JPH06153966A - New protein and gene coding the protein - Google Patents

Abstract

(57) [Summary] [Structure] A gene encoding a human serum-derived protease having an activity of converting a single-chain hepatocyte growth factor (HGF) into an active double-chain HGF is cloned. The DNA sequence was determined and the amino acid sequence of the protein was deduced. The same protein was produced in E. coli and CHO cells containing the same gene transformed with the vector. [Effect] The protein can be easily and stably obtained in a large amount from a culture solution obtained by culturing a transformant having the above-mentioned gene introduced therein.

Description

Detailed Description of the Invention

[0001]

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, and more specifically, hepatocyte growth factor (HGF). A protein having a protease activity that cleaves at a specific position to convert from an inactive single-chain form to an active double-chain form, a gene encoding the same, a transformant containing the gene, and The present invention relates to a protein production method using the same.

[0002]

BACKGROUND OF THE INVENTION It has been confirmed that the physiological activity of human HGF is not observed in a single-chain form in an in vitro experiment and is expressed only in a double-chain form. Has been done. It has also been confirmed that when human HGF is produced by a gene recombination technique, most of human HGF produced in serum-free culture is single-chain type. Since the presence or absence of addition of serum during the culture has a great influence on the production cost,
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 in mammalian serum into a double-chain form, and this protease is SDS-polyacrylamide gel electrophoresis. With a molecular weight of about 34,000
It was found that the protein is 0 dalton (Japanese Patent Application No. 3-271362). However, since the protein is present in serum in a very small amount, there is a problem that a great amount of labor is required to purify the protein to obtain a standard product.

[0004]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies for the purpose of easily preparing a protein having an activity equivalent to that of 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 by using a genetic engineering technique, and have completed the present invention.

That is, the gist of the present invention is a protein characterized by being represented by the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing, a gene encoding the same, a vector expressing a polypeptide encoded by the gene, It exists in transformants and a method for producing the protein using these transformants.
Hereinafter, the present invention will be described in detail.

[0006] The novel protein of the present invention is a protein having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and is partly within the range not impairing the protease activity for converting single chain HGF into double chain HGF. The present invention also includes those obtained by making changes such as removal, substitution, modification or addition of the amino acids of. Examples of the gene encoding the above-mentioned protein include those containing a nucleic acid sequence represented by the base sequence shown in SEQ ID NO: 2 in the sequence listing as a partial sequence, or the base sequence shown in SEQ ID NO: 3 in the sequence listing. Those represented are listed.

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 commercially available human liver prepared can be used. From this cDNA library, phagemid was prepared by the method of Saito et al. (Procedures of National Academy Science USA [Proc. Natl. Aca.
d. Sci. USA], 83 , 8664-8668 (1
986)) and culture. The colonies formed after the culture are subjected to a colony hybridization method ("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.

As a probe used in this colony hybridization method, a polymerase chain reaction (hereinafter abbreviated as "PCR") method (Science [Scie
No.], 239 , 487-491 (1988)), and a partial DNA fragment of the gene encoding the protein of the present invention is used. Specifically, the DNA fragment set forth in SEQ ID NO: 4 of the sequence listing (corresponding to a part of the amino acid sequence set forth in SEQ ID NO: 6 of the sequence listing) is used as a + strand DNA primer and SEQ ID NO: 5 of the sequence listing (set in the sequence listing PCR is performed using the DNA fragment described in (corresponding to a part of the amino acid sequence of SEQ ID NO: 7) as a-strand DNA primer, and the DNA fragment described in SEQ ID NO: 2 of the obtained sequence listing is prepared and used as a probe. . Also, an oligonucleotide synthesized based on the DNA sequence deduced from the amino acid sequence of the protein of the present invention can be used as a probe.

Further, from the above-mentioned screening positive colonies, T. DNA is prepared by the method of Maniatis et al. (“Molecular Cloning”, Cold Spring Harbor Laboratory, 85 (1982)) and cleaved with an appropriate restriction enzyme such as Bam HI.
Clone into a plasmid such as pUC18, Sang
er et al.'s dideoxy method (Proceedings of National Academy Sciences USA [Proc. Natl. Acad. Sci. USA]),
74 , 5463 (1977))
The base sequence of the A fragment can be determined.

