JP2963163B2 - Human hepatocyte growth factor - Google Patents

Description

The present invention relates to human hepatocyte growth factor.

(Prior art) The liver is the only regenerable organ in a living body. It has been suggested from liver transplantation experiments and body fluid exchange experiments that this liver regeneration phenomenon is due to some humoral factors. In recent years, the present inventors have taken hepatic parenchymal cells out of a living organism and promoted their proliferation in vitro, ie, a human-derived protein factor, that is, human hepatocyte growth factor (hereinafter, abbreviated as “hHGF”). From patient plasma (Biomedical Research (Biomed. Res.), Vol. 6, p. 231 (1985) and Experimental Cell Research (Exp. Cell. Res.), Vol. 166, p. 139 (198)
6)) and succeeded in purifying as a single protein for the first time in the world (JP-A-63-22526 and Journal of Clinical Investigation (J. Clin. Inves).
t.) 81: 414 (1988)). Furthermore, a gene encoding the hHGF protein has been isolated (patent pending (Japanese Patent Application No.
-209449) and Biochemical Biophysical Research Communication (Biochem. Biophys. Res. Comu)
n.) 163: 967 (1989)).

This hHGF protein is counted from the signal-like peptide sequence,
A protein comprising an H-chain peptide consisting of 494 amino acids and an L-chain peptide consisting of 234 amino acids, and having at least four sugar chain binding sites. These two peptide chains are linked by a disulfide bond (SS bond), and an activity of promoting the proliferation of hepatocytes in vitro has been recognized (Biochemical Biophysical Research Communication (Biochemical)). Biochem. Biophys. Res. Comun.) 163 967 (198
9)). The gene of each peptide chain is encoded in the form of an H chain and an L chain peptide connected in that order. So one
It is transcribed on RNA and simultaneously translated as a single protein. Thereafter, the signal-like sequence existing on the N-terminal side is cleaved and removed, and the subsequent one protein chain is cleaved into two. The resulting two peptide chains are SS
It is thought that a functional protein is formed through the binding.

(Problems to be Solved by the Invention) Elucidation of the biochemical and physiological functions of this hHGF protein not only elucidates the mechanism of liver regeneration, but also provides a stable supply of hepatic parenchymal cells in vitro and hepatic disease. It will play an important role in the development of therapeutics. However
In order to examine the detailed function of hHGF protein in a living body or the effect of hHGF protein on liver regeneration during liver injury, a large amount of hHGF protein is required.

However, up to the present time, as a method for obtaining hHGF protein, it has been necessary to purify a small amount of hHGF protein present in plasma of a patient with fulminant hepatitis as a material.
This method is not always easy in terms of man, time and cost, and it is extremely difficult to stably extract only a trace amount of hHGF protein from the plasma of a patient having an infectious source such as a virus. For these reasons, stable and large-scale purification of hHGF protein from plasma of fulminant hepatitis patients has not been performed.

(Means for Solving the Problems) Accordingly, the present inventors have conducted various studies to obtain a stable and large amount of hHGF protein by recombinant DNA technology, and found that a gene encoding hHGF protein useful for this purpose was obtained. A newly constructed expression vector containing the same was made possible to express the hHGF protein (Japanese Patent Application No. 2-88592).

Furthermore, the present inventors have studied the amino acid sequence of hHGF obtained by culturing host cells transformed with the above expression vector, and found that the N-terminal amino acid of secreted hHGF is pyroglutamic acid, The present invention has been completed.

That is, the gist of the present invention resides in a human hepatocyte growth factor having the amino acid sequence shown in FIG.

 Hereinafter, the present invention will be described.

For industrial production of hHGF protein, it is necessary to select a host-vector system in which the protein expression is stable, and that the expressed hHGF protein has a biological activity, that is, a hepatocyte proliferation activity. . Considering that the natural hHGF protein is a glycoprotein, and that the hHGF protein contains many cysteine residues, and the position of the thiol bond between the cysteine residues and the higher-order structure of the protein are important for maintaining the activity. There is a need to.

In such a case, as a host, yeast or E. coli, for example,
Saccharomyces cerevisiae strain and Escherichia coli YA-
Although microorganisms such as 21 strains can be used, it is desirable to express the above genes using animal cells such as CHO cells, COS cells, mouse L cells, mouse C127 cells, and mouse FM3A cells. When these cells are used as a host, the immature hHGF gene containing the signal-like sequence contained in the DNA sequence shown in FIG. Is expected to be secreted and produced extracellularly.

