JP3232714B2 - Novel proteins and genes encoding them - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel protein, a gene encoding the same, a transformant containing the gene, and a method for producing a protein using the same. More specifically, the present invention relates to hepatocyte growth factor (HGF). ) Is cleaved at a specific position to convert an inactive single-chain form into an active double-chain form, a novel protein having protease activity, a gene encoding the same, a transformant containing the gene, and The present invention relates to a method for producing a protein using the same.

[0002]

2. Description of the Related Art It has been confirmed that the biological activity of human HGF is not observed in a single-chain form in an in vitro experiment, but is expressed only in a double-chain form. Have been. It has also been confirmed that when human HGF is produced using a genetic recombination technique, most of the human HGF produced under serum-free culture is single-stranded. Since the presence or absence of serum during culture has a significant effect on production costs,
A method for producing HGF in serum-free culture has been desired.

[0003] Some of the present inventors have previously found a protease that converts single-chain HGF to double-chain form in the serum of mammals, and this protease is subjected to SDS-polyacrylamide gel electrophoresis. Has a molecular weight of about 34,000
It was found to be a 0 dalton protein (Japanese Patent Application No. 3-271362). However, since the protein is present in only a trace amount in serum, there has been a problem that a great deal of labor is required to obtain the protein by purification.

[0004]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies for the purpose of easily preparing a protein having the same activity as the protein, and as a result, obtained a gene encoding the protein for the first time. As a result, they have found that the protein can be mass-produced using genetic engineering techniques, and have completed the present invention.

That is, the gist of the present invention is to provide a protein characterized by the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, a gene encoding the same, a vector expressing the polypeptide encoded by the gene, Transformants and a method for producing the protein using them.
Hereinafter, the present invention will be described in detail.

New protein in [0006] The present invention is a protein comprising the amino acid sequence shown in SEQ ID NO: 1, within a range not to impair the protease activity to convert single chain HGF into two-chain type HGF, a part The present invention also includes those obtained by performing modifications such as removal, substitution, modification, or addition of amino acids. Examples of the gene encoding the protein include a gene containing a nucleic acid sequence represented by the nucleotide sequence of SEQ ID NO: 2 in the sequence listing as a partial sequence, and a gene having the nucleotide sequence of SEQ ID NO: 3 in the sequence listing. And the like represented.

The DNA fragment of such a gene can be obtained, for example, as follows. As the cDNA library encoding the protein of the present invention, a library prepared from a commercially available human liver can be used. From this cDNA library, a phagemid was prepared by the method of Saito et al. (Proceedings of National Academy Sciences USA [Proc. Natl. Aca.
d. Sci. USA], 83 , 8664-8668 (1
986)) and culture. Colonies formed after the cultivation were subjected to colony hybridization ("Molecular Cloning", Cold Spring Harbor Laboratory, 320-32) using a partial DNA fragment or a DNA fragment having a base sequence corresponding to the partial amino acid sequence of the protein of the present invention as a probe.
8 (1982)) to obtain the desired DNA fragment.

[0008] Probes used in this colony hybridization method include a polymerase chain reaction (hereinafter abbreviated as "PCR") method (Science [Science]).
239 ], 487-492 (1988)). Specifically, a DNA fragment described in SEQ ID NO: 4 in the sequence listing (corresponding to a part of the amino acid sequence described in SEQ ID NO: 6 in the sequence listing) was prepared by using a + strand DNA primer, and SEQ ID NO: 5 in the sequence listing (SEQ ID NO: 5). PCR is performed using the DNA fragment described in SEQ ID NO: 7 as a negative strand DNA primer, and a DNA fragment described in SEQ ID NO: 2 in the obtained sequence listing is prepared and used as a probe. . Oligonucleotides synthesized based on the DNA sequence deduced from the amino acid sequence of the protein of the present invention can also be used as probes.

Further, from the above-mentioned colonies positive for screening, T. DNA is prepared by the method of Maniatis et al. ("Molecular Cloning", Cold Spring Harbor Laboratory, 85 (1982)), and cut with an appropriate restriction enzyme such as Bam HI.
Cloned into a plasmid such as pUC18,
et al., dideoxy method (Proceedings of National Academy Sciences USA [Proc. Natl. Acad. Sci. USA],
74 , 5463 (1977))
The nucleotide sequence of fragment A can be determined.

The nucleotide sequence of the DNA fragment determined in this manner (for example, a fragment containing the nucleotide sequence of SEQ ID NO: 2 in the sequence listing as a partial sequence or represented by the nucleotide sequence of SEQ ID NO: 3 in the sequencing listing) ) Encodes the protein of the present invention shown in SEQ ID NO: 1 in the sequence listing. The DNA fragment of the present invention may be modified by removing, substituting, modifying or adding some bases within a range that does not impair the protease activity of converting single-stranded HGF to double-stranded HGF. Also included.

The DNA fragment obtained as described above is
The 5 ′ end is modified and inserted downstream of the promoter of the expression vector according to a conventional method, and E. coli, Bacillus subtilis, yeast,
It is introduced into cells such as animal cell hosts. Next, the method for producing the protein of the present invention will be described in detail. As the expression vector, those containing a promoter at a position where the DNA encoding the protein of the present invention obtained as described above can be transcribed are suitably used. For example, E. coli,
When a microorganism such as Bacillus subtilis is used as a host, the expression vector preferably comprises a promoter, a ribosome binding (SD) sequence, the protein gene, a transcription termination sequence, and a gene that controls the promoter.

Examples of the promoter include those derived from Escherichia coli and phage, such as tryptophan synthase (tr
p), lactose operon (lac), lambda phage _{P L, P R, P 25} , P 26 promoter, and the like is the promoter of the early gene of T ₅ phage. Also,
These include, for example, the pac promoter (Agricultural and Biological chemistry [Agri
c. Biol. Chem. ], 52 , 983-988
(1988)).

The ribosome binding sequence may be derived from Escherichia coli, phage or the like.
It may be a synthetic DNA sequence having a consensus sequence having a sequence complementary to the 3 'terminal region of A for 4 bases or more. Transcription termination sequences are not required, but may be independent, such as lipoprotein terminators, trp
It is preferable to have an operon terminator or the like.

Further, as for the sequence of the factors necessary for the expression on the expression plasmid, it is desirable to arrange the promoter, the SD sequence, the protein gene, and the transcription termination factor in this order from the 5 ′ upstream side. Further, a method of increasing the copy number of a transcription unit on a vector by inserting a plurality of units of an SD sequence on an expression vector and the protein gene in the same direction (JP-A-1-95798) is used. Can also.

Examples of the expression vector that can be used include pUAI2 (JP-A-1-95798), commercially available pKK233-2 (Pharmacia) and the like. Also, an expression vector pG to be expressed as a fusion protein
EX series (manufactured by Pharmacia) and the like can be used in the same manner. Transformation of the host cell can be performed according to a conventional method.

The transformant can be cultured according to the method described in Molecular Cloning (Cold Spring Harbor Laboratory, 1982). As described above, microorganisms such as Escherichia coli, Bacillus subtilis, and yeast can be used as host cells, but the protein of the present invention contains many cysteine residues, and the position of thiol bond between the cysteine residues and the protein Considering that the higher-order structure of A. may affect the maintenance of activity, animal cells such as CHO cells, COS cells, mouse L cells, mouse C127 cells, mouse FM3
It is desirable to express the protein gene using A cells or the like.

Various animal cell promoters have been reported. For example, SV40 promoter,
A metallothionein gene promoter or the like can be used. A secretion signal and a DNA fragment of the protein gene are inserted downstream of the promoter according to the transcription direction. In this case, the DNA fragment of the gene is
Up to three ligated DNAs may be inserted, or two or three ligated DNA fragments with a promoter bound to the 5 ′ upstream side of the DNA fragment of the gene in the same transcription direction may be inserted. May be.

It is desirable that the protein gene has a polyadenylation site downstream thereof. For example, one polyadenylation site derived from the SV40 DNA, β-globin gene, meterothionein gene or the like is present downstream of the protein gene. Further, a DNA fragment obtained by binding a promoter to the protein gene is 2-3
In the case of using the tandem insertion method, a polyadenylation site can be present on the 3 ′ side of each protein gene. SV40
Inserting a gene, a gene derived from an animal such as rabbit β-globin, a chemically synthesized exon, an intron splicing signal sequence, or the like upstream or downstream of the protein gene is also an effective means for high expression. .

When transforming animal cells, for example, CHO cells, using the above expression vector, it is desirable to use a selection marker. Inserting the selectable marker gene in the forward or reverse direction downstream of the polyadenylation site of the expression vector eliminates the need for double transformation of another plasmid containing the selectable marker gene when obtaining a transformant. . Examples of such a selection marker include a DHFR gene that confers methotrexate resistance (Journal of Molecular Biology [J. Mol. B]
iol. ], 159 , 601 (1982)), Neo gene conferring antibiotic G-418 resistance (Journal of Molecular Applied Genetics [J.
Mol. Appl. Genet. ], 1 , 327 (19
82)), an Ecogpt gene derived from Escherichia coli conferring mycophenolic acid resistance (Proceedings of National Academy Sciences USA [Proc. Natl. Acad. Sci. USA],
78 , 2072 (1981)), the hph gene conferring resistance to the antibiotic hygromycin (Molecular and Cellular Biology [Mol. Cell. Bio].
l. ], 5 , 410 (1985)).

A promoter, for example, the above-mentioned SV40-derived promoter is inserted 5 ′ upstream of each of these resistance genes, and the above-mentioned polyadenylation site is included 3 ′ downstream of each resistance gene. ing. Inv
Expression vector p commercially available from itorogen
cDNA / Neo originally has a neomycin resistance gene as a selection marker, and such a vector may be used.