The nucleotide sequence of the DNA fragment thus determined (for example, the nucleotide sequence containing the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing as a partial sequence, the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing) Which encodes the protein of the present invention shown in SEQ ID NO: 1 in the sequence listing. In addition, the DNA fragment of the present invention is modified by removing, substituting, modifying, or adding a part of bases as long as the protease activity for converting single-chain HGF into double-chain HGF is not impaired. Also included.

The DNA fragment obtained as described above is
The 5 ′ end is modified and inserted into the 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, one containing a promoter at a position where the DNA encoding the protein of the present invention obtained as described above can be transcribed is preferably used. 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 controlling the promoter.

The promoter is derived from Escherichia coli, phage, etc., such as tryptophan synthase (tr).
p), lactose operon (lac), lambda phages P _L , P _R , and T ₅ phage early gene promoters P ₂₅ and P ₂₆ . Also,
These include, for example, the pac promoter (agricultural and biological chemistry [Agri
c. Biol. Chem. ], 52 , 983-988
(1988)), the sequence may be uniquely modified and designed.

The ribosome-binding sequence may be derived from Escherichia coli, phage, etc., but 16S ribosomal RN
It may be a synthetic DNA sequence having a consensus sequence having 4 or more consecutive bases complementary to the 3'terminal region of A. Transcription termination sequences are not required, but are independent, eg lipoprotein terminators, trp.
It is preferable to have an operon terminator or the like.

Furthermore, as the sequence order of the factors necessary for the expression on the expression plasmid, the promoter, the SD sequence, the protein gene and the transcription termination factor are preferably arranged in this order from the 5'upstream side. In addition, a method of increasing the copy number of the transcription unit on the vector by inserting a plurality of units of the SD sequence on the expression vector and the protein gene in the same direction (JP-A-1-95798) You can also

Examples of the expression vector that can be used include pUAI2 (JP-A-1-95798) and commercially available pKK233-2 (Pharmacia). In addition, expression vector pG expressed as a fusion protein
EX series (Pharmacia) and the like can be used as well. The host cell can be transformed by a conventional method.

Cultivation of the transformant can be performed according to the method described in Molecular Cloning (Cold Spring Harbor Laboratory, 1982). As mentioned above, microorganisms such as Escherichia coli, Bacillus subtilis, and yeast can be used as the host cell, but the protein of the present invention contains many cysteine residues, and the position of the 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 promoters for animal cells have been reported. For example, SV40 promoter,
A metallothionein gene promoter and the like can be used. A secretion signal and a DNA fragment of the protein gene of interest are inserted downstream of this promoter according to the transcription direction. In this case, the DNA fragment of the gene
〜3 pieces may be inserted, or 2 to 3 pieces may be inserted by aligning the transcription direction with a DNA fragment having a promoter attached to the 5 ′ upstream side of the DNA fragment of the gene as a unit. You may.

It is desirable that the protein gene has a polyadenylation site downstream thereof. For example, one polyadenylation site derived from SV40 DNA, β-globin gene, metelothioneine gene, etc. is present downstream of the protein gene. In addition, a DNA fragment in which a promoter is bound to the protein gene is
When the method of inserting into individual tandem is used, it is possible to allow polyadenylation sites to exist on the 3'sides of the respective protein genes. Further SV40
Inserting a gene, an animal-derived gene 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 such as CHO cells with the above expression vector, it is desirable to use a selectable marker. When the selectable marker gene is inserted in the forward or reverse direction downstream of the polyadenylation site of the expression vector, it is not necessary to double-transform another plasmid containing the selectable marker gene to obtain a transformant. . As such a selection marker, a DHFR gene conferring resistance to methotrexate (Journal of Molecular Biology [J. Mol.
iol. ], 159 , 601 (1982)), the Neo gene conferring resistance to the antibiotic G-418 (Journal of Molecular Applied Genetics [J.
Mol. Appl. Genet. ], 1 , 327 (19
82)), an Ecogpt gene derived from Escherichia coli that confers resistance to mycophenolic acid (Proceedings of National Academy Science 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)) and the like.

A promoter such as the above-mentioned SV40-derived promoter is inserted 5'upstream of each resistance gene, and the polyadenylation site described above is contained 3'downstream of each resistance gene. ing. Inv
Expression vector p commercially available from itorogen
cDNA / Neo initially has a neomycin resistance gene as a selectable marker, and such a vector may be used.

When the above-mentioned selectable marker gene is not inserted into the expression vector, a vector having a selectable marker for transformants such as 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.Na
tl. Acad. Sci. USA], 78 , 2072
(1981)), pAd-D26-1 (Journal of Molecular Biology [J. Mol. Bio.
l. ], 159 , 601 (1982)) and the like are double-transformed with an expression vector of the protein gene, and transformants can be easily selected by the phenotype of the selectable marker gene.

It is preferable to change the selection marker for cells containing the protein gene to be selected and repeat double transformation by the above-mentioned method, because the expression level can be increased. The expression vector is introduced into animal cells by the calcium phosphate method (virology [V
irology], 52 , 456 (1973)), electroporation method (Journal of Membrane Biology [J. Menbr. Biol.], 1
0 , 279 (1972)) and the like.

Culturing of the transformed animal cells can be carried out by suspension culture or adherent culture by a conventional method. As a medium, a commonly used MEM, RPMI1640, or the like is used and cultured in the presence of 5 to 10% serum, in the presence of an appropriate growth factor, or in the absence of serum. Since animal cells producing the protein secrete the protein produced in the culture supernatant, it is possible to separate and purify the protein from the culture supernatant of this recombinant. Specifically, the produced culture supernatant containing the protein is purified by various chromatographies such as anion exchange resin, heparin-immobilized resin, hydrophobic chromatography resin, affinity chromatography resin and the like. 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, but the invention is not limited thereto unless it exceeds the gist. Example 1 Purification of 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 human serum into double chain HGF.
A protein having a protease activity for converting into a protein and having a molecular weight of about 34,000 daltons was obtained by SDS-polyacrylamide gel electrophoresis. This purified protein was added to buffer A (6M guanidine hydrochloride, 0.002M
After reduction with 2-mercaptoethanol at 40 ° C. for 2 hours in ethylenediamine tetraacetate and 1M Tris-hydrochloric acid buffer (pH 8.5), monoiodoacetic acid with the same concentration was added, and under nitrogen gas, at room temperature, under light shielding. The reaction was carried out for 1 hour to carry out carboxymethylation. After reaction, YMC
Pack C4 column (manufactured by WMC) and add acetonitrile / isopropyl alcohol (3 /
7) Gradient elution from a concentration of 10% to 70% for 30 minutes was performed to collect one main peak.

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

After vacuum drying these peptide fragments,
Dissolved in 60 μl of 50% trifluoroacetic acid, added to a glass filter treated with polybrene, and applied
The amino acid sequence was determined by Edman degradation with a $ 470A sequencer manufactured by Biosystems. Phenylthiohydantoin (PTH) amino acid was identified by MCI gel ODS IHU (0.46 × 1) manufactured by Mitsubishi Kasei.
5 cm) column, and elution of a single solvent with an 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 ℃, PTH amino acid detection is 269 nm
Absorbance of The amino acid sequences of two of these peptides are shown in SEQ ID NOs: 6 and 7 of the sequence listing.
Shown in. Example 2 Partial DN of protein of the present invention by PCR method
Preparation of A fragment As a substrate DNA, a commercially available human liver cDNA bank (Quick Clone ^™ Human River cDNA, manufactured by Clontech) was used. PCR is based on Perkin Elmer Cetus DNA Thermal Cycler (Perkin
Elmer Cetus DNA Thermal C
Gene Amplifier Kit (G
ene Amp DNA Amplification
Reagent Kit, manufactured by Takara Shuzo). Substrate DNA (corresponding 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)
% (W / v) gelatin) 10 μl and 10 m each
2 μl of M dGTP, dATP, dCTP, dTTP
Each of the positive-strand DNA primer described in SEQ ID NO: 4 in the sequence listing as primer # 1 and the negative-strand DNA primer described in SEQ ID NO: 5 in the sequence listing as primer # 2 is 0. 0.5 μl of Taq DNA polymerase is added to make 1 μM, and the system is made up to 100 μl with sterile deionized water. The reaction was pretreated 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 carried out at 72 ° C. for 7 minutes to complete the reaction.

The resulting reaction solution was extracted with phenol: chloroform = 1: 1 and ethanol precipitation was performed. This precipitate was dissolved in 21.5 μl of sterile deionized water and then 10
Double concentration of restriction enzyme buffer (50 mM Tris-HCl buffer (pH 7.5), 10 mM magnesium chloride, 100
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, a 323 bp band was cut out in accordance with a conventional method, a DNA fragment was recovered, and ethanol precipitation was performed.

This DNA fragment was cloned into B of pUC19 vector.
After inserting into the am HI site, the nucleotide sequence was determined according to a conventional method. The determined base sequence of the 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 Clones Containing Fragments 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 be used as a screening probe. As a library for screening, a premade lambda phage library (manufactured by Stratagene), a 49-year-old man-derived human liver cDNA library was used. Escherichia coli was infected with the above phage using XL1-Blue (manufactured by Stratagene) so that the number of plaques was about 5 million, grown in NZY medium overnight, and then transferred to GeneScreen Plus manufactured by DuPont. . The membrane was impregnated with 0.1M sodium hydroxide-0.5M sodium chloride and allowed to stand on paper for 2 minutes, followed by 1.5M sodium chloride-0.
5M Tris-hydrochloric acid buffer (pH 7.5) was soaked and left still on the paper for 5 minutes. After repeating this filter treatment twice more, the filter was washed with 2 × SSC (2 × SSC) and air-dried on a dry filter paper. Next, this membrane was irradiated with 120 mJ / cm ² of UV to fix the DNA transferred to the membrane.

5 membranes treated in this way were replaced with 50
It was immersed in 50 ml of a solution containing mM Tris-HCl buffer (pH 7.5), 1 M sodium chloride and 1% SDS, and kept at 65 ° C. for 2 hours. Next, the above P-labeled probe 5 ng / ml, salmon sperm DNA 100 μg / ml, 50
It was immersed in 40 ml of a solution consisting 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 with 2 × SSC at room temperature for 5 minutes and 0.1 × SSC at room temperature for 30 minutes twice in this order, and then autoradiography was performed according to a conventional method to obtain 40 positive clones. Example 4 Subcloning of DNA fragment and determination of nucleotide sequence The positive phage clone obtained in Example 3 was directly transformed into a plasmid by the excision method. 500 μl 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 chloroform extracted the phage. 200 μl phage extract and 200 μl XL1-Blue and 1 μl
l R408 helper phage was treated 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 00-fold diluted solution was added to 200 μl of XL1-Blue.
After mixing with and treating at 37 ° C. for 15 minutes, 2 μl of the mixture was plated on LB agar medium containing 40 μl / ml of ampicillin. Of the emerging colonies, 24 plasmids were acquired and analyzed, and the clone with the longest insert (pB
The base sequence of HGFAP) was determined. The determined nucleotide sequence is shown in SEQ ID NO: 3 in the sequence listing. Based on this nucleotide 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 is performed by Perkin Elmer Cetus DNA Thermal Cycler (Pe
rkin Elmer Cetus DNA Ther
Mal Cycler)
Kit (Gene Amp DNA Amplific
ation Reagent Kit, manufactured by Takara Shuzo). Substrate DNA 1 μl (0.5 μg equivalent amount),
10 times concentration of reaction buffer (500 mM KCl, 100 m
M Tris-HCl buffer (pH 8.3), 15 mM Mg
Cl ₂ and 0.1% (w / v) gelatin 10 μl and 1.25 mM 4dNTP 16 μl each, 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 100 μl system with sterile deionized water. Reaction is 94 ° C, 10
After pretreatment for 1 minute, at 94 ° C for 1 minute (denaturation step), 5
1.5 minutes at 2 ° C (annealing step) and 72
2 minutes (extension step) incubation at 2 ° C
Two cycles were performed. Finally, incubation was carried out at 72 ° C. for 7 minutes to complete the reaction. The obtained reaction solution was extracted with phenol: chloroform = 1: 1 and ethanol precipitation was performed. This precipitate was dissolved in 16 μl of sterile deionized water and subjected to 5% polyacrylamide gel electrophoresis, and the 862 bp band was cut out according to a conventional method.
The A fragment was recovered and ethanol precipitated.

