JP3036782B2 - Method for producing human hepatocyte growth factor and transformant producing the factor - Google Patents

Description

The present invention relates to an expression vector having at least a promoter sequence necessary for protein expression, a signal peptide-like sequence, a DNA sequence encoding human hepatocyte growth factor, and a terminator sequence. The present invention relates to a transformed transformant, and a method for producing such a human hepatocyte growth factor by culturing the transformant.

(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. 138 (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 (Bioche
m. Biophys. Res. Comun.) 163: 967 (1989)). The gene of each peptide chain is encoded in the form of an H chain and an L chain peptide connected in that order. Therefore, it is transcribed on a single RNA and translated at the same time 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 thought to form a functional protein via an SS bond.

(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 hHGF protein stably and in large quantities by recombinant DNA technology, and found that a gene encoding a useful hHGF protein can be used for this purpose. A new expression vector was constructed to allow expression of hHGF protein.

That is, the gist of the present invention is a method for producing hHGF, comprising transforming a host cell with an expression vector having the hHGF gene shown in FIG. 2, and culturing the resulting transformant to produce hHGF. And a transformant producing the factor.

 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 or Escherichiacoli YA-21
Although microorganisms such as strains can be used, animal cells such as CHO cells, COS cells, mouse L cells, mouse C127 cells,
It is desirable to express the above genes using mouse FM3A cells or the like. When these cells are used as a host, a signal-like sequence contained in the DNA sequence shown in FIG. 1, that is, an immature hHGF gene containing 1 to 87 and 1 to 93 is introduced into the cells. Another advantage is that the mature hHGF protein 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, a DNA fragment obtained by linking two or three DNA fragments of the hHGF gene may be inserted.
A DNA fragment having a promoter bound to the 5 'upstream side of the fragment may be used as a unit, and two or three DNA fragments linked in the same transcription direction 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 gives methotrexate resistance (Journal of Molecular Biology (J. Mol. Biol.) 159, 601 (1982)) and the Neo gene that gives antibiotic G-418 resistance (Journal). Obmolecular Applied Genetics (J.Mol.Appl.Genet.)
1, 327 (1982)), an Ecogpt gene derived from Escherichia coli conferring mycophenolic acid resistance (Proceeding and National Academy of Sciences (Proc. Netl. Acad. S.
ci. USA) 78, 2072 (1981)) and the hph gene that confers resistance to the antibiotic hygromycin (Molecular Cell Biology (Mol. Cell. Biol.) 5: 410 (1985)). A promoter, such as the aforementioned SV40-derived promoter, is inserted 5 ′ upstream of each of these resistance genes, and 3 ′ downstream of each resistance gene,
The polyadenylation sites described above are included.

When the above-mentioned selectable marker gene is not inserted into the expression vector, a vector having a selectable marker for the transformant, for example, pSV2neo (Journal of Molecular Applied Genetics (J. Mol. Appl. Gene.
t.) 1 volume 327 pages (1982)), pMBG (Nature
e) vol. 294, p. 228 (1981)), pSV2gpt (Proceedings and National Academy of Sciences (Prec. Nat)
l. Acad. Sci. USA) 78: 2072 (1981)), pAd-D26
-1 (Journal of Molecular Biology (J. Mol.
Biol.) 159, 601 (1982)) (J. Mol. Biol. 159, 601 (1)
982)) can be double-transformed with an expression vector for the hHGF gene, and transformants can be easily selected according to 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 because the expression level can be increased about 20-fold.

The expression vector is introduced into animal cells by the calcium phosphate method (Virology, 52, 456 (1973)),
An electroporation method (J. Membr. Biol., Vol. 10, p. 279 (1972)) may be mentioned, 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 5-10% serum or in the presence of an appropriate amount 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.

(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. A Bam HI-Kpm I fragment containing hHGF cDNA (Biochemical and Biophysical Research Communication (BBRC Vol. 163 (2) pp. 967-973 (1989)), that is, 27 bases upstream from the hHGF protein translation initiation point ATG. Bam
Plasmid pUCHGF1 DNA containing a Bam HI-Kpn I fragment of about 2.3 kb covering the region from the HI cleavage point to the Kpn I cleavage point 8 bases upstream of the termination codon TAG was prepared by a conventional method ("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 resulting DNA fragment was extracted with phenol / chloroform according to a conventional method, and the DNA fragment was precipitated with ethanol.
The A 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 synthetic linker of 32 bases containing a GA and a restriction enzyme Bam HI 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.
An hHGF DNA fragment containing ATG and a stop codon TGA was separated from a non-target DNA fragment such as a vector. A 2.3 kb Bam HI-Bam HI DNA fragment encoding the desired hHGF gene was prepared from an agarose gel fragment according to the method of Maniatis et al., "(Molecular Cloning), Cold Spring Harbor Laboratory, p. 164 (1982)". did. The end of the obtained DNA fragment was blunt-ended with T4 DNA polymerase according to a conventional method, followed by phenol / chloroform extraction, and the DNA fragment was purified by ethanol precipitation and dissolved in 10 µ of 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 SamI 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.
μ of water.

0.01 μg of the DNA fragment of the pKCR vector prepared as described above
And the above-mentioned blunt-ended hHGF cDNA Bam HI fragment 0.1 μm
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.

These recombinants were prepared according to the method of Maniatis et al. (“Molecular Cloning”, Cold Spring Harbor Harbor).
Laboratory, pp. 86-96 (1982)), a plasmid in which the hHGF gene was forward-linked to a restriction enzyme SmaI cleavage site located between the promoter and polyadenylation site of the expression vector pKCR, pK
The CRHGF-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 hHGFcDANs were inserted into the restriction enzyme BamHI cleavage site on page 27 (1981) was prepared by the method of Maniatis et al. (“Molecular Cloning”, Cold Spring Harbor Laboratory, pages 86 to 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, recombinant E. coli and pAd-D26- having plasmid pSV2neo (Journal of Applied Genetics, vol. 1, p. 327 (1982)) encoding a marker for transformant cell selection
1 (Journal of Molecular Biology (Jo
urnal of molecular biology) Vol. 159, p. 601 (1982))
The plasmid DNA was recovered and purified from recombinant Escherichia coli having E. coli according to the method of Maniatis et al.

Using the obtained three types of plasmid DNA, the method of Ausubel et al. (Current Protocols in Molecular Biolog
y), Green Publishing Associates and Wheelie Interscience
Associates and Wiley-Interscience) Chapter 9.1.1
Based on 9.1.1 (1987), CHO cells were double-transformed to transform CHO cells.

That is, first, CHO cells were cultured in an ERDF medium (manufactured by Kyokuto Pharmaceutical Co., Ltd.) containing 10% FCS (fetal calf serum) in a petri dish having a diameter of 9 cm until the cells 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, 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 DAN solution by vigorous mixing for several seconds using a vortex mixer. 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. 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 of about 3.1 cm ² for one culture hole,
Each was re-cultured for about 7 days in 1 ml of ERDF medium containing 10% FCS.

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 as a sample, SD
S-polyacrylamide gel electrophoresis was performed.

This was analyzed by Western blotting according to a conventional method to confirm the expression of hHGF protein.

Gohda et al.'S method (Experimental Cell
Research (Experimental Cell Rescerch) 166 pages 139-
HHGF activity was measured according to page 150 (1986) to confirm the presence of biological activity.

Furthermore, the obtained cell lines were individually 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 the 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
(Nature 294,228 (1981)) from the recombinant E. coli according to the method of Maniatis et al.
Was recovered and purified.

Using the obtained two types of plasmid DNAs, the method of Ausubel et al. (Current Protocols in Molecular Biolog
y), Green Publishing Associates and Wheelie Interscience
Assoiates and Wiley-Interscience) Chapter 9.1.1
Based on Section 9.1.4 (1987), three of the cell lines that successively express the hHGF protein obtained in Example 1- [II] and which have a high hHGF expression level (B) -1, B-27, B
-102) were isolated and they were individually double transformed 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
Each cell of m dish was divided into 10 pieces of 9 cm 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
Research (Experimental Cell Reserch) Volume 166 139-1
The hHGF activity was measured according to page 50 (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.

(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, it is possible to produce hHGF protein having a biological activity, which has been difficult so far, in a large amount, stably and easily. Becomes possible.

[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 deduced from the gene encoding human hepatocyte growth factor. 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. It is.

Continued on the front page (72) Inventor Yoshiko Yoshiyama 1000 Kamoshita-cho, Midori-ku, Yokohama-shi, Kanagawa Prefecture Within Mitsubishi Chemical Co., Ltd. (72) Inventor Takehisa Ishii 1000 Kamoshita-cho, Midori-ku, Yokohama-shi, Kanagawa Mitsubishi Stock (72) Inventor Kazunobu Takahashi 1000, Kamoshida-cho, Midori-ku, Yokohama-shi, Kanagawa Prefecture Mitsubishi Chemical Corporation (56) References JP-A-63-22526 (JP, A) JP-A-3-3 130091 (JP, A) Clin. Invest. , 1988, Vol. 81, p. 414-419 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15/00-15/90 BIOSIS (DIALOG) WPI (DIALOG) GenBank / EMBL / DDBJ

Claims (4)

(57) [Claims] 1. An animal cell or yeast is transformed with an expression vector having a gene encoding human hepatocyte growth factor represented by the following amino acid sequence, and the resulting transformant is cultured to grow human hepatocyte: A method for producing human hepatocyte growth factor, which comprises producing the factor. 2. An animal cell or yeast is transformed with an expression vector having a gene encoding human hepatocyte growth factor represented by the following nucleotide sequence, and the resulting transformant is cultured to grow human hepatocyte: A method for producing human hepatocyte growth factor, which comprises producing the factor. 3. A transformant obtained by transforming an animal cell or yeast with an expression vector having a gene encoding human hepatocyte growth factor represented by the following amino acid sequence: A method for producing human hepatocyte growth factor, comprising repeatedly transforming using an expression vector having a gene encoding a growth factor, and culturing the resulting transformant to produce human hepatocyte growth factor. . 4. An animal cell producing human hepatocyte growth factor obtained by transforming with an expression vector having a gene encoding human hepatocyte growth factor represented by the following amino acid sequence: