcc atgaagatta taaatcctct atgctgtgct 12120agctaacttt ttcttgacag atacattagg tatacttatt agagaaaaat attctctttc 12180tcatttccct gtatcagttt ttggtgagga aggcaaaggt aggaggaact gtaatagaga 12240aagatgaagg aagctgatgg atatattgac atgtgtatgt acatctagtg tgaacaatct 12300atagttggaa gaaaggtgtg gatgggtatg ctttttgagg gaagtttttg agaaaagaag 12360taatatgaac tatttctaaa tttcctgata aagttgtaaa tacagcatag tcttcacagg 12420agaatctatt tagtttatca tcatcattca gcaaatacag catgatgtta ggcactataa 12480aaggctaaga aaaatgattc tctctctctc ataaactaat ccaatttaga gatttagaag 12540acaacaaatc tggagaggac atgaaccttc taaataatga ccttcccttg ctttgggtat 12600cctggtttta aatattttta gtacagcttt aaatagatcc aaatgagata ttttcctctt 12660ttacaaaagc aattcaaaga tctaggtttt tgttgtacac tgagaattaa tacttttttc 12720tttaaaatcc ttaattgcaa atctttaaat tctataaata ttttgccttg tgatctcaga 12780aatataagcc aatttgggat atggatatct aatatattgc tacttgttac acgtgagtag 12840tgacagatgt ctgtccattt ctttctgaca ttccacaaag aaacactgaa gaaggaccag 12900tgcaatcaaa gaaatgactg atggcatcac aaaatatcac atcccatttg atgatctgat 12960tacctttttg tttagggtga tcagaaagtc acagtttcat ggcaccctcc acacccacac 13020accttgtatg acactggatc caactgcttt ctccaataga cacagcactt aaagatgtgg 13080cagttaggct tgaccccaag aaggccaaaa agccttctgt gagcatcact cagtgctcag 13140gttgactaag ctctatccag gcttgagaga atggttcata gctgacttct tggatccaaa 13200aaaaaaaaaa aaacacctag agttttatac agatatgata cgaacttaaa aggactgcac 13260taaaaactac caagattatg attcttattt ttggagagta aagaaaatag gctgcctttg 13320gagaggggtg caacagtttc tgatcctctt acaaactgct tgctgcccat cagtgggtag 13380gaggtcttag tgagaaccta cctgcatgct catcctgagg taggcactgt gaaggcgtta 13440acaggctctg aagctacatg gccctggttt cagtgaactc tgtggtgtca acttgggcaa 13500gtcacttcct cttctatgaa acgtgaataa tcatagtact caccttagag ggctgatttg 13560aaagcaaatg agctcaaaca caatgacatc tgtgcttggt gcatatatgg cagacaacag 13620tgattcccac tattataatt attacagtct taccaaggag gagctttcca caaataatca 13680attacctaaa atgtccaaaa acaggaaaaa aaaatctctt ccgataattc atgtgtaatt 13740ttcttttttc tctaggagca ttgatctcaa cctgatgtaa agcaagcact ttaaaaagtc 13800ttataaaatt ttcctggtaa atgcaaaact ttctgataaa taaattctca cctttttatc 13860aatttgttaa ttcaacaaaa atatactaca taccaacagc atgcaaagca ctatgctaga 13920ttttatagac tatgaaaaga taaattgcca tctctatgca taaagggttt gccatttaat 13980aaaagagact atatatttgc ataaatatat agtgaatata ttgcataaat atataatata 14040tgtttacatt aaagaataaa aggtataaga gggataagaa aaattgagac agagggaaga 14100caggtcagtt tgagattaac gaatatcccc aaagaaggta ttatctgaga ttggccttga 14160aggatagttg tgattcagga acacagaact tgcagaatga gaaggttgtt acagaccaaa 14220ggaacagcct gagaggcgtg agtatgcagg aaaatgaggg ccatgcctga aagtactggt 14280ggtgttgaag atggagccag gcaagttggt cacagaggga gaggaccttg aatgtctaac 14340attgtggaca gaggctcaaa ggctcaaatt ccctattttt accttgagtt caatccttgt 14400ggcaatgaaa cctcagtgaa gctttattta aggctaaaag tgtcttttaa aaatccctct 14460tatataatat cctttgcatg ttactcttgt tgtaattagg agaaagcaat aggatctaaa 14520gttttttttc acagcatggt tttggtttct ttaattctaa ggagctcacc tggtgttacg 14580ttggaaaaaa cagcttttat atctcattta tattccatat gccagtctgc agtgacatat 14640ctatctgagg tttacagtgt tagccacaaa acactcccta agtgaataca ttgactgctg 14700taaggggagc cagtcaggaa gcacctgcag agaaaagcag gcaacatgta taaacagagt 14760taattcagga atgaaagctg aatggctggg cgagtctgtt tgtttgagtt gacagcctct 14820ccctcactct ttcattaaat atccaactaa ccttcaattg ccctcttgga acttaatctc 14880agtgtaattt ccagcatgtc aaaattatca agcagaaaga gatactaccc tgaaagaggg 14940tcttttgttc aatgctagga gacaaactcc aactacaaaa ttctagaaat gccctaaaga 15000gagagatagg atagatttac aaattgctaa tgctattagg ttgtatagat aacaatagat 15060ttataacaac ctggcacaca gctttaaata tataagtttc tctgaaactt ctgggaactt 15120ggaatgccag aacgttggca aaaagaatgc ttctaataat gaaagccatc atctgccatg 15180gaaacaattt cagggtcttt agaaagctag tttatacata agctccattc tacaataaaa 15240cttatgttca tgttttttct gattttcctc ctgctgtaaa ttcattttat cagaattctt 15300tttaccagtc cctctgcccc atttctcaaa gcgttgtcct cagactacct gtatcaccta 15360aagattctaa ggcctcctcc gatgtagtaa atgagacttt tctagagaga gagtcctaga 15420attttataaa gaaggatcct ttttattatt gtgatcacca aagttacttc tgcctagatt 15480cttctcatgt tatttttaca gctcctatct tcccagacaa cctaacaatt caaagataaa 15540attggtgctt ggtttagaca ttcatagcag gcacggtgcc agattgatga tgtcatccag 15600agtcaaaaac ttcatccaat gccttcacca aaaagttaca aatggccagg aatcaaatgt 15660ggttgaactt attcagaggg taattacaaa acaaacttct ttaaataccc aactgctatt 15720tgcttttttc cttctaaatt gtatcacttc tctccctgtt ccattttgtt tgccttttta 15780ttttttggaa tccctcacct ccatactgag tagtagagct ggctgtgggt gatgagagag 15840aaattgttat aacaaagtca