Protein, DNA coding for same and method of producing the protein

A novel protein having inhibitory activity on the protease activity of hepatocyte growth factor (HGF) activator was purified and isolated, and its molecular weight (ca. 40,000 daltons) and its N-terminal and partial amino acid sequences were determined. A gene coding for the protein was cloned, and the gene DNA was incorporated into a vector, for transforming host cells. Cultivation of the transformant gave the desired protein. The protein can be used as an in vivo or in vitro control factor for HGF or HGF activator. It is also useful as an antigen to be used in producing an antibody to be used as means for kinetic studies of the protein, or as a standard in assay systems therefor.

FIELD OF THE INVENTION
 The present invention relates to a novel protein and a DNA coding for the
 same. More particularly, it relates to a novel protein having inhibitory
 activity on the protease activity of hepatocyte growth factor activating
 factor (HGF activator) (hereinafter this protein is sometimes referred to
 also as "HAI-I"), a gene coding for the protein, an expression vector
 containing the gene, a transformant as transformed with the expression
 vector, and a method of producing HAI-I using the transformant.
 BACKGROUND OF THE INVENTION
 It is already reported that thrombin activates the precursor of HGF
 activator (JP-A-5-103670, JP-A-6-141859, JP-A-6-153946 and JP-A-6-153966
 (the term "JP-A" as used herein means an "unexamined published Japanese
 patent application"); factor having activity to convert the single chain
 form of hepatocyte growth factor (HGF) to its double chain form) in the
 manner of positive activity control. However, human tissues-derived
 protease inhibitor capable of inhibiting, as a negative control factor,
 the physiological activity of HGF activator has not been known. Therefore,
 how HGF activator is controlled in human tissues remains unknown. Such a
 negative control factor might also influence indirectly on the activity of
 hepatocyte growth factor (HGF) on which HGF activator acts. Thus, for the
 analysis of the mechanism of action of HGF in vivo, as well, it has been
 demanded that such a human tissues-derived protease inhibitor be isolated
 and identified.
 By using such a protease inhibitor and an antibody to the protease
 inhibitor, it would become possible to know the in vivo physiological
 activity of HGF activator, analyze the mechanism of action thereof or
 analyze the mechanism of control of HGF activation, from a standpoint
 different from those of the prior art.
 Furthermore, for investigating the detailed in vivo function of HAI-I or
 the effect of HAI-I in hepatic disorder, for instance, HAI-I is required
 in large quantities. At present, however, there is only one method
 available for preparing HAI-I, which method comprises using, as a starting
 material, the culture supernatant obtained with a human cancer cell line
 such as MKN45 or A549 cells and purifying therefrom HAI-I occurring
 therein in trace amounts. This method is not always the best one from the
 labor, time and cost viewpoint. It encounters great difficulties in stably
 isolating the minor amount of HAI-I alone. Therefore, it has been desired
 that an expression system be constructed so that HAI-I can be obtained
 stably and in large quantities.
 SUMMARY OF THE INVENTION
 The present inventors have conducted screening of various cultured cell
 lines using, as an indicator, the inhibitory activity on the protease
 activity of hepatocyte growth factor activator and have found that a
 substance having the activity occurs in the culture supernatant of certain
 human cancer cell lines (MKN45 cells, A549 cells and like epithelial tumor
 cell lines). To reveal the nature of its inhibitory activity, they further
 attempted to purify the substance from the MKN45 cell culture supernatant
 using various column chromatography techniques. As a result, they have
 found a novel protein with a molecular weight of about 40,000 daltons as
 determined by SDS (sodium dodecyl sulfate)-polyacrylamide gel
 electrophoresis (PAGE) and they also have obtained an amino-terminal amino
 acid sequence of this protein by analyzing the protein on a protein
 sequencer. Further, they determined partial amino acid sequences by
 decomposing the protein using proteolytic enzymes, isolating the resultant
 peptides and subjecting each peptide to the same amino acid sequence
 analysis as mentioned above. Furthermore, they estimated DNA base
 sequences based on the partial amino acid sequences and conducted
 screening of a cDNA library using oligonucleotide probes prepared based on
 the sequences. As a result, they have succeeded in cloning a gene coding
 for the protein and have now completed the present invention.
 Furthermore, as a result of various investigations to produce the protein
 stably and in large quantities using the recombinant DNA technique and,
 the present inventors have constructed a novel expression vector coding
 for the protein and have enabled expression of the protein. Thus, by
 constructing a plasmid for protein expression by inserting a DNA fragment
 coding for part or the whole of the amino acid sequence of the protein
 into a plasmid vector such as the expression vector pME18S for use in
 animal cells or an expression vector for use in yeasts, Escherichia coli
 and the like, at a site downstream from the promoter thereof and using the
 thus-obtained recombinant plasmid to transform host cells, they have now
 completed the present invention in another aspect.
 The present invention thus relates to a protein having the following
 physico-chemical properties:
 (1) a molecular weight of about 40,000 daltons as determined by
 SDS-polyacrylamide gel electrophoresis;
 (2) inhibitory activity on the protease activity of hepatocyte growth
 factor activator; and
 (3) one of the amino acid sequences depicted in the sequence listing under
 SEQ ID NO:1 through 7 or an amino acid sequence substantially equivalent
 thereto; proteins respectively having the amino acid sequences depicted in
 the sequence listing under SEQ ID NO:l through 7 or amino acid sequences
 substantially equivalent thereto and having inhibitory activity on the
 protease activity of hepatocyte growth factor activator; a protein having
 the amino acid sequence depicted in the sequence listing under SEQ ID
 NO:18 or an amino acid sequence substantially equivalent thereto; and a
 protein having, as its amino acid sequence, that segment of the amino acid
 sequence depicted in the sequence listing under SEQ ID NO:18 which starts
 with the 36th amino acid (glycine) residue and ends with the 513th amino
 acid (leucine) residue, or an amino acid sequence substantially equivalent
 thereto; DNAs and genes coding for the proteins defined above; expression
 vectors respectively containing the DNA or genes; transformants obtained
 by transformation of host cells with the expression vectors; as well as a
 method of producing proteins having inhibitory activity on the protease
 activity of hepatocyte growth factor activator which comprises cultivating
 the transformants.
