JP2001017183A - New metal protease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new metal protease composed of a metal protease containing a specific amino acid sequence and having protease activity, highly possible to be usable as a drug-creating target molecule and useful e.g. for searching a treating drug for a disease caused by the enzymatic activity. SOLUTION: This new protease comprises a metal protease containing the amino acid sequence corresponding to the 1st to 740th amino acids of the amino acid sequence of formula I or corresponding to the 1st to 481st amino acids of the amino acid sequence of formula II and having protease activity or a protease having action similar to the metal protease. It is highly promising as a drug-creating target molecule and useful e.g. for the screening of a substance usable as a treating agent for cancer, arthritis, arthrosis deformans, etc. caused by abnormality such as promotion or lowering of metal protease activity or deformation. The metal protease can be produced by screening a cDNA library derived from human spinal marrow, integrating the obtained gene into an expression vector, transducing into a host cell and expressing the enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel metalloprotease, a gene encoding the metalloprotease, a method for producing the metalloprotease, and a screening method using the metalloprotease.

[0002]

2. Description of the Related Art Several hundred types of proteases have been reported so far. Some of these proteases include not only molecules that simply digest proteins and peptides, but also life phenomena such as the maturation of proteins, the expression of biological activities, the regulation of metabolism, the expression and transmission of information through the cleavage of peptide chains. It is known that there are a number of molecules that are involved in important direct roles.
Therefore, pharmaceutical applications of protease inhibitors have been promoted since ancient times. In the classification of proteases, those containing metalloproteases, particularly zinc, include those in which the inhibitors of angiotensin I exchange enzyme (ACE) are actually used in the medical field as drugs for treating cardiovascular diseases such as hypertension, and matrix metalloproteases. Group, endothelin converting enzyme,
Many molecules such as TNF (Tumor Necrosis Factor) converting enzyme (TACE) and procollagen C proteinase have become drug discovery target molecules, and clinical development of their inhibitors is underway (supervision: Osamu Hayaishi, protease and its inhibitors, Medical View, 1993). Here, as notable among metalloproteases, ADAM (A D isintegrin A nd M e
talloprotease) family. ADAM is, as its name implies, a molecule containing a disintegrin-like domain containing many Cys residues and a metalloproteinase-like domain, many of which have a recognition sequence for furin protease between the propeptide and the metalloproteinase-like domain. It is believed that they cleave off the propeptide and become the mature protein (Black, RA et al., Curr. Opi.
n. Cell Biol., 10, 654-659, 1998; Huovila APJ
et al., Curr.Opin.Cell Biol., 8,: 692-699, 199
6; Wolfsberg TG et al., J. CellBiol., 131, 275-
278, 1995). ADAM constitutes a metalloprotease subfamily independent of the matrix metalloprotease group due to the homology of its metalloprotease-like domains. Recently, thrombospondin in addition to transmembrane type
It has been reported that a type I repeat sequence (hereinafter referred to as a TSP-1 repeat sequence) and a sequence located between them have a type attached to a matrix (Kuno, K. et al., J. Bio).
l. Chem., 272, 556-562, 1997; Kuno, K. et al., J.
Biol. Chem., 273,13912-13917,1998; Tang, BL et
al., FEBS Lett., 445, 223-225, 1999).

[0003] Among the more than 20 known ADAM family molecules to date, TACE (ADAM17; Moss, ML et a
l., Nature, 385, 733-736, 1997; Black, RA et a
l., Nature, 385, 729-733, 1997), ADAM9 (MDC9; Wes
kamp, G. et al., J. Cell Biol., 132, 717-26, 199
6), ADAM10 (MADM; Howard, L. et al., Biochem. J.,
317, 45-50, 1996), ADAM12 (Meltrin alpha; Yagami-H
iromasa, T. et al., Nature, 377, 652-656, 1995), A
DAM15 (Metargidin; Kratzschmar, J. et al., J. Bio
l. Chem., 271; 4593-4596, 1996), ADAM20 (Hooft va
n Huijsduijnen, R., Gene, 206, 273-282, 1998), mouse ADAMTS-1 (Kuno, K. et al., J. Biol. Chem., 272,
556-562, 1997) has HExx in the metalloproteinase-like domain.
It has a zinc-binding consensus sequence consisting of H (HisGluXaaXaaHis). Among them, TACE analyzed TNF and amyloid precursor protein APP (B
uxbaum, JD et al., J. Biol. Chem., 273, 27765-2
7767, 1998), TNF receptor, L-selectin, TGF (Transfor
ming Growth Factor) -α. (Peschon, JJ et al., Sc
ience, 282, 1281-1284, 1998). Therefore, TACE has been implicated in shedding of many membrane proteins, including TNF converting enzyme (production of soluble TNF) and the activity of APP as α-secretase, and is believed to be responsible for various physiological functions. In fact, attention has been paid to the function as a TNF-converting enzyme, and application of the inhibitor to the inflammatory area is being explored (Conquering Airway Inflammation in the 21st Cen
tury (Part A), September 14-16, 1998). Also, ADAM1
0 indicates Notch (Blobel, CP, Cell, 90, 589-592, Notch) from the analysis of the Drosophila homolog Kuzbanian.
1997; Pan, D. et al., Cell, 90, 271-280, 1997), N
otch ligand delta (Qi, H. et al., Science, 283, 91
-94, 1999). In addition, APP (Lammich, S. et al., Pro
c. Natl. Acad. Sci. USA., 96 3922-3927, 1999), professional TNF (Lunn, CA et al., FEBS Lett., 400, 333-33).
5, 1997), type IV collagen (Millichip, MI et al.,
Biochem. Biophys. Res.Commun., 245, 594-598, 199
8) has been shown to cut. Therefore, ADAM10
Is involved in the regulation of Notch signaling and plays an important role in neurogenesis, and has also been suggested to be involved in the degradation of extracellular matrix components, as well as in the shedding of membrane proteins. In addition, aggreca, a type of aggrecanase that was recently considered to be the enzyme responsible for the degradation of the extracellular matrix aggrecan in arthritis and osteoarthritis
nase-1 has been purified, cloned, and reported to be a member of the ADAM family (Tortorella, MD et al.,
Science, 284, 1664-1666, 1999). Thus, ADAM
In addition to cell adhesion activity, the relationship between protease activity and physiological significance has been attracting attention (Black, RA et al.,
Curr. Opin. Cell Biol., 10, 654-659, 1998).

[0004] From such a viewpoint, the protease activity of ADAM has been examined, and it has been shown that ADAM9 and ADAM12 have a protein decomposability, although their physiological substrates are unknown. That is, ADAM9 cleaves the insulin B chain, which is a substrate for many proteases (Roghani, M. et al., J. Biol. Che.
m., 274,: 3531-3540, 1999), and ADAM12 was reported to produce a reaction product with α2 macroglobulin (hereinafter α2M), which reacts with a wide range of proteases (Loech).
el, F. et al., J. Biol. Chem., 273, 16993-16997, 1
998.). With regard to these molecules, a search for physiological substrates will be conducted in the future, and the relationship between protease activity and physiological significance will be explored. As a result, if there is a connection with a disease, it is expected that the inhibitor or the protease protein itself will be applied to pharmaceutical applications. In addition, Japanese Patent Application Laid-Open No. 11-46781 discloses a human ADAMTS-1 gene sequence and a sequence as an amino acid sequence, which are 727 amino acids compared to mouse ADAMTS-1 having 951 amino acids. Short, furin-like protease recognition sequence Arg Lys L
ysArg (the ninth to twelfth amino acids of SEQ ID NO: 1 in the sequence listing of the publication) has only 8 amino acids on the N side,
The secretion signal sequence present in AMTS-1 followed by the propeptide portion was absent. This suggested two possibilities. That is, the possibility that the gene that the inventors of the publication are human ADAMTS-1 is not a homolog of the mouse ADAMTS-1 gene, and in fact, the sequence determination of the gene containing the full-length ORF, the isolation has not been successful Signaled the possibility. That is, in any case, the full-length ORF sequence of human ADAMTS-1 was not known.

DISCLOSURE OF THE INVENTION The present invention is to isolate and identify genes encoding novel metalloproteinase family proteins, which have a very high potential as drug target molecules, and to construct expression and production systems for them. It is an object of the present invention to provide a recombinant protein required for searching for a substance that modifies their activity.

[0005]

Under these circumstances, the present inventors have made intensive studies and as a result, isolated a gene encoding a novel metalloprotease classified into the ADAM family, determined the full-length ORF sequence, and And succeeded in enabling the production of recombinant proteins. Further, the present inventors have identified a unit of an amino acid sequence having a protease activity, and have shown that a recombinant protein having the amino acid sequence has a protease activity at the site, thereby finding the present invention. Furthermore, a vector containing the gene, a host cell containing the vector, and a method for producing the same metalloprotease using the host cell have been established. This enabled screening of the metal protease and compounds, peptides and antibodies that modify the metal protease activity, and completed the present invention.

That is, the present invention relates to (1) an amino acid sequence of the first to 470th amino acid sequence of SEQ ID NO: 4,
Alternatively, a metal protease comprising the amino acid sequence of amino acids 1 to 481 of the amino acid sequence represented by SEQ ID NO: 10 and having protease activity, or a metal protease that is the same as the metal protease, (2 A) a protein having the amino acid sequence represented by SEQ ID NO: 4 or 10; (3) a gene encoding the amino acid sequence of the metalloprotease according to (1), or (2); A vector containing a gene, (5)
(4) a host cell containing the vector described in (4), (6) a method for producing the metalloprotease described in (1) and the protein described in (2) using the host cell described in (5), and (7) a metal described in (1). (8) a method of contacting the protease and an antibody against the protein according to (2), (8) contacting the metal protease according to (1) or the protein according to (2) with a test compound, and screening for a substance that modifies the activity of the metal protease; About.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Terms used in the present invention will be described below. As used herein, "metal protease" means a "metal protease" having a zinc consensus sequence (HExxH) and having protease activity. “Protease” represents “protein” unless otherwise specified. The novel metalloprotease of the present invention is the first to 470th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 4, or the first to 481th amino acid of the amino acid sequence represented by SEQ ID NO: 10. Any metal protease containing a sequence and having the ability to degrade proteins or a metal protease that is the same as the metal protease may be used. Here, the “equivalent of metalloprotease” means
Any one of the first to 470th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 4 or the first to 481th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 10 Or a metal protease having one to several amino acid residues in which substitutions, deletions, and / or insertions may be made at a plurality of sites, and having protein-degrading ability. As the "same effect of metalloprotease", preferably
The metal protease generated by amino acid substitution such as SNP is shown. The novel metalloprotease of the present invention is preferably a polypeptide having the amino acid sequence of SEQ ID NO: 4 or 10.

[0008] The gene encoding the novel metalloprotease of the present invention is a gene containing a base sequence encoding the above metalloprotease, that is, the first to 470th amino acid sequence represented by SEQ ID NO: 4. An amino acid sequence, or a metalloprotease represented by the first to 481st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 10,
Alternatively, any metal-protease gene containing a nucleotide sequence encoding the same effective substance and having the ability to degrade proteins may be used. As the gene encoding the novel metalloprotease of the present invention, preferably the first to 2850th of the nucleotide sequence of SEQ ID NO: 5, 1 of the nucleotide sequence of SEQ ID NO: 11
It is a gene having the numbers 3615 to 3615.

Here, the gene encoding the metalloprotease of the present invention, the vector of the present invention, the host cell of the present invention,
The metalloprotease of the present invention, a compound for modifying the activity of the metalloprotease of the present invention, a method for screening a peptide and an antibody, and a method for producing an antibody that reacts with the metalloprotease are described in the following 1) to 4). 1) Method for producing a novel metalloprotease gene a) First method for production-a method using PCR mRNA is extracted from human cells or tissues having the ability to produce the metalloprotease of the present invention. Then this mRNA
Using the metalloprotease mRNA or a part of the mRNA as a template
Two types of primers sandwiching the region are prepared. denatu
The re temperature, conditions for adding a denaturing agent, and the like are improved, and a reverse transcriptase-polymerase chain reaction (hereinafter referred to as RT-PCR) suitable for each of the proteins containing a part of the amino acid sequence represented by SEQ ID NO: 4 or 10 of the present invention. ) To obtain a full-length cDNA of the metalloprotease or a part thereof.
Alternatively, a polymerase chain reaction (hereinafter referred to as a template) using cDNA prepared by reverse transcriptase from mRNA prepared from human cells or tissues capable of producing the metalloprotease of the present invention or commercially available cDNA derived from the human cells or tissues. , PCR) to obtain the full-length cDNA of the metalloprotease or a part thereof. Furthermore, the metal protease can be produced by incorporating the full-length cDNA of the obtained metal protease or a part thereof into an appropriate expression vector and expressing it in a host cell. First, mRNAs including those encoding the protease are extracted from cells or tissues capable of producing the metalloprotease of the present invention by known methods. As the extraction method, guanidine / thiocyanate /
Hot phenol method, guanidine thiocyanate-
A guanidine / hydrochloric acid method and the like can be mentioned, and a guanidine / thiocyanate cesium chloride method is preferable.
Cells or tissues having the protease-producing ability are identified by a Northern blotting method using a gene having a nucleotide sequence encoding the protease or a part thereof, a Western blotting method using an antibody specific to the protease, or the like. be able to. mR
Purification of NA may be carried out according to a conventional method.
T) It can be adsorbed and eluted on a cellulose column and purified. Furthermore, the mRNA was subjected to sucrose density gradient centrifugation, etc.
Can be further fractionated. Instead of extracting mRNA, commercially available extracted and purified mRNA may be used. Next, the purified mRNA is subjected to a reverse transcriptase reaction in the presence of a random primer, an oligo dT primer, or a custom-synthesized primer to synthesize a first-strand cDNA. This synthesis can be performed by a conventional method. The obtained first-strand cDNA is subjected to PCR using two types of primers sandwiching a partial region of the target gene to amplify the target novel metalloprotease DNA. Alternatively, a commercially available cDNA may be used without synthesizing the cDNA. Obtained DNA
Is fractionated by agarose gel electrophoresis or the like. If desired, the target DNA fragment can be obtained by cutting the DNA with restriction oxygen or the like and connecting it.

B) Second Production Method The gene of the present invention can be produced by a conventional genetic engineering technique in addition to the above-mentioned production method. First, using the mRNA obtained by the above method as a template and a single-stranded cD
After synthesizing the NA, a double-stranded cDNA is synthesized from the single-stranded cDNA. The method includes the S1 nuclease method (Efstrati
adis, A. et al., Cell, 7, 279-288, 1976), Land method (L
and, H. et al., Nucleic Acids Res., 9, 2251-2266,
1981), O. Joon Yoo method (Yoo, OJ et al., Proc. Nat.
l. Acad. Sci. USA, 79, 1049-1053, 1983), Okayama-B
erg method (Okayama, H. and Berg, P., Mol.Cell.Biol.,
2,161-170, 1982). Next, the recombinant plasmid obtained by the above-mentioned method is
Strain, HB101 strain, JM109 strain, etc., and transformed, and a recombinant can be selected using drug resistance to tetracycline, ampicillin, kanamycin, or the like as an index. For example, when the host cell is Escherichia coli, the method of Hanahan (Hanahan, DJ, Mol. Bio
l., 166, 557-580, 1983 ), that is, carried out by a method of adding the recombinant DNA material into competent cells prepared in the coexistence of CaCl ₂ and MgCl ₂ or RbCl. Of course, commercially available competent cells may be used. In addition, a phage vector such as a lambda system can be used in addition to the plasmid. As a method for selecting a strain having the DNA of the desired novel metalloproteinase from the transformants obtained as described above, for example, the following various methods can be adopted.

Screening Method Using Synthetic Oligonucleotide Probes Oligonucleotides corresponding to all or a part of the metalloprotease of the present invention were synthesized (in this case, nucleotide sequences derived using codon usage or possible nucleotide sequences were combined. may be either of a plurality of nucleotide sequences, also in the latter case, it is also possible to reduce the types by including inosine), this probe ⁽³² P
Or labeling with ³³ P), and hybridizing with a nitrocellulose filter or a nylon filter in which the DNA of the transformed strain is denatured and fixed, and searching for the obtained positive strain,
Select this. Screening Method Using Probe Prepared by Polymerase Chain Reaction Oligonucleotides of sense primer and antisense primer corresponding to a part of the metalloprotease of the present invention are synthesized, and these are combined to perform polymerase chain reaction (Sai
ki, RK et al., Science 239, 487-491, 1988) to amplify a DNA fragment encoding all or part of the desired metalloprotease. As the template DNA used here,
CDNA or genomic DNA synthesized by reverse transcription from mRNA of a cell that produces the metalloprotease can be used. The DNA prepared in this manner was fragmented with ³² P or ³³
The target clone is selected by labeling with P and performing colony hybridization or plaque hybridization using this as a probe. Method for Screening by Producing a Novel Metal Protease in Other Animal Cells A transformant is cultured, the gene is amplified, and the gene is transfected into an animal cell (in this case, a plasmid capable of self-replication and containing a transcription promoter region). Or a plasmid which can be integrated into the chromosome of an animal cell), and the protein encoded by the gene is produced extracellularly. By detecting the metalloprotease using an antibody against the metalloprotease of the present invention, cD encoding the metalloprotease of interest can be obtained from the original transformant.
Select strains with NA. Method for selecting using an antibody against the metalloprotease of the present invention In advance, cDNA is incorporated into an expression vector, and a protein is produced in the culture supernatant of a transformant, intracellularly or on the cell surface. Using a secondary antibody to the antibody, a desired metalloprotease-producing strain is detected, and a target strain is selected. Method using selective hybridization / translation system cDNA obtained from the transformant, mRNA from the metalloprotease-producing cells of the present invention blotted on a nitrocellulose filter or the like, and then mRNA bound to the cDNA Is dissociated and recovered. The recovered mRNA is translated into protein by a protein translation system, for example, injection into Xenopus oocytes or a cell-free system such as rabbit reticulocyte lysate or wheat germ. The target strain is selected by detection using an antibody against the metalloprotease of the present invention. A method for collecting DNA encoding the metalloprotease of the present invention from the obtained desired transformant is a known method (Sambroo
k, J. et al., "Molecular Cloning-A Laboratory Manu
al ", Cold Spring Harbor Laboratory, NY, 1989), etc. For example, it can be carried out by separating a fraction corresponding to a plasmid DNA from a cell and cutting out a cDNA region from the plasmid DNA.

C) Third Production Method The metalloprotease gene of the present invention can also be produced by joining DNA fragments produced by a chemical synthesis method. Each DNA is used as a DNA synthesizer [for example, Oligo 1000M DNA Sy
nthesizer (Beckman) or 394 DNA / RNA Syn
thesizer (manufactured by Applied Biosystems)].

D) Fourth Production Method The metalloprotease gene of the present invention can be prepared, for example, by the phosphite-triester method (Hunkapiller, M. et al., Nature, 10, 105-111, 19) based on information on the metalloprotease.
It can also be produced by chemical synthesis of nucleic acids according to a conventional method such as 84). In addition, the codon for the desired amino acid is known per se, and the selection may be arbitrarily determined. , 9, r43
-r74, 1981). Furthermore, partial modification of the codons of these nucleotide sequences can be performed by a conventional method using site-specific mutagenesis (site specific mutase) using a primer composed of a synthetic oligonucleotide encoding the desired modification.
agenesis) (Mark, DF et al., Proc. Natl. Acad. S
ci. USA, 81, 5662-5666, 1984).

As described above, sequencing of the DNA obtained by a) to d) is performed, for example, by the Maxam-Gilbert chemical modification method.
(Maxam, AM and Gilbert, W., "Methods in Enzymol
ogy ", 65, 499-559, 1980) and dideoxynucleotide chain termination (Messing, J. and Vieira, J., Gene, 19, 269-2).
76, 1982). The vector of the present invention, the host cell of the present invention, and the metalloprotease of the present invention can be obtained by the following methods. 2) Method for Producing Recombinant Protein of the Vector of the Present Invention, the Host Cell of the Present Invention, and the Metalloprotease of the Present Invention The isolated fragment containing the gene encoding the metalloprotease of the present invention is re-integrated into an appropriate vector DNA. Thus, eukaryotic and prokaryotic host cells can be transformed. Furthermore, by introducing an appropriate promoter and a sequence related to expression into these vectors, the gene can be expressed in each host cell. For example, eukaryotic host cells include cells of vertebrates, insects, yeasts, and the like, and vertebrate cells include monkey COS cells (Gluzman,
Natl. Acad. Y. Cell, 23, 175-182, 1981) and a dihydrofolate reductase-deficient strain of Chinese hamster ovary cells (CHO) (Urlaub, G. and Chasin, LA, Proc. Natl. Acad.
Sci. USA, 77, 4216-4220, 1980), human fetal kidney-derived HE
K293 cells and 293-EBNA cells (Invitrogen) into which Epstein BarrVirus EBNA-1 gene has been introduced are often used, but not limited thereto. As a vertebrate cell expression vector, those having a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence, and the like, which are usually located upstream of the gene to be expressed, can be used. May have. Examples of the expression vector include S
PSV2dhfr with the early promoter of V40 (Subraman
i, S., et al. Mol. Cell. Biol., 1, 854-864, 198
1), pEF with human elongation factor promoter
-BOS (Mizushima, S. and Nagata, S., Nucleic Acids
Res., 18, 5322, 1990) and pCEP4 (manufactured by Invitrogen) having a cytomegalovirus promoter, but are not limited thereto. As an example, when a COS cell is used as a host cell, the expression vector has an SV40 origin of replication and is capable of autonomous propagation in the COS cell.
In addition, transcription promoter, transcription termination signal and RNA
Those having a splice site can be used. For example, pME18S (Maruyama, K. and Takebe, Y., Med.
nol., 20, 27-32, 1990), pEF-BOS (Mizushima, S. and
Nagata, S., Nucleic Acids Res., 18, 5322, 1990),
pCDM8 (Seed, B., Nature, 329, 840-842, 1987). The expression vector is a DEAE-dextran method (Lut
hman, H. and Magnusson, G. ,, Nucleic Acids Res., 1
1, 1295-1308, 1983), calcium phosphate-DNA coprecipitation method
(Graham, FL and van der Ed, AJ ,, Virology, 5
2, 456-457,1973), FuGENE ^TM 6 Transfection Reagent
(Boeringer Mannheim) and electric pulse perforation (Neumann, E. et al. ,, EMBO J., 1, 841-
845, 1982) and the like, and thus a desired transformed cell can be obtained.

When a CHO cell is used as a host cell, a vector capable of expressing a neo gene functioning as a G418 resistance marker together with an expression vector, for example, pRSVn
eo (Sambrook, J. et al., "Molecular Cloning-A Labor
atory Manual ", Cold SpringHarbor Laboratory, NY, 1
989) and pSV2-neo (Southern, PJ and Berg, P., J., Mo.
l. Appl. Genet., 1, 327-341, 1982) and the like, and by selecting G418-resistant colonies, a transformed cell stably producing a novel metalloprotease can be obtained. When 293-EBNA cells are used as host cells, pCEP4 (Invitrogen) which has an Epstein Barr Virus replication origin and is capable of self-replication in 293-EBNA cells
Desired transformants can be obtained using an expression vector such as The desired transformed cells obtained above can be cultured according to a conventional method, and the culture produces the metalloprotease of the present invention extracellularly. The medium used for the culture can be appropriately selected from various conventional ones depending on the host cell employed.
If it is a cell, use a medium such as RPMI-1640 medium or Dulbecco's Modified Eagle's Minimum Essential Medium (DMEM) if necessary.
Those containing serum components such as BS) can be used. Also,
If the above 293-EBNA cells, Dulbecco's Modified Eagle Minimum Essential Medium (DME) supplemented with serum components such as fetal bovine serum (FBS)
A medium obtained by adding G418 to a medium such as M) can be used. As described above, the metalloprotease of the present invention produced extracellularly in transformed cells can be obtained by various known separation procedures utilizing the physical properties and biochemical properties of the metalloprotease.
It can be separated and purified. As the method, specifically, for example, a culture solution containing the metal protease is treated with a usual protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchanger chromatography, affinity Examples include various types of liquid chromatography such as chromatography and high performance liquid chromatography (HPLC), dialysis methods, and combinations thereof. The expression and fusion of the metal protease of the present invention can be confirmed by in-frame fusion with the marker sequence and expression of the metal protease. As the marker sequence, for example, FLAG epitope, Hexa-Histidi
There are ne tag, Hemagglutinin tag and myc epitope.
In addition, by inserting a specific amino acid sequence recognized by a protease such as enterokinase, factor Xa, or thrombin between the marker sequence and the metalloprotease, the marker sequence can be cleaved and removed by these proteases. is there.

3) Method for Screening Compound, Peptide and Antibody that Modifies the Activity of Metal Protease of the Present Invention The screening method of the present invention uses at least the metal protease prepared as described above, The system includes a means for adding a test substance to a system for measuring an index of modification of metalloprotease activity in accordance with the property, and measuring the index. Here, as the measuring system, various known protease measuring systems (Dainori Tsuru, Masaru Funatsu, Biochemistry Experiment Method 30, “Protease I”, Gakkai Shuppan Center, 1993, and 31 “Protease I,” Gakkai Shuppan Center , 1993), and a test substance can be screened by carrying out according to, according to, or according to the treatment method described in the literature. Screening can also be performed using the protease activity measurement method described in Example 4-2 described later. As a test substance, a compound or peptide which is conventionally known to have a metalloprotease inhibitory activity but is not known to modify the activity of the novel metalloprotease,
Alternatively, various known compounds and peptides registered in the chemical file, combinatorial chemistry technology (Terrett, NK et al., Tetrahedron, 51, 8135-81)
37, 1995) and the phage display method (Felici, F. et al., J. Mol. Biol., 222,
301-310, 1991)
Peptides can be used. In addition, microbial extracts and culture supernatants, natural components derived from plants and marine organisms, animal tissue extracts, and the like are also targets for screening. Alternatively, a compound or peptide obtained by chemically or biologically modifying the compound or peptide selected by the screening method of the present invention can be used. In screening for compounds, peptides and antibodies that modify the activity of the metalloprotease of the present invention, any compounds that can serve as substrates for the protease of the present invention or its partial peptides can be used. For example, proteins such as casein, collagen, fibronectin and gelatin, physiologically active peptides and synthetic peptides such as insulin, fluorescent or radiolabeled gelatin, collagen and synthetic peptides, and synthetic substrates having a fluorophore and a chromophore are used. The term "synthetic peptide" used herein includes those containing unnatural amino acids. Commercially available substrates such as Knight's substrate can also be used. As a system for measuring an index of modification of the metal protease activity according to the biochemical properties of the metal protease, the above substrate and the metal protease protein of the present invention are mixed in an appropriate buffer, and reacted, Protease activity is detected by a method appropriate for each substrate. For example, when using an unlabeled substrate, it is possible to detect a degradation product by SDS-PAGE, HPLC, Zymography, or the like, and when using a radiolabeled substrate, a fluorophore, or a substrate having a luminophore, Protease activity can be detected by using an appropriate detector such as a liquid scintillation counter, a fluorescence detector, and a luminescence detector.

4) Method for Producing Antibodies Reacting with the Metalloprotease of the Present Invention Antibodies that react with the metalloprotease of the present invention, such as polyclonal antibodies and monoclonal antibodies, can be prepared by directly applying the novel metalloprotease and fragments of the metalloprotease to various animals It can be obtained by administration. Further, a DNA vaccine method (Raz, E. et al., Proc. Natl.
Acad. Sci. USA, 91, 9519-9523, 1994; Donnelly, J.
J. et al., J. Infect. Dis., 173, 314-320, 1996)
Can also be obtained by A polyclonal antibody is prepared by emulsifying the metal protease or a fragment thereof in a suitable adjuvant such as Freund's complete adjuvant and immunizing the peritoneal cavity, subcutaneously or intravenously, for example, a rabbit,
Manufactured from serum or eggs of rats, goats or chickens. The polyclonal antibody thus produced can be separated and purified by conventional protein isolation and purification methods.Examples of conventional protein isolation and purification methods include centrifugation, dialysis, salting out with ammonium sulfate, DEAE- Chromatography using cellulose, hydroxyapatite, protein A agarose and the like. Monoclonal antibodies were obtained using the Kohler and Milstein cell fusion method (Kohler, G. and Milstein, C., Nature, 256, 495-49).
7, 1975) can be easily manufactured by those skilled in the art. That is, immunization is carried out by repeatedly inoculating a mouse intraperitoneally, subcutaneously or intravenously several times with an emulsion obtained by emulsifying the metalloprotease of the present invention or a fragment thereof in an appropriate adjuvant such as Freund's complete adjuvant every few weeks. After the final immunization, the spleen cells are removed and fused with myeloma cells to produce a hybridoma. Myeloma cells for obtaining hybridomas include myeloma cells having markers such as hypoxanthine-guanine-phosphoribosyltransferase deficiency and thymidine kinase deficiency, for example, mouse myeloma cell line P3X63Ag8.U
1, use. In addition, polyethylene glycol is used as a fusion agent. Further, as a medium for preparing a hybridoma, a commonly used medium such as Eagle's minimum essential medium, Dulbecco's modified minimum essential medium, and RPMI-1640 is used by appropriately adding 10 to 30% fetal bovine serum. The fusion strain is selected by the HAT selection method. Hybridoma screening is performed by using well-known methods such as ELISA method and immunohistochemical staining method or the above-mentioned screening method using the culture supernatant, and a clone of the hybridoma secreting the desired antibody is selected. In addition, the monoclonality of the hybridoma is guaranteed by repeating subcloning by the limiting dilution method. The hybridoma thus obtained is cultured in a medium for several days or in the abdominal cavity of a BALB / c mouse pretreated with pristane for 10 to 20 days to produce a purifiable amount of the antibody. The monoclonal antibody thus produced can be separated and purified from the culture supernatant or ascites by a conventional protein isolation and purification method.

With respect to the antibody separated and purified as described above,
Proteolytic enzymes such as pepsin, papain, etc.
Therefore, digestion is performed, and then the protein isolation and purification method is continued.
By separating and purifying more,
Antibody fragments, e.g., F (ab ') _Two, Fab, Fab ', Fv
Can be. Furthermore, it reacts with the metalloprotease of the present invention.
Antibodies are used in the method of Clackson et al.
n, T. et al., Nature, 352, 624-628, 1991; Zebedee,
 S. et al., Proc. Natl. Acad. Sci. USA, 89, 3175-3.
179, 1992) to obtain single chain Fv or Fab.
Both are possible. In addition, the mouse antibody gene is replaced with a human antibody
Transgenic mice (Lonber
g, N. et al., Nature, 368, 856-859, 1994).
Thus, it is also possible to obtain a human antibody.

The present invention includes a drug containing as an active ingredient a compound, a peptide or an antibody which significantly modifies the activity of a metal protease or a metal protease selected by the above screening method. Formulations containing the metalloprotease activity-modifying compound, peptide, antibody or antibody fragment of the present invention as an active ingredient may be, depending on the type of the active ingredient, carriers, excipients, and other additives that are commonly used in the formulation thereof. Can be prepared using For administration, tablets, pills, capsules, granules, fine granules, powders, oral liquids, etc., or injections such as intravenous and intramuscular injections, suppositories, transdermal preparations, transmucosal preparations And parenteral administration. In particular, parenteral administration such as intravenous injection is desired for peptides digested in the stomach. A solid composition for oral administration according to the present invention may comprise one or more active substances in at least one inert diluent, such as lactose, mannitol,
It is mixed with glucose, microcrystalline cellulose, hydroxypropyl cellulose, starch, polyvinylpyrrolidone, magnesium aluminate metasilicate and the like. The composition may contain additives other than the inert diluent, such as a lubricant, a disintegrant, a stabilizer, and a solubilizing or solubilizing agent, according to a conventional method. Tablets and pills may be coated with sugar coating or a film of a gastric or enteric substance, if necessary.

Liquid compositions for oral use include emulsions, solutions, suspensions, syrups and elixirs, and include commonly used inert diluents such as purified water and ethanol. The composition may contain additives other than inert diluents, such as wetting agents, suspending agents, sweetening agents, flavoring agents, preservatives. Parenteral injections include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The water-soluble solutions and suspensions include, for example, distilled water for injection, physiological saline, and the like as diluents. Examples of diluents for non-aqueous solutions and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, and polysorbate 80. The composition may further contain a wetting agent, an emulsifier, a dispersant, a stabilizer, a solubilizing or solubilizing agent, a preservative and the like. The composition is sterilized by, for example, filtration through a bacteria-retaining filter, blending of a bactericide, or irradiation. In addition, a sterile solid composition can be produced and dissolved in sterile water or another sterile injectable medium before use. The dose is appropriately determined in consideration of the activity intensity, symptoms, age, sex, and the like of the active ingredient selected by the screening method.

[0021]

Hereinafter, the present invention will be described more specifically. Unless otherwise noted, known methods (Sambrook, J. et al., "M
olecular Cloning-A Laboratory Manual ", Cold Spring
Harbor Laboratory, NY, 1989), etc., but the present invention is not limited to the examples.

(Example 1) Determination of full-length ORF sequence of novel human ADAM-type metalloprotease (MDTS4) The novel human ADAM-type metalloprotease MDTS4 full-length protein translation region (hereinafter referred to as ORF) sequence is a cDNA clone derived from human spinal cord. (Manufactured by Clontch) and rapid amplification of cDNA ends (hereinafter, RACE
) Were determined by connecting the sequences obtained in the above. For screening of a human spinal cord-derived cDNA library, a DNA fragment corresponding to nucleotides 848 to 1144 of SEQ ID NO: 3 was used as a probe. ³² P of probe DNA fragment
Labeling and purification, respectively, BcaBEST ^TM Labeling Kit
(Manufactured by Takara Shuzo) and NICK ^™ column (manufactured by Amersham Pharmacia Biotech), and the specific activity was 5 × 10 ⁸ cpm /
More than ³² μg of ³² P-labeled probe was prepared. First, a human spinal cord-derived cDNA library (human Brain spinal cord 5-st
rech plus; Clontch, catalog number: HL5001a, lot number: 46027) About 1.2 million clones were screened. Specifically, first, Hybond N ⁺ in accordance with (Amersham Pharmacia Biotech Ltd.) attached instructions, transferring the plaques to Hybond N ⁺ nylon membrane, after fixing the DNA by alkali treatment, 18 hours ^{3 2} at 65 ° C. Hybridization with P-labeled probe was performed and 2 × SSPE,
0.1 × SDS, room temperature twice for 15 minutes, 1 × SSPE, 0.1 × SDS, 65 ° C15
Washing was performed twice, 0.1 × SSPE, 0.1 × SDS, twice at 65 ° C. for 15 minutes. Subsequently, after exposing the nylon membrane to the X-ray film for 2 days, the X-ray film was developed and a positive signal was detected. Secondary screening was performed on the phage recovered from the position of the positive signal under the same conditions as described above. As a result, a positive clone 24-A was obtained. Clone 24-A was found to correspond to the nucleotide sequence of positions 766 to 2382 of SEQ ID NO: 5. The remaining 3'-side sequence is a cDNA clone containing the 3'-side partial sequence of MDTS4 (from position 2383 of SEQ ID NO: 5):
IMAGE clone number 163187 (ATCC Cat. No. 401100) was purchased, and the nucleotide sequence of this clone was determined to obtain the portion of SEQ ID NO: 5 from position 2383 to position 2853. The sequence on the 5 'side was determined by direct nucleotide sequence analysis of the DNA fragments generated by the two sets of RACE. RACE was performed using Clontech human placenta Marathon-Ready ^™ cDNA as a template and two rounds of PCR as one set. First PCR of the first set
The human placenta Marathon-Ready ^TM cDNA template (Clontch
Using the oligo DNA represented by SEQ ID NO: 6 and the oligo DNA represented by SEQ ID NO: 1 (hereinafter referred to as AP-1) as primers using LA-Taq ^™ (manufactured by Takara Shuzo) at 94 ° C. After a minute, 9
The cycle was performed at 8 ° C for 20 seconds, 72 ° C for 2 minutes 5 times, 98 ° C for 20 seconds, 70 ° C for 2 minutes 5 times, 98 ° C for 20 seconds, and 68 ° C for 2 minutes 30 times. Oligo D represented by SEQ ID NO: 7 was prepared using 5 μl of a solution obtained by diluting the first reaction solution 50-fold with TE buffer as a template.
A second PCR reaction was performed using the oligo DNA represented by SEQ ID NO: 2 (hereinafter referred to as AP-2) as a primer in the same cycle as the first cycle. The first PCR of the second set was performed using the oligo DNA represented by SEQ ID NO: 8 and AP-1 as primers, and the second PCR was performed by using the oligo DNA represented by SEQ ID NO: 9 and AP-2.
Was used as a primer under the same conditions as in the first set. By determining the nucleotide sequence of the fragment thus generated, the codon corresponding to the translation initiation Met was finally reached in-frame, and a portion of the nucleotide sequence from No. 1 to No. 765 of SEQ ID NO: 5 was obtained. By joining the sequences obtained as described above, the MDTS4 full-length ORF was found to consist of 2853 bp (SEQ ID NO: 5) and encode a protein consisting of 950 amino acids (SEQ ID NO: 4).

(Example 2) Determination of full-length ORF sequence of novel human ADAM-type metalloprotease (MDTS5) The nucleotide sequence of novel human ADAM-type human metalloprotease MDTS5 is represented by nucleotides 1 to 1443 of MDTS5 shown in SEQ ID NO: 11. A DNA fragment A containing the sequence and a DNA fragment B containing the nucleotide sequence from Nos. 1354 to 3618 were generated by PCR and the nucleotide sequences were determined. Specifically, the DNA fragment A was prepared by using a human brain Marathon-Ready ^™ cDNA (manufactured by Clontech) as a template, an oligo DNA represented by SEQ ID NO: 18 and an oligo DNA represented by SEQ ID NO: 19 as primers, and Native pfu DNA After 2 minutes at 94 ° C, use polymerase (Stratagene)
35 cycles of 8 ° C for 30 seconds, 65 ° C for 30 seconds, and 74 ° C for 3 minutes, 74 ° C for 10 seconds
It was generated by PCR under the conditions of minutes. DNA fragment B is from human brain Mara
thon-Ready ^™ cDNA (Clontech) as template, SEQ ID NO: 2
Oligo DNA represented by 1 and oligo DN represented by SEQ ID NO: 22
PyroBest ^™ DNA (Takara Shuzo) using A as a primer
After 2 minutes at 94 ° C, cycle at 98 ° C for 10 seconds and at 68 ° C for 3 minutes.
It was generated by PCR for 40 times at 68 ° C. for 7 minutes. MD from this
The full-length TS5 ORF consists of 3618 bp (SEQ ID NO: 11) and was found to encode a protein consisting of 1205 amino acids (SEQ ID NO: 1).
0). MDTS, a novel ADAM protein obtained by the above procedure
4.MDTS5 shows homology to each other and reported ADAM
It shows the highest homology at the amino acid sequence level with mouse ADAMTS-1 in the family, and its domain structure is N
From the end, secretory signal sequence, propeptide, metalloprotease-like domain, disintegrin-like domain, TSP-
It was the same as one repeat sequence.

(Example 3) Expression of novel human ADAM-type metalloprotease in animal cells The novel human ADAM-type metalloprotease includes a secretory signal sequence, a propeptide, a metalloprotease-like domain, a disintegrin-like domain, and a TSP-1 repeat sequence. Has a domain structure of It is considered that protein expression from at least a secretory signal to a metalloproteinase-like domain is required for the display of metalloprotease activity. Thus, HEK293EBNA cells were used as host cells for the expression and production of proteins from the N-terminus to the Gln residue just before the first Cys residue of the disintegrin-like domain for MDTS4 and MDTS5. The details will be described below.

(Example 3-1) Improvement of expression vector pCEP4d (manufactured by Invitrogen) was digested with restriction enzymes ClaI and NsiI, blunt-ended, and then self-ligated to remove the expression vector pCEP4d. Was prepared. This vector was digested with restriction enzymes NheI and BamHI, and a heavy chain oligonucleotide obtained by annealing the nucleic acid represented by SEQ ID NO: 12 and the nucleic acid represented by SEQ ID NO: 13 to a fragment of about 7.7 Kba extracted by agarose gel was inserted. A clone having the desired sequence was selected and named pCEP4d-FLAG. Using this vector as a template, oligo DNA represented by SEQ ID NO: 14, SEQ ID NO: 1
PyroBest D using the oligo DNA shown in 5 as a primer
A PCR reaction was performed using NA polymerase. About 0.4
The kbp DNA fragment was cut with the restriction enzyme SpeI and cut with XbaI.
Insert into pCEP4d-FLAG (about 7.7 Kbp) and cloning sites XbaI, NheI, NotI, BamH from the promoter as desired.
A clone in the order of I recognition sequence and FLAG tag was selected to complete pCEP4dE2-FLAG.

In the following Examples 3-2 to 3-3,
DNA fragments designed to add a FLAG tag to the C-terminus in frame were inserted into the restriction enzyme sites of these expression plasmids. (Example 3-2) Construction of MDTS4 Protease Expression Plasmid To examine the protease activity of MDTS4, MDTS4 contains a secretory signal, propeptide, and protease domain of MDTS4
It was decided to express a protein corresponding to amino acids 1 to 470 of SEQ ID NO: 4 of MDTS4. The DNA fragment containing the nucleotide sequence from No. 1 to No. 1410 of SEQ ID NO: 5
Obtained by PCR using oBest ^™ DNA polymerase.
Using a human placenta Marathon-Ready ^™ cDNA (manufactured by Clontech) as a template, the oligo DNA of SEQ ID NO: 16 and the oligo DNA of SEQ ID NO: 17 as primers, at 94 ° C. for 1 minute,
40 cycles of 98 ° C for 10 seconds, 60 ° C for 30 seconds, and 72 ° C for 2 minutes, 72 ° C for 7 seconds
PCR was performed under the conditions of minutes. XbaI on the 5 'side generated in this way
Recognition sequence and Kozak sequence, after confirming the sequence by subcloning the target fragment having a BamHI recognition sequence added to the 3 ′ side into PCR-Blunt, cutting with restriction enzymes XbaI and BamHI, pCEP4E2
-Insert into the XbaI and BamHI sites of FLAG, pCEP-MDTS4PR-FLA
G completed.

(Example 3-3) Construction of MDTS5 Protease Expression Plasmid DNA fragment A generated in (Example 2) is represented by SEQ ID NO: 10.
It contains the nucleotide sequence of No. 1 to No. 1443 of SEQ ID NO: 11 necessary for expressing the amino acid No. 1 to No. 481 of the amino acid sequence of MDTS5, the NheI recognition sequence and Kozak sequence on the 5 ′ side, 3 The NotI recognition sequence is added to the 'side. This fragment A
After subcloning into pCR-Blunt and confirming the sequence, digestion with restriction enzymes NheI and NotI, NheI-NotI of pCEP4dE2-FLAG
The expression plasmid pCEP-MDTS5PR-FLAG was completed.