The expression vector used in the present invention has a DNA fragment encoding a part or all of the amino acid sequence of the hHGF protein downstream of the promoter.

As the promoter, various promoters have been reported, but in the present invention, the SV40 promoter or the metallothionein gene promoter is preferable.
Downstream of this promoter, a DNA fragment of the immature hHGF gene containing the aforementioned signal-like sequence is inserted in the direction of transcription. In this case, the hHGF gene DNA fragment is
-3 ligated DNA may be inserted, or Dg with a promoter bound to the 5 'upstream of the DNA fragment of the hHGF gene.
2-3 units in tandem with the transcription direction aligned in units of NA fragments
Individually joined ones may be inserted.

The hHGF gene needs to have a polyadenylation site downstream thereof. For example, SV40 DNA, β-
It is necessary that one polyadenylation site derived from the globin gene or metallothionein gene is present downstream of the hHGF gene. When a method of inserting 2-3 DNA fragments each having a promoter linked to the hHGF gene in tandem is used, it is possible to have a polyadenylation site on the 3 'side of each hHGF gene.

When transforming animal cells such as CHO cells using the above expression vectors, it is desirable to use a selection marker. When the selectable marker gene is inserted in the forward or reverse direction downstream of the polyadenylation site of the expression vector, there is no need to double-transform another plasmid containing the selectable marker gene when obtaining a transformant. . Examples of such selectable markers include the DHFR gene that confers methotrexate resistance (Journal of Molecular Biology (J. Mol. Biol.) 159, 601 (1982)) and the Neo gene that confers resistance to the antibiotic G-418. (Journal of Molecular Applied Genetics (J. Mol.
Appl. Genet., 1: 327 (1982)), an Ecogpt gene derived from Escherichia coli conferring mycophenolic acid resistance (Proceding and National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 78). Volume 2072 (198
1)), the hph gene that confers resistance to the antibiotic hygromycin (Molecular Cell Biology (Mol. Cell. Bio
l.) Vol. 5, p. 410 (1985)). A promoter, for example, the aforementioned SV40-derived promoter is inserted 5 'upstream of each of these resistance genes, and the polyadenylation site described above is included 3' downstream of each resistance gene.

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 Aprite Genetics (J.Mol.App
l. Genet.) 1: 327 (1982)), pMBG (Nature (N
294: 228 (1981)), pSV2gpt (Proceding and National Academy of Sciences (Prec. Natl. Acad. Sci. USA) 78: 2072 (198)
1)), pAd-D26-1 (Journal of Molecular Biology (J. Mol. Biol.) 159, 601 (1982))
(J. Mol. Biol. 159, 601 (1982)) and the like are double transformed with an expression vector for the hHGF gene, and transformants can be easily selected based on the phenotype of the selectable marker gene.

It is preferable to repeat the double transformation by changing the selection marker for the cell containing the selected hHGF protein gene by the method as described above, since the expression level is increased by about 20 times.

The expression vector is introduced into animal cells by the calcium phosphate method (Virology, vol. 52, p. 456 (1973)), and the electroporation method (Journal of Membrane Biology (J. Membr. Biol.), Vol. 10, p. 279). (197
2)) and the like, but the calcium phosphate method is generally used.

Culture of the transformed animal cells can be performed by suspension culture or adherent culture by a conventional method. As a medium,
Using MEM, RPMI1640 or the like, the cells are cultured in the presence of 5 to 10% serum, in the presence of appropriate amounts of insulin, dexamethasone, transferrin, or in the absence of serum.

Since animal cells producing hHGF protein secrete the produced hHGF protein in the culture supernatant, it is possible to separate and purify the hHGF protein using the culture supernatant of this recombinant. Specifically, the culture supernatant containing the produced hHGF protein is purified by various types of chromatography, for example, S-sepharose, heparin sepharose, hydroxyapatite or sulfated cellulofine, etc. Can be isolated and purified.

In the present invention, pre-hHGF starting from Met shown in FIG. 2 is first expressed in a host. Then, after modification in the host, hydrolysis occurs between the 31st Gly and the 32nd Gln to cleave the 31 signal peptides. Next, N-terminal Gln is deammonified and hHGF converted to pyroglutamic acid is secreted.

Thus, hHGF of the present invention having the N-terminus modified with pyroglutamic acid is obtained.

(Examples) Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to the following Examples as long as the gist of the present invention is not exceeded.