ccctttcaaa aacatgtctt ccaaaagaat tttgtttcta 15900gcagataaac cccacaccac ctcagctaaa tggggctttc tttatttaag taccaataaa 15960gacatatttt ggatactagc aatttatttt ccaaatgcta tctttgatct taagtttaag 16020gctattacca aatctatatc tctacaagtt ttatacttta ggtcaataaa ttacttgata 16080acttattact atgtgttcta caaaagaaac cgaagtaaaa tttacatcac atttaacagg 16140gtggttgtgt gattgagtgg gaagaggcgg accctacaga tagaagactt gggtttcagt 16200cccagcttac tagtatctgc gtgatgccag ggaaattcac ataatgcctc tgagtcacag 16260atttctaaca ggaatgaaga tacttcttcg cagaattgtc attagagtta aagaagataa 16320caaataatgt ggttcctgat gaggtattta tgaattcctg agcatgctaa ggaagttata 16380acttgtttct tgatccctga aacagctttc cctatatttg tgtgtgtgtg tgtgtgtgtg 16440tttcagtcat gcaagttggt ttttcttctc attccttgag aatttaggat attttgtgcg 16500cacatttggt tcttctgtcc aacatgaact gtagtacctt acccacattg agatgacact 16560atttctacca agtgagtgct aggggatact gcaagccgaa tgccaggtgt gagagaccac 16620agcatcacaa taccgtggca gtagattaaa gctgtgcata tggactaaaa gcagtggctt 16680tgcttctcct accttggtga cataaactga gtaacaaatt tgacctaata ctggaatacc 16740acctaattct tttttcctcc ctgatttacc ctagagtcca caattgacaa taatttaaaa 16800attttggctc tctcttaaat ccctaatgcc tcctccttac accttacaag caaagacctg 16860cagagctaag acctgtaatg ccaggatgga ggctagagga ccatcagcaa ttaactacca 16920aaacttaccc aacattttat atctgtttaa ccttcatagc cttatgagta gcagatcaat 16980atctttgttt tacaggttag aaaactgagg ctcaaattga ttcagtaact ttgccaagat 17040tgcccagttt gggaaaagta gtatacgctc aaatccagga ctgaggcagg gttttctttg 17100tcaccactca aagcctctct gaatatccta tctctgctct gtatctctct gctactcctt 17160ctatggtgtt ttagcaagat atcttctact ccagaaacct actctagcac agtagaatta 17220cttgggtagg ttttttaaaa atatgagtgc ctaggtcccc tctagaccaa tcgaaaccaa 17280aattcttgga gaggatccct ggcatccata aattttttta attcatcaaa tgattctgtt 17340gcactgtgaa agctgagatc caccaattta aataatgatg ttagttctgt gaaaaaattt 17400ttgattgctt taacatttaa tcaaggatat attcctatta taaaatatat tattaacaca 17460tagtttctct cttgttgtgt aacaggtgga tgagatattt atagattcag cctggaagaa 17520aaattatatc aagaaccaag tagtcagact gacgtatgta tgtttgggca aaggtggaat 17580cacaagactg gagggaaaag gaacaaagga gacagggact ctcatgtatt gtatgtctcc 17640atggactagg cttttggcta gaatttttca taaacattac ctttaaagca gtcttgaagt 17700atagggctga ccaccgtttt gtcaacaaaa agactaagat tcaggaaggg taagaaatat 17760gttcaaagtt caccaactga cagtttccca aagtgacaga accaggaatc aaaccccatt 17820aacttattgt gaggcctgga acctaccaga acccatgacg tggggaaaac ccagcagctt 17880gtcgttgcat gcaccaagtt atattatgtt gacaattata ttatttcaac cacgttaagc 17940aggcaaactt ggctataaaa tgggttcaca aattttacct gtaatgtaac cgaatgacat 18000aaggcatgcc taaacaaaaa gatattcctg ttgtaataaa ttttctttct gtcatggtgg 18060agggggaaga ctcatatcag ttgcagatat tgctcagaag tttcaattgt gttattttga 18120aaaactacat agcagaacac gcatgtcata tacacaaatc catgagcctg tatgactcat 18180atttcttaaa gataaagaaa aataatatat tcagattttg atttatttga agaaaataat 18240tatccctttc tcaccaatag actaataatg ctttgttggc aggtgtactc aaagttctct 18300atgtcttgac tgagtaacta gtgacttccg taaggatttt ataacataaa ttgggtaatt 18360cctacaatac ttaggaggga aaaagcatat aaatgctaga actttctaga tttcatgttt 18420tctgttttca aattctcctt taccatatta ttgtagcaac attattatac tcctgtgaac 18480tcctttggat ggtagccatc actatataat acctggtaaa aatgttaatt cctcagattt 18540aagaagtaaa attagtcatc tgtttgccaa tttgacataa aattctagtt atttagatct 18600ttatattcca gagcctaaat gaacaaaaat acataaattg tctcagaatt tccttttagc 18660caaaagattc agggagatgg gcctctagag tttttcacag tttttttttt ttttgtaaaa 18720aaaaaaaaaa aaaaaaaaag gagagataac agatcaatat atattagttt caaggttttt 18780tgtttttttt tttaaacaaa aacctgtaat tgcttttcct attttaacag tatttaaaag 18840tttagttcct caggtaacag aacttgaacc tgtttatatg atcaaagttc aagaaattgg 18900gcatgtttaa tttggagaag actcggggac cacaatattg ttgtcttcaa atatttgggc 18960tagaggagga aattatttta tgtatgttcc aactggtaga cctaagcctt atggaatggg 19020agatataggg agacatattt caactcaaaa tgatgaactc ttaaaagcag agctgaccaa 19080agagaaacaa gcctctttag aaaattaaac ttactatctt tttaattact gcactgtcat 19140tagagggcca attgtcatgg accctgtaga agtgattcag gtatcaaata tacaattgat 19200tagcctaaga aaacatgaag gcttcttcta actctcagag cttgtaattt tgatgatgat 19260tttttatatc tgtcattcct agctgctgta acaatccttc aaattaatgg gggaaatgca 19320ctgaaaacat aatgaaagct agaagaggga acatatgaaa tgaccttggg tcagaatgac 19380atgagaggat cagcacttga cactctcagc aactgaggga tcattcaggg gaggaagata 19440caggtaagac tgaaggacaa ttccaggtgt attctttgaa aatgtacctt tcttttgtgt 19500gtcacagtcc agaggaagat ggtgtgaaag tagatgtcat tatggtgttc cagttcccct 19560ctactgaaca aagggcagta agagagaaga aaatccaaag catcttaaat cagaagataa 19620ggaatttaag agccttgcca