The DNA fragment thus obtained was blunt-ended with T4 DNA polymerase according to a conventional method and then cut with a restriction enzyme Xba I. This DNA fragment was recovered again, and 15 ng was mixed with 5 ng of the synthetic DNA described in SEQ ID NO: 10 of the sequence listing and 20 ng of plasmid vector pUC18 cleaved with Eco RI and Xba I, and a ligation kit (Takara Shuzo) was used. Ligation was performed and E. coli JM105 was transformed. Competent cells are
NT HIGH, manufactured by Toyobo Co., Ltd.) was used and the attached protocol was followed. The thus obtained recombinant was subjected to mini-screening by a conventional method to obtain the desired 926
A plasmid pSHGFAP having a bp insert was obtained. Next, pSHGFAP 8 μg was added to the restriction enzyme Bam H
Cleavage with I and Xho I followed by phenol / chloroform treatment and ethanol precipitation. This precipitate was dissolved in 16 μl of sterile deionized water and subjected to 5% polyacrylamide gel electrophoresis to cut out a 920 bp band according to a conventional method to recover a DNA fragment, followed by ethanol precipitation. 10 ng of DNA thus obtained
And pcDNAI which had been digested with the restriction enzymes Bam HI and Xho I in advance and treated with alkaline phosphatase.
5 ng of / Neo plasmid was ligated with a ligation kit to transform E. coli DH5. Competent high was used as the competent cell, and the attached protocol was followed. Mini-screening was performed from the thus obtained recombinants by a conventional method to obtain the desired plasmid pNSHGFAP. Example 6 Expression of the Protein of the Invention by E. coli E. 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 (12-1).
After culturing for 6 hours, 10 ml of LB (50 μL) was added to this culture solution.
1/100 volume (0.1 ml) was added to a medium containing g / ml of ampicillin and the mixture was cultured at 37 ° C. for 2 hours, and then isopropyl β-D thiogalactoside (IPTG), which is a transcription inducer of the lac promoter present on the vector. )
Was added so that the final concentration was 1 mM, and the mixture was further cultured at 37 ° C. for 6 hours. When cells were collected from 1 ml of the culture broth by centrifugation and the protein expressed in the cells was detected by Western blotting according to a conventional method, it was revealed that the protein was indeed expressed.
On the other hand, no expression was confirmed when IPTG was not added or when the Escherichia coli strain containing no expression plasmid pSHGFAP was used. Example 7 Acquisition of animal cell line that continuously expresses the protein of the present invention Expression vector pcDNAI / Ne prepared in Example 5
Plasmid pNHGFAP in which the protein cDNA was inserted at the restriction enzyme cleavage site of
Et al. (“Molecular Cloning”, Cold Spring Harbor Laboratory, 86-96 (1
982)) and recovered and purified from recombinant Escherichia coli to obtain a large amount of the protein expression plasmid.