 The base sequence shown in the sequence listing under SEQ ID NO:8 contains
 only one strand, with the other complementary base sequence being omitted.
 Starting with this gene and using the recombinant DNA technology, it is
 possible to cause expression of, for example, the protein having the amino
 acid sequence shown in the sequence listing under SEQ ID NO:18. On that
 occasion, the protein translated from MRNA coding for the protein contains
 a signal sequence. After extracellular excretion, however, the signal
 sequence has been cleaved off and the protein obtained has an amino acid
 sequence comprising the 36th amino acid (glycine) residue and the
 subsequent amino acid residues of the amino acid sequence shown in the
 sequence listing under SEQ ID NO:18. Signal sequences of other proteins
 may also be used as the signal sequence. For signal sequence-free mature
 protein expression in host cells, a gene having that portion of the base
 sequence shown in the sequence listing under SEQ ID NO:8 which comprises
 the 106th nucleotide (guanine) residue and the subsequent nucleotide
 residues may be used as the gene coding for the relevant protein and
 joined to the ATG codon of a vector. The present invention further
 includes, within the scope thereof, modifications of the proteins or DNAs
 mentioned above as derived therefrom by deletion, substitution and/or
 addition of one or more amino acid or nucleotide residues within limits
 not harmful to the inhibitory activity on the protease activity of HGF
 activator, namely those proteins or DNAs that respectively have
 "substantially equivalent amino acid sequences" or "substantially
 equivalent base sequences".

DETAILED DESCRIPTION OF THE INVENTION
 In the following, the present invention is described in further detail. The
 novel protein of the present invention which has protease inhibitor
 activity can be obtained by proceeding via such purification steps as
 mentioned below. For example, a human cancer cell line (MKN45 cells or
 A549 cells, deposited with the Japanese Cancer Research Resources Bank
 under the deposit numbers JCRB0254 and JCRB0076, respectively, or like
 epithelial tumor cell line) is cultivated in a serum-free medium for
 several days, the culture supernatant is recovered and, after removal of
 cells therefrom and concentration, submitted to a heparin-Sepharose column
 (available e.g. from Pharmacia). The non-adsorbed fraction is submitted to
 a ConA-Sepharose column (available-e.g. from Pharmacia) and separated into
 an adsorbed fraction and an non-adsorbed fraction. The adsorbed fraction
 is subjected to hydrophobic chromatography using Phenyl-5PW (available
 e.g. from Tosoh Corp.). The thus-obtained fraction containing the desired
 protein is chromatographed on a DEAE ion exchange column (available e.g.
 from Polymer Laboratory), then submitted to a hydroxyapatite column
 (available e.g. from Mitsui Toatsu Chemicals or Seikagaku Corp.), and
 further to gel filtration column chromatography (using e.g. Asahi Chemical
 Industry's GS520) to give the protein in question. The purification steps
 may further include reversed phase column chromatography and/or other
 appropriate means, as necessary.
 Upon SDS-polyacrylamide gel electrophoresis, the thus-purified protein of
 the present invention migrates as a smear band or several fragments
 presumably resulting from differences in sugar chain, amino acid residue
 modification and/or C-terminal side mutation and having a molecular weight
 of about 40,000 daltons. When reacted with HGF activator, the protein
 shows inhibitory activity on the protease activity of HGF activator. This
 protein of the present invention contains the amino acid sequence shown in
 Table 1 below.
 A DNA fragment of the gene coding for the novel protein of the present
 invention can be obtained in the following manner. By analyzing the novel
 protein purified in the above manner using a gaseous phase protein
 sequencer (available e.g. from Applied Biosystems), its amino-terminal
 amino acid sequence can be determined. Further, the protein is decomposed
 using lysyl endopeptidase (e.g. Achromobacter protease I), the resulting
 peptide fragments are separated by reversed phase high-performance liquid
 chromatography (using e.g. a YMC's column) and each fragment is subjected
 to amino acid sequence analysis in the same manner as mentioned above,
 whereby the amino acid sequence of an intermediate portion of the protein
 can be revealed.
 A DNA base sequence is deduced from the amino acid sequence thus determined
 and, correspondingly, appropriate oligonucleotides are synthesized and
 used as probes. Human-derived liver, spleen and placenta cDNA libraries
 (available e.g. from Clonetec), among others, can be used as the cDNA
 library for screening out the gene coding for the desired protein. In
 addition, a cDNA library may be constructed in the conventional manner
 from a cell line or tissue material in which the protein is expressed.
 Escherichia coli is transfected with .lambda. phage containing such cDNA
 incorporated therein (the method of Maniatis et al.: "Molecular Cloning")
 and then cultivated. The plaques formed are subjected to selection by
 plaque hybridization using oligonucleotide probes prepared based on the
 base sequence deduced from the amino acid sequence of a portion of the
 protein in question, whereby a certain number of different .lambda. phage
 clones having the amino acid sequence of the desired protein and
 containing, in addition, those segment base sequences of the protein that
 correspond to other regions than the probes can be obtained with ease.
 Then, the phage from each positive plaque obtained in the above screening
 is then allowed to replicate by the method of Maniatis et al., and DNA is
 purified therefrom by the glycerol gradient method and, after appropriate
 restriction enzyme cleavage, submitted to cDNA subcloning into a plasmid
 vector such as pUC18 or pUC19 or a single chain phage vector such as
 M13mp18 or M13mp19. Thereafter, the base sequence of the desired cDNA
 fragment can be determined by the method of Sanger et al. The base
 sequences of the clones obtained are analyzed and synthesized and, as a
 result, a gene totally corresponding to the whole amino acid sequence of
 the desired protein as shown in the sequence listing under SEQ ID NO:18
 can be derived from a group of cDNAs coding for respective portions of the
 protein. It is also possible to obtain a gene containing the whole of the
 cDNA in question, a gene containing the cDNA with deletion of a partial
 base sequence thereof, a gene containing the cDNA with insertion of some
 other base sequence, a gene containing the cDNA with substitution of some
 other base sequence for a partial base sequence of the cDNA, or the like
 gene from a variety of cDNA libraries by the PCR method using portions of
 the cDNA in question as probes. Such site-specific mutation, inclusive of
 base sequence deletion, addition or substitution, can be readily realized
 by the methods described in the literature (e.g. Methods in Enzymol., 217,
 218 (1993); ibid., 217, 270 (1993)).
 The group of cDNAs obtained in the above manner are joined together so that
 the order of the base sequences is fit to the amino acid sequence of the
 protein, to give a DNA fragment covering the whole region of the protein.
 The DNA fragment is inserted into a plasmid, such as pCDL-SR.alpha.296, at
 a site downstream from the promoter thereof and matched in phase with the
 translation initiation codon ATG, to thereby construct a protein
 expression vector. Then, the protein can be expressed in a host, for
 example animal cells, transformed with the plasmid. Thereafter, the
 protein expressed can be recovered by purification by a conventional
 method.
 Thus, each of the thus-obtained cDNAs is inserted into a plasmid, such as
 pME18S, at a site downstream from the promoter thereof to thereby
 construct a plasmid for protein expression. The protein or a protein
 derived therefrom by partial amino acid sequence deletion, insertion or
 substitution can be expressed in a host, such as animal cells, transformed
 with the expression plasmid. More concretely, CHO cells, COS cells, mouse
 L cells, mouse C127 cells, mouse FM3A cells and the like can be used as
 the animal cells for protein expression. When these animal cells are used
 as the host, the use, as a signal sequence, of that portion of the DNA
 base sequence shown in the sequence listing under SEQ ID NO:8, namely the
 gene for the protein, which starts with the 1st nucleotide and ends with
 the 35th nucleotide, or the use of an existing signal sequence is expected
 to be conducive to extracellular secretory production of the protein or
 production thereof on the cell membrane.
 The expression plasmid for use in animal cell hosts is constructed in the
 following manner. As the promoter, use can be made of any of the existing
 promoters, for example the SR.alpha. promoter, SV40 promoter or
 metallothionein gene promoter. A DNA containing the whole gene for the
 protein, inclusive of the above-mentioned signal-like sequence, a DNA
 containing the gene with a partial base sequence deletion, a DNA
 containing the gene with insertion of a base sequence or a DNA containing
 the gene with substitution of some other sequence for a partial base
 sequence thereof is inserted into a site downstream from the promoter in
 the direction of transcription. In constructing the expression plasmid for
 the protein, two or three pieces of the DNA fragment of the gene coding
 for the protein may be joined together and used for insertion downstream
 from the promoter. It is also possible to join such a promoter as the SV40
 promoter to the 5' upstream side of the DNA fragment of the gene coding
 for the protein to give a unit insert and insert, into a vector, two or
 three such units joined together in the same direction of transcription. A
 polyadenylation site is added to the downstream side of the gene coding
 for the protein. For example, the polyadenylation site derived from the
 SV40 DNA, .beta.-globin gene or metallothionein gene can be joined to the
 downstream side of the gene coding for the protein. When a DNA fragment
 comprising a promoter and the gene coding for the protein as joined
 together is duplicated or triplicated, each unit may contain a
 polyadenylation site on the 3' side of the gene coding for the protein. In
 transforming animal cells, for example CHO cells, with such expression
 vector, a drug resistance gene can be used for the purpose of expression
 cell selection. As the drug resistance gene, there may be mentioned the
 DHFR gene which provides resistance to methotrexate (J. Mol. Biol., 159,
 601 (1982)), the Neo gene which provides resistance to the antibiotic
 G-418 (J. Mol. Appl. Genet., 1, 327 (1982)), the Escherichia coli-derived
 Ecogpt gene which provides resistance to mycophenolic acid (Proc. Natl.
 Acad. Sci. U.S.A., 78, 2072 (1981)) and the hph gene which provides
 resistance to the antibiotic hygromycin (Mol. Cell. Biol., 5, 410 (1985)),
 among others. Each resistance gene contains a promoter, such as the
 above-mentioned SV40-derived promoter, inserted on the 5' upstream side
 and a polyadenylation site as mentioned above on the 3' downstream side of
 each resistance gene. In inserting such resistance gene into the
 expression vector for the protein, the gene may be inserted at a site
 downstream from the polyadenylation site of the gene coding for the
 protein, in either direction, the same or opposite. These expression
 vectors make it unnecessary to perform double transformation with another
 plasmid containing a selective marker gene for the purpose of transformant
 isolation. When the expression vector for the protein does not contain
 such a selective marker gene insert, a vector having a marker suited for
 transformant selection, for example pSV2neo (J. Mol. Appl. Genet., 1, 327
 (1982)), pMBG (Nature, 294, 228 (1981)), pSV2gpt (Proc. Natl. Acad. Sci.
 U.S.A., 78, 2072 (1981)) or pAd.cndot.D26.cndot.1 (J. Mol. Biol., 159, 601
 (1982)), may be used, in combination with the expression vector for the
 gene coding for the protein, for transformation to thereby make it easy to
 perform transformant selection based on phenotypic expression of the drug
 resistance gene.
 The expression vector can be introduced into animal cells by the calcium
 phosphate method (Virology, 52, 456 (1973)) or the electroporation method
 (J. Membr. Biol., 10, 279 (1972)), for instance. The transformed animal
 cells can be cultivated in the conventional manner in the manner of
 suspension culture or adhesion culture. They are cultivated in a medium
 such as MEM or RPMI 1640 in the presence of 5 to 10% of serum or in the
 presence of an appropriate amount of insulin, transferrin or the like, or
 under serum-free conditions. Further, it is also possible to produce the
 protein using microorganisms such as yeasts or Escherichia coli, for
 example strains of Saccharomyces cerevisiae or the strain Escherichia coli
 YA-21. Since the cells express the protein in the culture supernatant or
 on the cell surface, it is possible to recover and purify the protein
 using the culture supernatant or cells of this transformant. More
 specifically, the protein can be isolated and purified by subjecting the
 culture supernatant or cell extract containing the protein produced to an
 appropriate chromatography procedure, for example chromatographic
 treatment using heparin-Sepharose, ConA-Sepharose, hydroxyapatite and the
 like in combination.
 The protease inhibitor activity-endowed protein of the present invention
 has inhibitory activity on the protease activity of HGF activator and,
 therefore, is useful as a in vitro or in vivo regulatory factor for HGF
 activator or, indirectly, as a HGF activity regulating factor. The protein
 as well as an antibody to the protein or a gene coding for the protein is
 further useful as a tool or means for function analysis of the factors.
 Furthermore, by introducing an expression vector carrying a gene coding for
 the protein into animal cells, it becomes possible to produce part or the
 whole of the protein or a protein equivalent thereto, which is
 biologically active, in a stable manner and in large quantities. This has
 so far been difficult to attain.
 The present invention is now illustrated in greater detail with reference
 to the following Examples. However, it is not intended that the present
 invention be limited to these Examples.
 EXAMPLE 1
 (Purification of the Protein Using an MKN45 Cell Culture Supernatant)
 MKN45 cells (Naito et al., Gan to Kagaku-ryoho (Cancer and Chemotherapy),
 5, 89 (1978)) (obtained from Meneki Seibutsu Kenkyusho) were seeded into
 eRDF medium containing 5% FBS (fetal bovine serum) as placed in a roller
 bottle 850 and allowed to multiplicate until a confluent state was
 attained. Then, the FBS-containing culture supernatants were removed and
 the cells were washed with two portions of serum-free eRDF medium. After
 removing the washing medium, 500 ml of serum-free eRDF medium was added
 and incubation was carried out at 37.degree. C. for 3 to 6 days. After
 incubation, the culture supernatants were recovered, 500 ml of fresh
 serum-free eRDF medium was added, and incubation was again conducted. This
 procedure was repeated several times. The culture supernatants thus
 recovered were combined and concentrated about 20-fold using a YM30
 ultrafiltration membrane (Amicon).
 This concentrate was submitted to a heparin-Sepharose column (equilibrated
 with PBS) and the non-adsorbed fraction was recovered. This fraction was
 submitted to a ConA-Sepharose column (equilibrated with PBS) and separated
 into the non-adsorbed fraction and an adsorbed fraction eluted with a PBS
 solution containing 200 mM .alpha.-methyl-D-mannoside. The ConA adsorbed
 fraction was concentrated using YM30, followed by buffer substitution to
 10 mM phosphate buffer (pH 6.8) containing 1 M ammonium sulfate. The new
 solution was subjected to HPLC using Phenyl-5PW (Tosoh Corp.; equilibrated
 with 10 mM phosphate buffer (pH 6.8) containing 1 M ammonium sulfate),
 followed by linear concentration gradient elution with 1 M ammonium
 sulfate to 0 M ammonium sulfate. A fraction containing the desired
 protease inhibitor activity was thus recovered.
 The fraction was dialyzed against 20 mM Tris-hydrochloride buffer (pH 8)
 containing 0.05% CHAPS and then subjected to HPLC using DEAE (equilibrated
 with 20 mM Tris-hydrochloride buffer (pH 8) containing 0.05% CHAPS),
 followed by linear concentration gradient elution with 0 M to 500 mM NaCl,
 whereby a fraction showing the desired protease inhibitor activity was
 recovered. The fraction was dialyzed against 5 mM phosphate buffer (pH
 6.8) containing 0.05% CHAPS and then subjected to HPLC using a HCA A-4007
 column (product of Mitsui Toatsu Chemicals) (equilibrated with 5 mM
 phosphate buffer (pH 6.8) containing 0.05% CHAPS), and the non-adsorbed
 fraction was recovered. The fraction was submitted to GS-520 (equilibrated
 with PBS containing 0.05% CHAPS) and an active fraction (fraction of about
 50 to 30 kDa) was recovered. For eliminating minor bands, the fraction was
 applied to a YMC pack C4 column (obtained from YMC), linear concentration
 gradient elution was carried out over 30 minutes using
 acetonitrile-isopropyl alcohol (3/7) containing 0.1% TFA and varying the
 concentration thereof from 10% to 50%, and the active fraction was
 neutralized with 1 M Tris-hydrochloride buffer (pH 8) and then dried under
 reduced pressure. After drying, the solid obtained was dissolved in PBS
 containing 0.05% CHAPS to give a purified protein solution.
 EXAMPLE 2
 (Amino-terminal Amino Acid Sequence and Partial Amino Acid Sequence
 Determination of the Protein)
 The protein having protease inhibitor activity as purified as in Example 1
 and eluted by reversed phase HPLC was dried under reduced pressure without
 neutralization. This was dissolved in 60 .mu.l of 50% TFA (trifluoroacetic
 acid), added to a polybrene-treated glass filter and subjected to Edman
 degradation on an Applied Biosystems model 470A sequencer, and the amino
 acid sequence of an N-terminal region was determined. Phenylhydantoin
 (PTH)-amino acids were identified using a Mitsubishi Chemical's MCI gel
 ODS IHU column (0.46.times.15 cm) and conducting single solvent elution
 with acetate buffer (10 mM acetate buffer (pH 4.7), 0.01% SDS, 38%
 acetonitrile) at a flow rate of 1.2 ml/minute and a temperature of
 43.degree. C. PTH-amino acids were detected based on the absorbance at 269
 nm.
 As a result, the N-terminal amino acid sequence shown below in Table 1 was
 identified.
 Then, the same protein having protease inhibitor activity as purified as in
 Example 1 and eluted by reversed phase HPLC was dissolved in 100 .mu.l of
 50 mM Tris-hydrochloride buffer (pH 9.0) containing 4 M urea, lysyl
 endopeptidase (Achromobacter protease I) was added to the solution, and
 the reaction was carried out at 37.degree. C. for 8 hours. The resulting
 peptide mixture was separated by reversed phase HPLC using a YMC pack C8
 column (YMC) to give respective peptide fragments. Six peptides were
 subjected to amino acid analysis using a gaseous phase sequencer (Applied
 Biosystems model 1470A). The sequences shown in Table 1 were found.
 Table 1
 Amino Acid Sequences of Peptides
 N-terminal:
 Gly-Pro-Pro-Pro-Ala-Pro-Pro-Gly-Leu-Pro-Ala-Gly-Ala-Asp-Cys-Leu-Asn-Ser-Ph
 e-Thr-Ala-Gly-Val-Pro-Gly-Phe-Val-Leu-Asp-Thr-Xaa-Ala-Ser-Val-Ser-Asn-Gly-A
 la-Thr-Phe (SEQ ID NO:1 in the sequence listing)
 Partial Amino Acid Sequences
 1: Val-Gln-Pro-Gln-Glu-Pro-Leu-Val-Leu-Lys (SEQ ID NO:2 in the sequence
 listing)
 2:
 Asp-Val-Glu-Asn-Thr-Asp-Trp-Arg-Leu-Leu-Arg-Gly-Asp-Thr-Asp-Val-Arg-Val-Gl
 u-Arg-Lys (SEQ ID NO: 3 in the sequence listing)
 3: Ala-Trp-Ala-Gly-Ile-Asp-Leu-Lys (SEQ ID NO:4 in the sequence listing)
 4: Ser-Xaa-Val-Tyr-Gly-Gly-Xaa-Leu-Gly-Asn-Lys (SEQ ID NO:5 in the sequence
 listing)
 5: Asp-Pro-Asn-Gln-Val-Glu-Leu-Trp-Gly-Leu-Lys (SEQ ID NO:6 in the sequence
 listing)
 6: Asn-Asn-Tyr-Leu-Arg-Xaa-Xaa-Xaa-Xaa-Ile-Leu-Ala-Xaa-Arg-Gly-Val-Gln (SEQ
 ID NO:7 in the sequence listing)
 (Xaa: amino acid residue not yet identified)
 EXAMPLE 3
 (Purification of the Protein Using an A549 Cell Culture Supernatant and
 Amino Acid Sequence Analysis)
 A culture supernatant was prepared by cultivating A549 cells (obtained from
 the Japanese Cancer Research Resources Bank) in the same manner as in
 Example 1. Using the culture supernatant and proceeding in the same manner
 as in Example 1, a protein having the inhibitory activity on the protease
 activity of HGF activator was obtained. Upon SDS-PAGE, this protein showed
 the same molecular weight as that derived from MKN45 cells. When subjected
 to the same N-terminal amino acid sequence determination as in Example 1,
 this protein gave the same sequence as that of the MKN45 cell-derived
 protein. This suggested the possibility of the protein being identical
 with the MKN45-derived protein.
 EXAMPLE 4
 (Method of Assaying the Activity of the Protein Inhibiting the Protease
 Activity of HGF Activator as Well as the Activity)
 One to ten .mu.l of the sample to be assayed was added to 30 to 40 .mu.l of
 PBS-0.05% CHAPS solution containing 2 to 5 ng of serum-derived HGF
 activator. After 30 minutes of incubation at 37.degree. C., 5 to 10 .mu.g
 of single chain HGF was added and incubation was further continued for 2
 hours. This incubation mixture was subjected to SDS-polyacrylamide gel
 electrophoresis under reducing conditions. After electrophoresis,
 Coomassie Brilliant Blue R250 (CBB) staining was performed and the
 proportions of single chain HGF and double chain HGF were compared for
 activity detection.
 The purified protein (10 ng) and 5 ng of serum-derived HGF activator were
 incubated in 30 to 40 .mu.l of PBS-0.05% CHAPS solution at 37.degree. C.
 for 30 minutes, then 10 .mu.g of single chain HGF was added, and
 incubation was further continued for 2 hours. The incubation mixture was
 subjected to SDS-polyacrylamide gel electrophoresis under reducing
 conditions followed by staining with CBB. The results are shown in FIG. 1.
 In the figure, the numeral 1 indicates the case where neither of HGF
 activator and the protein was added, 2 indicates the case where HGF
 activator was added but the protein was not added, and 3 indicates the
 case where HGF activator and the protein were added. Addition of the
 protein resulted in suppression of the activity of HGF activator
 converting single chain HGF to double chain HGF.
 EXAMPLE 5
 (SDS-polyacrylamide Gel Electrophoresis)
 For determining the apparent molecular weight of the protein having
 protease inhibitor activity as purified from the MKN45 cell culture
 supernatant or A549 cell culture supernatant in Example 1 or Example 2,
 the protein was subjected to SDS-polyacrylamide gel electrophoresis. The
 protein finally purified was subjected to SDS-polyacrylamide gel
 electrophoresis using 12.5% polyacrylamide slab gels, which was conducted
 under nonreducing conditions. The molecular weight markers used were
 Molecular weight markers "Daiichi" III for Laemmli method (Daiichi Pure
 Chemicals). After electrophoresis, color development was performed using a
 silver stain reagent (Kanto Chemical). Upon relative comparison in
 migration distance between the protein and the molecular weight markers,
 the protein obtained from the MKN45 cell culture supernatant or A549 cell
 culture supernatant showed several fragments or a smear band, presumably
 due to differences in sugar chain, amino acid residue modification or
 terminal region, at positions around an apparent molecular weight of about
 40,000 daltons as determined by SDS-polyacrylamide gel electrophoresis.
 EXAMPLE 6
 (Cloning of a Gene Coding for the Protein and Base Sequence Determination)
 Two oligonucleotide primers (primer 1 and primer 2) were prepared which
 were estimable from the sequences Gly-Ala-Asp-Cys-Leu-Asn and
 Gly-Phe-Val-Leu-Asp-Thr contained in the N-terminal amino acid sequence
 (SEQ ID NO:1 in the sequence listing) of the protein obtained in Example
 2. Two further oligonucleotide primers (primer 3 and primer 4) were
 synthesized which were estimable from the sequences
 Ser-Phe-Val-Tyr-Gly-Gly (SEQ ID NO:5) and Gln-Val-Glu-Leu-Trp-Gly (SEQ ID
 NO:6) in the partial amino acid sequence (Sequence listing 1) of the
 protein.
 The sequences of these primers are shown below:
 Primer 1: mixture of 5'-GGNGCNGAYTGYTTRAA-3' (SEQ ID NO:9 in the sequence
 listing) and 5'-GGNGCNGAYTGYCTNAA-3' (SEQ ID NO:10 in the sequence
 listing);
 Primer 2: mixture of 5'-GTRTCYAANACRAANCC-3' (SEQ ID NO:11 in the sequence
 listing) and 5'-GTRTCNAGNACRAANCC-31 (SEQ ID NO:12 in the sequence
 listing);
 Primer 3: mixture of 5'-CCNCCRTANACRAANGA-3' (SEQ ID NO:13 in the sequence
 listing) and 5'-CCNCCRTANACRAARCT-3' (SEQ ID NO:14 in the sequence
 listing);
 Primer 4: mixture of 5'-CCCCANAGYTCNACYTG-3' (SEQ ID NO:15 in the sequence
 listing) and 5'-CCCCAYAAYTCNACYTG-3' (SEQ ID NO:17 in the sequence
 listing).
 (In the above sequences, N indicates A, G, C or T, Y indicates C or T, and
 R indicates A or G.)
 Separately, total RNA was prepared from MKN45 cells (a stomach cancer cell
 line) by the method described in Anal. Biochem., 162, 156 (1987) and
 applied to an oligo(dT) column, whereby poly(A)+RNA was prepared.
 Then, an attempt was made to obtain a cDNA fragment corresponding to the
 N-terminal amino acid sequence of the protein. Using, as the template, the
 poly(A)+RNA prepared from MKN45 cells and using the two oligonucleotide
 primers (primer 1 and primer 2) prepared as mentioned above, RT-PCR
 (reverse transcription-polymerase chain reaction; cf. K. Hayashi (ed.):
 "PCR Ho no Saishin Gijutu (State-of-art Techniques of PCR)", page 44 and
 page 52, published Feb. 5, 1995 by Yohdosha) was carried out. The reaction
 mixture obtained by this RT-PCR was analyzed by polyacrylamide gel
 electrophoresis, whereupon a DNA fragment of about 56 bp was detected.
 Therefore, this DNA fragment was extracted from the polyacrylamide gel,
 followed by phenol-chloroform extraction and ethanol precipitation,
 whereby the DNA fragment was recovered. The base sequence of the DNA
 fragment was determined by the dideoxy method.
 Then, a primer comprising part of this base sequence, 5'-AACAGCTTTACCG-3'
 (primer 5) (SEQ ID NO:16 in the sequence listing), was synthesized and
 used for obtaining the cDNA, as follows.
 PCR was carried out using the poly(A)+RNA (as template) prepared previously
 from MKN45 cells and the primer 3 and primer 5. Further, using the
 thus-obtained PCR-amplified DNA, the primer 4 and the primer 5, PCR was
 conducted. As a result, a 480 bp DNA fragment specific to the protein was
 obtained. The thus-obtained 480 bp DNA fragment was labeled with .sup.32 P
 by the method described in "Molecular Cloning" (Cold Spring Harbor
 Laboratory, 1982) and used as a screening probe.