ataaatgcct catcagttca agttaatggt aaggaggtcc 19680ccttctatgt gatatgaagt tgtctattag gtccatgttt tgacgaatct caaatttatt 19740tgtcattatt tccatttcaa ataatagcta gaattcagat gaaaaaattc aagttaaaga 19800tgtgacattt caaggtgtat tagtctctaa cgtaagcatg tctgaagtta gtcatccagt 19860ggttttcccg acagtaattg attggcactc atcccaaaat ataggcaagc atttacaact 19920aacagagagt taatcccacc caggcactgc ctccatgact aagcaagtga aaatactagg 19980ggtttagcaa taattgtttt tctgggtggg accttcctaa aacacaaatt catgtgttgc 20040catactttta ttgatagttt ctatatatgg tgatatacaa tttttgttag ctttttttcc 20100tatgggcatt tgggaaaatg gcaagccaac tttgaagttg ttagagtcat tttaccatta 20160atgctttaaa aatcacagtc taggaaaaca tcactgaaac tatgtgtaca ttgttccact 20220tttctctttt tttttgttca cccttagccc attataccat tatcacttcc ctcaattaag 20280gagaacaaac ctttatcaag gtctatctct atggccttta ccttaagtaa ctaatttctt 20340tttatattcc agtgacgtac gcaaattcac ctttatagaa gtgaaattca cacaaaaaga 20400gttgaggaat tcagtaatta aaaggagcta agaatcaaat ttaaatctct aatttcttaa 20460aaggctccaa ttaaaaaagg tttctatagt caaacacatc ttaaaaattc tggctttgat 20520actcgtttct tggaaattct tccttatagt gtcatattaa aaattctaag gcagccagct 20580agagagaaac ttgtttaccc tcgtccgcta agctgtttgc acagcatctt cttccaacag 20640acaagtatag atttctccta caaatttcaa tggataccag acctaagtgt tacagaagag 20700attcagggca agcgattttt atcagacatg aaacaggaca ctctgccctt gtaagggtct 20760agctgacact tcaagaggaa accagataag gaagtaaaaa atgtgaggta atggaatggg 20820cagatgtttg ctgatgtgag aacgagtcag ctacttaggg aataaagctg aggacctctc 20880ccagccagaa gggaggaacc tgacaagtgc ttaatccatc ttctttgtta gatggggaag 20940caaatgaata gaagttgtga aacaatgggc attctgataa tttacatgat gctttctgtg 21000taatttccaa taaatagtta atttgtcagg aatgtaaaag cctgaactat ctgaaaccag 21060agtaaagcat aaattgttca ttggctgcct ggtctttttg ttttttgtag gctcagcttc 21120taaacttcag cttattttaa taattgtact aaattaaatg gtaggatatg ctaatggaga 21180acctgatttg agagtcacct gaggctgggc atggtggctc aagcctataa ttccagcact 21240ttgggaggcc gaggcgggtg gatcacctga ggtcaggagt tcaagaccag cctggccaat 21300atggtgaaac cccgtctctt ctaaaaatac aaaatattag tcaggcctgg tgacgggcac 21360ctgtaatccc agctacttgg gagactgagg gggaagaatc acttgaaccc gggaggcgga 21420ggttgcagtg agccaagatc gcgccactgc actcaagcct gggcttgaca gagcaagact 21480ccatctccaa aaaaataaaa aataaaagag ttacctgacc aattctaact ccactaagtc 21540accacaggac cacccaaata attggctcat gcctttgtct tcattttctc atctgtaaaa 21600ttccaatggt aatgtttgtt cttcctgaaa tcacagagag attataacga tatacaagga 21660aatagaaaac acaatgtgaa ataaagaggc tgttactaat gagaaaacta ttatgttgtg 21720catatgcttt ggaaacctga aatcattaat ttgagtgatt gactagtagc agaaagatag 21780atccttgaaa gtttcagaat gttcaatgta gaaagaacag tgtttgttag tgatatggga 21840gcctaggggg tgttgctttt ctggccagaa acctctgtgg ccagtggttg gtgcctttgc 21900ccaagttttg ctctggccca ctgggcttgt tctgcccact tgacctggca gactgtgccc 21960accttccgct accagcctgg atcccatgcc caccaaggcc aacccaggca tggagctgtg 22020agggttgtct gagcgagcac agggtctggc cactgcccac agccaggcac actggctgca 22080gcatgacggg cagctccagg cactggcaca ggtgtgctgt ctctctgtga ggctgtggct 22140ggacaaagct cactgcaagc agcttccctg gcaggcacct gggaatgtgg tggcacccag 22200gaagcttgga gatgccagga actgcagggt cccaaagagg gagtcacaac cctggcttgg 22260ggagctccca ggtctgggat ccctaaaggg ctgcagcttt tctctctttt tacccacaat 22320gtggccagca aggggtatgt ttcattcctg tttgtgttac agctctttta gtcttgctat 22380ttggcaggtc ctgagttctt gtcctgagac caagaagaat gaggtatgca gacaagtgga 22440gggtgagcaa gacgaagaaa ggtttactga gcaagagaac agctcacagg agacccacag 22500tgggcagctc ctcttcatag ccagggtgtc ccaacaagtg tccagctcct agcaaagagg 22560aggccctgga ggtagaagct cctctctgca ggcaggttgt cctgttgagt gttcagcttt 22620cagcacacag taggcagtag gccctagagt ggtctatctc ctctctgcag gcaggtagtc 22680ccatggtctc ccagtcacct ctccatctgc aagggtccaa tgctgcctcc agcacctctc 22740tgcccacccc tccgtgcctg accaagctgc tcccccacca gtgggcaact cagcccagcc 22800ccattgtggt agctcccagg gtggcaggct ctggggggct cccagggatg ggctccaagg 22860actgtccacc ttctccccac gccctccctg cagtggccat ggtcaagaat ggcaatgtgg 22920ggccaggttc cggagcagga gaggctccag gcctgggagc aggtcctgcc tggtcacgtg 22980aggttggggg tggcacagtc ggctgcctca gggatgtggg acacagggga cccaccacca 23040tcactgctac tcccgcatcc gctcctgcta ccactgctcc agacagcctg tagctgccat 23100cactagcact taagaaaggc acattcagtg gacagctcag gaaaatcttt acgtcaattt 23160tttataggca aaaacattgt ttcctgggca aacaaaattt atggactacc aataaataga 23220aaactgtaga gattctagat taagtctaga aataatcctg tagcccaaga tttatttata 23280atttgtcaag aatctgtatt ttgttttgac aaaaaaaaaa ctgtgtggtg tgggtccttc 23340aggagacaca gtgtgacaaa gcaaagctaa aatcaacttc tttgcattgc aaacaccaag 23400gctgtagtca agcagctcac