Example 3-4 Expression of MDTS4 and MDTS5 Proteases in Animal Cell Lines Regarding both MDTS4 and MDTS5, an expression plasmid prepared using pCEP4dE2-FLAG as a backbone in (Examples 3-2 to 3-3) Of FuGENE ^TM 6 Transfection Reagent (Boeringer Ma
nnheim) according to the attached instructions. After introduction of the plasmid, the culture supernatant obtained by continuing the culture for 2-3 days, or 200 μg / ml of Hygromycin B
The presence of the target protein in the culture supernatant of the cells selected in the medium supplemented with the medium was confirmed by Western blotting using an antibody against the FLAG tag added to the C-terminus (mouse anti-FLAG monoclonal antibody (M2; manufactured by Sigma)). Confirmed.
That is, the above culture supernatant was subjected to electrophoresis using SDS / 10% to 20% acrylamide gel (manufactured by Daiichi Kagaku) and then transferred to a PVDF membrane using a blotting device. PV after transfer
After blocking by adding Block Ace (manufactured by Dainippon Pharmaceuticals) to the DF membrane, mouse anti-FLAG monoclonal antibody (M2; manufactured by Sigma), horseradish peroxidase-labeled rabbit anti-mouse IgG polyclonal antibody (Zymed or (Manufactured by TAGO). Alternatively, after blocking, biotinylated M2 antibody (manufactured by Sigma) and horseradish peroxidase-labeled streptavidin (manufactured by Amasham) were sequentially reacted. After the reaction, ECL western blotting detection system (Amersham Pharmacia)
Was used to confirm the expression of the protease.

(Example 4) MDT expressing animal cells in a host
Detection of enzyme activity of S4 and MDTS5 protease (Example 4-1) Purification of MDTS4 and MDTS5 protease (Example 3-4) Expression of HEK293-EBNA cells in a host from host culture supernatant containing target protein produced and produced Purification of the target protein of
Affinity purification was performed using the fact that a FLAG tag was added to the C-terminus. That is, the culture supernatant was applied to M2-agarose (manufactured by Sigma) packed in a column, and 20 mM Trial was added.
After washing with s-HCl (pH 7.4) / 150 mM NaCl (hereinafter referred to as “TBS”), T containing 100 μg / ml FLAG peptide (manufactured by Sigma) was used.
Eluted and fractionated with BS. After electrophoresing purified HEK293-EBNA cells expressing MDTS4 and MDTS5 protease proteins using SDS / 10% -20% acrylamide gel, silver staining and anti-FL
As a result of the confirmation experiment by Western blotting using the AG antibody (M2), in each case, a molecule considered to be produced by processing with a furin-like protease and a molecule not processed were detected. The apparent molecular weight of SDS / 10% -20% acrylamide gel is MD
TS4 was about 34KDa and about 53KDa, and MDTS5 was about 32KDa and about 60KDa. MDTS4 processed with furin protease is estimated to be the 236th to 470th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 4, and MDTS5 processed with furin protease has the amino acid sequence represented by SEQ ID NO: 10. Two
Presumed to be the amino acid sequence of positions 50 to 481. These molecular weights were consistent with the values deduced from the amino acid sequence.

Example 4-2 Detection of Enzyme Activity of MDTS4 and MDTS5 Proteases The enzymatic activity was examined using synthetic peptide A represented by SEQ ID NO: 20 as a substrate. That is, the purified protease protein was reacted with the synthetic peptide in TBS at room temperature, and analyzed by HPLC using a YMC packed R & D RODS-5 (4.6 IDX 25 cm) column. Using a detection wavelength of 220 nm, eluent A: 0% acetonitrile / 0.085% trifluoroacetic acid B: 14
% Acetonitrile / 0.085% trifluoroacetic acid, flow rate 1 ml
Separation was performed with a linear gradient of A → B at 20 ml / min. As a result, in MDTS4 and MDTS5 proteases,
The peak of A shrank or disappeared, and the peak of the degradation product was detected (FIG. 1). Also, this phenomenon is due to EDTA at a final concentration of 10 mM.
Was completely inhibited by adding to the reaction solution. From these, it was shown that MDTS4 and MDTS5 proteases actually have metalloprotease activity.

[0031]

The novel metalloprotease and protein obtained by the present invention are characterized by selectively controlling the activity of the metalloprotease, and the use of the protein as a medicament is to increase or decrease the metalloprotease activity. , Diseases caused by abnormalities such as degeneration or expressing / combining the abnormalities,
For example, cancer, arthritis, osteoarthritis and the like can be mentioned.

[0032]

[Brief description of the drawings]

FIG. 1 shows the results of a peptide cleavage experiment of control, MDTS4 and MDTS5 proteases.

[Sequence List] SEQUENCE LISTING <110> Yamanouchi Pharmaceutical Co., Ltd. <120> Novel metalloprotease <130> 0000002897 <160> 22 <170> PatentIn Ver. 2.0 <210> 1 <211> 27 <212> DNA <213> Homo sapiens <400> 1 ccatcctaat acgactcact atagggc 27 <210> 2 <211> 23 <212> DNA <213> Homo sapiens <400> 2 actcactata gggctcgagc ggc 23 <210> 3 <211> 2670 <212> DNA <213> Homo sapiens <400> 3 atgctccccg cccccgccgc cccccggtgg cctccgctcc tgctgctgct gctgctgctg 60 ctgccgctgg cccgcggcgc cccggcccgg cccgcagccg gggggcaggc ctcggagctg 120 gtggtgccca cgcggttgcc cggcagcgcg ggcgagctcg cgctccacct gtccgccttc 180 ggcaagggct tcgtgctgcg cctggcgccc gacgacagct tcctggcgcc cgagttcaag 240 atcgagcgcc tcgggggctc cggccgggcg accgggggcg agcgggggct gcgcggctgc 300 ttcttctccg gcaccgtgaa tggggagccc gagtcgctgg cggcggtcag cctgtgccgc 360 gggctgagcg gctccttcct gctggacggc gaggagttca ccatccagcc gcagggcgcg 420 gggggctccc tggctcagcc gcaccgcctg cagcgctggg gtcccgccgg agcccgcccc 480 ctcccgcgg gg ggcagggag gg aggca ggagagagga 540 gaccaccagg aggacagcga ggaggagagc caagaagagg aggcagaagg cgctagcgag 600 ccgccaccgc ccctgggggc cacgagtagg accaagcggt ttgtgtctga ggcgcgcttc 660 gtgaagacgc tgctggtggc cgatgcgtcc atggctgcct tctacggggc cgacctgcag 720 aaccacatcc tgacgttaat gtctgtggca gcccgaatct acaagcaccc cagcatcaag 780 aattccatca acctgatggt ggtaaaagtg ctgatcgtag aagatgaaaa atggggccca 840 gaggtgtccg acaatggggg gcttacactg cgtaacttct gcaactggca gcggcgtttc 900 aaccagccca gcgaccgcca cccagagcac tacgacacgg ccatcctgct caccagacag 960 aacttctgtg ggcaggaggg gctgtgtgac accctgggtg tggcagacat cgggaccatt 1020 tgtgacccca acaaaagctg ctccgtgatc gaggatgagg ggctccaggc ggcccacacc 1080 ctggcccatg aactagggca cgtcctcagc atgccccacg acgactccaa gccctgcaca 1140 cggctcttcg ggcccatggg caagcaccac gtgatggcac cgctgttcgt ccacctgaac 1200 cagacgctgc cctggtcccc ctgcagcgcc atgtatctca cagagcttct ggacggcggg 1260 cacggagact gtctcctgga tgcccctgct gcggccctgc ccctccccac aggcctcccg 1320 ggccgcatgg ccctgtacca gctggaccag cagtgcaggc agatctttgg gccggatt tc 1380 cgccactgcc ccaacacctc tgctcaggac gtctgcgccc agctttggtg ccacactgat 1440 ggggctgagc ccctgtgcca cacgaagaat ggcagcctgc cctgggctga cggcacgccg 1500 tgcgggcctg ggcacctctg ctcagaaggc agctgtctac ctgaggagga agtggagagg 1560 cccaagcccg tggtagatgg aggctgggca ccgtggggac cctggggaga atgttctcgg 1620 acctgtggag gaggagtaca gttttcacac cgtgagtgca aggaccccga gcctcagaat 1680 ggaggaagat actgcctggg tcggagagcc aagtaccagt catgccacac ggaggaatgc 1740 ccccctgacg ggaaaagctt cagggagcag cagtgtgaga agtataatgc ctacaattac 1800 actgacatgg acgggaatct cctgcagtgg gtccccaagt atgctggggt gtccccccgg 1860 gaccgctgca agttgttctg ccgagcccgg gggaggagcg agttcaaagt gttcgaggcc 1920 aaggtgattg atggcaccct gtgtgggcca gaaacactgg ccatctgtgt ccgtggccag 1980 tgtgtcaagg ccggctgtga ccatgtggtg gactcgcctc ggaagctgga caaatgcggg 2040 gtgtgtgggg gcaaaggcaa ctcctgcagg aaggtctccg ggtccctcac ccccaccaat 2100 tatggctaca atgacattgt caccatccca gctggtgcca ctaatattga cgtgaagcag 2160 cggagccacc cgggtgtgca gaacgatggg aactacctgg cgctgaagac ggctgatggg 222 0 cagtacctgc tcaacggcaa cctggccatc tctgccatag agcaggacat cttggtgaag 2280 gggaccatcc tgaagtacag cggctccatc gccaccctgg agcgcctgca gagcttccgg 2340 cccttgccag agcctctgac agtgcagctc ctgacagtcc ctggcgaggt cttcccccca 2400 aaagtcaaat acaccttctt tgttcctaat gacgtggact ttagcatgca gagcagcaaa 2460 gagagagcaa ccaccaacat catccagccg ctgctccacg cacagtgggt gctgggggac 2520 tggtctgagt gctctagcac ctgcggggcc ggctggcaga ggcgaactgt agagtgcagg 2580 gacccctcca gccaggcctc tgccacctgc aacaaggctc tgaaacccga ggatgccaag 2640 ccctgcgaaa gccagctgtg ccccctgtga 2670 <210> 4 <211> 950 <212> PRT <213> Homo sapiens <400> 4 Met Gly Asn Ala Glu Arg Ala Pro Gly Ser Arg Ser Phe Gly Pro Val 1 5 10 15 Pro Thr Leu Leu Leu Leu Ala Ala Ala Leu Leu Ala Val Ser Asp Ala 20 25 30 Leu Gly Arg Pro Ser Glu Glu Asp Glu Glu Leu Val Val Pro Glu Leu 35 40 45 Glu Arg Ala Pro Gly His Gly Thr Thr Arg Leu Arg Leu His Ala Phe 50 55 60 Asp Gln Gln Leu Asp Leu Glu Leu Arg Pro Asp Ser Ser Phe Leu Ala 65 70 75 80 Pro Gly Phe Thr Leu Gln Asn Val Gly Arg Lys Ser Gly Ser Glu Thr 85 90 95 Pro Leu Pro Glu Thr Asp Leu Ala His Cys Phe Tyr Ser Gly Thr Val 100 105 110 Asn Gly Asp Pro Ser Ser Ala Ala Ala Leu Ser Leu Cys Glu Gly Val 115 120 125 Arg Gly Ala Phe Tyr Leu Leu Gly Glu Ala Tyr Phe Ile Gln Pro Leu 130 135 140 Pro Ala Ala Ser Glu Arg Leu Ala Thr Ala Ala Pro Gly Glu Lys Pro 145 150 155 160 Pro Ala Pro Leu Gln Phe His Leu Leu Arg Arg Asn Arg Gln Gly Asp 165 170 175 Val Gly Gly Thr Cys Gly Val Val Asp Asp Glu Pro Arg Pro Thr Gly 180 185 190 Lys Ala Glu Thr Glu Asp Glu Asp Glu Gly Thr Glu Gly Glu Asp Glu 195 200 205 Gly Pro Gln Trp Ser Pro Gln Asp Pro Ala Leu Gln Gly Val Gly Gln 210 215 220 Pro Thr Gly Thr Gly Ser Ile Arg Lys Lys Arg Phe Val Ser Ser His 225 230 235 240 Arg Tyr Val Glu Thr Met Leu Val Ala Asp Gln Ser Met Ala Glu Phe 245 250 255 His Gly Ser Gly Leu Lys His Tyr Leu Leu Thr Leu Phe Ser Val Ala 260 265 270 Ala Arg Leu Tyr Lys His Pro Ser Ile Arg Asn Ser Val Ser Leu Val 275 280 285 Val Val Lys Ile Leu Val Ile His Asp Glu Gln Ly s Gly Pro Glu Val 290 295 300 Thr Ser Asn Ala Ala Leu Thr Leu Arg Asn Phe Cys Asn Trp Gln Lys 305 310 315 320 Gln His Asn Pro Pro Ser Asp Arg Asp Ala Glu His Tyr Asp Thr Ala 325 330 335 Ile Leu Phe Thr Arg Gln Asp Leu Cys Gly Ser Gln Thr Cys Asp Thr 340 345 350 Leu Gly Met Ala Asp Val Gly Thr Val Cys Asp Pro Ser Arg Ser Cys 355 360 365 Ser Val Ile Glu Asp Asp Gly Leu Gln Ala Ala Phe Thr Thr Ala His 370 375 380 Glu Leu Gly His Val Phe Asn Met Pro His Asp Asp Ala Lys Gln Cys 385 390 395 400 Ala Ser Leu Asn Gly Val Asn Gln Asp Ser His Met Met Ala Ser Met 405 410 415 Leu Ser Asn Leu Asp His Ser Gln Pro Trp Ser Pro Cys Ser Ala Tyr 420 425 430 Met Ile Thr Ser Phe Leu Asp Asn Gly His Gly Glu Glu Cys Leu Met Asp 435 440 445 Lys Pro Gln Asn Pro Ile Gln Leu Pro Gly Asp Leu Pro Gly Thr Ser 450 455 460 Tyr Asp Ala Asn Arg Gln Cys Gln Phe Thr Phe Gly Glu Asp Ser Lys 465 470 475 480 His Cys Pro Asp Ala Ala Ser Thr Cys Ser Thr Leu Trp Cys Thr Gly 485 490 495 Thr Ser Gly Gly Val Leu Val Cys Gln Thr Lys H is Phe Pro Trp Ala 500 505 510 Asp Gly Thr Ser Cys Gly Glu Gly Lys Trp Cys Ile Asn Gly Lys Cys 515 520 525 Val Asn Lys Thr Asp Arg Lys His Phe Asp Thr Pro Phe His Gly Ser 530 535 540 Trp Gly Met Trp Gly Pro Trp Gly Asp Cys Ser Arg Thr Cys Gly Gly 545 550 555 560 Gly Val Gln Tyr Thr Met Arg Glu Cys Asp Asn Pro Val Pro Lys Asn 565 570 575 Gly Gly Lys Tyr Cys Glu Gly Lys Arg Val Arg Tyr Arg Ser Cys Asn 580 585 590 Leu Glu Asp Cys Pro Asp Asn Asn Gly Lys Thr Phe Arg Glu Glu Gln 595 600 605 Cys Glu Ala His Asn Glu Phe Ser Lys Ala Ser Phe Gly Ser Gly Pro 610 615 620 Ala Val Glu Trp Ile Pro Lys Tyr Ala Gly Val Ser Pro Lys Asp Arg 625 630 635 640 Cys Lys Leu Ile Cys Gln Ala Lys Gly Ile Gly Tyr Phe Phe Val Leu 645 650 655 Gln Pro Lys Val Val Asp Gly Thr Pro Cys Ser Pro Asp Ser Thr Ser 660 665 670 Val Cys Val Gln Gly Gln Cys Val Lys Ala Gly Cys Asp Arg Ile Ile 675 680 685 Asp Ser Lys Lys Lys Phe Asp Lys Cys Gly Val Cys Gly Gly Asn Gly 690 695 700 Ser Thr Cys Lys Lys Ile Ser Gly Ser Val Thr SerAla Lys Pro Gly 705 710 715 715 720 Tyr His Asp Ile Ile Thr Ile Pro Thr Gly Ala Thr Asn Ile Glu Val 725 730 735 Lys Gln Arg Asn Gln Arg Gly Ser Arg Asn Asn Gly Ser Phe Leu Ala 740 745 750 Ile Lys Ala Ala Asp Gly Thr Tyr Ile Leu Asn Gly Asp Tyr Thr Leu 755 760 765 Ser Thr Leu Glu Gln Asp Ile Met Tyr Lys Gly Val Val Leu Arg Tyr 770 775 780 Ser Gly Ser Ser Ala Ala Leu Glu Arg Ile Arg Ser Phe Ser Pro Leu 785 790 795 800 Lys Glu Pro Leu Thr Ile Gln Val Leu Thr Val Gly Asn Ala Leu Arg 805 810 815 Pro Lys Ile Lys Tyr Thr Tyr Phe Val Lys Lys Lys Lys Glu Ser Phe 820 825 830 Asn Ala Ile Pro Thr Phe Ser Ala Trp Val Ile Glu Glu Trp Gly Glu 835 840 845 Cys Ser Lys Ser Cys Glu Leu Gly Trp Gln Arg Arg Leu Val Glu Cys 850 855 860 Arg Asp Ile Asn Gly Gln Pro Ala Ser Glu Cys Ala Lys Glu Val Lys 865 870 870 875 880 Pro Ala Ser Thr Arg Pro Cys Ala Asp His Pro Cys Pro Gln Trp Gln 885 890 895 Leu Gly Glu Trp Ser Ser Cys Ser Lys Thr Cys Gly Lys Gly Tyr Lys 900 905 910 Lys Arg Ser Leu Lys Cys Leu Ser His Asp Gly GlyVal Leu Ser His 915 920 925 Glu Ser Cys Asp Pro Leu Lys Lys Pro Lys His Phe Ile Asp Phe Cys 930 935 940 Thr Met Ala Glu Cys Ser 945 950 <210> 5 <211> 2853 <212> DNA <213> Homo sapiens <400> 5 atggggaacg cggagcgggc tccggggtct cggagctttg ggcccgtacc cacgctgctg 60 ctgctcgccg cggcgctact ggccgtgtcg gacgcactcg ggcgcccctc cgaggaggac 120 gaggagctag tggtgccgga gctggagcgc gccccgggac acgggaccac gcgcctccgc 180 ctgcacgcct ttgaccagca gctggatctg gagctgcggc ccgacagcag ctttttggcg 240 cccggcttca cgctccagaa cgtggggcgc aaatccgggt ccgagacgcc gcttccggaa 300 accgacctgg cgcactgctt ctactccggc accgtgaatg gcgatcccag ctcggctgcc 360 gccctcagcc tctgcgaggg cgtgcgcggc gccttctacc tgctggggga ggcgtatttc 420 atccagccgc tgcccgccgc cagcgagcgc ctcgccaccg ccgccccagg ggagaagccg 480 ccggcaccac tacagttcca cctcctgcgg cggaatcggc agggcgacgt aggcggcacg 540 tgcggggtcg tggacgacga gccccggccg actgggaaag cggagaccga agacgaggac 600 gaagggactg agggcgagga cgaagggcct cagtggtcgc cgcaggaccc ggcactgcaa 660 ggcgtaggac agcccacagg aactggaagc ataaga aaga agcgatttgt gtccagtcac 720 cgctatgtgg aaaccatgct tgtggcagac cagtcgatgg cagaattcca cggcagtggt 780 ctaaagcatt accttctcac gttgttttcg gtggcagcca gattgtacaa acaccccagc 840 attcgtaatt cagttagcct ggtggtggtg aagatcttgg tcatccacga tgaacagaag 900 gggccggaag tgacctccaa tgctgccctc actctgcgga acttttgcaa ctggcagaag 960 cagcacaacc cacccagtga ccgggatgca gagcactatg acacagcaat tcttttcacc 1020 agacaggact tgtgtgggtc ccagacatgt gatactcttg ggatggctga tgttggaact 1080 gtgtgtgatc cgagcagaag ctgctccgtc atagaagatg atggtttaca agctgccttc 1140 accacagccc atgaattagg ccacgtgttt aacatgccac atgatgatgc aaagcagtgt 1200 gccagcctta atggtgtgaa ccaggattcc cacatgatgg cgtcaatgct ttccaacctg 1260 gaccacagcc agccttggtc tccttgcagt gcctacatga ttacatcatt tctggataat 1320 ggtcatgggg aatgtttgat ggacaagcct cagaatccca tacagctccc aggcgatctc 1380 cctggcacct cgtacgatgc caaccggcag tgccagttta catttgggga ggactccaaa 1440 cactgccccg atgcagccag cacatgtagc accttgtggt gtaccggcac ctctggtggg 1500 gtgctggtgt gtcaaaccaa acacttcccg tgggcggatg gcacc agctg tggagaaggg 1560 aaatggtgta tcaacggcaa gtgtgtgaac aaaaccgaca gaaagcattt tgatacgcct 1620 tttcatggaa gctggggaat gtgggggcct tggggagact gttcgagaac gtgcggtgga 1680 ggagtccagt acacgatgag ggaatgtgac aacccagtcc caaagaatgg agggaagtac 1740 tgtgaaggca aacgagtgcg ctacagatcc tgtaaccttg aggactgtcc agacaataat 1800 ggaaaaacct ttagagagga acaatgtgaa gcacacaacg agttttcaaa agcttccttt 1860 gggagtgggc ctgcggtgga atggattccc aagtacgctg gcgtctcacc aaaggacagg 1920 tgcaagctca tctgccaagc caaaggcatt ggctacttct tcgttttgca gcccaaggtt 1980 gtagatggta ctccatgtag cccagattcc acctctgtct gtgtgcaagg acagtgtgta 2040 aaagctggtt gtgatcgcat catagactcc aaaaagaagt ttgataaatg tggtgtttgc 2100 gggggaaatg gatctacttg taaaaaaata tcaggatcag ttactagtgc aaaacctgga 2160 tatcatgata tcatcacaat tccaactgga gccaccaaca tcgaagtgaa acagcggaac 2220 cagaggggat ccaggaacaa tggcagcttt cttgccatca aagctgctga tggcacatat 2280 attcttaatg gtgactacac tttgtccacc ttagagcaag acattatgta caaaggtgtt 2340 gtcttgaggt acagcggctc ctctgcggca ttggaaagaa ttcgcagctt tagccctctc 2400 aaagagccct tgaccatcca ggttcttact gtgggcaatg cccttcgacc taaaattaaa 2460 tacacctact tcgtaaagaa gaagaaggaa tctttcaatg ctatccccac tttttcagca 2520 tgggtcattg aagagtgggg cgaatgttct aagtcatgtg aattgggttg gcagagaaga 2580 ctggtagaat gccgagacat taatggacag cctgcttccg agtgtgcaaa ggaagtgaag 2640 ccagccagca ccagaccttg tgcagaccat ccctgccccc agtggcagct gggggagtgg 2700 tcatcatgtt ctaagacctg cgggaagggt tacaaaaaaa gaagcttgaa gtgtctgtcc 2760 catgatggag gggtgttatc tcatgagagc tgtgatcctt taaagaaacc taaacatttc 2820 atagactttt gcacaatggc agaatgcagt taa 2853 <210> 6 <211> 25 <212> DNA <213> Homo sapiens <400> 6 acctgtcctt