 A human placenta cDNA library (Clonetec) was used as a library for
 obtaining the full-length cDNA for the protein. First, Escherichia coli
 Y-1090 was infected with a phage prepared in the form of a human placenta
 cDNA library (.lambda.gt11, Clonetec) to give about 4.times.10.sup.5
 plaques and incubation in NZY medium was performed overnight at 42.degree.
 C. Then, the plaques were transferred to a Gene Screening Plus membrane
 (du Pont). The membrane was placed on a filter paper impregnated with 0.1
 M sodium hydroxide-0.5 M Tris hydrochloride buffer (pH 7.5) and allowed to
 stand for 2 minutes and then placed on a filter paper impregnated with 1.5
 M sodium chloride-0.5 M Tris hydrochloride buffer (pH 7.5) and allowed to
 stand for 5 minutes. After two more repetitions of this series of
 treatments, the membrane was washed with 2.times.SSC (two-fold
 concentrated SSC) and air-dried on a dry filter paper. This membrane was
 irradiated with UV light at a dose of 20 mJ/cm.sup.2 for fixation of the
 DNAs transferred to the membrane. The thus-treated membrane was immersed
 in 50 ml of a solution comprising 50 mM Tris hydrochloride buffer (pH
 7.5), 1 M sodium chloride and 1% SDS and maintained in that state at
 65.degree. C. for 2 hours.
 Then, the membrane was immersed in 40 ml of a solution comprising 5 ng/ml
 of the above-mentioned .sup.32 P-labeled probe, 100 .mu.g/ml of salmon
 sperm, 50 mM Tris hydrochloride buffer (pH 7.5), 1 M sodium chloride and
 1% SDS and maintained in that state at 65.degree. C. for 16 hours.
 Thereafter, this membrane was washed with 2.times.SSC at room temperature
 over 5 minutes and then with two portions of 0.1.times.SSC at room
 temperature over 30 minutes, and subjected to autoradiography, which gave
 84 positive clones supposedly containing cDNA for the protein.
 The phage was extracted from each clone with 500 .mu.l of SM buffer (50 mM
 Tris hydrochloride buffer (pH 7.5), 100 mM sodium chloride, 10 mM
 magnesium sulfate and 0.01% gelatin) and 20 .mu.l of chloroform. A
 100-.mu.l portion of the phage extract and Escherichia coli Y-1090 cells
 suspended in 100 .mu.l of 10 mM magnesium sulfate were mixed together, and
 the mixture was sown, together with 3 ml of top agar medium, onto LB agar
 medium (9 cm) and incubated overnight at 37.degree. C. After confirmation
 of plaque formation, 3 ml of SM buffer and several drops of chloroform
 were added and the whole was allowed to stand at room temperature for 1
 hour. This phage-containing SM buffer was recovered and centrifuged at
 8,000 rpm for 10 minutes, and the supernatant was recovered. A 10-.mu.l
 portion of the thus-obtained phage solution and 300 .mu.l of an
 Escherichia coli Y-1090 cell suspension were mixed together, 10 mL of LB
 medium containing 10 mM magnesium sulfate was added, and shake culture was
 conducted at 37.degree. C. After bacteriolysis, several drops of
 chloroform was added, and the mixture was further shaken for 10 minutes
 and centrifuged at 10,000 rpm for 5 minutes. The supernatant was
 recovered, DNase I (final concentration 5 .mu.g/ml) and RNase (final
 concentration 2 .mu.g/ml) were added, and the mixture was allowed to stand
 at 37.degree. C. for 30 minutes. Then, 5 g of sodium chloride and 1.1 g of
 PEG 6000 were added, the mixture was allowed to stand at 4.degree. C. for
 1 hour and then centrifuged at 10,000 rpm for 15 minutes, and the
 precipitate was recovered. This precipitate was suspended in 400 .mu.l of
 SM buffer. The suspension was further subjected to phenol-chloroform
 treatment and ethanol precipitation, and a phage DNA containing cDNA for
 the protein was recovered. This DNA was cleaved with the restriction
 enzyme EcoRI and the cDNA fragments were analyzed by agarose gel
 electrophoresis. The longest cDNA fragment band in this agarose gel
 electrophoresis was separated and extracted, and the cDNA fragment was
 inserted into the plasmid vector pUC19 at its EcoRI site. A plasmid
 vector, pHAI, containing cDNA for the protein was thus constructed. The
 base sequence of the cDNA insert in the thus-obtained plasmid vector pHAI
 was analyzed and the base sequence of the whole gene coding for the
 protein was determined (SEQ ID NO:8 in the sequence listing).
 EXAMPLE 7
 (Preparation of an Expression Plasmid for the Protein)
 By treating the plasmid (pHAI) containing cDNA for the protein as obtained
 in Example 6 with the restriction enzyme EcoRI, it is possible to separate
 the full-length cDNA fragment coding for the protein (full-length cDNA
 fragment for the protein, including the translation initiation codon base
 sequence ATG and the termination codon TGA) from the plasmid vector pUC19.
 Thus, agarose gel electrophoresis was carried out by the method of
 Maniatis et al. ("Molecular Cloning", Cold Spring Harbor Laboratory, page
 164 (1982)), and an about 2.4 kb EcoRI-EcoRI DNA fragment containing the
 cDNA for the protein was separated and extracted. The thus-obtained DNA
 fragment was rendered blunt-ended by the conventional method using T4 DNA
 polymerase and, then, the DNA fragment was purified by phenol-chloroform
 extraction and ethanol precipitation and dissolved in 10 .mu.l of water.
 Separately, 0.05 .mu.g of the expression vector pME18S (Medical
 Immunology, 20, 27 (1990)) was cleaved in advance with the restriction
 enzyme XhoI, and the DNA fragment obtained was rendered blunt-ended by the
 conventional method using T4 DNA polymerase and then purified by
 phenol-chloroform extraction and ethanol precipitation. This was dissolved
 in 400 .mu.l of a 1 MM MgCl.sub.2 solution in 50 mM Tris-HCl (pH 8), 1
 unit of bacterial alkaline phosphatase (Toyobo, BAP-101) was added, and
 dephosphorylation treatment was conducted at 65.degree. C. for 30 minutes.
 Then, the DNA fragment was purified from this reaction mixture by
 phenol-chloroform extraction and ethanol precipitation and dissolved in 10
 .mu.l of water. Ligation reaction was carried out in 20 .mu.l of a
 reaction mixture (66 mM Tris-HCl, pH 7.6, 6.6 mM MgCl.sub.2, 10 mM
 dithiothreitol, 66 AM ATP) containing 0.01 .mu.g of the pME18S
 vector-derived DNA fragment prepared as mentioned above and 0.1 .mu.g of
 the above-mentioned blunt-ended EcoRI fragment of cDNA for the protein in
 the presence of T4 DNA ligase (Toyobo LGA-101) at 14.degree. C. for 12
 hours. A 10-.mu.l portion of this T4 DNA ligase reaction mixture was used
 to transform Escherichia coli HB101 (Takara Shuzo) according to the manual
 attached thereto. The microorganism was cultured on a medium containing 50
 .mu.g/ml of ampicillin and scores of ampicillin-resistant strains were
 obtained. These transformants were analyzed by the method of Maniatis et
 al. ("Molecular Cloning", Cold Spring Harbor Laboratory, pages 86-96
 (1982)) and, as a result, a plasmid, pME18S-HAI, containing the gene
 coding for the protein as inserted-at the XhoI restriction enzyme cleavage
 site occurring between the promoter and polyadenylation site of the
 expression vector pME18S could be obtained. Its structure is shown in FIG.
 2.
 EXAMPLE 8
 (Obtaining of a Cell Line Expressing the Protein)
 The plasmid pME18S-HAI constructed in Example 7 and containing the cDNA for
 the protein as inserted at the XhoI restriction enzyme cleavage site of
 the expression vector pME18S was recovered and purified from the
 recombinant Escherichia coli by the method of Maniatis et al. ("Molecular
 Cloning", Cold Spring Harbor Laboratory, pages 86-96 (1982)) and thus a
 large amount of the expression plasmid DNA for the protein was obtained.
 COS cells were transformed by transfection thereof with the expression
 plasmid DNA. Thus, COS cells were first cultured in eRDF medium containing
 10% FBS (fetal bovine serum) in tissue culture dishes 9 cm in diameter
 until a semiconfluent condition. Then, the medium was removed from the
 dishes, and a DNA solution prepared as mentioned below was added dropwise
 thereto as mentioned below. First, for each dish 9 cm in diameter, a
 solution was prepared in an Eppendorf centrifuge tube by adding thereto
 300 .mu.l of 2.times.HEBS solution (2.times.HEBS solution: 1.6% sodium
 chloride, 0.074% potassium chloride, 0.05% disodium hydrogen phosphate
 dodecahydrate, 0.2% dextrose, 1% HEPES (pH 7.05)) and 10 .mu.g of the
 plasmid DNA and making the volume 570 .mu.l with sterilized water. Then,
 while adding 30 .mu.l of 2.5 M calcium chloride solution to the DNA
 solution, the tube contents were stirred vigorously using a vortex mixer
 for several seconds. The resulting mixture was allowed to stand at room
 temperature for 30 minutes, with occasional stirring at intervals of about
 10 minutes using a vortex mixer. The thus-prepared DNA solution was laid
 on the cells mentioned above and the whole was allowed to stand at room
 temperature for 30 minutes. Then, 9 ml of eRDF medium (Kyokuto
 Pharmaceutical) supplemented with 10% FBS was added to each dish and
 incubation was performed at 37.degree. C. for 4 to 5 hours in the presence
 of 5% CO.sub.2. Then, the medium was removed from each dish and the cells
 were washed with 5 ml of 1.times.TBS++ solution (1.times.TBS++ solution:
 25 mM Tris-hydrochloride (pH 7.5), 140 mM sodium chloride, 5 mM potassium
 chloride, 0.6 mM disodium hydrogen phosphate, 0.08 mM calcium chloride,
 0.08 mM magnesium chloride). After removing the 1.times.TBS++ solution,
 the cells were covered with 5 ml/dish of 1.times.HEBS solution containing
 10% DMSO (dimethyl sulfoxide) and allowed to stand at room temperature for
 1 to 2 minutes. The supernatant was then removed. The cells were again
 washed with 5 ml of 1.times.TBS++ solution, 10 ml of eRDF medium
 supplemented with 10% FBS was added to each dish and incubation was
 performed at 37.degree. C. in the presence of 5% CO.sub.2. After the lapse
 of 48 hours, the medium was recovered. The supernatant recovered was
 20-fold concentrated and assayed for inhibitory activity against HGF
 activator in the same manner as in Example 4. The inhibitory activity was
 confirmed.
 While the invention has been described in detail and with reference to
 specific embodiments thereof, it will be apparent to one skilled in the
 art that various changes and modifications can be made therein without
 departing from the spirit and scope thereof.