Example 1 [I] Preparation of hHGF protein expression plasmid FIG. 3 shows a method of preparing an hHGF protein expression plasmid. HHGFcDNA (Biochemical And Biophysical Research Communications (BBRC # 163 Volume (2) 967 pp -973 (1989)) Bam H I- Kpn I fragment i.e. hHGF protein translation initiation ATG than 27 bases upstream of including Bam
Conventional method plasmid pUCHGF1DNA containing Bam H I- Kpn I fragment of about 2.3kb covering the region from HI cleavage point to the Kpn I cleavage point of 8 bases upstream of the termination codon TAG ( "Molecular Cloning", Cold Spring Harbor
Laboratory, page 93 (1982)).

Next, 10 μg of the plasmid DNA was digested with the restriction enzyme Kpn according to a conventional method.
The DNA fragment obtained was digested with phenol / chloroform according to a conventional method, and ethanol-precipitated.
The N fragment was purified and dissolved in 10 µ of water.

In addition, both ends shown in FIG. 3 have a restriction enzyme Kpn I cleavage site and a termination codon T inside the Kpn I cleavage site of this DNA fragment.
A 32-base synthetic linker containing GA and the restriction enzyme BamHI cleavage point was introduced according to the method of Maniatis et al. ("Molecular Cloning", Cold Spring Harbor Laboratory, pp. 396-397 (1982)).

Using this, Escherichia coli was transformed according to a conventional method, and plasmid DNA was prepared from the resulting transformant by a conventional method ("Molecular Cloning", Cold Spring Harbor Laboratory, p. 93 (1982)).

Next, 10 μg of the plasmid DNA was digested with the restriction enzyme Bam
After digestion with HI, this restriction enzyme reaction solution is electrophoresed on a 1.0% agarose gel to obtain the desired start codon.
The hHGF DNA fragment containing ATG and the termination codon TGA was separated from non-target DNA fragments such as vectors. Maniatis et al method Bam H I- Bam HI DNA fragment of about 2.3kb which encodes the hHGF gene of interest from an agarose gel fragment according ( "Molecular Cloning", Cold Spring Harbor Laboratory, 164 pp. (1982)) Prepared.
The end of the obtained DNA fragment was blunt-ended with T4 DNA polymerase according to a conventional method, extracted with phenol / chloroform, and the DNA fragment was purified by ethanol precipitation to obtain 10 μl.
Dissolved in water.

On the other hand, the expression vector pKCR (Proceeding and Natural Academy of Science (Proc.
At. Acad. Sci.) 78: 1527 (1981)) 0.05 μg was cut in advance with a restriction enzyme SmaI which generates blunt ends, extracted with phenol / chloroform, and purified by ethanol precipitation. This was mixed with 400 µm of 50 mM Tris-HCl (pH
8) After dissolving in 1 mM magnesium chloride solution, 1 unit of bacterial alkaline phosphatase (Toyobo, BAP-101) was added, and a reaction was performed at 65 ° C. for 30 minutes to perform a dephosphorylation treatment. Next, the DNA fragment was purified from the reaction solution by phenol / chloroform extraction and ethanol precipitation to obtain a 10 μl DNA fragment.
Dissolved in water.

0.01 μg of the DNA fragment of the pKCR vector prepared as described above
The aforementioned Bam H I fragment blunt-ended HHGFcDNA 0.1 [mu]
Reaction solution containing 66 g (66 mM Tris-HCl pH 7.6, 6.6 mM magnesium chloride 10 mM dithiothreitol, 66 μMATP) 20 μ
T4 DNA ligase (Toyobo LGA-101) at 14 ° C for 12 hours
Was performed. This T4 DNA ligase reaction solution 10
E. coli HB101 strain (Takara Shuzo) was transformed using μ according to the instructions, and cultured on a medium containing ampicillin at a concentration of 50 μg / ml, thereby obtaining several tens of ampicillin-resistant strains.

By analyzing these recombinants according to the method of Maniatis et al. ("Molecular Cloning", Cold Spring Harbor Laboratory, pp. 86-96 (1982)), the promoter and polyadenylation site of the expression vector pKCR were obtained. A plasmid in which the hHGF gene is forward-linked to the restriction enzyme Sma I cleavage site present in the middle,
pKCRHGF-2 plasmid was obtained.

 The structure is shown in FIG.