When the above-mentioned selection marker gene is not inserted into the expression vector, a vector having a selection marker for the transformant, for example, pSV2Neo (Journal of Molecular Applied Genetics [J. Mol. Appl. Genet. ], 1 ,
327 (1982)), pMBG (Nature [Nat
ure], 294 , 228 (1981)), pSV2g
pt (Proceedings of National Academy Science USA [Proc.
tl. Acad. Sci. USA], 78 , 2072
(1981)), pAd-D26-1 (Journal of Molecular Biology [J. Mol. Bio]
l. , 159 , 601 (1982)) together with an expression vector for the protein gene, and transformants can be easily selected according to the phenotype of the selectable marker gene.

In the manner described above, the selected tank
Change the selectable marker for cells containing the protein gene.
Repeated double transformation further increases expression levels.
It is preferable because it can be obtained. Expression vector animals
Transfection into cells is performed by the calcium phosphate method (Virology [V
irology],52, 456 (1973)),
Cutroporation method (Journal of Membrane
 Biology [J. Menbr. Biol. ],1
0, 279 (1972)).

Culture of the transformed animal cells can be performed by suspension culture or adherent culture according to a conventional method. As a medium, a commonly used MEM, RPMI1640 or the like is used, and the cells are cultured in the presence of 5 to 10% serum or an appropriate growth factor, or in the absence of serum. Since the animal cell producing the protein secretes the protein produced in the culture supernatant, the protein can be separated and purified from the culture supernatant of the recombinant. Specifically, the culture supernatant containing the produced protein is purified by various types of chromatography, for example, an anion exchange resin, a heparin-immobilized resin, a resin for hydrophobic chromatography, a resin for affinity chromatography, or the like, where appropriate. By doing so, the protein can be isolated and purified.

[0024]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the scope of the invention. Example 1 Purification of the protein of the present invention and determination of partial amino acid sequence According to the method described in Example 2 of Japanese Patent Application No. 3-271362, single-chain HGF was converted from double-chain HGF from human serum.
A protein having a protease activity of converting to 34,000 and having a molecular weight of about 34,000 daltons by SDS-polyacrylamide gel electrophoresis was obtained. The purified protein was added to buffer A (6 M guanidine hydrochloride, 0.002 M
After reduction with 2-mercaptoethanol at 40 ° C. for 2 hours in ethylenediamine tetraacetate and 1M Tris-HCl buffer (pH 8.5), monoiodoacetic acid of the same concentration was added, and the mixture was kept under nitrogen gas at room temperature under light shielding. The reaction was carried out for 1 hour to carry out carboxymethylation. After the reaction, YMC
pack C4 column (manufactured by YMC) and acetonitrile / isopropyl alcohol (3 /
7) A concentration gradient elution was performed for 30 minutes from a concentration of 10% to 70%, and one major peak was collected.

This peak was dissolved in a 0.1% ammonium bicarbonate solution containing 2 M urea, and TPCK-trypsin (manufactured by Miles Laboratories) or TLCK-chymotrypsin (manufactured by Miles Laboratories) was used at an enzyme / substrate ratio of 1:50. The reaction was performed at 37 ° C. for 16 hours. It was added to high performance liquid chromatography (HPLC) and acetonitrile / isopropyl alcohol (3 /
7) A concentration gradient elution was performed for 1 hour from a concentration of 0% to 80% to obtain a plurality of peptide fragments.

After drying these peptide fragments under vacuum,
Dissolved in 60 μl of 50% trifluoroacetic acid, added to a glass filter treated with polybrene, and applied.
The product was subjected to Edman degradation using a Biosystems # 470A sequencer, and the amino acid sequence was determined. Phenylthiohydantoin (PTH) amino acid was identified by MCI gel ODS IHU (0.46 × 1
5 cm) column, elution with a single solvent with acetate buffer (10 mM acetate buffer, pH 4.7, 0.01% SDS and 38% acetonitrile) at a flow rate of 1.2 ml / min.
Performed at a temperature of 43 ° C., and the detection of PTH
The absorbance was measured. The amino acid sequences of two of these peptides are shown in SEQ ID NOs: 6 and 7 in the sequence listing.
Shown in Example 2 Partial DN of protein of the present invention by PCR method
Preparation of fragment A As a substrate DNA, a commercially available human liver cDNA bank (Quick Clone ^™ Human River cDNA, manufactured by Clontech) was used. PCR was performed on a Perkin Elmer Cetus DNA Thermal Cycler (Perkin
Elmer Cetus DNA Thermal C
ycler) using the Gene Amp Kit (G
ene Amp DNA Amplification
Reagent Kit, manufactured by Takara Shuzo). Substrate DNA (equivalent to 1 ng), 10-fold concentration of reaction buffer (500 mM KCl, 100 mM Tris-HCl buffer (pH 8.3), 15 mM MgCl ₂ and 0.1 mM
% (W / v) gelatin) 10 μl and 10 m each
2 μl of M dGTP, dATP, dCTP, dTTP
In each case, one kind (one sequence) of a + strand DNA primer described in SEQ ID NO: 4 in the sequence listing as a primer # 1 and a minus strand DNA primer described in SEQ ID NO: 5 in the sequence listing as a primer # 2 was 0. Add 0.5 μl of Taq DNA polymerase to 1 μM and make up to 100 μl with sterile deionized water. The reaction was pre-treated at 94 ° C for 10 minutes, followed by 30 cycles of incubation at 94 ° C for 1 minute (denaturation step), 37 ° C for 2 minutes (annealing step) and 72 ° C for 3 minutes (extension step). Finally, incubation was performed at 72 ° C. for 7 minutes to complete the reaction.

The obtained reaction solution was extracted with phenol: chloroform = 1: 1 and precipitated with ethanol. The precipitate was dissolved in 21.5 μl of sterile deionized water,
Double-concentration restriction enzyme buffer (50 mM Tris-HCl buffer (pH 7.5), 10 mM magnesium chloride, 100 mM
mM potassium chloride and 1 mM DTT) 2.5 [mu] l and restriction enzyme Bgl II1myueru (15 units) was added,
After reacting at 37 ° C. for 2 hours, 5% polyacrylamide gel electrophoresis was performed, and a 323 bp band was cut out according to a conventional method to recover a DNA fragment, followed by ethanol precipitation.

This DNA fragment was ligated to the pUC19 vector B
After insertion into the am HI site, the nucleotide sequence was determined according to a conventional method. The nucleotide sequence of the determined PCR fragment is shown in SEQ ID NO: 2 in the sequence listing. Example 3 Full-length DNA encoding the protein of the present invention
Screening of Fragments Containing the Fragment The 323 bp PCR fragment prepared in Example 2 was labeled with ³² P according to the method described in Molecular Cloning (Cold Spring Harbor Laboratory, 1982) to obtain a screening probe. As a library for screening, a premade lambda phage library (manufactured by Strategene), a human liver cDNA library derived from a 49-year-old man was used. Escherichia coli was infected with the above-described phage so as to form about 5 million plaques using XL1-Blue (manufactured by Strategene), grown overnight in NZY medium, and then transferred to GeneScreen Plus manufactured by DuPont. . The membrane was impregnated with 0.1M sodium hydroxide-0.5M sodium chloride and left on paper for 2 minutes, followed by 1.5M sodium chloride-0.
A 5M Tris-HCl buffer (pH 7.5) was impregnated and left on paper for 5 minutes. After repeating this filter treatment twice more, the filter was washed with 2 × SSC (2 × SSC) and air-dried on dry filter paper. Next, the membrane was irradiated with UV light of 120 mJ / cm ² to fix the DNA transferred to the membrane.

Five membranes treated in this way are
It was immersed in 50 ml of a solution composed of mM Tris-HCl buffer (pH 7.5), 1 M sodium chloride and 1% SDS, and kept at 65 ° C. for 2 hours. Next, 5 ng / ml of the above-mentioned P-labeled probe, 100 μg / ml of salmon sperm DNA, 50
It was immersed in 40 ml of a solution composed of mM Tris-HCl buffer (pH 7.5), 1 M sodium chloride and 1% SDS, and kept at 65 ° C. for 16 hours. Thereafter, the membrane was taken out, washed twice with 2 × SSC at room temperature for 5 minutes and then with 0.1 × SSC at room temperature for 30 minutes twice, and then subjected to autoradiography according to a conventional method to obtain 40 positive clones. Example 4 Subcloning of DNA Fragment and Determination of Base Sequence From the positive phage clone obtained in Example 3, a plasmid was directly converted into an plasmid by the extrusion method. 500 μl of S from single plaque
M buffer (50 mM Tris-HCl buffer (pH 7.
5), 100 mM sodium chloride, 10 mM magnesium sulfate and 0.01% gelatin) and 20 μl of chloroform to extract phage. The above 200 μl phage extract, 200 μl XL1-Blue and 1 μl
After treating 1 liter of R408 helper phage at 37 ° C. for 15 minutes, 5 ml of 2 × YT medium was added, and shaking culture was performed at 37 ° C. for 3 hours. Subsequently, the mixture was treated at 70 ° C. for 20 minutes and then centrifuged at 4000 g for 5 minutes to obtain a supernatant. 1 of supernatant
20 μl of the 00-fold diluted solution was added to 200 μl of XL1-Blue
After treatment at 37 ° C. for 15 minutes, 2 μl of the mixture was seeded on an LB agar medium containing 40 μl / ml ampicillin. From the emerged colonies, 24 plasmids were obtained and analyzed, and the clone having the longest insert (pB
HGFAP). The determined base sequence is shown in SEQ ID NO: 3 in the sequence listing. Based on this base sequence, the amino acid sequence of the protein of the present invention (SEQ ID NO: 1 in the sequence listing) was determined. Example 5 Preparation of Expression Plasmid FIG. 1 shows a method for preparing the protein expression plasmid of the present invention.

The plasmid obtained in Example 3 (pBHG
FAP) was used as the substrate DNA. PCR was performed on a PerkinElmer Cetus DNA Thermal Cycler (Pe
rkin Elmer Cetus DNA Ther
Using a Mal Cycler)
Kit (Gene Amp DNA Amplifier)
ation Reagent Kit, manufactured by Takara Shuzo). 1 μl of substrate DNA (equivalent to 0.5 μg),
10 times concentration of reaction buffer (500 mM KCl, 100m
M Tris-HCl buffer (pH 8.3), 15 mM Mg
16 μl each of 10 μl of Cl ₂ and 0.1% (w / v) gelatin and 1.25 mM of 4dNTP, 20 μM of primer # 3 (+ strand DNA primer: SEQ ID NO: 8 in the sequence listing) and primer # 4 (− Strand DNA primer: 5 μl each of SEQ ID NO: 9) in the sequence listing
Add 0.5 μl of Taq DNA polymerase and make up to 100 μl with sterile deionized water. The reaction is at 94 ° C, 10
1 minute (denaturation step) at 94 ° C after 5 minutes of pretreatment.
1.5 minutes at 2 ° C (annealing step) and 72
Incubate for 2 minutes (extension step) at
Two cycles were performed. Finally, incubation was performed at 72 ° C. for 7 minutes to complete the reaction. The obtained reaction solution was extracted with phenol: chloroform = 1: 1, followed by ethanol precipitation. The precipitate was dissolved in 16 μl of sterile deionized water, and subjected to 5% polyacrylamide gel electrophoresis. The 862 bp band was cut out according to a conventional method to obtain DN.
The A fragment was recovered and subjected to ethanol precipitation.

The DNA fragment thus obtained was blunt-ended with T4 DNA polymerase according to a conventional method, and then cut with the restriction enzyme XbaI . The DNA fragment was recovered again, and mixed with synthetic DNA5ng, and plasmid vectors pUC18 20 ng cleaved with Eco RI and Xba I according to SEQ ID NO: 10 in the sequence listing with 15 ng, using a ligation kit (Takara Shuzo Co., Ltd.) After ligation, E. coli JM105 was transformed. Competent cells are competent high
NT HIGH, manufactured by Toyobo Co., Ltd.) and according to the attached protocol. Mini-screening was performed on the thus obtained recombinant by a conventional method to obtain the desired 926
A plasmid pSHGFAP having a bp insert was obtained. Then limit the pSHGFAP 8μg enzyme Bam H
Cleavage with I and Xho I was followed by phenol / chloroform treatment and ethanol precipitation. The precipitate was dissolved in 16 μl of sterilized deionized water, and then subjected to 5% polyacrylamide gel electrophoresis to cut out a 920 bp band by a conventional method to collect a DNA fragment, followed by ethanol precipitation. 10 ng of the DNA thus obtained
And pcDNA I which had been cut with restriction enzymes Bam HI and Xho I and treated with alkaline phosphatase in advance.
5 ng of the / Neo plasmid was ligated using a ligation kit, and Escherichia coli DH5 was transformed. Competent cells used competent high and followed the attached protocol. Mini-screening was performed on the thus obtained recombinant by a conventional method to obtain a target plasmid pNSHGFAP. Example 6 Expression of the protein of the present invention by Escherichia coli The Escherichia coli carrying the plasmid pSHGFAP prepared in Example 5 was inoculated into 10 ml of LB (containing 50 µg / ml ampicillin) medium and incubated at 37 ° C overnight (12 to 1).
6 hours), and this culture was added to 10 ml of LB (50 μl).
g / ml of ampicillin) medium, and cultured at 37 ° C. for 2 hours. Then, isopropyl β-D thiogalactoside (IPTG) which is a transcription inducer of the lac promoter present on the vector was added. )
Was added to a final concentration of 1 mM, and the cells were further cultured at 37 ° C. for 6 hours. The cells were collected from 1 ml of the culture solution by centrifugation, and the protein expressed in the cells was detected by Western blotting according to a conventional method. As a result, it was revealed that the protein was certainly expressed.
In contrast, no expression was confirmed in the case where IPTG was not added or in the case where an Escherichia coli strain not containing the expression plasmid pSHGFAP was used. Example 7 Acquisition of an animal cell line that continuously expresses the protein of the present invention Expression vector pcDNAI / Ne prepared in Example 5
Plasmid pNHGFAP having the protein cDNA inserted at the restriction enzyme cleavage site of
(Molecular cloning, Cold Spring Harbor Laboratory, 86-96 (1
982)), the protein was recovered and purified from Escherichia coli to obtain a large amount of the protein expression plasmid.

Using the plasmid DNA obtained, Aus
ubel et al. (Current Protocol in Molecular Biology [Current Prot
ocols in Molecular Bilog
y], Green Publishing Associates and Wheelie Interscience [Green Pu
blushing Associates and W
iley-InterScience], 9.1.1-1-9.4.1 (1987)). That is, first, in a petri dish having a diameter of 9 cm, F
The CHO cells were cultured in an ERDF medium (manufactured by Far East Pharmaceutical Co., Ltd.) containing 10% BS (fetal calf serum) until they became semi-confluent. Next, the medium was removed from the Petri dish, and a DNA solution was added dropwise thereto. The DNA solution was prepared in advance according to the following procedure. First, 300 μl of a 2 × HEBS solution (1.6% sodium chloride, 0.074% potassium chloride, 0.05% disodium hydrogen phosphate decahydrate, 0.2% dextrose and 1% HEPES (pH 7.05)) and 10
μg of plasmid DNA and add 570 with sterile water.
Prepare μl of the solution in an Eppendorf centrifuge tube. Next, 30 μl of a 2.5 M calcium chloride solution is added dropwise to the DNA solution using a vortex mixer for several seconds while adding dropwise. This is left at room temperature for 30 minutes.

The thus prepared DNA solution was spread on the above-mentioned cells and allowed to stand at room temperature for 30 minutes. Then FBS
9% of an ERDF medium containing 10% was placed in a Petri dish, and cultured at 37 ° C. for 4 to 5 hours in the presence of 5% CO ₂ .
Next, the medium was removed from the Petri dish, and 5 ml of 1 × TBS ++ was removed.
Solution (25 mM Tris-HCl buffer (pH 7.5),
140 mM sodium chloride, 5 mM potassium chloride, 0.1 mM
The cells were washed with 6 mM disodium hydrogen phosphate, 0.08 mM calcium chloride and 0.08 mM magnesium chloride), and the 1 × TBS ++ solution was removed. Then, 5 ml of a 1 × TBS ++ solution containing 20% glycerol was applied to the cells at room temperature for 1 hour. The mixture was allowed to stand for ２2 minutes, and the supernatant was removed. Then 5
Wash the cells again with 1 ml of 1 × TBS ++ solution and add FBS
Was placed in a Petri dish and cultured at 37 ° C. in the presence of 5% CO ₂ . After 48 hours of culture, the medium was removed and 5 ml of 1 × TBS ++ was removed.
After washing the cells with the solution, the cells are peeled off from the Petri dish by performing trypsin treatment, the cells of one 9 cm Petri dish are divided into ten 9 cm Petri dishes, and the drug G418 (G41
Trisulfate (GENETICIN, manufactured by GIBCO) was added to a concentration of 200 μg / ml, and the culture was continued. Ten days later, G418-resistant cells that survived were isolated, and 10% using a 24-well plate.
The cells were cultured in 1 ml of ERDF medium containing FBS for 7 days.
Thereafter, the culture was continued in place of the serum-free ERDF medium, and after 72 hours, the individual cultures were collected and concentrated by ultrafiltration.
SDS-polyacrylamide gel electrophoresis was performed. This was subjected to Western blotting according to a conventional method to confirm the expression of the protein.

[0034]

Industrial Applicability According to the present invention, a novel DNA fragment encoding a polypeptide having a protease activity for converting single-chain HGF to active double-chain HGF was obtained, and its nucleotide sequence was clarified. By introducing an expression vector into which the protein gene has been inserted into a host cell, it is considered that the protein can be produced in large quantities, stably, and easily.

[0035]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 300 Sequence type: amino acid Number of chains: double-stranded Topology: linear Sequence type: protein Origin Organism name: human Sequence Ala Leu Ser Trp Glu Tyr Cys Arg Leu Glu Ala Cys Glu Ser Leu Thr 1 5 10 15 Arg Val Gln Leu Ser Pro Asp Leu Leu Ala Thr Leu Pro Glu Pro Ala 20 25 30 Ser Pro Gly Arg Gln Ala Cys Gly Arg Arg His Lys Lys Arg Thr Phe 35 40 45 Leu Arg Pro Arg Ile Ile Gly Gly Ser Ser Ser Leu Pro Gly Ser His 50 55 60 Pro Trp Leu Ala Ala Ile Tyr Ile Gly Asp Ser Phe Cys Ala Gly Ser 65 70 75 80 Leu Val His Thr Cys Trp Val Val Ser Ala Ala His Cys Phe Ser His 85 90 95 Ser Pro Pro Arg Asp Ser Val Ser Val Val Leu Gly Gln His Phe Phe 100 105 110 Asn Arg Thr Thr Asp Val Thr Gln Thr Phe Gly Ile Glu Lys Tyr Ile 115 120 125 Pro Tyr Thr Leu Tyr Ser Val Phe Asn Pro Ser Asp His Asp Leu Val 130 135 140 Leu Ile Arg Leu Lys Lys Lys Gly Asp Arg Cys Ala Thr Arg Ser Gln 145 150 155 160 Phe Val Gln Pro Ile Cys Leu Pro Glu Pro Gly Ser Thr Phe Pr o Ala 165 170 175 Gly His Lys Cys Gln Ile Ala Gly Trp Gly His Leu Asp Glu Asn Val 180 185 190 Ser Gly Tyr Ser Ser Ser Leu Arg Glu Ala Leu Val Pro Leu Val Ala 195 200 205 Asp His Lys Cys Ser Ser Pro Glu Val Tyr Gly Ala Asp Ile Ser Pro 210 215 220 Asn Met Leu Cys Ala Gly Tyr Phe Asp Cys Lys Ser Asp Ala Cys Gln 225 230 235 240 Gly Asp Ser Gly Gly Pro Leu Ala Cys Glu Lys Asn Gly Val Ala Tyr 245 250 255 Leu Tyr Gly Ile Ile Ser Trp Gly Asp Gly Cys Gly Arg Leu His Lys 260 265 270 Pro Gly Val Tyr Thr Arg Val Ala Asn Tyr Val Asp Trp Ile Asn Asp 275 280 285 Arg Ile Arg Pro Pro Arg Arg Leu Val Ala Pro Ser 290 295 300

SEQ ID NO: 2 Sequence length: 329 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA Origin Organism name: Human Direct origin: Quick-cloneTM Human liver cDNA (Clonet
ech) Sequence GGATCC CAG ATT GCG GGC TGG GGC CAC TTG GAT GAG AAC GTG AGC GGC 48 Gln Ile Ala Gly Trp Gly His Leu Asp Glu Asn Val Ser Gly 15 10 TAC TCC AGC TCC CTG CGG GAG GCC CTG GTC CCC CTG GTC GCC GAC CAC 96 Tyr Ser Ser Ser Leu Arg Glu Ala Leu Val Pro Leu Val Ala Asp His 15 20 25 30 AAG TGC AGC AGC CCT GAG GTC TAC GGC GCC GAC ATC AGC CCC AAC ATG 144 Lys Cys Ser Ser Pro Glu Val Tyr Gly Ala Asp Ile Ser Pro Asn Met 35 40 45 CTC TGT GCC GGC TAC TTC GAC TGC AAG TCC GAC GCC TGC CAG GGG GAC 192 Leu Cys Ala Gly Tyr Phe Asp Cys Lys Ser Asp Ala Cys Gln Gly Asp 50 55 60 TCA GGG GGG CCC CTG GCC TGC GAG AAG AAC GGC GTG GCT TAC CTC TAC 240 Ser Gly Gly Pro Leu Ala Cys Glu Lys Asn Gly Val Ala Tyr Leu Tyr 65 70 75 GGC ATC ATC AGC TGG GGT GAC GGC TGC GGG CGG CTC CAC AAG CCG GGG 288 Gly Ile Ile Ser Trp Gly Asp Gly Cys Gly Arg Leu His Lys Pro Gly 80 85 90 GTC TAC ACC CGC GTG GCC AAC TAT GTG GAC TGG AT GGATCC 329 Val Tyr Thr Arg Val Ala Asn Tyr Val Asp Trp 95 100 105

SEQ ID NO: 3 Sequence length: 970 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA origin Organism name: human Direct origin: Pre-made Lambda phage Library , human liver (49 years old, male) (manufactured by Stratagene Co.) cDNA Library sequence GCGCGCTCTC CTGGGAGTAC TGCCGCCTGG AGGCCTGCGA ATCCCTCACC AGAGTCCAAC 60 TGTCACCGGA TCTCCTGGCG ACCCTGCCTG AGCCAGCCTC CCCGGGGCGC CAGGCCTGCG 120 GCAGGAGGCA CAAGAAGAGG ACGTTCCTGC GGCCACGTAT CATCGGCGGC TCCTCCTCGC 180 TGCCCGGCTC GCACCCCTGG CTGGCCGCCA TCTACATCGG GGACAGCTTC TGCGCCGGGA 240 GCCTGGTCCA CACCTGCTGG GTGGTGTCGG CCGCCCACTG CTTCTCCCAC AGCCCCCCCA 300 GGGACAGCGT CTCCGTGGTG CTGGGCCAGC ACTTCTTCAA CCGCACGACG GACGTGACGC 360 AGACCTTCGG CATCGAGAAG TACATCCCGT ACACCCTGTA CTCGGTGTTC AACCCCAGCG 420 ACCACGACCT CGTCCTGATC CGGCTGAAGA AGAAAGGGGA CCGCTGTGCC ACACGCTCGC 480 AGTTCGTGCA GCCCATCTGC CTGCCCGAGC CCGGCAGCAC CTTCCCCGCA GGACACAAGT 540 GCCAGATTGC GGGCTGGGGC CACTTGGATG AGAACGTGAG CGGCTACTCC AGCTCCCTGC 600 GGGAGGCCCT GGTCCCCCTG GTCGCCGACC ACAAGTGCAG CAGCCCTGAG GTCTACGGCG 660 CCGACATCAG CCCCAACATG CTCTGTGCCG GCTACTTCGA CTGCAAGTCC GACGCCTGCC 720 AGGGGGACTC AGGGGGGCCC CTGGCCTGCG AGAAGAACGG CGTGGCTTAC CTCTACGGCA 780 TCATCAGCTG GGGTGACGGC TGCGGGCGGC TCCACAAGCC GGGGGTCTAC ACCCGCGTGG 840 CCAACTATGT GGACTGGATC AACGACCGGA TACGGCCTCC CAGGCGGCTT GTGGCTCCCT 900 CCTGACCCTC CAGCGGGACA CCCTGGTTCC CACCATTCCC TGCCTTGCTG ACAATAAAGA 960 TATTTCCAAG 970

SEQ ID NO: 4 Sequence length: 26 Sequence type: nucleic acid Topology: linear Sequence type: other nucleic acid (synthetic DNA) Origin Organism name: human sequence CTCGGATCCC ARATNGCNGG NTGGGG 26 R: G or A, N: A, G, C or T

SEQ ID NO: 5 Sequence length: 26 Sequence type: nucleic acid Topology: linear Sequence type: other nucleic acid (synthetic DNA) Origin Organism name: human sequence CTCGGATCCA TCCARTCNAC RTARTT 26 R: G or A, N: A, G, C or T

SEQ ID NO: 6 Sequence length: 7 Sequence type: amino acid Topology: linear Sequence type: peptide Fragment type: intermediate fragment Origin Organism name: human

SEQ ID NO: 7 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide Fragment type: intermediate fragment Origin Organism name: human

SEQ ID NO: 8 Sequence length: 19 Sequence type: nucleic acid Topology: linear Sequence type: other nucleic acid (synthetic DNA) Origin Organism name: human Sequence GTCCAACTGT CACCGGATC 19

SEQ ID NO: 9 Sequence length: 19 Sequence type: nucleic acid Topology: linear Sequence type: other nucleic acid (synthetic DNA) Origin Organism name: human sequence CGCTCGAGGG TCAGGAGGG 19

SEQ ID NO: 10 Sequence length: 71 Sequence type: number of nucleic acid chains: double-stranded Topology: linear: Sequence type: other nucleic acid (synthetic DNA) Origin Organism name: human sequence AATTCGGATC CATGAAGGTT CTGTGGGCTG CGTTGCTGGT CACATTCCTG GCAGGATGCC 60 GCCTAG GTACTTCCAA GACACCCGAC GCAACGACCA GTGTAAGGAC CGTCCTACGG AGGCCAAGGT G 71 TCCGGTTCCA C

[Brief description of the drawings]

FIG. 1 is a diagram showing a method for preparing a protein expression plasmid of the present invention. In the figure, P _lac E. coli lac promoter, P _CMV is cytomegalovirus (CMV) promoter, A _P ^r is an ampicillin resistant gene, the N _m ^r represents a neomycin resistance gene.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI C12R 1:19) C12R 1:91) (C12N 9/64 (C12N 9/64 C12R 1:91) C12R 1:19) (C12N 9/64 C12N 15/00 ZNAA C12R 1:19) 5/00 B (72) Inventor Naomi Kitamura 2-82-21-910, Yakumo Higashi-cho, Moriguchi-shi, Osaka (72) Inventor Keiji Miyazawa 3-1 Takahamacho, Ashiya-shi, Hyogo Prefecture -524 (56) References JP-A-6-153946 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/57 C12N 9/50-9/76 BIOSIS (DIALOG) WPI (DIALOG) Genbank / EMBL / DDBJ / GeneSeq SwissProt / PIR / GeneSeq

Claims (7)

(57) [Claims] [Claim 1] comprising the amino acid sequence set forth in SEQ ID NO: 1, protein characterized by having protease activity to convert single chain HGF inactive to active two-chain form of HGF. 2. A gene encoding the protein according to claim 1. 3. The gene according to claim 2, which comprises, as a partial sequence, a nucleic acid sequence represented by the nucleotide sequence of SEQ ID NO: 2 in the sequence listing. 4. The gene according to claim 2, which is represented by the nucleotide sequence of SEQ ID NO: 3 in the sequence listing. 5. An expression vector comprising the gene according to claim 2 incorporated downstream of a promoter so as to express a polypeptide encoded by the gene. 6. A transformant obtained by transforming a host cell with the vector according to claim 5. 7. A method for producing a protein represented by the amino acid sequence of SEQ ID NO: 1 by culturing the transformant according to claim 6.