tggtgagacg ccagc 25 <210> 7 <211> 26 <212> DNA <213> Homo sapiens <400 > 7 atctggctgc caccgaaaac aacgtg 26 <210> 8 <211> 30 <212> DNA <213> Homo sapiens <400> 8 tcccccagca ggtagaaggc gccgcgcacg 30 <210> 9 <211> 30 <212> DNA <213> Homo sapiens <400 > 9 ccgagctggg atcgccattc acggtgccgg 30 <210> 10 <211> 1205 <212> PRT <213> Homo sapiens <400> 10 Met Val Leu Leu Ser Leu Trp Leu Ile Ala Ala Ala Leu Val Glu Val 1 5 10 15 Arg Thr Ser Ala Asp Gly Gln Ala Gly Asn Glu Glu Met Val Gln Ile 20 25 30 Asp Leu Pro Ile Lys Arg Tyr Arg Glu Tyr Glu Leu Val Thr Pro Val 35 40 45 Ser Thr Asn Leu Glu Gly Arg Tyr Leu Ser His Thr Leu Ser Ala Ser 50 55 60 His Lys Lys Arg Ser Ala Arg Asp Val Ser Ser Asn Pro Glu Gln Leu 65 70 75 80 Phe Phe Asn Ile Thr Ala Phe Gly Lys Asp Phe His Leu Arg Leu Lys 85 90 95 Pro Asn Thr Gln Leu Val Ala Pro Gly Ala Val Val Glu Trp His Glu 100 105 110 Thr Ser Leu Val Pro Gly Asn Ile Thr Asp Pro Ile Asn Asn His Gln 115 120 125 Pro Gly Ser Ala Thr Tyr Arg Ile Arg Lys Thr Glu Pro Leu Gln Thr 130 135 140 Asn Cys Ala Tyr Val Gly Asp Ile Val Asp Ile Pro Gly Thr Ser Val 145 150 155 160 Ala Ile Ser Asn Cys Asp Gly Leu Ala Gly Met Ile Lys Ser Asp Asn 165 170 175 Glu Glu Tyr Phe Ile Glu Pro Leu Glu Arg Gly Lys Gln Met Glu Glu 180 185 190 Glu Lys Gly Arg Ile His Val Val Tyr Lys Arg Ser Ala Val Glu Gln 195 200 205 Ala Pro Ile Asp Met Ser Lys Asp Phe His Tyr Arg Glu Ser A sp Leu 210 215 220 Glu Gly Leu Asp Asp Leu Gly Thr Val Tyr Gly Asn Ile His Gln Gln 225 230 235 240 Leu Asn Glu Thr Met Arg Arg Arg Arg His Ala Gly Glu Asn Asp Tyr 245 250 255 Asn Ile Glu Val Leu Leu Gly Val Asp Asp Ser Val Val Arg Phe His 260 265 270 270 Gly Lys Glu His Val Gln Asn Tyr Leu Leu Thr Leu Met Asn Ile Val 275 280 285 Asn Glu Ile Tyr His Asp Glu Ser Leu Gly Val His Ile Asn Val Val 290 295 300 Leu Val Arg Met Ile Met Leu Gly Tyr Ala Lys Ser Ile Ser Leu Ile 305 310 315 320 Glu Arg Gly Asn Pro Ser Arg Ser Leu Glu Asn Val Cys Arg Trp Ala 325 330 335 Ser Gln Gln Gln Arg Ser Asp Leu Asn His Ser Glu His His Asp His 340 345 350 Ala Ile Phe Leu Thr Arg Gln Asp Phe Gly Pro Ala Gly Met Gln Gly 355 360 365 Tyr Ala Pro Val Thr Gly Met Cys His Pro Val Arg Ser Cys Thr Leu 370 375 380 380 Asn His Glu Asp Gly Phe Ser Ser Ala Phe Val Val Ala His Glu Thr 385 390 395 400 400 Gly His Val Leu Gly Met Glu His Asp Gly Gln Gly Asn Arg Cys Gly 405 410 415 Asp Glu Thr Ala Met Gly Ser Val Met Ala Pro Leu Val GlnAla Ala 420 425 430 Phe His Arg Tyr His Trp Ser Arg Cys Ser Gly Gln Glu Leu Lys Arg 435 440 445 Tyr Ile His Ser Tyr Asp Cys Leu Leu Asp Asp Pro Phe Asp His Asp 450 455 460 Trp Pro Lys Leu Pro Glu Leu Pro Gly Ile Asn Tyr Ser Met Asp Glu 465 470 475 480 480 Gln Cys Arg Phe Asp Phe Gly Val Gly Tyr Lys Met Cys Thr Ala Phe 485 490 495 Arg Thr Phe Asp Pro Cys Lys Gln Leu Trp Cys Ser His Pro Asp Asn 500 505 510 Pro Tyr Phe Cys Lys Thr Lys Lys Gly Pro Pro Leu Asp Gly Thr Glu 515 520 525 Cys Ala Ala Gly Lys Trp Cys Tyr Lys Gly His Cys Met Trp Lys Asn 530 535 540 Ala Asn Gln Gln Lys Gln Asp Gly Asn Trp Gly Ser Trp Thr Lys Phe 545 550 555 560 Gly Ser Cys Ser Arg Thr Cys Gly Thr Gly Val Arg Phe Arg Thr Arg 565 570 575 Gln Cys Asn Asn Pro Met Pro Ile Asn Gly Gly Gln Asp Cys Pro Gly 580 585 585 590 Val Asn Phe Glu Tyr Gln Leu Cys Asn Thr Glu Glu Cys Gln Lys His 595 600 605 Phe Glu Asp Phe Arg Ala Gln Gln Cys Gln Gln Arg Asn Ser His Phe 610 615 620 Glu Tyr Gln Asn Thr Lys His His Trp Leu Pro Tyr Glu His ProAsp 625 630 635 640 Pro Lys Lys Arg Cys His Leu Tyr Cys Gln Ser Lys Glu Thr Gly Asp 645 650 655 Val Ala Tyr Met Lys Gln Leu Val His Asp Gly Thr His Cys Ser Tyr 660 665 670 Lys Asp Pro Tyr Ser Ile Cys Val Arg Gly Glu Cys Val Lys Val Gly 675 680 685 Cys Asp Lys Glu Ile Gly Ser Asn Lys Val Glu Asp Lys Cys Gly Val 690 695 700 Cys Gly Gly Asy Asp Asn Ser His Cys Arg Thr Val Lys Gly Thr Phe Thr 705 710 715 720 Arg Thr Pro Arg Lys Leu Gly Tyr Leu Lys Met Phe Asp Ile Pro Pro 725 730 735 Gly Ala Arg His Val Leu Ile Gln Glu Asp Glu Ala Ser Pro His Ile 740 745 750 Leu Ala Ile Lys Asn Gln Ala Thr Gly His Tyr Ile Leu Asn Gly Lys 755 760 765 Gly Glu Glu Alu Lys Ser Arg Thr Phe Ile Asp Leu Gly Val Glu Trp 770 775 780 Asp Tyr Asn Ile Glu Asp Asp Ile Glu Ser Leu His Thr Asp Gly Pro 785 790 795 800 Leu His Asp Pro Val Ile Val Leu Ile Ile Pro Gln Glu Asn Asp Thr 805 810 815 Arg Ser Ser Leu Thr Tyr Lys Tyr Ile Ile His Glu Asp Ser Val Pro 820 825 830 Thr Ile Asn Ser Asn Asn Val Ile Gln Glu Glu Leu Asp Thr Phe Glu 835 840 845 Trp Ala Leu Lys Ser Trp Ser Gln Cys Ser Lys Pro Cys Gly Gly Gly 850 855 860 Phe Gln Tyr Thr Lys Tyr Gly Cys Arg Arg Lys Ser Asp Asn Lys Met 865 870 875 875 880 Val His Arg Ser Phe Cys Glu Ala Asn Lys Lys Pro Lys Pro Ile Arg 885 890 895 Arg Met Cys Asn Ile Gln Glu Cys Thr His Pro Leu Trp Val Ala Glu 900 905 910 Glu Trp Glu His Cys Thr Lys Thr Cys Gly Ser Ser Gly Tyr Gln Leu 915 920 925 Arg Thr Val Arg Cys Leu Gln Pro Leu Leu Asp Gly Thr Asn Arg Ser 930 935 940 Val His Ser Lys Tyr Cys Met Gly Asp Arg Pro Glu Ser Arg Arg Pro 945 950 955 960 Cys Asn Arg Val Pro Cys Pro Ala Gln Trp Lys Thr Gly Pro Trp Ser 965 970 975 Glu Cys Ser Val Thr Cys Gly Glu Gly Thr Glu Val Arg Gln Val Leu 980 985 990 Cys Arg Ala Gly Asp His Cys Asp Gly Glu Lys Pro Glu Ser Val Arg 995 1000 1005 Ala Cys Gln Leu Pro Pro Cys Asn Asp Glu Pro Cys Leu Gly Asp Lys 1010 1015 1020 Ser Ile Phe Cys Gln Met Glu Val Leu Ala Arg Tyr Cys Ser Ile Pro 1025 1030 1035 1040 Gly Tyr Asn Lys Leu Cys Cys Glu Ser Cys Ser Lys Ar g Ser Ser Thr 1045 1050 1055 Leu Pro Pro Pro Tyr Leu Leu Glu Ala Ala Glu Thr His Asp Asp Val 1060 1065 1070 Ile Ser Asn Pro Ser Asp Leu Pro Arg Ser Leu Val Met Pro Thr Ser 1075 1080 1085 Leu Val Pro Tyr His Ser Glu Thr Pro Ala Lys Lys Met Ser Leu Ser 1090 1095 1100 Ser Ile Ser Ser Val Gly Gly Pro Asn Ala Tyr Ala Ala Phe Arg Pro 1105 1110 1115 1120 Asn Ser Lys Pro Asp Gly Ala Asn Leu Arg Gln Arg Ser Ala Gln Gln 1125 1130 1135 Ala Gly Ser Lys Thr Val Arg Leu Val Thr Val Pro Ser Ser Pro Pro 1140 1145 1150 Thr Lys Arg Val His Leu Ser Ser Ala Ser Gln Met Ala Ala Ala Ser 1155 1160 1165 Phe Phe Ala Ala Ser Asp Ser Ile Gly Ala Ser Ser Gln Ala Arg Thr 1170 1175 1180 Ser Lys Lys Asp Gly Lys Ile Ile Asp Asn Arg Arg Pro Thr Arg Ser 1185 1190 1195 1200 Ser Thr Leu Glu Arg 1205 <210> 11 <211> 3618 <212> DNA <213 > Homo sapiens <400> 11 atggttctcc tgtcactttg gttgatagca gccgctctgg tagaggttag gacttcagct 60 gatggacaag ctggtaatga agaaatggtg caaatagatt taccaataaa gagatataga 120 gagtatgagc tggtgactcc agtca gcaca aatctagaag gacgctatct ctcccatact 180 ctttctgcga gtcacaaaaa gaggtcagcg agggacgtgt cttccaaccc tgagcagttg 240 ttctttaaca tcacggcatt tggaaaagat tttcatctgc gactaaagcc caacactcaa 300 ctagtagctc ctggggctgt tgtggagtgg catgagacat ctctggtgcc tgggaatata 360 accgatccca ttaacaacca tcaaccagga agtgctacgt atagaatccg gaaaacagag 420 cctttgcaga ctaactgtgc ttatgttggt gacatcgtgg acattccagg aacctctgtt 480 gccatcagca actgtgatgg tctggctgga atgataaaaa gtgataatga agagtatttc 540 attgaaccct tggaaagagg taaacagatg gaggaagaaa aaggaaggat tcatgttgtc 600 tacaagagat cagctgtaga acaggctccc atagacatgt ccaaagactt ccactacaga 660 gagtcggacc tggaaggcct tgatgatcta ggtactgttt atggcaacat ccaccagcag 720 ctgaatgaaa caatgagacg ccgcagacac gcgggagaaa acgattacaa tatcgaggta 780 ctgctgggag tggatgactc tgtggtccgt ttccatggca aagagcacgt ccaaaactac 840 ctcctgaccc taatgaacat tgtgaatgaa atttaccatg atgagtccct cggagtgcat 900 ataaatgtgg tcctggtgcg catgataatg ctgggatatg caaagtccat cagcctcata 960 gaaaggggaa acccatccag aagcttggag aatgtgtgtc gct gggcgtc ccaacagcaa 1020 agatctgatc tcaaccactc tgaacaccat gaccatgcaa tttttttaac caggcaagac 1080 tttggacctg ctggaatgca aggatatgct ccagtcaccg gcatgtgtca tccagtgaga 1140 agttgtaccc tgaatcatga ggatggtttt tcatctgctt ttgtagtagc ccatgaaacg 1200 ggccatgtgt tgggaatgga gcatgatgga caaggcaaca ggtgtggtga tgagactgct 1260 atgggaagtg tcatggctcc cttggtacaa gcagcattcc atcgttacca ctggtcccga 1320 tgcagtggtc aagaactgaa aagatatatc cattcctatg actgtctcct tgatgaccct 1380 tttgatcatg attggcctaa actcccagaa cttcctggaa tcaattattc tatggatgag 1440 caatgtcgtt ttgattttgg tgttggctat aaaatgtgca ccgcgttccg aacctttgac 1500 ccatgtaaac agctgtggtg tagccatcct gataatccct acttttgtaa gactaaaaag 1560 ggacctccac ttgatgggac tgaatgtgct gctggaaaat ggtgctataa gggtcattgc 1620 atgtggaaga atgctaatca gcaaaaacaa gatggcaatt gggggtcatg gactaaattt 1680 ggctcctgtt ctcggacatg tggaactggt gttcgtttca gaacacgcca gtgcaataat 1740 cccatgccca tcaatggtgg tcaggattgt cctggtgtta attttgagta ccagctttgt 1800 aacacagaag aatgccaaaa acactttgag gacttcagag cacagcagt g tcagcagcga 1860 aactcccact ttgaatacca gaataccaaa caccactggt tgccatatga acatcctgac 1920 cccaagaaaa gatgccacct ttactgtcag tccaaggaga ctggagatgt tgcttacatg 1980 aaacaactgg tgcatgatgg aacgcactgt tcttacaaag atccatatag catatgtgtg 2040 cgaggagagt gtgtgaaagt gggctgtgat aaagaaattg gttctaataa ggttgaggat 2100 aagtgtggtg tctgtggagg agataattcc cactgccgaa ccgtgaaggg gacatttacc 2160 agaactccca ggaagcttgg gtaccttaag atgtttgata taccccctgg ggctagacat 2220 gtgttaatcc aagaagacga ggcttctcct catattcttg ctattaagaa ccaggctaca 2280 ggccattata ttttaaatgg caaaggggag gaagccaagt cgcggacctt catagatctt 2340 ggtgtggagt gggattataa cattgaagat gacattgaaa gtcttcacac cgatggacct 2400 ttacatgatc ctgttattgt tttgattata cctcaagaaa atgatacccg ctctagcctg 2460 acatataagt acatcatcca tgaagactct gtacctacaa tcaacagcaa caatgtcatc 2520 caggaagaat tagatacttt tgagtgggct ttgaagagct ggtctcagtg ttccaaaccc 2580 tgtggtggag gtttccagta cactaaatat ggatgccgta ggaaaagtga taataaaatg 2640 gtccatcgca gcttctgtga ggccaacaaa aagccgaaac ctattagacg aatg tgcaat 2700 attcaagagt gtacacatcc actctgggta gcagaagaat gggaacactg caccaaaacc 2760 tgtggaagtt ctggctatca gcttcgcact gtacgctgcc ttcagccact ccttgatggc 2820 accaaccgct ctgtgcacag caaatactgc atgggtgacc gtcccgagag ccgccggccc 2880 tgtaacagag tgccctgccc tgcacagtgg aaaacaggac cctggagtga gtgttcagtg 2940 acctgcggtg aaggaacgga ggtgaggcag gtcctctgca gggctgggga ccactgtgat 3000 ggtgaaaagc ctgagtcggt cagagcctgt caactgcctc cttgtaatga tgaaccatgt 3060 ttgggagaca agtccatatt ctgtcaaatg gaagtgttgg cacgatactg ctccatacca 3120 ggttataaca agttatgttg tgagtcctgc agcaagcgca gtagcaccct gccaccacca 3180 taccttctag aagctgctga aactcatgat gatgtcatct ctaaccctag tgacctccct 3240 agatctctag tgatgcctac atctttggtt ccttatcatt cagagacccc tgcaaagaag 3300 atgtctttga gtagcatctc ttcagtggga ggtccaaatg catatgctgc tttcaggcca 3360 aacagtaaac ctgatggtgc taatttacgc cagaggagtg ctcagcaagc aggaagtaag 3420 actgtgagac tggtcaccgt accatcctcc ccacccacca agagggtcca cctcagttca 3480 gcttcacaaa tggctgctgc ttccttcttt gcagccagtg attcaatagg tgcttcttct 3540 caggcaagaa cctcaaagaa agatggaaag atcattgaca acagacgtcc gacaagatca 3600 tccaccttag aaagatga 3618 <210> 12 <211> 50 <212> DNA <213> Homo sapiens <400> 12 ctagcgcggc cgcaggatcc gactacaagg <10> <50> DNA <ca> <213> Homo sapiens <400> 13 gatcttatca tttgtcatcg tcgtccttgt agtcggatcc tgcggccgcg 50 <210> 14 <211> 34 <212> DNA <213> Homo sapiens <400> 14 ggactagtct agaagctggg taccagctgc tagc 34 <210> 152 <21> 212> DNA <213> Homo sapiens <400> 15 ggactagtgt cgaccggtca tggctgcgc 29 <210> 16 <211> 37 <212> DNA <213> Homo sapiens <400> 16 gctctagacc atggggaacg cggagcgggc tccgggg 37 <210> 17 <211> 30 <212> DNA <213> Homo sapiens <400> 17 ggatccctgc cggttggcat cgtacgaggt 30 <210> 18 <211> 42 <212> DNA <213> Homo sapiens <400> 18 gtggctagcg ccatggttct cctgtcactt tggttgatag ca 42 <210> 19 <211 > 41 <212> DNA <213> Homo sapiens <400> 19 agagcggccg cttgctcatc catagaataa ttgattccag g 41 <210> 20 <211> 11 <212> PRT <213> Homo sapiens <400> 20 Asn Ile Thr Glu Gly Gl u Ala Arg Gly Ser Val 1 5 10 <210> 21 <211> 27 <212> DNA <213> Homo sapiens <400> 21 tcctatgact gtctccttga tgaccct 27 <210> 22 <211> 35 <212> DNA <213> Homo sapiens <400> 22 gcggatcctc atctttctaa ggtggatgat cttgt 35

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12N 9/50 9/50 C12Q 1/37 C12Q 1/37 C12N 5/00 A (72) Inventor Miho Sasamata 21 Miyukigaoka, Tsukuba City, Ibaraki Pref. BA02 CA33 CA44 CA46 4H045 AA11 AA20 BA10 CA40 DA76 EA50 FA72

Claims (8)

[Claims] (1) the first amino acid sequence represented by SEQ ID NO: 4;
The amino acid sequence from No. 470 to No. 470, or SEQ ID NO: 10
A metal protease having the amino acid sequence of the 1st to the 481st amino acid of the amino acid sequence represented by and having protease activity, or a metal protease which is an equivalent of the metal protease. 2. A protein having an amino acid sequence represented by SEQ ID NO: 4 or 10. [3] a gene encoding the amino acid sequence of the metalloprotease of [1] or the protein of [2]; A vector comprising the gene according to claim 3. (5) A host cell comprising the vector according to (4). [6] The host cell according to [5], wherein the host cell is used.
A method for producing the metalloprotease according to claim 1 and the protein according to claim 2. 7. An antibody against the metalloprotease according to claim 1 and the protein according to claim 2. 8. A method for screening a substance that modifies the activity of the metal protease by bringing the metalloprotease of claim 1 or the protein of claim 2 into contact with a test compound.