[II] Acquisition of a cell line that continuously expresses hHGF protein Example 1-Expression vector pKCR prepared by [I]
(Proceding and Natural Academy
Science of Proc. Nat. Acad. Sci. 78 (2) 15
The plasmid pKCRHGF-2 in which two hHGF cDNAs were inserted into the restriction enzyme Bam HI cleavage sites on page 27 (1981) was prepared by the method of Maniatis et al. (“Molecular Cloning”, Cold Spring Harbor Laboratory, pp. 86-96). (198
The HGF expression plasmid DNA was obtained in a large amount by collecting and purifying from recombinant Escherichia coli according to 2)).

On the other hand, plasmid pSV2neo (Journal of Applied Genetics) Vol. 327, which encodes a marker for selecting transformed cells.
(1982)) and the recombinant E. coli and pAd-D26.
-1 (Journal of molecular biology, vol. 159, p. 601 (1
982)) from the recombinant E. coli having the aforementioned Maniatis
The plasmid DNA was recovered and purified according to these methods.

Using the obtained three kinds of plasmid DNAs, the method of Ausubel et al. (Current Protocols in Molecular Bio
Theory Publishing, Green Publishing Associates and Wheelie Interscience
ng Associates and Wiley-Inter science) 9.1
CHO cells were transformed by double transformation into CHO cells based on Chapters 1 to 9.1.4 (1987).

First, FCS (fetal calf serum) in a 9 cm diameter petri dish
Were cultured in an ERDF medium (manufactured by Kyokuto Pharmaceutical Co., Ltd.) containing 10% of CHO cells until they reached a semi-confluent state. Next, the medium is removed from the Petri dish, and the DNA solution is added dropwise thereto.
The NA solution was prepared in advance according to the following procedure.

First, 300μ 2 × HEBS per 9cm diameter petri dish
Solution (2 × 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 μg of plasmid DNA and 1 μg of pSV2neo plasmid DNA,
Add 1 μg of pAd-D26-1 plasmid DNA and prepare a solution adjusted to 570 μl with sterile water in an Eppendorf centrifuge tube. Next, 30 μm of a 2.5 M calcium chloride solution is added dropwise to the DNA solution using a vortex mixer while mixing vigorously for several seconds. This is left at room temperature for 30 minutes, during which it is mixed with a vortex mixer approximately every 10 minutes.

The DNA solution thus prepared was applied to the above-mentioned cells and allowed to stand at room temperature for 30 minutes. Then, ERDF medium containing 10% FCS 9
ml in a Petri dish and 4-5 at 37 ° C in the presence of 5% CO _2.
Cultured for hours. Next, remove the culture medium from the Petri dish and 5 ml of 1 × T
BS ++ solution (1 × TBS ++ solution; 25 mM Tris-HCl (pH 7.
5) Wash the cells with 140 mM sodium chloride, 5 mM potassium chloride, 0.6 mM disodium hydrogen phosphate, 0.08 mM calcium chloride, 0.08 mM magnesium chloride), remove the 1 × TBS ++ solution, and then add 1 × containing 20% glycerol. TBS ++ solution 5
The ml was spread on the cells, allowed to stand at room temperature for 1-2 minutes, and the supernatant was removed. Thereafter, the cells were washed again with 5 ml of 1 × TBS ++ solution, and 10 ml of ERDF medium containing 10% of FCS was placed in a Petri dish and cultured at 37 ° C. in the presence of 5% CO ₂ . After 48 hours from the culture, the medium was removed, and the cells were washed with 5 ml of 1 × TBS ++ solution. Then, 2 ml of trypsin-EDTA solution (Sigma) was applied to the cells, and the cells were allowed to stand at room temperature for 30 seconds. Thereafter, the trypsin-EDTA solution was removed, and 5 minutes later, 10 ml of ERDF medium containing 10% FCS was placed in a Petri dish to detach the cells, and 9c
m Divide cells for one petri dish into 10 9 cm petri dishes
200 μg / ml of G418 (G418 sulfate (GENETICIN); GIBCO)
And the culture was continued. After 10 days, survived G418 resistant cells were isolated,
Using a 24-well culture dish in which one culture well has a size of about 3.1 cm ² , each culture well is about 7 ml in 1 ml of ERDF medium containing 10% FCS.
The culture was re-cultured for days.

The culture of the above cells was replaced with ERDF medium not containing FCS, and culturing was continued, and 2 ml of the culture medium of each cell after 72 hours was individually collected, and collected with a centricon concentrator (Millipore) to 50 μl.
Centrifugal concentration to about 15μ of that as a sample,
SDS-polyacrylamide gel electrophoresis was performed.

This was analyzed by Western blotting according to a standard method, and hHGF
The expression of the protein was confirmed.

Gohda et al.'S method (Experimental Cell
The hHGF activity was measured by Research (Experimental Cell Rescerch), Vol. 166, pp. 139-150 (1986) to confirm the presence of biological activity.

Furthermore, the obtained cell lines were individually isolated and subjected to enzyme immunoassay to quantify hHGF protein.

As a result, the cell lines B-1, B-27, B
-102 was obtained.

Example 2 [I] Acquisition of a cell line that continuously expresses hHGF protein obtained by repeatedly transforming with an expression vector having an hHGF gene Example 1-Expression of hHGF gene obtained by [I] Plasmid pMBG encoding the vector pKCRHGF-2 and a marker for selection of transformed cells resistant to mycophenolic acid (Na
294,228 (1981)), the plasmid DNA was recovered and purified according to the method of Maniatis et al.

Using the obtained two types of plasmid DNAs, the method of Ausubel et al. (Current Protocols in Molecular Biology)
Theory, Green Publishing Associates and Wheelie Interscience (Greene Publishi)
ng Associates and Wiley-Inter science) 9.1
Example 1 based on Chapters 1 to 91.4 (1987))
Among the cell lines that successively express the hHGF protein obtained by [II], three cell lines expressing a large amount of hHGF (B-1,
B-27, B-102) were isolated and double transformed individually to transform the cells.

That is, first, cell lines expressing the above-mentioned hHGF protein subcultured in an ERDF medium containing 10% FCS in a 9 cm-diameter dish were individually cultured 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 the same as in Example 1- [II] except that 10 μg of pKCRHGF-2 plasmid DNA and 1 μg of pMBG plasmid DNA were used. Prepared by the procedure.

The DNA solution thus prepared was applied to the above-mentioned cells and allowed to stand at room temperature for 30 minutes. Then, ERDF medium containing 10% FCS 9
ml in a Petri dish and 4-5 at 37 ° C in the presence of 5% CO _2.
Cultured for hours. Next, remove the culture medium from the Petri dish and 5 ml of 1 × T
After washing the cells with a BS ++ solution (described above) and removing the 1 × TBS ++ solution, 5 ml of a 1 × TBS ++ solution containing 20% glycerol was applied to the cells, allowed to stand at room temperature for 1 to 2 minutes, and the supernatant was removed. Thereafter, the cells were washed again with 5 ml of 1 × TBS ++ solution, 10 ml of ERDF medium containing 10% FCS was placed in a Petri dish, and cultured at 37 ° C. in the presence of 5% CO _2. After removing the cells and washing the cells with 5 ml of 1 × TBS ++ solution, 2 ml of trypsin-EDTA solution (Sigma) was added to the cells.
And allowed to stand at room temperature for 30 seconds. Then trypsin-ED
After removing the TA solution, 5 minutes later, α-M containing 10% FCS
10 ml of EM (-) medium was placed in a Petri dish to detach cells, and 9c
m Petri dish was divided into 10 pieces of 9 cm petri dishes, and the drug mycophenolic acid (manufactured by Sigma) was added at a concentration of 1 μg / ml and xanthine (manufactured by Sigma) at a concentration of 250 μg / ml, and the culture was continued. . After 10 days had passed, the surviving mycophenolic acid-resistant cells were isolated, and one culture hole was used in a 24-well culture dish of approximately 3.1 cm ² for each.
The cells were re-cultured in 1 ml of ERDF medium containing 10% FCS for about 7 days.

The culture of the above cells was replaced with ERDF medium not containing FCS, and culturing was continued. 2 ml of the culture medium of the cells after 72 hours were collected individually, and they were collected by a centricon concentrator (Millipore) to 50 μl
Centrifugal concentration to about 15μ of the sample as SD
S-polyacrylamide gel electrophoresis was performed.

This was analyzed by Western blotting according to a standard method, and hHGF
The expression of the protein was confirmed.

Gohda et al.'S method (Experimental Cell
HHGF activity was measured by Research (Experimental Cell Research), Vol. 166, pp. 139-150 (1986) to confirm the presence of biological activity.

 The results are shown in FIG.

Furthermore, as a result of individually isolating some of the obtained cell lines and confirming the expression level of the hHGF protein by enzyme immunoassay, the expression level of the cell line B-102 before double transformation was approximately 20-fold. A cell line BD-24 showing the amount was obtained.

Example 3 The hHGF producing strain BD-24 obtained in Example 2 was ERD containing 10% FCS.
Culture in F medium (Kyokuto Pharmaceutical) and add 500 ml of the culture supernatant
Obtained. 10 ml of S-Sepharose Fast Flow (F
(Pharmacia), then adsorbed to a column packed with 10 mM
Sodium chloride in a pH 7.5 buffer containing sodium phosphate
Of the adsorbed protein by increasing the
Was. Recombinant hHGF protein has a sodium chloride concentration of about 0.7
Eluted in the fraction. This hHGF fraction is subjected to SDS-polyacrylic acid.
Analysis by luamide gel electrophoresis showed no reduction
Under conditions, a broad band with a molecular weight of about 76,000 to 92,000
And under reducing conditions, about 60,000-6
5,000 broad band, less than 56,000 molecular weight
Bands (these correspond to the H chain of the hHGF protein).
), And two bands with a molecular weight of about 32,000 to 35,000.
(These correspond to the light chain of the hHGF protein). like this
The multiplicity and broadness of the band
This is caused by heterogeneity of added sugar chains.

After substituting the buffer solution of this purified hHGF protein solution with an aqueous 0.1 mol ammonium bicarbonate solution, 1 / l of the hHGF protein mass was replaced.
Staphylococc aureus in 50 volumes
us Aureus ) V8 protease (Miles Laboratory) was added and incubated at 37 ° C overnight to decompose the peptide mixture. The mixture was subjected to reverse phase high performance liquid chromatography equipped with a C8 column (manufactured by Bacarbond, 4.6 × 250 mm) to reduce the acetonitrile concentration to 0%.
% To 60% (1% / min) and separated. Amino acid composition analysis was performed on about 10 eluted peptide peaks, and the amino acid composition of the peptide eluted at about 18 minutes was as shown in Table 1. This composition is hH
Among the amino acid sequences deduced from the nucleotide sequence of the cDNA encoding GF, the theoretical composition almost coincided with the theoretical composition of the peptide starting from glutamine at the 32nd position and ending with glutamic acid at the 41st position, counted from the read methionine.

This peptide was subjected to mass analysis by Fast Atom Bombardment Mass Spectroscopy (HX-100, manufactured by JEOL Ltd.). As a result, a peak was obtained at a molecular weight of 1321, and the molecular weight of the peptide was determined to be 1320. Since the theoretical molecular weight of the peptide ranging from glutamine 32 to glutamic acid 41 is 1337, it can be concluded that glutamine at the amino terminus has been deammonified and changed to pyroglutamic acid. That is, it was found that the N-terminal amino acid of the secreted hHGF protein was pyroglutamic acid generated by modification of glutamine at position 32.

(Effect of the Invention) By introducing an expression vector into which a hHGF gene according to the present invention is inserted into a host cell, a large amount, stable and easy expression of hHGF protein having a biological activity, which has been difficult so far, can be achieved. As a result, the hHGF protein of the present invention in which the N-terminus is modified with pyroglutamic acid is purified and purified.
You can get it.

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of the gene encoding human hepatocyte growth factor. FIG. 2 shows the amino acid sequence of human hepatocyte growth factor. In the sequence, the first to 31st (-) represent a signal peptide, Z represents G1n before modification, and represents pyroglutamic acid after modification. FIG. 3 shows the steps for constructing a vector expressing human hepatocyte growth factor. FIG. 4 shows the structure of an expression vector having DNA encoding human hepatocyte growth factor of the present invention. FIG. 5 shows the biological activity of a culture supernatant containing human hepatocyte growth factor produced by CHO cells having an expression vector having a DNA encoding human hepatocyte growth factor of the present invention. is there.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12P 21/02 C12R 1:91) (72) Inventor Takehisa Ishii 1000 Kamoshita-cho, Midori-ku, Yokohama-shi, Kanagawa Prefecture Mitsubishi Chemical Corporation Within the Research Institute (72) Inventor Kazunobu Takahashi 1000, Kamoshita-cho, Midori-ku, Yokohama-shi, Kanagawa Pref. Mitsubishi Chemical Corporation Research Institute (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 15/00-15 / 90 C12P 1/00-41/00 BIOSIS (DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] A human hepatocyte growth factor represented by the following amino acid sequence (wherein Xaa represents pyroglutamic acid).