tgcctatgtg tcagatgact ttgcttcatt tttcatcatg 23460atacttgtag tctatagagc cctgaatatt aactagcttt ctcccaactc agaaccgtgt 23520taggaggtgg ttgctttcaa aactaaagtg ttaatgttta tttccatttc tataccagga 23580aagtaaaaat ctttggtcaa aattagaaat ctttaacaac tagttacttg tgtattgaca 23640gtttgtttcc aggtgtaatc attctccctt aaaatccggt tatattcacg accattatac 23700ttatcctggt atcattcctg gaaatggcta acttgcatcc tgctcagact aagttgacaa 23760agtttcaatt gaagaattct aactttatgc tattttccac tttattgcat tacaaaggac 23820aaaatatata gttttcttaa aaatgaaata aatttactgc cttaaactac atttgacggt 23880aaactgagtt ccttccatag aataaccact aacagcaatc gatggtcctg agcaattgac 23940tcttcaccat acaatgattt gggatgcctt taagggtata tttgaattga atattttcaa 24000aagctcccac tttgtagagt ttatcatcac tagtttcccc agtggaattt gtagaaagtt 24060agtagaatga aacaatctta ttttgtataa tgaggaatag aatactgaga atgtgtctga 24120gaaacatggc actggtagga aaaagtaaac agtttattct catctgctca ataagctaag 24180tcattttaac ttgaaaatca tcaaaatttt catgaaacct tccaccaact ttatttttcc 24240ccagctttag taagatataa ttgacaaata aaaattgtat actgtataca acatgatgct 24300ttgatacatg tatacaagtt taaatatttg tgtttcctta gtcaaactcc tcactttttt 24360ggaagttgac agaatttaat cttggattgt gtccaataac tagcttttac cactattcag 24420tatattttgg ataagaaaca cataacagtt tattctttaa aaaagcaatt ttactattta 24480ggaactgtgt ttaaaaagca ttttaaatat catttatgca agagttttca aggttttttc 24540attctaaacc ctttaaccaa aaaaaaaaaa aaaaagattt atgtgaaatt cgaagtaaat 24600agaagagatc aaagcagatc tgttctggct gaggctgagt ttgagacctg taagacagtc 24660tacttgccat atggcttggc tgtgtcccca cccaaatctc atctcgaatt gtagccccca 24720taattcccac atgttgtgag agggacctgg tgggagataa attaaatcat gggtgcagtt 24780tcccccatac tgttctatgg tagtgaatga gatctgatgg ttttataaga ggcttcccct 24840ttcacttggc tcacattctc tgacttgctt gccaccatgt aagacatgcc ttttgccttc 24900ctccatgatt gtgaggcctc cccagccaca tggaactctg agtccattaa acctcttttt 24960ctttataaat tacccagtct cagatatgtc tttatcagca gtgtgaaaac aaactaatat 25020aacctgtttc ctctgtccca tttatccatc ttctgaagtg gaatgcaaag aagctttacc 25080ccgaactgct ggaaaaccat agttctctat taatacaaac tatttgtggg ctttagtcat 25140ccactatttg tgccttactc acccattgct tgtgatagta tccacctaat tagaggctgc 25200ctataagtct ctacaaaaac tgtacacaga tgttgttata tcagatagcc attctcctaa 25260ttaatctata tgttcaactg tctagaatcc atatatggtc agtatcctct gattattcct 25320ggtcattgag accaaccagg aaaatatcaa attatcacta tttgttttat cttctttttc 25380agcaatgagc tcatcaacag gggagttaac tgtccaagca agtaagtcaa gttagcttat 25440ataaacaagt tcaattttca catcagaaag gacattttca aatatttgct catacttgcc 25500catctgtcct ccagattttc tttgagagat aataactatt tgtacgatag atttaaatac 25560attttttttc taactcatgg actgatcttt tagtcatgtt caagaaaaaa attgccatgg 25620taaccttctg gggcaatttg aagaaagcat ttatttttga ttgggaatat tggacttgtt 25680tttctaattt ttaaaaatgc cataaaatgt actttctgct acaaaataaa ataataagaa 25740agtaatcaat aggaaggaca taaaacccat tgtctgtgac tgacaatttg tctgtgaaat 25800atgctaaggt caggagttcg agaccagcct gaccaacatg gagaagaaaa cccatctcta 25860ttaaaaatac aaaaattagc caggtgcggt ggcaggtgcc tgtagtccca gctacttggg 25920aggctgaggc aggagaatca cttgaacctg ggaggcagag gttgcagtga gccaagattg 25980caccactgca ctccagcctc agcgacagag tgagactcca tctcaaaaaa gaagaaaaaa 26040atatgcttaa tagattcatc ttaatcgcta acagtggctt cattaaatca cttcaaatca 26100ctgtggccta aattttgaaa gattttacaa aaaacagtga tgaatttgag caatgatgtt 26160catgcatttg cctctgtgac ttgcaaacac cctaagtatt tttatccatg tgtttattca 26220ttcaacaata tcttttaaca tctaccaagt gccagaaatt agaccaggag ttggtggtac 26280cattgtgaat aaaacatgat ccctgctcta aaattagaat tccaaagtag agaaagatat 26340aaataaatca ggaagtatga aaataatgtg attaatgcta tgacagagga agtgcatagt 26400gctatgagag ttgatcagag agtcagctaa cctgttctca cacagtaaga aagtgaaccc 26460tgaaatgtga gagagaagag gccatgaatc cagtgacagg tggggtaagt gtcctgggca 26520ggaggagtag tatacgaaaa tgtcttcagg caagtaagaa tggggtcatt tcctgtaatt 26580acaagatgtt tcttataact taatgatctc atcttttttc aggttgtggt aaacgagttg 26640ttccattaaa cgtcaacaga atagcatctg gagtcattgc acccaaggcg gcctggcctt 26700ggcaagcttc ccttcagtat gataacatcc atcagtgtgg ggccaccttg attagtaaca 26760catggcttgt cactgcagca cactgcttcc agaagtaagt tattgacctt aagttagaac 26820ccacttctgc taaaaagccc tgagttttgt catattcttg gtaacaatta atgtctcaaa 26880tattactgaa gtaaaataag aaaaagttat ttcaggttct tttctaaaat aatgttacac 26940ttgcatactt aatcagaaat ttgatgggaa taagtaacag tcattatcct agtatccatc 27000aatcatttcc tcaaagtttt taataaggaa actgtgtaaa gaaatcagaa ctattttgtg 27060acatcctaac acaaaatatt cactaataac atgtaccatt aatcttttgt caaacaatgc 27120tctccactta aaactagtgt ctgtttctgc caaacacttg ggccagtctc atactgatct 27180taaataatca aactaattcc aaagtaaaat ggaaattttc aataaatgcc ggaagttggt 27240aaccgtgatg atggagaact gcagatcaaa tttagagcat tgacatatga agatctgtgg 27300aatcagaaca gtttacaacc aaaatgagag attgctagca tgataaagac aggcacttca 27360aaagagattc ctcggagtat caaaggattc atagaggccc ttgggccact caatgtgacc 27420ttcccataat agagcatctc ttcacaatag tgacacaaaa gacaaagctg aagtgaagaa 27480tagcaaattg tgctatccta taattgtttc tgaatgcata cattttatta aatatatgat 27540taaatgactt tttataactt ttaatcttac ttttcaagat aataaccagt catttttatc 27600actattacat ttagaatttt agatttgttt ctaagtagat taactgtatc gcctttcttc 27660ttcattgcca attattacag taataacaaa gacttcttga gtatctctat ataataggtg 27720gcagcaggat ttagtgggaa aaatatgtcc caggcagttg gagagctggg caaattattg 27780aaccttagtg tattaggtaa tagataggct agatcttttc acattctttt tgacctataa 27840aattctaact tttgttacta taataaattt catttgccta ggagcataaa tctttataga 27900gactcttaat attccaaaga atatacatat taagaatcta ggcttggcat ggtggctcat 27960gcctgtaatc ccagcatttt gggaggccga ggcaagagga ccacttgagc tcaggagttc 28020aagaccagct tgggcaagat agtgaaaccc cattgggcat ggtggtgcat acctatcatc 28080ccagctactt gggaggctaa cgcaggagga tcccttaagc ccaggagttt gaggctcctg 28140caagctatga ttgcaccact gcactccagc ctgagtgaca atgcaagacc ccatcttaaa 28200aaaatagtaa tatattttta aaaataatct acataaattc ttaatgtttg aaagatgtga 28260gagctcagta agctgatata ttagaaagcc agaaatccct tatgctggtg tctggttttt 28320caaagtaatg ggaaacttac tttgccaaag ttagccattt ttgtggtaga tagttctatt 28380tttgcaaata tctttatagc attgaacacc aaatctatac tctattaact tctaccatca 28440atatttgttt ttcttttaat ctggaacaac aggaaccaat tttatttctt cattcatata 28500acagctattc tttagtttct ctttttcaga ccaaacataa aatgagggag aatatccaaa 28560ccataagtga aaataaatat cattactgtg agctttagtt tgctaaggat aatgacctcc 28620agccctatcc atgtccctgc aaagggcatg attttgttct ttttatggct gcatagcatt 28680cccatggtgt atgtatacca cattttcttt atccagtcta tcactaatgg gcatttaggt 28740tgattctatg tctttgctat accgaagagt gctagaggga gaggatcagg aaaaataact 28800aatgggtact aggcttaata cctgggtgat gaaataatat gtacaacaaa accccatgac 28860acaagtttac ctgtgtaaca aacctgcaca tgtaaccctg aactttgaaa aaagtatata 28920tatgcacaca catatatatg catacatata tatgtgtgta tatatatgca tatatgtgtg 28980tgtgtatata taaaaaaaaa tatatatata tatatatata tataattacc tcatttttcc 29040agaaccaact tccagatgcc ctaccacatt ggttcttatt ctctgaacat tcgagacttt 29100gtcagtgtct tccttaaaat atgcttccaa taactaaata caccaagaca gatgtgtgac 29160tagtgtcaca cataacaaaa taaagcagga agtcttctga aaaatacaaa taatgtaaat 29220tggtgggaga cagtgtttta taaagggaag agcagagaga ggcaggcaga tatgtgatgt 29280gaatcaaata gtttaaccta tccaggcttt attttcctta agtataaaac acagtcttta 29340ctagatgatc tttcattgct actaaatgat ttttccgatt cctgtatgta ccataatcca 29400cccattgccc aagcccacaa gctagaagtc aaccgcattt accacatttg atcatctctc 29460aaaggactat gcagtcatct aatagacttt accacatcca ttcttgacct tcaagaatct 29520actccccaga aagaacaaac atgtttttta aaaatgtaaa tgagactaca ttattctctg 29580gcttaattat ccagtagatt cccatatcac ttcaataaaa tttaagcact ttatcatgac 29640ctataaaaca ctctaaaatc tagtccctgc ttacctctcc aagctcaccc ccaaccattc 29700tttcccttgt gttctgactg cagcccatcc aacccaagac cttgggattt ttgcctggaa 29760acttgtttcc ctcatctcct cacactgacc ctcttttact atgtcttagc ccaaatgcgt 29820tatcaaaata atcataatga cctgttagta ctctattccg ttaccctatt ttattttgtt 29880catagccttt atcaatgttt aagattattt atctatttgt ttgcttgctt tgatcctttt 29940ccttctctgg aatcttatac tcctgtgagc aggcacctta ggtcctgttc atcactttat 30000ccccagcagt tcagataagg ctcagcacac agatgctcag taaatatttg tggaagggat 30060aaatgaatga tattttatgt gtattacagt tctaaaattc aatagttttg tattaaatat 30120cagttctaat atggcattta tatgatttta tctttcaaaa cattagcaat agattatatt 30180taaatgataa aagaaaacta taactgcagc caagtattct caggattgta tttctcttat 30240attagcctaa atgcaattaa tctagctcat atactttggg cagcttatat atattctgtt 30300aatttctaac cttttccagg tataaaaatc cacatcaatg gactgttagt tttggaacaa 30360aaatcaaccc tcccttaatg aaaagaaatg tcagaagatt tattatccat gagaagtacc 30420gctctgcagc aagagagtac gacattgctg ttgtgcaggt ctcttccaga gtcacctttt 30480cggatgacat acgccagatt tgtttgccag aagcctctgc atccttccaa ccaaatttga 30540ctgtccacat cacaggattt ggagcacttt actatggtgg tgggtatctc aggatagcta 30600acagagcgct aagccctgtc taaggcaatg tgatttcatc tccatcaata ttatcctgac 30660agccatttcc acacagtctg gttggattag ttagggttct tactttgtgt gacagaaatt 30720caattcacat taaccagtgc agaataaaaa acaaagaaac aaaaacttcc acaaatttgg 30780ctcatgtaat ttggaagtca aaaaagtgta gtaagtttca cttcagacac aggggtttat 30840atgatgtcat ctggctctgt gtctctgaat ttgaattttt tgccccttct tttctctatg 30900ttggcttcat tcagagggat gctagcttca cctagtgtca gaggtggcta acaacacctc 30960aacacatcat cctcaacaaa gaaaaaatac atagaaagga atatttattt cttttctttg 31020ccagaattca cattaatttc tattgttcca gctgtgtcta ggaggactca gattgagtgg 31080ctaactcaaa tattctttat gcctatgtag caaaatttgc ttcagtactg aagaagctaa 31140tttaagtgtg atggtgaata agaatagtgt agagataaat tgtcaaacta tttgtcccct 31200ctaaaagtat tcaacttgat atactaactt agtcttgtaa gaaataatga tgatttagtt 31260actgaatgtt ctaggcaatc ttagtgagac acgctctgga ttctaacatg tggtccaggt 31320acatatgtat aacaaagcta gaaagtttct ttaacactgg gcttgagaaa atgcaaaagg 31380gctttctgag aatgactaaa tctatttgca ggattctata caatttattt acatacaaga 31440aattataaag aataagcttt tgattctcag tctaccatta aggaactagg aataaccttt 31500cactcacata ggcaggaatc ggttttaggg tctctagatt ttttccagat gtcccatgtg 31560gttttgtttt atcttataca gagtgagaca tgcattgctt tctttaaggt tgtattacca 31620atcacagaaa atattaccta tggtttatta attctagtag atccagtgct gctgtaagcc 31680tgacacctcc ctaggtctgc actctcttgg atggattttc tctgaagata gggcttgcat 31740tctctgcttc atagtggtgg gaaagacatc acaaatcccc tttggcttgg tgggaaaaat 31800cactttcagg agtttgagac tggcacagaa acatacctgt cataatgcgc tgtgagtggc 31860aacagaatct gacacttata gagcactcca ccctacttga acacggcctc tcttggtgag 31920tgacccacag gtgcttttaa tctattaaat agattaaatt aacctatcat tcttaatctg 31980ttaagtacat taatagatta aaagcagcca ttcgttactc accaagagag gctatattca 32040agtctgtaaa gcaaacctta agaagttttt taaaattgaa attgtacaaa gtatattctc 32100tgatcataat ggaatctaac tagacatcag taacagaaag ataacataaa aatccccaaa 32160tgcttaccaa ttaaaaaaca tatgtaaata aagagaatat ctcgaagaaa tttgtaaaaa 32220caaatagaac taaatgaaaa caaaaatata taaatatatg ccagatgctg ctaaaatagt 32280gtagaaaggg aaatttatag aaaatgcata ttataaggaa agatatcaaa tcaataatta 32340agttctcact tcaagaaact agaaaaataa aaaataaacc taaaacaaac ataaggaagg 32400aaataataag aataagaata gaaatgaata aaattaaaaa taaactatag aaaattgata 32460aataaaaagc tgattatttg ataaaatcaa tattttgcta gaaatgtcat taagcatttt 32520tacagaagat gagatatagc tcagggatgt ccagaattta tgggctatgc ttttcatgac 32580ttggaataca ttttaccaac cagtttagtt tgctgaagaa gttgtggatt tgcactgtca 32640cctacttaca atacttagat tgtcagtttc accttactct tctcaccatt attttatttt 32700tatttttatt tttattttta ttttgaaaca gagtctcgct ctgtctccca ggctggagtg 32760cagtggcgtg atctcggctc actgcaaact ccgcctcccg ggttcacgcc attctcctgc 32820ctcagcctcc cgagtagctg ggactgcagg cgcccaccac catgcccggc taattgtttt 32880gtagttttag taaagaaggg gtttcaccgt gttagccagg atggttttga tctcctgacc 32940tcgtgatcca cctgcctcgg cctcccaaag tgctgggatt acaggcgtga gccaccgcgc 33000gccaggccat gaatgttttt aattgatgat atagtaggca atataaatgt gtgtgtgtgt 33060gtgtgtgtgt gtgtataata tatataaacc aattgtattc aaataacaga ataatttgaa 33120aaatctctta gcatatttct gagttacaca cttaaatctt ccgagcactt ttaaatatgt 33180gtttacaaac atttcttcag aaataaatct tggaaatcgt cttctaaaga aactggtgta 33240ttagggtttt ttcaaatgta cttagttttt tttttaattg atgtataaaa ttgcatgtac 33300ttaccatgtg caacataatg tgttgaagta tagtatatgt acactgtgag tgttaaatct 33360agttaactaa gaagcgtctt attttacata attatcattt ttgtggcaag aacacttaat 33420atctactctt gtagcgtttc tcaagaatac gatatatcaa cagtaggcaa ccagaagctg 33480ggggtcttta caggggaagg agttagggag atgctggtca acaaattcat atttgcagtt 33540aggaagaaaa agttcaagag atctctcatc catcatggtg actatagctg atgatatatc 33600gtattcttgt attagttttt tataaatgtg taacaaataa tcacaaacag ttaaaacagc 33660actcatttat ttttatctca ctgttttcat gagtcagacg ttcagacaca gcttagttga 33720gtcctcttct cagggtctca ccaaactgta atcaaggtgt cagctggggt tgtggccaca 33780tctgtggctc ctttgaaggt ctcctcaagg tttgctggca gaattccttt actcgcagct 33840gtagaatgca tgccagcttg ctgctttaac tctttaggaa agtgtctcaa ctccagcaag 33900gctcgccctt tttgaaatgg ctcagctgat taggtcaggc ccacctttga taatctcctt 33960ttgatgaatt caaagtcaaa ctcattagag gtcttaatcg catctgtaaa attccctcat 34020cttggccata taacataacc taatcatgag aatggcatcc ctcatattca cagatcctgc 34080ccatatttgg gaggagggga atcacacagg aatcttgggg actatcctag aattctgcca 34140accatggggt catggtttcc caatcaatat atggtttggt ataaagaatc cctgaatgct 34200tgtgctattc ttagttttct acgtagcctg ccataataat ggtttctaaa actcagaacc 34260tagcttacag tctgcagcca ccaacttgta atacattgga agtgaaatca ttgccgttta 34320atgcatttat atatatatga tgtataatat atgtatattt cacatatatc ttatatatgt 34380gaaagctcat cataaacttt aaataataaa ataaatgtac atagtattat aggcatttta 34440tcaagccaat ggagaaaacc atctaggcat gcagagtttc tgggaacaat ctggaaccca 34500caaataaaag ctttacaaaa gataaaaggc cttcctgaaa tatataagct gattattttt 34560aaggttagat tttaccagga aaaagaatcc aaatggcttt cttgctttga gaagttttta 34620taaaaatgtg attggacaat aattatcgtt agatgtgcca gatttaacca gaaattcttt 34680tttctagaaa ctgcttatat taacttcatt ctgtattgac aattttacca tgaaaaaaat 34740attaggaaag tcttctcact tcactctagc caaagatgct gattgtaaat actagaataa 34800ctctattttt ccttaagggg aatcccaaaa tgatctccga gaagccagag tgaaaatcat 34860aagtgacgat gtctgcaagc aaccacaggt gtatggcaat gatataaaac ctggaatgtt 34920ctgtgccgga tatatggaag gaatttatga tgcctgcagg gtaagttgga gggatttttt 34980tatattacta actcaaaaat ttgtatctgg cttagaatat attatatgtt ctttacataa 35040ggacaaaaca tagatatcat gtcagctcaa aaaagttaca aatgcaaatt tcacagcaca 35100aaatactttt aaatgtttta ttaagataaa tgaagtaaga gtttctctga tgctatcaaa 35160caaacaaaat tagaatttct taaccagaaa tccaaagatt aataaagcag tttattttct 35220caagcggctc acattcaaga aagaaaataa tcataaacag agaagtataa agtgatgtta 35280tgaataatat aatgaaaagc aaatattttt cttgaaggaa acatttttgg aacaagtatc 35340agagagatga gacgtaaata aggcctgaag aataaataac atccaatttc agaataagaa 35400aataatgtta tagaaaagac aaaaagcata gccaaaatta tgaaggtgtg aaattacaat 35460tcatatctga gggaactcca agtaattggt tgggtctcag catgaggagg atgagaagag 35520aaacaagtag ataaccatga gaaggtggat taggccatgt tgtgattcca tgggccctcc 35580ccagtgccct catctgcctt ctaacatgga tgttttccag cgaaggtacg tttcttcctg 35640gagacacttg ctttttaaca tgagatactt tagaactcta aggaggccac tctatgtgga 35700aatgatggaa tggtattgat atcaggtggc agaaagtcct gtccagagtc ccacaaactg 35760taccacatgt gcgacctcta tcagaaaagg agcagggacc tatgtgacat agaggctggg 35820caaaagcagg atctggtcca cagccagcct cggttgctaa taatgtggag ggaggcaggc 35880agaatttagg gattccaaca aaaggtccat accacgggga acaggtggaa ggtgcaggag 35940tcttggagca gacaggaccg gggaattcag gtgaaccatg acattactga aaagccttag 36000gagggattgg tggtcataga gatgcttcac tggattgggg agcagaggta aacttgctgc 36060ctaactgtgc aaagtaagtg ataaaacaag gctttagtca tagaaaaata cagtaagtta 36120tcagggcagc ggttcaggta caaggatcca agacaggaat acagtgattg taattggggc 36180acatggtgag gggcctagtc tgatacaaca gaagtgcaag caccaccaac acctcgtctt 36240tctccataag tctttctctc cagagccctc atgacctaat cacctcttct taagtcccat 36300ctctcaacac tattgtattg gagattaagt ttccccaacc tatgaactct tgggctcaca 36360ttcaaaccat agcaccaccc agcacaaaag cacagagctt ccaatctggt ttctagctcc 36420ataccctaga accaaacagt aagaatcacc tctggaaatg tagcaataat ataatcataa 36480tttttaaaat ccagtggaag gattggaaga taaaatcaag gaaatctctc agaaagaaca 36540acaacaacaa aaaagacaca gaggagaaaa ataatcagaa aaattaagaa aactagagga 36600taagctcagg agatccaaca ccaaatgaat aggagctctg aaaacataaa acgcgagtgt 36660acaatataaa aaaaaataaa gaatgctcct agttctgaag cttacatgca tcctattgaa 36720gaaaaggtcc aagtagtgct gggcacaata aatgaagtac ttctttccaa gacataccat 36780cataaagggt cagaagccag ggataaggag aacaatctta aaactttgaa ggaagaacca 36840tcagaactac atagaactcc tcaacagtaa ctctagaagg tagacgatgg tggaaaacac 36900attcaaattt caaagggaag attatttcaa cctagattcc tacccatgct aactaaatat 36960caactgtgag ggtggaatta agaagtttag acaagcaatg actgaaaaaa atgtacttct 37020gataccctac ttcttaggaa actacttgag agggtacctc agcaaaatga gggaataaat 37080caagaaagtg gaagacgtaa gacctgaaac tgttagtcca acactaaaga gtggtatcag 37140ataatcccaa caccatagct ctgcaccagg cttaaagtaa ccagctcgaa tttgagcaga 37200agtaagaaaa gattgtgtgt atgtgtatgt gtatgtgtgt atgtgtgtgt gtgtgtgtgt 37260gtgtgttgat atggtggaac agcttcagag gaagtaaaag aactaacaag ctatctgatg 37320tccttgaaca ttagtaaaca ttattgtgag gtgttggtag atcttttgga gcattcagca 37380tttaccaggt acatagaaaa ctatccacat gaaaaaaaga gttgtgttat taattctagg 37440aaagcaaaaa aagatttctg taatccaaat atgttacttg actcttcaat taataaaatt 37500tacacactgg tactaaatgt aggctgttaa tttaaccaaa aatagagatg ctataatgta 37560aagatgtggt gtggaaaagt tgcaaagaag ttgtaaaaca actaaatccc taactacgta 37620agagaaaata aatatttact gtctaaacct agaagctgta atttgagcat attatctagt 37680gataaggagt tagatactat aagaaatcat taaacaagca tgaagtggct acctcttgga 37740gaacagcttg cgtgaggtaa catgggacat aactgctttt caagcctctt catgtttttt 37800cgtttttgcc ttttttaact aagtgctgtt tactctaaca aaataaattt tattttttaa 37860atgtgaaagt tgaaccttaa ggctctttgt aatattaaaa tccatgtctc aattaattat 37920tctgtgttga tagtctatac atgtactgtc tagtaacaaa atatgtgatt catcaaaata 37980tcttaaataa tgagctttat gtttagctaa ttttctttct tttttcttat gtttttattt 38040ttagggtgat tctgggggac ctttagtcac aagggatctg aaagatacgt ggtatctcat 38100tggaattgta agctggggag ataactgtgg tcaaaaggac aagcctggag tctacacaca 38160agtgacttat taccgaaact ggattgcttc aaaaacaggc atctaattca cgataaaagt 38220taaacaaaga aagctgtatg caggtcatat atgcatgaga attcaactat ttagtgggtg 38280tagtacaaca aagtgatatt aaattactgg atctagtaac atgaaacaca caacgtaagt 38340tatttagaat cactttaatc aaccaataat ccttagccaa tttataaggg acttttattt 38400gtaaagtaat ggatctggct tgaaaaatac ggtagagata cttagctctt taaatcacga 38460atgttgaagt accagtgaga ctcaatacat atttttgaag atagtccatg ggatttttag 38520aatgtcgttg tcaagggtct ccttttaact gagaaacttt ttgaactcac aaagtgttca 38580agaaaccctt gtataattcc ctacatttct ctcgagctca caaatacttt tttttctttt 38640tccttattca atcagatttt ccaaagtacc tttccaccat aagaaatgaa ttttctactt 38700ctacacccat ttgagagaca ccaataaaag aaagtcatat gtaggaaaca aagtctgata 38760gtaaaacaag ccagagatct tctaactttt tttagttata aaacctctaa tttttggtga 38820cttttctaca cacacacaca cata 38844 4 407 PRT Human 4Glu Pro Trp Val Ile Gly Leu Val Ile Phe Ile Ser Leu Ile Val Leu 1 5 10 15Ala Val Cys Ile Gly Leu Thr Val His Tyr Val Arg Tyr Asn Gln Lys 20 25 30Lys Thr Tyr Asn Tyr Tyr Ser Thr Leu Ser Phe Thr Thr Asp Lys Leu 35 40 45Tyr Ala Glu Phe Gly Arg Glu Ala Ser Asn Asn Phe Thr Glu Met Ser 50 55 60Gln Arg Leu Glu Ser Met Val Lys Asn Ala Phe Tyr Lys Ser Pro Leu65 70 75 80Arg Glu Glu Phe Val Lys Ser Gln Val Ile Lys Phe Ser Gln Gln Lys 85 90 95His Gly Val Leu Ala His Met Leu Leu Ile Cys Arg Phe His Ser Thr 100 105 110Glu Asp Pro Glu Thr Val Asp Lys Ile Val Gln Leu Val Leu His Glu 115 120 125Lys Leu Gln Asp Ala Val Gly Pro Pro Lys Val Asp Pro His Ser Val 130 135 140Lys Ile Lys Lys Ile Asn Lys Thr Glu Thr Asp Ser Tyr Leu Asn His145 150 155 160Cys Cys Gly Thr Arg Arg Ser Lys Thr Leu Gly Gln Ser Leu Arg Ile 165 170 175Val Gly Gly Thr Glu Val Glu Glu Gly Glu Trp Pro Trp Gln Ala Ser 180 185 190Leu Gln Trp Asp Gly Ser His Arg Cys Gly Ala Thr Leu Ile Asn Ala 195 200 205Thr Trp Leu Val Ser Ala Ala His Cys Phe Thr Thr Tyr Lys Asn Pro 210 215 220Ala Arg Trp Thr Ala Ser Phe Gly Val Thr Ile Lys Pro Ser Lys Met225 230 235 240Lys Arg Gly Leu Arg Arg Ile Ile Val His Glu Lys Tyr Lys His Pro 245 250 255Ser His Asp Tyr Asp Ile Ser Leu Ala Glu Leu Ser Ser Pro Val Pro 260 265 270Tyr Thr Asn Ala Val His Arg Val Cys Leu Pro Asp Ala Ser Tyr Glu 275 280 285Phe Gln Pro Gly Asp Val Met Phe Val Thr Gly Phe Gly Ala Leu Lys 290 295 300Asn Asp Gly Tyr Ser Gln Asn His Leu Arg Gln Ala Gln Val Thr Leu305 310 315 320Ile Asp Ala Thr Thr Cys Asn Glu Pro Gln Ala Tyr Asn Asp Ala Ile 325 330 335Thr Pro Arg Met Leu Cys Ala Gly Ser Leu Glu Gly Lys Thr Asp Ala 340 345 350Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Ser Ser Asp Ala Arg Asp 355 360 365Ile Trp Tyr Leu Ala Gly Ile Val Ser Trp Gly Asp Glu Cys Ala Lys 370 375 380Pro Asn Lys Pro Gly Val Tyr Thr Arg Val Thr Ala Leu Arg Asp Trp385 390 395 400Ile Thr Ser Lys Thr Gly Ile 405

Claims

1. An isolated nucleic acid molecule encoding a serine protease, wherein the isolated nucleic acid molecule consists of a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence that encodes a serine protease comprising the amino acid sequence of SEQ ID NO:2; (b) a nucleotide sequence consisting of SEQ ID NO:1; and (c) a nucleotide sequence consisting of SEQ ID NO:3.

2. A nucleic acid vector comprising a nucleic acid molecule of claim 1.

3. A host cell containing the vector of claim 2.

4. A process for producing a serine protease comprising culturing the host cell of claim 3 under conditions sufficient for the production of said serine protease, and recovering the peptide from the host cell culture.

5. An isolated polynucleotide consisting of a nucleotide sequence set forth in SEQ ID NO:1.

6. An isolated polynucleotide consisting of a nucleotide sequence set forth in SEQ ID NO:3.

7. A vector according to claim 1, wherein said vector is selected from the group consisting of a plasmid, virus, and bacteriophage.

8. A vector according to claim 2, wherein said isolated nucleic acid molecule is inserted into said vector in proper orientation and correct reading frame such that the serine protease comprising the amino acid sequence of SEQ ID NO: 2 may be expressed by a cell transformed with said vector.

9. A vector according to claim 8, wherein said isolated nucleic acid molecule is operatively linked to a promoter sequence.

10. An isolated nucleic acid molecule consisting of a nucleotide sequence that is completely complementary to a nucleotide sequence of claim 1.

11. An isolated nucleic acid molecule encoding a serine protease, wherein the isolated nucleic acid molecule consists of a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence that encodes a serine protease comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2; (b) a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:1; and (c) a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:3.

12. An isolated nucleic acid molecule consisting of a nucleotide sequence that is completely complementary to a nucleotide sequence of claim 11.