Using the obtained plasmid DNA, Aus
The method of ubel et al. (Current Protocol in Molecular Biology [Current Protocol
ocols in Molecular Biolog
y], Green Publishing Associates and Wheelie Interscience [Green Pu
blushing Associates and W
CHO cells were transformed based on iley-InterScience], 9.1.1-1-9.1.4 (1987)). That is, first, in a petri dish with a diameter of 9 cm, F
CHO cells were cultured in an ERDF medium (manufactured by Kyokuto Pharmaceutical Co., Ltd.) containing 10% of BS (fetal bovine serum) until a semi-confluent state was reached. Next, the medium was removed from the dish and the DNA solution was added dropwise thereto, and the DNA solution was prepared in advance according to the following procedure. First, 300 μl of 2 × HEBS solution (1.6% sodium chloride, 0.074% potassium chloride, 0.05% disodium hydrogen phosphate dodecahydrate, 0.2% dextrose and 1%) was used for each dish having a diameter of 9 cm. HEPES (pH 7.05)) and 10
Add μg of plasmid DNA and 570 with sterilized water
Prepare the μl-matched solution in an Eppendorf centrifuge tube. Then, to the DNA solution, 30 μl of a 2.5 M calcium chloride solution is added dropwise and mixed vigorously for several seconds using a vortex mixer. This is left at room temperature for 30 minutes.

The DNA solution thus prepared was applied to the above-mentioned cells and allowed to stand at room temperature for 30 minutes. Then FBS
9 ml of ERDF medium containing 10% of was added to a Petri dish and cultured at 37 ° C. for 4 to 5 hours in the presence of 5% CO ₂ .
Then remove the medium from the dish and add 5 ml of 1 × TBS ++
Solution (25 mM Tris-HCl buffer (pH 7.5),
140 mM sodium chloride, 5 mM potassium chloride, 0.
After washing the cells with 6 mM disodium hydrogen phosphate, 0.08 mM calcium chloride and 0.08 mM magnesium chloride and removing the 1 × TBS ++ solution, 5 ml of 1 × TBS ++ solution containing 20% glycerol was applied to the cells at room temperature for 1 hour. Allow to stand for ˜2 minutes and remove the supernatant. Then 5
Wash cells again with ml 1 × TBS ++ solution and wash with FBS
10 ml of ERDF medium containing 10% of was added to a Petri dish and cultured at 37 ° C. in the presence of 5% CO ₂ . After 48 hours of culturing, the medium was removed and 5 ml of 1 × TBS ++
After washing the cells with the solution, trypsin treatment is performed to separate the cells from the petri dish, and one 9 cm petri dish cell is divided into 10 9 cm petri dishes to obtain the drug G418 (G41).
Trisulfate (GENETICIN, manufactured by GIBCO) was added at a concentration of 200 μg / ml to continue the culture. After 10 days, G418-resistant cells that survived were isolated and were isolated in a 24-well plate by 10%.
The cells were cultured in 1 ml of ERDF medium containing FBS for 7 days.
After that, the culture was continued in place of the serum-free ERDF medium, and after 72 hours, individual culture solutions were collected, 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 a single chain HGF into an active double chain HGF was obtained, and its nucleotide sequence was clarified. By introducing an expression vector into which the protein gene is inserted into a host cell, it is considered possible to produce the protein in large quantities, stably and easily.

[0035]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 300 Sequence type: Amino acid Number of chains: Double chain 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 strand Topology: Linear Sequence type: cDNA Origin organism name: human Direct origin: Quick-cloneTM human liver cDNA (Clonet
ech) 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 1 5 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 strand 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 Biological 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: Nucleic acid Number of strands: Double strand 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 drawings]

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

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location C12N 5/10 C12P 21/02 C 8214-4B // (C12N 1/21 C12R 1:19) ( (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:91) (72) Inventor Naomi Kitamura 2-82-21-910 Yakumohigashi-cho, Moriguchi City, Osaka Prefecture (72) Keiji Miyazawa Ashiya City, Hyogo Prefecture 3-1-5 Takahamacho

Claims (7)

[Claims] 1. A protein having the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing. 2. A gene encoding the protein according to claim 1. 3. The gene according to claim 2, comprising the nucleic acid sequence represented by the nucleotide sequence set forth in SEQ ID NO: 2 in the sequence listing as a partial sequence. 4. The gene according to claim 2, which is represented by the nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing. 5. An expression vector in which the gene according to claim 2 is inserted 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 culturing the transformant according to claim 6 to produce the protein represented by the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing.