JPH03204899A - Human liver-regenerating factor, dna base sequence thereof, plasmid and transformant containing the same sequence and recombinant liver regenerating factor - Google Patents

Abstract

NEW MATERIAL:A liver-regenerating factor having a molecular weight (SDS-PAGE analysis) of 96000-82000 and divided into subunits alpha, beta1 and beta2 when reduced in the presence of mercapto ethanol. The subunit alpha: having a molecular weight of approximately 62000 and at least amino acid sequences of formulas I to VI; the subunit beta1: having a molecular weight of 34000 and a sequence of formula VII as the amino acid sequence ranging from the N terminal to the twenty fifth amino acid; the subunit beta2: having a molecular weight of 32000, the amino acid sequence ranging from the N terminal to the twenty fifth amino acid being coincident with that of the beta1. USE:A remedy for the patients of hepatic diseases and for liver-resected patients, and a reagent for researches relating to hepatic cells. PREPARATION:For example, a patient plasma prepared from a fulminant hepatitis patient by a plasmapheresis therapy is diluted with a buffer solution, filtered with a glass filter and gel-filtered. The separated active fraction is applied to an immobilized heparin column, eluted with a linear gradient and subsequently purified by a reverse phase high performance liquid chromatography to provide the liver-regenerating factor.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to human liver regeneration factor (hepatocyte growth factor, HEPA).
tocyte growth factor; h-
HGp), more specifically, a new proteinaceous substance derived from human serum that enables the proliferation of hepatocytes, a novel NA base sequence (gene) encoding this, a plasmid for expressing h-HGF containing the gene, The present invention relates to a transformant transformed with the plasmid, recombinant h-HGF obtained by culturing the transformant, and a method for producing the same.

Conventional technology and its problems The liver is an organ with a strong regenerative ability. For example, even if approximately two-thirds of a rat's liver is removed, the remaining tissue begins to rapidly proliferate, and after about two weeks, the liver will grow. It is known that it returns to its original size, and this fact can be used in humans to wait for the growth of normal liver tissue left behind after partial resection of liver tissue in patients with fulminant hepatitis or liver cancer. Treatment is in progress. Regarding the liver proliferation (liver regeneration) mechanism mentioned above,
Various studies have been conducted in the past, and it has been suggested that some humoral liver regeneration factors appear in the blood of rats after liver resection.
wth factor: r-HGF) (7) Various examples of successful partial purification have also been reported. It has also been reported that the above factors are present in the serum of rats with liver damage caused by carbon tetrachloride, thioacetamide, etc.

Although the existence of a humoral liver regeneration factor similar to that in rats is presumed to exist in humans, the existence of this factor has not yet been confirmed or proven.

The present inventors have been conducting intensive research on the human liver regeneration factor, and in the process, they have obtained the knowledge that serum from patients with certain liver diseases such as fulminant hepatitis has high hepatocyte proliferation activity. Based on this, the active substance (h-HGF) was successfully isolated and purified.

Further, as a result of subsequent research, we elucidated the partial amino acid sequence of h-HGF, synthesized an oligonucleotide encoding it, and used the oligonucleotide as a probe.
We have newly succeeded in isolating h-HGF cDNA, and here we have h-HGF cDNA.
We have elucidated the DNA sequence (gene) that encodes HGF, and have succeeded in a series of research and development efforts to produce an h-HGF expression plasmid containing the sequence, a transformant carrying the plasmid, and recombinant h-HGF by culturing the same. ,
The present invention has now been completed.

Means for Solving the Problems According to the present invention, (1) the molecular weight (according to SDS-PAGE analysis) is in the range of 96,000 to 82,000 and is mainly between 92,000 and 84,000, and (2) in the presence of mercaptoethanol. By the reduction treatment, the following subunit α and subunit β1 or β2 are separated, and (3) p
Asp l Q 6Thr 48Ser 51Gl whose amino acid composition is as follows based on h e (22 mol)
u 87 Gly 76Ala 31Val
33 Met 17 11e 4QLe
u 5Q Tyr 3(3Phe 22LY
S 58 H4S 27 Arg 52
A liver regeneration factor is provided.

Subunit α: (1) Molecular weight (according to SDS-PAGE analysis) is approximately 62
000; (2) has at least the following amino acid sequence; (i) C
ys-Gin-Arg-Trp(ii) Asn-Me
t-G 1u-Asp-Le u-Hi s-Arg-
His-I1e-Phe-Trp-Glu-P
ro-Asp-Ala-3er-Lys (plane A sn-
Tyr-Me t-G 1 y-Asn-Leu-Se
r-G l n -Th r-Arg-5er-Gly
-Leu-Thr-Cys-Ser-Met-Trp-
Asp-Lys($As p-Leu-Arg-G
1 u-Asn-Tyr-Cys-Arg-Asn-P
ro-Asp-Gly-5er-Glu-5er-Pr
o-Trp-Cys-Phe-ThrThr-Asp-
Pro-Asn-I 1e-Arg-Val-Gly-
Tyr-Cys-5er-Gln-11e-Pro-A
sn(y)Gly-Phe-Asp-Asp-Asn-
Tyr-Cys-Arg-Asn-Pr.

Asp-Gly-Gln-Pro-Arg-Pro-T
rp-Cys-Tyr-ThrLeu-Asp-Pro
-His-Thr-Arg-Trp-Glu-Tyr-
Cys-Ala-11e-Lys (y9 Leu-Asn-Glu-Asn-Tyr-C
ys-Arg-Pro-Asp-Asp-Asp-Al
a-Hls-Gly-Pro-Trp-Cys-Tyr
-Thr-GlyAsn-Pro-Leu-I 1e-
Pro-Trp-Asp-Tyr-Cys-Pr.

11e-5er-Arg-C:ys-Glu-Gly(
3) Co+c having the following amino acid composition based on Phe (18 mol) 38. 5 Asp 87. 5T
hr 37. l Ser 32. 7Gl
u60. 8 G')' 43. 0Ala
14. 9 Val 13. 3Met
IQ, 4 11e 22.4Leu 23.
7 Tyr 23. 5Ph” 18.OL
ys 41.4His 20. 8Arg 36
．． 8 subunit β1; (1) Molecular weight (according to SDS-PAGE analysis) is approximately 34
(1) The molecular weight (according to SDS-PAGE analysis) is as follows: Val-Val-Asn-Gly-I 1e-Pro-
Thr-Arg-Thr-Asn-Ile-Gly-
Trp-Met-Val-5er-Leu-Arg-T
yr-ArgAsn-Lys-His-11e-Cys
-(3) Cmc 12. having the following amino acid composition based on Phe (4 mol). 2 Asp 28. 7Th
r12. 3 Ser 19. 4Glu
28. I G'Y 41. IAla
15. 3 Val 22. QMet
3.2 He 15.7Leu 26.
I T)'r 14. 6Phe 4. Q
Lys IB, 5His 9.2Arg
19°0 subunit β2: (1) Molecular weight (according to SDS-PAGE analysis) is approximately 32
000, (1) The molecular weight (by SDS-PAGE analysis) is as follows and matches that of β1, Val-Val-A
sn-Gly-11e-Pro-Thr-Arg-Th
r-AsnI le-Gly-Trp-Met-Val
-5er-Leu-Arg-Tyr-Arg-Asn-
Cmc 15. having the following amino acid composition based on Lys-His-11e-Cys-(3)Phe (4 mol). 0 Asp 32. 4th
r13. Q Ser 21. 0 Glu
31. OG'Y, 44. 2Ala 15.
7 Val 24. 1Met 3.9
11e 17.8Leu 29. 5 Ty
r15. 7Phe 4. OLys 21.
6His 10. 3 Arg 21. 3 The abbreviations for amino acids, peptides, base sequences, nucleic acids, etc. in this specification above and below are IUPAC, I
It shall comply with the UB regulations or the display method commonly used in the field.

The above characteristics and other properties of the liver regeneration factor (hereinafter abbreviated as rh-HGFJ) of the present invention will be described in detail in Examples below.

The h-HGF of the present invention can be used as a therapeutic agent for liver diseases such as acute hepatitis, chronic hepatitis, cirrhosis, and fulminant hepatitis, or as a therapeutic agent after hepatectomy, and as an antigen for establishing immunological diagnosis of each of the above-mentioned diseases. It is useful as such. Furthermore, by using the h-HGF, hepatocytes derived from various animals including humans can be treated with the h-HGF.
Hepatocytes that can be extremely easily grown and maintained in vitro in the presence of HGF, and thus grown and maintained, can be used for basic research such as liver function, and for studying the effects of various hormones or drugs on hepatocytes. They are useful for research, screening tests for drugs for treating liver diseases, etc., and are also useful as host cells for carcinogenesis tests and in vitro culture of hepatitis viruses. The present invention provides such useful physiologically active substances.

Hereinafter, the method for producing h-HGF of the present invention will be described in detail.

The h-HGF of the present invention can be isolated more efficiently and in a higher yield than the serum of patients with certain liver diseases, particularly from the serum of patients with fulminant hepatitis. The serum used as a raw material here can be obtained according to a conventional method.

The production of the h-HGF of the present invention from the above-mentioned raw materials is basically the same as the usual method commonly used for separating proteinaceous substances from this type of biological material. , can be carried out according to various processing operations using chemical properties. The processing operations include, for example, treatment with a normal protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), centrifugation, electrophoresis, ion exchange chromatography, affinity chromatography, reversed phase chromatography, and adsorption. Examples include chromatography, dialysis, and combinations thereof. As a particularly preferred example of the above-mentioned operation, first, the raw serum is subjected to cation exchange chromatography to collect active peaks, and then this is subjected to affinity chromatography using, for example, heparin, which specifically binds to HGF. The active peak can be purified by subjecting it to reverse phase chromatography. The operations, conditions, etc. of each of the above-mentioned treatments can be the same as those in ordinary methods of this type, and thus the present invention h-
HGF has been isolated and purified, and is characterized by the properties described above.

In addition, the h-HGF of the present invention is purified as a single product by the above-mentioned purification operation, and then purified with 2-mercaptoethanol (2-ME
), dithiothreitol (DTT), etc., subunit α and subunit β are
1 or β2, and from this, α
It is confirmed that it is composed of two subunits: chain and β chain.

The reduction treatment for isolation of each of the above subunits was performed using 2-M
It can be carried out according to conventional methods using various drugs including E and DTT. More preferably, DTT is reduced in an amount of about 50 times the molar amount of the disulfide bonds of h-HGF in a solution of a denaturing agent such as guanidine hydrochloride, and then about twice the amount of the denaturing agent is used. After blocking the free sulfide groups using iodoacetamide,
Each subunit can be separated and obtained by various chromatographic operations, more preferably by reverse phase chromatography.

The physical properties (molecular weight, amino acid sequence, amino acid composition, etc.) of the h-HGF of the present invention and its subunits thus obtained are determined according to the method detailed in the Examples below.

Furthermore, the activity of h-HGF of the present invention can be measured, for example, by the following method. That is, hepatocytes separated from the liver are spread on a plate and cultured.

Various mammalian livers such as rat liver and human liver can be used as the liver, but considering its ease of availability and the fact that HGF research has started from rats, it is recommended to use rat liver. preferable. The above-mentioned separation of hepatocytes from the liver can be carried out by, for example, a conventional method using a suitable enzyme such as collagenase. The initial culture of the liver is preferably carried out for about 6 to 24 hours, and the cell density on the plate is preferably about 5 x 104/cm2. As the medium for the above-mentioned culture, any ordinary cell culture medium can be used, and it is particularly preferable to use Williams E medium or the like. Add the sample to the plate that was initially cultured above. The amount added is 50μ! It is better to set it as a degree.

After culturing the sample-added plate for about 6 to 24 hours, preferably about 12 hours, under the same conditions as above, incubate the plate with a nucleobase labeled with a radioactive element, such as deoxyuridine labeled with ① or thymidine labeled with H. The degree of increase in DNA synthesis in hepatocytes after addition of the sample is measured by adding such substances and measuring the degree of their incorporation into the cell nucleus. The amount of the labeled nucleobase added is preferably about 1 μCi.

Further, according to the present invention, it is expressed by the following formula (1), and the present invention h
- represented by the following formula (2) containing the DNA base sequence of formula (1) together with a new NA base sequence related to HGF,
A novel NA base sequence encoding h-HGF of the present invention (
The present invention provides an expression plasmid and a transformant containing the gene (hereinafter simply referred to as "the gene of the present invention"), and a recombinant h-HGF produced by culturing the transformant.

Formula (1): % formula %: GTTCAATGTGGGACAAGAACATGGA
AGACTTACATCGTCATATCTTCTGG
GAACCAGATGCAAGTAAGCTGAATG
AGAATTACTGCCGAAATCCAGATGA
TGATGCTCATGGACCC:TGGTGCTA
CACGGGAAATCCACTCATTCCTTG
GATTATTGCCCTATTTCTCGTTGTG
AAGGTGATAACCACACCTACAATAGT
CAATTTAGACCATCCCGTAATATCT
TGTGCCAAACGAAACAATTGCGAG
TTGTAAATGGGATTCCAACACGAAC
AAACATAGGATGGATGGTTAGTTTG
AGATACAGAAATAAAACATATCTGCG
GAGGATCATTGATAAAAGGAGAGTTG
GGTTCTTACTGCACGACAGTGTTTC
CCTTCTCGAGACTTGAAAAGATTATG
AAGCTTGGCTTGGAATTCATGATGT
CCACGGAAGAGGGAGATGAGAAATG
AAACAGGTTCTCAATGTTTCCCAGC
TGGTATATGGCCCTGAAGGATCAGA
TCTGGTTTTAATGAAGCTTGCCAGG
CCTGCTGTCCTGGATGATTTTGTTA
GTACGATTGATTTACCTAATTATGG
ATGCACAATTCCTGAAAAGACCAGT
TGCAGTGTTTATGGCTGGGGCTACA
CTGGATTGATCAACTATGATGGCCT
ATTACGAGTGGCACATCTCTATATA
ATGGGAAATGAGAAATGCAGCCAGC
ATCATCGAGGGGAAGGTGACTCTGAA
TGAGTCTGAAAATATGTGCTGGGGCT
GAAAAGATTGGTCAG Formula (2): % formula % AATGAGAAATGCAGCCAGCATCATC
GAGGGAAGGTGACTCTGAATGAGTC
TGAAAATATGTGCTGGGGCTGAAAAG
ATTGGATCAGGACCATGTGAGGGGGG
ATTATGGTGGCCCACTTGTTGTGA
GCAACATAAAATGAGAATGGTTCTT
GGTGTCATTGTTCCTGGTCGTGGAT
GTGCCATTCCAAATCGTCCTGGTAT
TTTTGTCCGAGTAGCATATTATGCA
AAATGGATACACAAAAATTATTTTAA
CATATAAGGTACCACAGTCATAGCT
GAAGTAAGTGTGTCTGAAGCACCCA
CCAATACAACTGTCTTTTACATGAA
GATTTCAGAGAATGTGGAATTTAAA
ATGTCACTTACAACAATCCTAAGAC The DNA base sequence represented by the above formula (1) is h-HG
It is related to F, that is, it encodes at least a part of the amino acid sequence of h-HGF of the present invention, and it also has the formula (
The gene represented by 2) is the gene of the present invention. All of these contain genetic information that enables the production of recombinant h-HGF by genetic engineering techniques, and from this point they can be advantageously used in the production of recombinant h-HGF.

In particular, the gene of the present invention as well as the DNA of the above formula (1)
The base sequence includes a sequence encoding the active center of h-HGF, and by genetic engineering techniques using this, active or combinant h-HGF can be produced.

In the following, the production of the DNA base sequence of the present invention (gene of the present invention) will be explained in detail.
It can be prepared from mRNA isolated from the cells.

Isolation of mRNA from the above cells is basically carried out according to normal extraction procedures. More specifically, the above cells are
First, for example, CEM medium, CMRL-1066 medium, DM
-160 medium, Eagle's minimum essential medium (Eagle'
s MEM), - Fisher's medium (Fisher's medium)
's Medium), F-10 medium, F-12 medium, L-15 medium, NCTC-109 medium, RPMI-
1640 medium etc. or fetal bovine serum (FCS) as necessary.
) and serum components such as albumin, at a concentration range of about 1 x 104 to 1 x 10 cells/xll, according to normal culture methods such as carbon dioxide gas culture, at about 30 to 40 °C, The culture is preferably carried out at about 37° C. for 1 to 5 days. Next, when the target substance is produced and accumulated in the culture supernatant, the cultured cells are treated with a suitable surfactant such as SDS, NP-40, Triton X100, deoxycholic acid, etc., or by using a homogenizer. After partially or completely destroying and solubilizing the chromosomal DNA by physical methods such as freezing and thawing,
(7n(POLYTRON, KinematicaS
The mixture is sheared to a certain extent using a mixer (such as a syringe (e.g., Witzerland), etc.) or a syringe, and then the protein and nucleic acid fractions are separated and total RNA is extracted. This extraction operation requires
Guanidinium/cesium chloride method [Guanid
inium / Cesium Chloride m
ethod.

T, Maniatis, E, F, Fr1tsc.
h and J, Sambrook.

Molecular Cloning, p194-1
96 (Cold Spring Harbor Lab
Oratory), 19823 etc. are available for general use.

In addition, in each of the above methods, in order to prevent RNA degradation by RNase, for example, RNase inhibitors, heparin,
Diethylpyrocarbonate, vanadium complex, etc. can also be used in addition.

Separation of mRNA from RNA obtained according to the above procedure,
Purification can be carried out using, for example, oligo dT-cellulose [Collaborative Research Co., Ltd.
It can be carried out by an adsorption column method or a batch method using polyU-Sepharose, manufactured by Pharmacia Co., Ltd., etc.

Alternatively, commercially available mRNA may be used instead of such extraction procedure.

That is, it is also possible to use human placental poly(A)'' RNA.

Purification, concentration, and identification of mRNA for h-HGF of interest can be accomplished, for example, by fractionating the obtained mRNA by sucrose density gradient centrifugation, etc., and injecting the fraction into a protein translation system, such as African frog oocytes. This can be carried out by injecting the protein into a protein using a cell-free system such as rabbit reticulocyte lysate or wheat germ, and then examining the h-HGF activity of the protein. In this way, the presence of the target mRNA can be confirmed. Furthermore, the target mRNA can be confirmed by an immunization method using an antibody against h-HGF instead of the above h-HGF activity measurement.

Since the purified mRNA obtained above is usually unstable,
This is converted into stable cDNA and connected to a replicon derived from a microorganism to enable amplification of the target gene. The in vitro conversion of mRNA into cDNA, that is, the synthesis of the gene of the present invention, can be carried out using general methods, such as the Okayama Berg method [H. Okayama and P., Be.
rg, Molecular and Cellular
rBiology, vol, 3. p280 (19
83)) and the Doubler-Hoffman method (V, Gubler a
nd B, J, Hoffman, Gene, vol.
.

25, p. 263-269 (1983) 3, etc. More specifically, first use oligo dT as a primer (this primer may be a vector primer with poly dT added), then use mRNA as a template to generate dN.
In the presence of T P (dATP, dGTP, dcTP or dTTP), reverse transcriptase is used to synthesize complementary single-stranded cDNA from mRNA. The next step differs as follows depending on whether oligo dT or vector primers were used in the above.

In the former case, the template mRNA is degraded and removed by alkaline treatment, etc., and then the double-stranded DNA is converted into double-stranded DNA using reverse transcriptase or DNA polymerase (■) using the double-stranded DNA as a template.
Create A. Next, both ends of the double-stranded DNA obtained are treated with exonuclease, an appropriate linker DNA or a plurality of bases in an annealing possible combination are added to each of them, and this is inserted into an appropriate vector, such as an EK-based plasmid vector or a λgt Incorporate into a phage vector, etc.

In the latter case, an open circular plasmid with a plurality of annealing-capable combinations of bases added to it while leaving mRNA as a template, and a linker DNA (L
After annealing a DNA fragment containing a region that can autonomously replicate in animal cells and an mRNA transcription promoter region to form a closed circle, RNase H and DNA polymerase I are allowed to coexist in the presence of dNTPs. replace the mRNA with a DNA strand to create a complete plasmid DNA.
A can be created.

The DNA obtained as described above is transferred to a vector host, such as Escherichia coli (Escherichia coli).
ia coli), Bacillus subtilis (Ba
cillus 5ubtilis), Saccharomyces cerevisi 7
evisiae) and transform the host. The introduction of this DNA into a host and the transformation method using the same can be carried out by a commonly used method, for example, by collecting cells mainly in the logarithmic growth phase and injecting CaCl2
It is possible to employ methods such as processing the DNA to make it easy to naturally incorporate the plasmid.

In the above method, MgCl2 or RbCA' may be further coexisting in order to further improve the efficiency of transformation, as is generally known. It is also possible to adopt a method in which host cells are transformed into sphnyloplasts or protoplasts and then transformed.

From the transformed strain obtained above, the desired h-HGF
In order to select a strain having the cDNA, for example, various methods shown below can be employed.

(1) Screening method using synthetic oligonucleotide probes When all or part of the amino acid sequence of the target protein has been elucidated (the sequence may be from any region of the target protein as long as it is a specific sequence with multiple contiguous sequences) ), to synthesize an oligonucleotide corresponding to the amino acid, which may be either a base sequence derived using codon usage frequency or a plurality of possible combinations of base sequences, and in the latter case, inosine may be included and the type Using this as a probe (labeled with P or S), the DNA of the transformed strain is hybridized to a denatured and fixed nitrocellulose filter, and the resulting positive strains are searched and selected. do.

(2) Method for screening by producing h-HGF in animal cells. A transformed strain is cultured, the gene is amplified, and the gene is transfected into animal cells. In this case, h-HGF is capable of self-replication and contains the mRNA transcription promoter region. Either a plasmid or a plasmid that infects animal cell chromosomes), produces the protein encoded by the gene, and extracts h-H from the culture supernatant or cell extract.
A strain having a cDNA encoding the desired h-HGF is selected from the original transformed strain by measuring GF activity or detecting h-HGF using an antibody against h-HGF.

(3) Method of selection using an antibody against h-HGF Introduce the cDNA in advance into a vector that can express the protein in the transformed strain, produce the protein in the transformed strain, and
Using an antibody against HGF and a second antibody against the antibody, an h-HGF producing strain is detected to obtain a target strain.

(4) Method using selective hybridization/translation system cDNA obtained from the transformed strain is plotted on a nitrocellulose filter, and mR from h-HGF producing cells is
After hybridization of NA-1, mRNA corresponding to cDNA is recovered. The recovered mRNA is translated into protein using a protein translation system, such as injection into African frog oocytes or a cell-free system such as rabbit reticulocyte lysate or wheat germ, and the h-HGF activity of the protein is examined. or detect using an antibody against h-HGF to obtain the desired strain.

The method of collecting the gene of the present invention from the obtained transformed strain of interest is a general method, for example, separating a fraction corresponding to plasmid DNA from cells, and extracting cDNA from the plasmid DNA.
This can be done by cutting out area A.

Thus, the DNA base sequence of formula (1) and formula (2)
The gene of the present invention can be obtained. These are each expressed by the following formula (3
) encodes an h-HGF-related polypeptide with a 588 amino acid sequence shown in the following formula (4) and an h-HGF polypeptide with a 728 amino acid sequence shown in the following formula (4).

Formula (3): %Formula% Arg-Trp-Glu-Tyr-Cys-Ala-1
1e-Lys-Thr-Cys-Ala-Asp-As
n-Thr-Met-Asn-Asp-Thr-Asp
-Val-Pro-Leu-Glu-Thr-Thr-
Glu-Cys-11e-Gln-Gly-Gln-G
ly-Glu-Gly-Tyr-Arg-Gly-Th
r-Val-Asn-Thr-Ile-Trp-Asn
-Gly(le-Pro-Cys-Gln-Arg-T
rp-Asp-Ser-Gln-Tyr-Pro-Hi
s-Glu-His-AspMet-Thr-Pro-
Glu-Asn-Phe-Lys-Cys-Lys-A
spLeu-Arg-Glu-Asn-Tyr-Cys
-Arg-Asn-Pro-Asp-Gly-5er-
Glu-5er-Pro-Trp-Cys-Phe-T
hr-Thr-Asp-Pro-Asn-Ile-Ar
g-Val-Gly-Tyr-Cys-5er-Gln
-11e-Pro-Asn-Cys-Asp-Met-
3er-His-Gly-Gln-Asp-Cys-T
yr-Arg-Gly-Asn-Gly-Lys-As
nTyr-Met-Gly-Asn-Leu-8er-
Gln-Thr-Arg-SerGly-Leu-Th
r-Cys-Ser-Met-Trp-Asp-Lys
-Asn-Met-Glu-Asp-Leu-Hls-
Arg-)(is-11e-Phe-Trp-Glu-
Pro-Asp-Ala-5er-Lys-Leu-A
sn-Glu-Asn-Tyr-Cys-Arg-As
n-Pro-Asp-Asp-Asp-Ala-His
-Gly-Pro-Trp-Cys-Tyr-Thr-
Gly-Asn-Pro-LeuAsn-Glu-Ly
s-Cys-5er-Gin-His-His-Arg
-GlyLys-Val-Thr-Leu-Asn-G
lu-5er-Glu-Ile-Cys-Ala-Gl
y-Ala-Glu-Lys-11e-Gly-3er
(4): Met-Trp-Val-Thr-Lys-Leu-L
eu-Pro-Ala-Leu-Leu-Leu-Gl
n-His-Val-Leu-Leu-His-Leu
-Leu-Leu-Leu-Pro-11e-Ala-
11e-Pro-Tyr-Ala-Glu-Gly-G
ln-Arg-Lys-Arg-Arg-Asn-Th
r-11e-His-Glu-Phe-Lys-Lys
-3er-Ala-Lys-Thr-Thr-Leul
le-Lys-11e-Asp-Pro-Ala-Le
u-Lys(le-Lys-Thr-Lys-Lys-
Val-Asn-Thr-Ala-Asp-Gln-C
ysAla-Asn-Arg-Cys-Thr-Arg
-Asn-Lys-Gly-Leu-Pro-Phe-
Thr-Cys-Lys-Ala-Phe-Val-P
he-Asp-Lys-Ala-Arg-Lys-Gl
n-Cys-Leu-Trp-Phe-Pro-Phe
-Asn-3er-Met-5er-5er-Gly-
Val-Lys-Lys-Glu-Phe-Gly-H
is-Glu-Phe-Asp-Leu-Tyr-Gl
u-Asn-Lys-Asp-Tyr-11e-Arg
-Asn-Cys-11e-11e-Gly-Lys-
Gly-Arg-5er-Tyr-Lys-Gly-T
hr-Val-Ile-Pro-Trp-Asp-Ty
r-Cys-Pro-11e-5er-ArgCys-
Glu-Gly-Asp-Thr-Thr-Pro, -
Thr-11e-Val-Asn-Leu-Asp-H
is-Pro-Val-11e-3er-Cys-Al
aLys-Thr-Lys-Gln-Leu-Arg-
Val-Val-Asn-Gly-Ile-Pro-T
hr-Arg-Thr-Asn-Ile-Gly-Tr
p-Met-Val-3er-Leu-Arg-Tyr
-Arg-Asn-Lys-His-Ile-Cys-
Gly-Gly-8er-Leu-I 1e-Lys-
Glu-3er-Trp-Val-Leu-Thr-A
la-Arg-Gln-Cys-Phe-Pro-5e
t-Arg-Asp-Leu-Lys-Asp-Tyr
-Glu-Ala-Trp-Leu-Gly-I 1e
-Hls-Asp-Val-His-Gly-Arg-
Gly-Asp-Glu-Lys-Cys-Lys-G
ln-Val-Leu-Asn-Val-5er-Gl
n-Leu-Val-Tyr-Gly-Pro-Glu
-Gly-Ser-Asp-Leu-Val-Leu-
Met-Lys-Leu-Ala-Arg-Pro-A
Ia-Val-Leu-Asp-Asp-Phe-Va
l-5er-Thr-11e-Asp-Leu-Pro
-Asn-Tyr-Gly-Cys-Thr(le-P
ro-Glu-Lys-Thr-Ser-Cys-Se
r-Val-Tyr・Gly-Trp-Gly-Tyr
-Thr-Gly-Leu-Ile-Asn-Tyr-
Asp-Gly-Leu-Leu-Arg-Val-A
la-His-Leu-Tyr-11e-Met-Gl
y-Ser-11e-Thr-Lys-5er-Gly
-11e-Lys-Cys-Gln-Pro-Trp-
5er-8er-Met-11e-Pro-His-G
lu-His-3er-Phe-Leu-Pro-5e
r-9er-Tyr-Arg-Gly-Lys-Asp
-Leu-Gln-Glu-Asn-Tyr-Cys-
Arg-Asn-Pr.

Arg-Gly-Glu-Glu-Gly-Gly-P
ro-Trp-Cys-Phe-Thr-5er-As
n-Pro-Glu-Val-Arg-Tyr-Glu
-Val-Cys-Asp-Ile-Pro-Gln-
Cys-5er-Glu-Val-Glu-Cys-M
et-Thr-Cys-Asn-Gly-Glu-Se
r-Tyr-Arg-Gly-Leu-Met-Asp
-His-Thr-Glu-5er-Gly-Lys-
11e-Cys-Gln-Arg-Trp-Asp-H
is-Gln-Thr-Pro-His-Arg-Hi
s-Lys-Phe-Leu-Pro-Glu-Arg
-Tyr-Pro-Asp-Lys-Gly-Phe-
Asp-Asp-Asn-Tyr-Cys-Arg-A
sn-Pro-Asp-Gly-Gln-Pro-Ar
g-Pro-Trp-Cys-Tyr-Thr-Leu
-Asp-Pro-His-Thr-Arg-Trp-
Glu-Tyr-Cys-Ala-11e-Lys-T
hr-Cys-Ala-Asp-Asn-Thr-Me
t-Asn-Asp-Thr-Asp-Val-Pro
-Leu-Glu-Thr-Thr-Glu-Cys-
11e-Gln-Gly-Gln-Gly-Glu-G
ly-Tyr-Arg-Gly-Thr-Val-As
n-Thr-11e-Trp-Asn-Gly-Ile
-Pro-Cys-Gln-Arg-Trp-Asp-
5er-Gln-Tyr-Pro-Hls-Glu-H
is-Asp-Met-Thr-Pro-Glu-As
n-Phe-Lys-Cys-Lys-Asp-Leu
-Arg-Glu-Asn-Tyr-Cys-Arg-
Asn-Pro-Asp-Gly-5er-Glu-5
er-Pro-Trp-Cys-Phe-Thr-Th
r-Asp-Pro-Asn(Ie-Arg-Val-
Gly-Tyr-Cys-5er-Gln-11e-P
ro-Asn-Cys-Asp-Met-5er-Hi
s-Gly-Gln-AspCys-Tyr-Arg-
Gly-Asn-Gly-Lys-Asn-Tyr-M
et-Gly-Asn-Leu-5er-Gln-Th
r-Arg-Ser-Gly-Leu-Thr-Cys
-5er-Met-Trp-Asp-Lys-Asn-
Met-Glu-Asp-Leu-His-Arg-H
is-11e-Phe-Trp-Glu-Pro-As
p-Ala-Ser-Lys-Leu-Asn-Glu
-Asn-Tyr-Cys-Arg-Asn-Pro-
Asp-Asp-Asp-Ala-His-Gly-P
ro-Trp-Cys-Tyr-Thr-Gly-As
n-Pro-Leu(le-Pro-Trp-Asp-
Tyr-Cys-Pro(le-5er-Arg-Cy
s-GluGly-Asp-Thr-Thr-Pro-
Thr-Ile-Val-Asn-LeuAsp-Hi
s-Pro-Val-11e-3er-Cys-Ala
-Lys-Thr-Lys-Gln-Leu-Arg-
Val-Val-Asn-Gly-11e-Pro-T
hr-Arg-Thr-Asn-11e-Gly-Tr
p-Met-Val-Ser-Leu-Arg-Tyr
-Arg-Asn-Lys-)(is-11e-Cys
-GlyGly-5er-Leu-I 1e-Lys-
Glu-5er-Trp-Val-Leu-Thr-A
la-Arg-Gln-Cys-Phe-Pro-Se
r-Arg-AspLeu-Lys-Asp-Tyr-
Glu-Ala-Trp-Leu-Gly-I 1e-
His-Asp-Val-Hls-Gly-Arg-G
ly-Asp-Glu-Lys-Cys-Lys-Gl
n-Val-Leu-Asn-Val-5er-Gln
-Leu-Val-Tyr-Gly-Pro-Glu-
Gly-3er-Asp-Leu-Val-Leu-M
et-Lys-Leu-Ala-Arg-Pro-Al
a-Val-Leu-Asp-Asp-Phe-Val
-Ser-Thr-Ile-Asp-Leu-Pro-
Asn-Tyr-Gly-Cys-Thr-11e-P
ro-Glu-Lys-Thr-3er-Cys-5e
r-Val-Tyr-Gly-Trp-Gly-Tyr
-Thr-Gly-Leu-I 1e-Asn-Tyr
-Asp-Gly-Leu-Leu-Arg-Val-
Ala-Hls-Leu-Tyr-11e-Met-G
ly-Asn-Glu-Lys-Cys-3er-Gl
n-3(is-Hls-Arg-Gly-Lys-Va
l-Thr-Leu-Asn-Glu-5er-Glu
ile-Cys-Ala-Gly-Ala-Glu-L
ys-Ile-Gly-9er-Gly-Pro-Cy
s-GluGly-Asp-Tyr-Gly-Gly-
Pro-Leu-Val-Cys-Glu-Gin-H
ls-Lys-Met-Arg-Met-Val-Le
u-Gly-Valle-Val-Pro-Gly-
Arg-Gly-Cys-Ala-11e-Pro-A
sn-Arg-Pro-Gly(le-Phe-Val
-Arg-Val-AlaTyr-Tyr-Ala-L
ys-Trp-11e-Hls-Lys-11e-11
e-Leu-Thr-Tyr-Lys-Val-Pro
-Gln-8er gene of the present invention (DNA base sequence):
Based on the information shown in the above formulas (3) and (4), for example, the phosphite triester method (Nature 3
10, 105. (1984)), etc., and can also be produced by chemical synthesis of nucleic acids, or by using DNA encoding a polypeptide having the amino acid sequence shown in each formula as a raw material, using conventional methods including the chemical synthesis methods described above. It can also be produced, and the latter method is particularly convenient and suitable.

In this latter method, some chemical synthesis of DNA or DNA
Various operations and means such as enzymatic treatment for the purpose of cutting, deleting, adding, or binding the A chain, and DNA isolation, purification, replication, and selection can all be carried out in accordance with conventional methods. Any of the various methods well known in the art regarding genes or DNA strands can be employed. For example, the above-mentioned DNA isolation and purification can be carried out by agarose gel electrophoresis, etc., and part of the codons of the nucleic acid sequence can be modified by site-specific mutagenesis.
ts) [Proc, Natl, Acad, Sci,,
81.5662-5666 (1984)) etc. In addition, the selection of the genetic code corresponding to the desired amino acid in the above is not particularly limited, and can be determined according to a conventional method considering the codon usage frequency of the host cell to be used (Nucl, Ac1ds Res,
, 9.43-74 (1981)).

In addition, the determination and confirmation of the DNA sequence obtained according to the above,
For example, Maxam-Gilbe
rt) (7) Chemical modification method [Meth, Enzym,
65.499-560 (1980)] and dideoxynucleotide chain termination method using M13 phage (Mess
ing, J., and Vieira, J., Ge.
ne, 19.269-276 (1982)).

By utilizing the thus obtained gene of the present invention, recombinant h-HGF can be easily produced and obtained in large quantities by genetic recombination technology.

This method for producing recombinant h-HGF requires the use of the above-mentioned gene (DNA) of the present invention, and can basically follow various known gene recombination techniques6 (Mo
regular Cloning, T, Mania
tiset al,, Co1d Spring Ha
See Rbor Laboratory (1982), etc.].

More specifically, a recombinant DNA (expression plasmid, etc.) that allows the gene of the present invention to be expressed in a host cell is created,
This may be introduced into host cells for transformation, and the transformed strain may be cultured.

As the host cell, both eukaryotes and prokaryotes can be used. The eukaryotic cells include cells of mammals, yeast, etc. Examples of eukaryotic cells include COS cells (Y, Gluz), which are monkey cells.
man, Ce1l, 23.175-182 (198
1)) and a dihydrofolate reductase-deficient strain of Chinese hamster ovary cells (G, Urlauband
L., A., Chasin, Proc., Natl.
Acad, Sci.

U, S, A, , mutual, 4216-4220 (198
0)) etc. are often used, but are not limited to these. As expression vectors for animal cells, those containing promoter RNA splice sites, polyadenylation sites, transcription termination sequences, etc., which are usually located upstream of the gene to be expressed, can be used, and if necessary, a replication origin can be used. may hold. An example of such an expression vector is S y 2dhfr [S.

Subramani, R. Mulligan and
P., Berg, Mol.

Ce11. Biol,, 1.854-864 (1
Examples include, but are not limited to, 981)) and the like.

Yeast is commonly used as a eukaryotic microorganism.
Among them, yeast of the genus Satucharomyces can be advantageously used. Examples of expression vectors for eukaryotic microorganisms such as yeast include p
AM82 [A. Miyanohara et al.
Proc, Natl, Acad, Sci.

U, S, A, 80.1-5 (1983)] and the like can be preferably used.

Escherichia coli and Bacillus subtilis are commonly used as prokaryotic hosts. When the host is used as a host, the present invention uses, for example, a plasmid vector that can be replicated in the host,
In order to express the gene of the present invention in this vector, an expression plasmid is provided upstream of the gene with a promoter, an SD (Shine and Dalgarno) base sequence, and an initiation codon (for example, ATG) necessary for starting protein synthesis. It is preferable to use it. The Escherichia coli used as the above host is Escherichia coli (Escherichia coli).
A coli) K12 strain is often used, and pBR322 is commonly used as a vector, but the present invention is not limited thereto, and any of various known strains and vectors can be used. Examples of promoters that can be used include the tryptophan (trp) promoter, lpp promoter, lac promoter, and PL promoter, all of which can express the gene of the present invention.

In detail, one method using a prokaryotic organism such as E. coli as a host is to use the vector pTM1 [Konmotobo 2 Metabolism, Vol. 22.289 (1985)] having a tryptophan promoter and an SD sequence as an expression vector. and the restriction enzyme C1a I located downstream of the SD sequence.
A gene encoding a desired polypeptide to which ATG has been added, if necessary, may be linked to the site. The expression of the gene of the present invention is not limited to the direct expression system as described above, but can also be expressed in a fusion protein expression system using, for example, β-galactondase or β-lactamase.

An example of the above-mentioned fusion protein expression system is a method in which a target polypeptide is secreted and expressed outside the cytoplasmic membrane by using a signal peptide. Examples of the signal peptide include Lpp and OmpA.

Outer membrane proteins such as OmpF and PhoE, and PhoA
Various known proteins such as periplasmic proteins such as Bla can be used.

Another example of the production of recombinant h-HGF using the gene of the present invention is a method using CO8 cells as host cells. In this method, an expression vector that possesses the SV40 replication origin, is capable of autonomous replication in CO8 cells, and is further equipped with a transcription promoter, a transcription termination signal, an RNA splice site, etc. can be used, for example, in a state capable of transformation by DNA uptake. The desired expression plasmid can be obtained by incorporating the gene of the present invention into (competent) E. coli.

Various commonly used methods can be used to introduce the desired recombinant DNA thus obtained into the host cell and to transform it. For example, the expression plasmid into which the target gene has been inserted can be prepared using the alkaline lysis method (Molecul).
ar Cloning-A LaboratoryM
annual (Cold Spring Harbor
Laboratory), p36B (1982))
It can be prepared by the DEAE-dextran method, the calcium phosphate-DNA co-precipitation method, etc., and taken into CO8 cells, and thus the desired transformed cells can be easily obtained.

Another method for producing recombinant h-HGF using the gene of the present invention includes, for example, a method using a dihydrofolate reductase-deficient cell line of Chinese hamster ovary cells as the host cell.

The desired transformant thus obtained can be cultured according to a conventional method, and the desired h-HGF is produced and accumulated by the culture. The medium used for this culture is
Various commonly used ones can be appropriately selected depending on the host cell employed. For example, for culturing transformants using E. coli etc. as host cells, LB medium, E medium, M9 medium,
M63 medium etc. can be used, and if necessary, various commonly known carbon sources, nitrogen sources, inorganic salts, vitamins, natural product extracts, physiologically active substances, etc. can be added to the secondary medium. can. In addition, transformants using CO8 cells as hosts can be prepared using, for example, RPM I-1640 medium, Dulbecco's modified Eagle's minimum essential medium (Dulbecco's modified Eagle's minimum essential medium).
modified Eagle's MEMDMEM)
Culture can be carried out using a medium to which serum components such as fetal calf serum (FCS) are added as necessary. As the culture conditions for the above transformant, conditions suitable for the growth of host cells can be adopted, and in the case of E. coli, for example, the pH is about 5 to 8.
, preferably at or around 7°C, a temperature of 20 to 43°C, preferably at or around 37°C.

As a result of the above, human h-HGF, which is characterized by being contained intracellularly or extracellularly in the transformant, is produced, accumulated, and secreted. The recombinant h-HGF has been subjected to various separation operations utilizing its physical properties, chemical properties, etc. ("Biochemical Data Book II J, pp. 1175-1259,
1st edition 1st printing, June 23, 1980 Published by Tokyo Kagaku Doujin Co., Ltd., Biochemistry, vol. 2
5°No, 25.8274-8277 (1986)
; Eur, J., Biochem.

313-321 (1987), etc.). Specifically, the method includes, for example, ordinary reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock method, ultrasonic disruption, ultrafiltration, molecular sieve chromatography (gel ), adsorption chromatography, ion exchange chromatography, abinitake chromatography, high performance liquid chromatography (H
Examples include various liquid chromatography methods such as PLC), dialysis methods, and combinations thereof. In a particularly preferred separation method, the target substance is first partially purified from the culture supernatant. This partial purification can be carried out using, for example, ammonium sulfate,
This can be carried out by a treatment using a salting-out agent such as sodium sulfate or sodium phosphate, and/or an ultrafiltration treatment using a dialysis membrane, flat plate membrane, hollow fiber membrane, or the like. The operations and conditions for each of these treatments may be the same as those for ordinary methods of this type.

Next, the target substance can be isolated by subjecting the crude product obtained above to adsorption chromatography, affinity chromatography gel filtration, ion exchange chromatography, reversed phase chromatography, etc., or by a combination of these operations. Both components exhibiting activity can be obtained, and thus the target substance can be isolated as a homogeneous substance. By the above method, the desired paste combination h-HGF can be easily produced in high yield and high purity.
can be manufactured on an industrial scale.

The glue combination h-HGF of the present invention thus obtained can be advantageously used in various uses similar to the above-described h-HGF of the present invention.

EXAMPLES The present invention will be described in more detail by examples below.
The present invention is not limited thereto.

Example 1 (1) Measurement of hepatocyte proliferation activity (HGF activity) Seg 17
(7) Methods
n Cell Biology, vol, 13. p2
9. Academic Press, New York
(1976)), 0.05% collagenase (Thailand 1) was administered from Wistar male rats (body weight 150 g).
Hepatic parenchymal cells were isolated using 1,9 Fufu Co., Ltd.). This hepatic parenchymal cell has a diameter of 2. 5 in a multiwell plastic plate (Nunc, Inc.) with 4 wells.
x104 pieces / 1 xi / cm 20:) Concentration, 30% CO2 + 70% 02 mixed gas gas phase, 3
Monolayer culture was carried out at 7°C [Tomita, Y, etal
,, Exp, Ce11. Res,, 135.3
63-371 (1981)).

The culture medium was 5% bovine serum (FBS, Flow
LabNarth Ryde, Au5tralia
), 2 μM insulin, 1 μM dexamethasone, 100
Williams E medium (Flow Laboratories, hereinafter referred to as "basal medium") supplemented with U/y/penicillin and 100 μg/xll streptomycin was used. 24 hours after the start of culture, the medium was replaced with a basal medium not containing FBS, and at the same time, an appropriate amount (50 μl or less) of the test sample was added.

h-HGF activity is determined by the addition of the test sample to the DNA of the test cells.
This was investigated by increasing A synthesis. This is 25. 12 hours after adding the above test sample, 1 μCi/50 μl of I-deoxyuridine (manufactured by Amajam) was added, and the culture was continued for another 24 hours. After this continued culture, the test cells were incubated twice with PBS. , 5% TCA (manufactured by Wako Tsumugi Kogyo Co., Ltd.)
The cells were washed once with INN a OH12A7, and the amount of I-deoxyuridine incorporated into DNA25 in the cell nucleus was measured using a gamma counter (manufactured by Aloka).

Incorporated into liver parenchymal cell DNA by the test sample125
- The amount of aoxyuridine was determined as the difference in counts from the test sample-free group, and this was calculated as the DNA synthesis activity (c
pm/well) and was used as an index of HGF activity of the test sample.

(2) Production of h-HGF of the present invention (1) Patient plasma obtained during plasma exchange therapy of a patient with fulminant hepatitis was used as a raw material.

First, raw plasma 11 was mixed with 50mM) lithium-hydrochloric acid buffer (pH
8,5), filtered using a N004 glass filter, and the filtrate was directly added to an S-Sepharose Fast Claw (manufactured by Pharmacia) column. The column (4 x 25 cm) was prepared with 0.15 M NaCl, Q, 1% CHAPS (manufactured by Katayama Chemical Industry Co., Ltd.) (pH 8.5) (hereinafter referred to as "A buffer") containing 50 mM) lithium-hydrochloric acid.
Equilibrate the sample with 31! After addition at a rate of 1/min, a linear gradient (A,
B was eluted with 400yA').

Active fraction obtained above (0.5-0.6 MNaC/)
was diluted 3 times with distilled water, the pH was adjusted to 7.8 with diluted hydrochloric acid, and the mixture was applied to a heparin-Sepharose column (Pharmacia, 0.8N7cm) equilibrated with A buffer.

After adding the sample at a rate of 0.5 x 1 min for 1 min, it was eluted using a linear gradient using B buffer (A
, 50 zl each of B buffers.

The active fraction (1-1.2M NaCA') obtained above was subjected to C4-reversed phase high performance liquid chromatography (HP L
A single h-HGF was obtained by fractionation in C).

That is, it was eluted by a linear gradient (equilibration and eluate removal 50 zl) using a C4 reverse phase column (Highbore RP304.240X4/6InIn1 manufactured by Bio-Rad) equilibrated with 0.1% TFA=water.

In this way, pure h-HGF that was 300,000 times more purified than the raw plasma was obtained.

■ Measuring the molecular weight of h-HGF by 5DS-PAGE Laemmli (7) method [Nature,
227.680-685 (1970)), 3
% stacking gel and 10% separating gel (thickness 1.5 m
5DS-PAGE was performed using h-HGF, and after silver staining, the molecular weight of h-HGF was determined by comparing with a band of a molecular weight marker that was electrophoresed at the same time.

As a result, the molecular weight of h-HGF was found to be approximately 84,000D.

■ Amino acid composition of h-HGF Approximately 20 μg of the h-HGF solution purified in the above (■) was poured into a hard glass sample tube (manufactured by Nikkei Rika Glass Co., Ltd., 6X40
+ nm), put it in a reaction vial for hydrolysis (manufactured by Pierce), and after vacuum drying, put 200 μl of 6N-hydrochloric acid (containing 1% phenol) into the reaction vial, seal it under reduced pressure,
Hydrolysis reaction was carried out at 130°C for 4 hours.

After the reaction was completed, 200 μl of NaSTM solution (manufactured by Beckman) was added to the sample tube, transferred to a sample tube for amino acid analysis, and 50 μl of the solution was automatically injected into a 6300E amino acid analyzer (manufactured by Beckman) for amino acid analysis.

Note that the ninhydrin method was used as the detection method.

Tryptophan cannot be detected by this method.

Each separated and fixed amino acid was quantified using a calibration curve prepared using standard amino acids (1 nanomol) at three concentrations, and phenylalanine was
The composition ratio was calculated assuming that two of them were included.

The amino acid composition ratio of the h-HGF of the present invention according to the above was as shown in Table 1 below.

Table 1 (3) Production of h-HGF subunit ■ Production of subunits α, β1, and β2 The pure h-HGF purified in (2) above was concentrated in a centrifugal evaporator, and then dissolved in 4M guanidine hydrochloride. Containing 0.5M) Lis-HCl buffer (pH 8,0) 3xll, 1 nanomol/z
The solution was dissolved to a concentration of 1 ml, 6 micromoles of dithiothreitol (DTT) was added, and a reduction reaction was performed at 50° C. for 4 hours.

After adding 12 micromoles of iodoacetamide and reacting at room temperature for 1 hour, fractionation was performed by C4-reverse phase HPLC (model, conditions, etc. were the same as in (2) above) to obtain subunits α, β1 and β2 were obtained.

■ Determination of the molecular weight of subunits by 5DS-PAGE The molecular weight of each subunit obtained in (1) above is determined by (1) above.
Determined by 5DS-PAGE in the same manner as above.

As a result, the molecular weight of subunit α was approximately 62,000.

The molecular weight of subunit β1 was approximately 34,000.

The molecular weight of subunit β2 was approximately 32,000.

■ Determination of the partial amino acid sequence of subunit α (3)
) to an amount equivalent to about 60 μg of the subunit α obtained by ①, 0.0 μg of lysyl endopeptidase (manufactured by Sigma).
2 mmol was allowed to react overnight at 37°C to obtain an enzyme-treated fragment solution.

The enzyme-treated fragment solution was fractionated by C18-reversed phase HPLC, divided into five fractions, and again subjected to C8-reversed phase HPLC (chromatography model and conditions described in (2) above).
).

As a result, the following sequence was identified from the obtained fragment.

(i) Cys-Gin-Arg-Trp (ii) Asn
-Met-G 1u-Asp-Leu-His-A
rg-His-Ile-Phe-Trp-Glu-
Pro-Asp-Ala-5er-Lys(iii)A
sn-Tyr-Me t-G 1y-Asn-Leu-
5e r-G 1 n-Thr-Arg-3er-Gl
y-Leu-Thr-Cys-5er-Met-Trp
-Asp-Lys(pJ Asp-Leu-Arg-G
1 u-A s n-Tyr-Cys-Arg-As
n-Pro-Asp-Gly-5er-Glu-5er
-Pro-Trp-Cys-Phe-Thr-Thr-
Asp-Pro-Asn-Ile-Arg-Val-G
ly-Tyr-CysSer-Gin-11e-Pro
-Asn(y) G 1y-Ph e -A 5p-A
sp-Asn-Tyr-Cys-A rg-As n-
Pro-Asp-Gly-Gln-Pro-Arg-P
ro-Trp-Cys-Tyr-Thr-Leu-As
p-Pro-His-Thr-Arg-Trp-Glu
-Tyr-CysAla-11e-Lys iDLeu-Asn-Glu-Asn-Tyr-Cys
-Arg-Pro-Asp-Asp-Asp-Ala-
H45-Gly-Pro-Trp-Cys-Tyr-T
hr-Gly-Asn-Pro-Leu-Ile-Pr
o-Trp-Asp-Tyr-Cys-Pro-I l
e-5er-Arg-Cys-Glu-Gly Partial amino acid sequence (i) to (Vl
The following amino acid sequence (1) Cys-Gln
-Arg-Trp.

(2) Asn-Tyr-Cys-Asn-Pro-
Asp and (3) Pro-Trp-Cys Kringle structure (Ma
Gnusson.

S. et al., In Proteolysis
and Physiological Regulation
ion, edited by D, W, R
ibbons and K.

Brew, pp203-238. Academic
(Press New York) Contains two characteristic sequences, and from this, subunit α contains
It is estimated that at least three kringle structures are included.

The N-terminal amino acid sequence of HGF subunit β is Va.
1-val and that the subunit α has the above-mentioned kringle structure are similar to factors such as plasminogen and urokinase, and from this,
It is presumed that GF is single-chain and can be activated by a process similar to plasminogen.

Furthermore, the characteristics of these factors are useful for elucidating the evolution and mechanism of action of HGF molecules.

■ Identification of the N-terminal amino acids of subunits β1 and β2 Approximately 50% of subunit β1 obtained in (3) above
Using an amount equivalent to 00 picomole, the sequence of 25 amino acid residues from the N-terminus of subunit β1 was determined using a gas phase protein sequencer (manufactured by Applied Biosystems, model 470A).

The PTH-amino acid solution obtained in each reaction cycle was dried in vacuo, and then dissolved in a 33% acetonitrile aqueous solution.
It was separated and fixed using reverse phase high performance liquid chromatography for TH-amino acid analysis (manufactured by Beckman).

The N-terminal amino acid sequence of subunit β1 determined above was as follows.

Val-Val-Asn-Gly-I 1e-Pro-
Thr-Arg-Thr-Asn-Ile-Gly-
Trp-Met-Val-5er-Leu-Arg-T
yr-Arg-Asn-Lys-His-I 1e-C
ys-subunit β2 obtained by (3) above
The sequence of 25 amino acid residues from the N-terminus was similarly determined for an amount equivalent to about 500 picomole of , and the results were as follows, which were confirmed to be identical to subunit β1.

Val-Val-Asn-Gly-I 1e-Pro-
Thr-Arg-Thr-Asn-Ile-Gly-
Trp-Met-Va 1-5er-Leu-Arg-
Tyr-Arg-Asn-Lys-Hls-11e-C
ys-■ Amino acid composition of subunits Approximately 2 μg equivalent of each of the subunits α, β1, and β2 obtained above was placed in a hard glass sample tube (manufactured by Nikkei Rika Glass Co., Ltd., 6×4 mm), and placed in a reaction vial for hydrolysis ( Pierce), and after vacuum drying, 200μ of 6N hydrochloric acid (containing 1% phenol) was added to the reaction vial,
The container was sealed under reduced pressure and a hydrolysis reaction was carried out at 130° C. for 4 hours.

After the reaction is complete, add 200al! of NaSTM solution (manufactured by Beckman) to the sample tube. was added, transferred to a sample tube for amino acid analysis, and 50 μl was added to a 6300E amino acid analyzer (
Amino acid analysis was performed by automatically injecting the sample into a tube (manufactured by Beckman).

The amino acid composition of subunit α determined according to the above method is shown in Table 2 below.

Table 3 shows the amino acid composition of subunit β1 and Table 4 shows the amino acid composition of subunit β2, which were determined in the same manner as in Table 2.

Example 2 This example shows the production of the DNA base sequence of formula (1), and was carried out as follows.

(1) Preparation of cDNA library Using 3 μg of human placental poly(A)'' RNA (manufactured by Clontic), cDNA was synthesized using a cDNA synthesis system plus (manufactured by Amarjam) using random primers as a template.

Eco RI linkers were ligated to the ends of the obtained cDNA (1 μg) using the cDNA cloning system λgtlO (manufactured by Amajam), and the ligated product was introduced into random phage λgtlo to obtain a cDNA library. The synthesis of this cDNA and its introduction into λgtlO were performed in accordance with the AmaJam manual.

(2) Isolation of h-HGF cDNA About 170 of the cDNA libraries obtained in (1) above
Screening for h-HGF cDNA was performed on 10,000 clones by the plaque hybridization method described below. As a probe, purified h-HGF
Oligonucleotides corresponding to the following peptides (peptides I and ■) obtained from the amino acid sequence analysis of the α chain were synthesized and used. These oligonucleotides are synthesized by conventional methods (for example, according to Japanese Patent Application Laid-Open No. 152398/1983).
(see Publication No.).

<Peptide ■> Asn-Met-Glu-Asp-Leu-His-A
rg-His-11e-PheTrp-Glu-Pro
-Asp-Ala-3er-Lys〈Peptide ■〉 Asp-Leu-Arg-Glu-Asn-Tyr-C
ys-Arg-Asn-Pr.

Asp-Gly-Ser-Glu-8er-Pro-T
rp-Cys-Phe-Thr-Thr-Asp-Pr
o-Asn-Ile-Arg-Val-Gly-Ty
r-Cys-9er-Gln (le-Pro-Asn Each probe obtained from each of the above peptides (probe 550
The DNA sequences of ~555) are as follows.
.

Peptide I → Probe 550 (447-32 types) Asn
-Met-Glu-Asp-Leu-His-Arg-
His-I 1e-Phe peptide ■ → Probe 554
(657-256 species) Glu-Asn-Tyr-Cys
-Arg-Asn-Pro-Asp-Gly-3er-
Trp-Glu-Pro-Asp-AlaTGG G
AG CCT GAT GC-3'Peptide→
Probe 551 (17mer His-11e-Phe-T
rp-Glu-Pr.

8 types) Peptide → Probe 552 (177-8 types) His-
11e-Phe-Trp-Glu-Pr.

Peptide → Probe 553 (177-8 species) His-
Tie-Phe-Trp-Glu-Pr.

Glu-5er-Pro-Trp-Cys-Phe-T
hr-Thr-Asp-Pr.

GT Asn-11e AACAT-3' Peptide ■ → Probe 555 (457-32 species) Asn
-I le-Arg-Val-Gly-Tyr-Cys
-5er-Gln-I 1e-Pro-Asn-Cys
-Asp-MetCCCAAACTGT GACATG
-LB agar medium prepared in a 3'15 cm petri dish (
1% Bactodryptone, 0.5% Bacto Yeast Extract, 1% NaCl, 1.5% Bacto Agar, p
Top agarose (1% Bactodryptone, 0.5% yeast extract, 0.5 %NaC
j? , 0.125% MgSO4, 0.7% agarose).

Prepare 34 of these plates and culture them at 37°C for 7 hours.
Cooled at ℃ for 1 hour. A nylon filter (Biodyne A Menplan, Pa1l Ult) was placed on the surface of this plate.
rafine Fjltration Cooprat
ion) for 1 minute, and then immerse the filter in denaturing solution [0.5M NaOH, 1.5M NaC/] for 5 minutes.
minutes, then neutralizing solution [0,5M Tris-HCl (pH 7,0)]
, treated with 1.5M NaC1+0 for 5 minutes, respectively,
2xSSC [1xSSC=0.15M NaC1+0
．． After washing with 015M sodium citrate solution and air drying for 30 minutes, baking was performed at 80° C. under vacuum for 1 hour. Note that two nylon filters were obtained from the same plate by repeating the same operation.

Add this nylon filter to the prehybridization solution [2 x 33 C, 10) <Denhardt's solution (I
．． 02% Ficoll), 100 μg/z/heat-denatured sperm DNA] at 50°C for 2 hours.

Next, hybridization solution [2X SSC.

IQ)<Denhardt's solution, probe] Transfer the filter to 65°C for 30 minutes, and then gradually incubate at 65°C.
Hybridization was carried out by lowering the temperature from 45°C to 45°C and treating overnight. As a probe, probe 550 was labeled at the 5' end with γ-32P-ATP and purified using a nick column (manufactured by Pharmacia) to give 3 ng/11
was added to the hybridization solution at a concentration of 1. After hybridization, the filters were washed a total of three times in 2X SSC for 5 minutes at room temperature and an additional 50 min in 2X SSC.
℃ for 2 minutes, rinsed in ZXSSC, air-dried for 30 minutes, and then exposed to X-ray film (X
AR5 (manufactured by Kodak) was subjected to autoradiography at -70°C for 3 days.

Select 27 cells that strongly hybridized with the probe at the same position on two nylon filters prepared from the same plate, scrape off the agarose containing secondary plaques from the original plate, and add 8M solution [100mM NaCl, 0.0
1% gelatin, Tris-HCl (pH 7.5), 8mM
It was suspended in MgSO4 to prepare a phage suspension.

This phage suspension was diluted and the plaque hybridization described above was repeated to finally obtain a single phage suspension.

The above single phage suspension was added to an LB agar medium overlaid with a mixture of E. coli NM514 strain and top agarose at 0.0%.
5 μl each was spotted separately and cultured at 37° C. for 12 hours. The above plaque hybridization was performed using this plate. Probe 55 as a probe
A mixture of 1.552 and 553, probe 554 and probe 555 were used separately. In the case of mixed probes, hybridization was performed at room temperature for at least 1 hour by gradually lowering the temperature from 65°C to room temperature, and washing with 2×SSC was performed at 40°C.

As a result, the mixed probes (551, 552 and 553
), probe 554, and probe 555. 24 clones were obtained that hybridized with all of probes 554 and 555.

(3) Base sequence determination of h-HGF cDNA Among the cDNAs obtained in (2) above, [λh-H
The cDNA base sequence of GF-4J was determined as follows.

After cutting λh-HGF-4 DNA with the restriction enzyme Eco R1, approximately 780 bp, approximately 740 bl] and approximately 350 bp
Each cDNA fragment of bl' was separated by agarose gel electrophoresis, and the cDNA fragments of BL' were separated using agarose gel electrophoresis.
Each fragment was isolated and purified using plasmid pUc118 (manufactured by Takara Shuzo Co., Ltd.) and purified using restriction enzyme Eco.
was inserted into the RI site, and two types of E. coli JM109 strain transformant species with different insertion directions were obtained.

Such as “E, coli JM109 / ph-HGF
-4J, [E.

coli JM109/ph-HGF-4LRJ,
“E. coli JM109 /ph-1 (GF-4M
J, [E, coli JM109/ph-H
GF-4MRJ, [E, coli JM109/p
h-HGF-4SJ and “E.coli JM109
/ph-HGF-4SRJ.

From these transformants, alkaline lysis [T
, Maniatis, E., F., Fr1tsch.
and J.

Sambrook, Mo1ecular Cloni
ng, p36B (ColdSpring Habo
r Laboratory), 19B2) Niyo? )
Prepare plasmid DNA and run it with 5equenase Uni using a kilosequencing kit.
The base sequence of the cDNA was determined using tedStates Biochemica 1 (manufactured by tedStates Biochemica 1) according to the chilatono manual.

As a result, λh-HGF-4 cDNA has the formula (3)
The deduced amino acid sequence encoded by this formula (1) was the same as the partial amino acid sequence of the h-HGFα chain. It also matched the amino terminal amino acid sequence of the β chain.

Example 3 This example is an example showing details of the production of recombinant h-HGF using the gene of the present invention and genetic engineering techniques,
It was carried out as follows.

(1) Isolation of h-HGF cDNA Human placenta cDNA library (manufactured by Clontic)
About 1.3 x 106 λ phages were extracted from cDNA by the plaque hybridization method shown below.
A was isolated and screened. As a probe, the cDNA region of λh-HGF-4 was digested with restriction enzyme Ec.
o Of the three DNA fragments obtained by cutting with RI, 5
736 bp on the 3' side (hereinafter referred to as the ``5'probe'') and 351 bp on the 3' side (hereinafter referred to as the 13' probe) were converted into 32P using a nick translation kit (manufactured by AmaJam). The one labeled with was used.

In the same manner as in Example 2, the nylon filter on which the phage DNA was immobilized was soaked in the prehybridization solution [5
0% formamide, 5x Denhardt's solution, 5xSSPE (20xSSPE"3M NaCA
' 10o, 2M-NaH2PO4 10.02M ED
TA (pH 7,4)), 0.1% SDS and 100μ
g/xll heat-denatured salmon sperm DNA1 at 42°C for 4 h. Then, hybridization solution [5' probe and 3' probe were added at 106C1
) The filter was transferred to the above prehybridization solution containing 111/ill, and incubated at 42°C for 16 hours. After hybridization, filters were washed three times in 2x SSC-0.1% SDS for 5 minutes at room temperature, followed by an additional 0.1x 5 SC wash.
Washed a total of two times in −0.1% SDS at 62° C. for 30 minutes. After air-drying the filter, use an intensifying screen to apply -70 to X-ray film (XAR5, manufactured by Kodak).
Autoradiography was performed overnight at °C.

As a result, 10 clones showing positive signals for both the 5' and 3' probes were obtained. Repeat the above plaque hybridization for these,
Each was finally isolated as a single clone.

Among the above 10 clones, there is a clone that hybridizes only with the 5' probe and has a 1.6 kb cDNA insert (clone 8), and a clone that hybridizes with both the 5' and 3' probes and has a 1.5 kb cDNA insert. A clone (clone 12) having the following was selected, and the nucleotide sequence of each of these cDNA regions was determined.

The results are shown in FIG. 1 along with the restriction enzyme cleavage sites.

In the figure, the number in parentheses after the restriction enzyme is the number of bases counted from the 5' end, the white part shows the coding region of h-HGF, and the solid line part shows the non-coding part.

Further, the entire DNA sequence shown in FIG. 1 and the entire amino acid sequence (consisting of 728 amino acid residues) of its coding region are shown in FIG. 2 (FIGS. 2-1 to 2-3).

From the above figure, it is clear that both clone 8 and clone 12 contain the entire coding region of h-HGF.

(2) Preparation of phHGF This outline is shown in FIG.

Derived from the cDNA insert of clone 8 obtained in (1) above (7) l 47bp no Bam HI - Eco RI
DNA fragment and 721 bp Eco RI-Sc
The a I[)NA fragments were each isolated and inserted between Bam Hl and Hinc HI of plasmid pUc118 to obtain phHGFBam-5ca.

Next, pstr-Eco RI of 771bl) derived from the cDNA insert of clone 8 obtained in (1) above
DNA fragment and cDNA insert derived from the same clone 12 (y) 555 bp (y) Eco RI-old nd
IIIl) NA fragment was isolated and inserted between plasmid pUc118(y)Pstl and HindIII to obtain phHGFPst-Hind.

Furthermore, BglI of 427bl derived from clone 12 above
I-Eco RI DNA fragment and the same clone 12-derived (7) 513 bp(y) Eco RI-Dra
The IDNA A fragment was isolated and
pUC118(7) was inserted between BamHI and HincHI to obtain ph)(GpBgl-Dra).

Regarding the above three plasmids, 1) hHGFBamSc
331 bp (y) Bam HI-Nco I from a
[) NA fragment as phHGp' Pst-Hin
8Q 8bp (7) Nco I-Xh from d.

I DNA fragment and pHHG F Bgl-Dr
From a is 658 bp no Xho l-5ph ID
Isolate the NA fragment and transform it into plasmid pU.
The target phHGF was obtained by inserting between Bam HI and Sph I of c118.

(3) Production of pR8Vs-hHGF-dhfr17) Plasmid phHGF obtained in (2) above was injected with restriction enzyme Ban.
Cut using HI and sph I to obtain approximately 2.3 kb h
-HGF DNA fragments were isolated by agarose gel electrophoresis.

After both ends of the obtained DNA fragment were blunted using T4 DNA polymerase, it was ligated with an S81 linker [GGTCGACC, manufactured by Zenshuzo Co., Ltd.] using T4 DNA ligase, and then cut with the restriction enzyme Sal. By this, h-HG having Sal I cleavage sites at both ends
F DNA fragment was obtained.

On the other hand, plasmid I) RS V S -dhfr [
JP-A-2-2391] is cut at the Sal I site located downstream of the R8V-LTR promoter,
The h-HGF DNA fragment having Sal I cleavage sites at both ends obtained above was ligated to the obtained fragment using T4 DNA ligase to construct the desired h-HGF expression vector pR8Vs-hHGF-dhfr. .

The thus obtained h-HGF expression vector pR8VS-
Escherichia coli HBIOI strain harboring hHGF-dhfr is
“Escherichia coli T (BIOI
/pR5VS-hHGF-dhfr J has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology (FEI), and its deposit number is FEI Microbiology Deposit No. 11733 (FEI).
RMP-11733) J.

(4) Obtaining a CHO cell line that produces recombinant h-HGF 3X108 cells (y) dhfr-deficient CHO cell line DXB
11 [G, Urlaub and L, A, C
hasin, Proc.

Natl, Acad, Sci,, U, S, A,,
77, 4216 (1980), the h-HGF expression vector pRS V S -h H obtained in (3) above was used.
20 μg of G F -dhfr(7) was subjected to electroporation method [Somatic Hybridiz
erSSH-1 was introduced using a Shimadzu Co., Ltd., and the transduced cells were placed in a complete medium consisting of DMEM (manufactured by Gibco) containing 10% dialyzed serum (manufactured by Gibco) and 1x non-essential amino acid solution (manufactured by Flow). dhfr transformed cell lines were screened, and clones were obtained by cloning using the limiting dilution method.

Twenty-four of the obtained clones were cultured for 4 days using a 24-well plate, and the amount of h-HGF in the culture supernatant was measured by ELISA using an antibody against h-HGF.

The ELISA method was performed as follows.

That is, first, h-HGF monoclonal antibody (H4-2)
(see JP-A No. 64-27491), whose concentration is 1μ
This was adjusted with 0.1M NaHCOs to give 100 μl/well of the solution, and this was added to the plate at a rate of 100 μl/well. After standing at 4°C overnight, phosphate buffered saline (PB) was added. S) Wash with buffer and add 1% fetal bovine serum albumin (
250μII/well of BSA) solution was added and left overnight at 4°C.

Next, containing 1% BSA and 0.4M NaCA';
, IMIJ acid buffer (pH 6,5) 80
ttll and 20 μl of the test sample were added, and after incubation for 2 hours, 0.05% Tween 2Q (Twee
Washed three times with a PBS solution containing n20 (manufactured by Sigma),
Furthermore, 100 μII/well of monoclonal antibody (H4-2) diluted to 0.1 MIJ acid buffer containing 0.1% BSA was added at 100 μII/well, and after incubation for 2 hours, PBS containing 0.05% Tween was added. O, containing 1% BSA and 0.15 M NaCA'.
An antibody (anti-rat IgG-pox, manufactured by Kappel) diluted 3000 times in 1M phosphate buffer was added and incubated for 2 hours. PBS containing 0.05% Tween
After washing three times with water, add 100μ of orthophenylenediamine (OPD, Wako Tsumugi Kogyo Co., Ltd.) 25μ/100z/solution!
was added to develop color. After 10 minutes, 100 μl of 2N sulfuric acid was added to stop the reaction, and measurement was performed at a wavelength of OD 492.

From the standard curve prepared in advance by similar measurements, h-HG
The production amount of F was determined by immunoreactive HGF (Immuno-reactive HGF).
reactive HGF ng/ll).

Based on the above h-HGF production measurement results, the desired glue combination h-HGF producing strain CHO-hHGF2-1 strain was selected.

5X105/25cm2 of the CHO-hHGF2-1 strain
/ 10xll of complete medium containing 200 nM methotrexate (MTX) [non-essential amino acid solution (X
100 Non-Essential Amino Acid Solution (manufactured by Flow), 11■/
illll radiumate (manufactured by Flow), 10% tallow serum (FCS, manufactured by Gibco) and 200 nM M
DMEM (manufactured by Gibco) medium containing TX], about 3
After culturing for weeks, the obtained MTX-resistant cells were cloned by the limiting dilution method, and then cultured in a 24-well plate for 4 days, and the amount of h-HGF in each culture supernatant obtained was determined in the same manner as above. It was measured as follows.

The results are shown in Table 5.

The medium was replaced on the fifth surface, and MTX-resistant strains were screened on this medium.

After screening the above 1 μM MTX resistant strain, the amount of h-HGF produced in the culture supernatant of the strain was measured under the same conditions as above.

The results are shown in Table 6 below.

Table 6 h-HGF high producing strain CHO-hH shown in Table 5 above
GF2-1 clone 0.2'-16 was further added to 200 nM
After culturing in a complete medium containing MTX for about 2 weeks,
Complete medium containing 1 μM MTX As shown in Table 6 above, this method yielded clones (CHO-hHGF2-1 clone 1-6 and clone 1-44) that produced h-HGF at 2 μg/xi or more. Ta.

(5) Purification of recombinant h-HGF ■ Purification using Mono S column 1000 zA' of the culture supernatant obtained in (4) above was preliminarily treated with 0.15 M NaCl, 10 mM N-2-hydroxyethylpiperazine-N'- 2-ethanesulfonic acid (
Mono S column (Mono S; manufactured by Pharmacia) equilibrated with 50 mM) Lis-HEPES) and 2 mM Ca CA'2 (pH 8,5).
After washing with the same buffer, 0.1 to 1. OM
It was eluted with a linear concentration gradient of NaC/(flow rate 11
//min, 211/tube).

The results are shown in FIG.

In the figure, the vertical axis (1) is the absorbance at 2801 m (
A 280 nm), (2) HGF activity (DNA synthesis activity, cpmxlo'), (3) immune activity (immunoreactive HGF amount, ng 7 xl!), (4
) indicate the NaCl concentration (M), and the horizontal axis indicates the fraction number.

From the figure, it can be seen that the elution positions of HGF activity and immune activity of the mono S column-FPLC of the culture supernatant containing recombinant h-HGF are both around 0.8M.

(2) Purification using a heparin-Sepharose CL-6B column.
and diluted 3 times with distilled water. This is 0.3M
50mM) Lis-HCl buffer (pH 7, containing NaC/
Heparin-Sepharose CL-6B equilibrated with 9)
(manufactured by Pharmacia, Vetsudvolium 2.2zl) and after washing with the same buffer, 0.3 to 2.0M
Elution was performed with a linear concentration gradient of NaCl (flow rate 0.
5yl1 min, 111/tube).

The above elution pattern is shown in FIG.

In the figure, the horizontal axis is the fraction number, and the vertical axis is 280.
Absorbance at 1 m (A2801m, curve (1)),
HGF immune activity (curve (2)), DNA synthesis activity (C
pm X 104 , curve (3)) and Na CA' concentration (curve (4)) are shown, respectively.

From the figure, the recombinant h-HGF of the present invention is NaC
It can be seen that the sample is eluted at a concentration of about 1.2M.

(6) Identification of recombinant h-HGF ■ 5DS-
Measurement of molecular weight by PAGE Using the h-HGF purified in step (5) above, the method of Laemme et al.
li et, al,, Nature.

227, 680=685 (1970))
5DS-PAGE was performed.

As a result, the molecular weight of the recombinant h-HGF of the present invention is about 84 kd under non-reducing conditions, about 62 kd heavyweight (subunit α) and 32 kd and 3
Two 4kd light chains (subunits β1 and β2) were detected.

■ Amino acid composition Purified in (5) above or combined h-HGF
An amount equivalent to about 20 μg of the solution was placed in a hard glass sample tube (manufactured by Nippon Rika Glass Co., Ltd., 6 x 40 mm), placed in a reaction vial for hydrolysis (manufactured by Pierce Co., Ltd.), and after vacuum drying, 6N-
200 μl of hydrochloric acid (containing 1% phenol) was placed in the reaction vial, the vial was sealed under reduced pressure, and a hydrolysis reaction was carried out at 130° C. for 4 hours.

After the reaction, 200 μl of NaSTM solution (manufactured by Beckman) was added to the sample tube, transferred to a sample tube for amino acid analysis, and 50 μl was automatically injected into a 63'00 E-type amino acid analyzer (manufactured by Beckman) for amino acid analysis. I did it.

Note that the ninhydrin method was used as the detection method.

Tryptophan cannot be detected by this method.

Each separated and fixed amino acid was quantified using a calibration curve prepared using standard amino acids (1 nanomol) at three concentrations.
The composition ratio was calculated assuming that 8 pieces were included.

The amino acid composition ratio of the recombinant h-HGF of the present invention according to the above was as shown in Table 7 below.

Furthermore, Table 7 shows the purified natural h-
The same results for HGF are also listed.

Table ■ HGF activity (DNA synthesis activity) Recombinant h- of the present invention purified in (5) above
The DNA synthesis activity of HGF was investigated according to the method described in Example 1 (1).

The results are shown in FIG. 6 as line (1).

In addition, FIG. 6 shows the purified natural h-
The same results for HGF (line (2)) are also shown.

In the figure, the horizontal axis is the concentration of the test sample (ng/well/11
), and the vertical axis is DNA synthesis activity (CI)" x 104
) are shown respectively.

The figure shows that the recombinant h-HGF of the present invention exhibits approximately the same DNA synthesis activity as the natural h-HGF of the present invention.

[Brief explanation of drawings]

Figure 1 shows clone 8 and clone 12 obtained in Example 3.
FIG. 2 is a schematic diagram of a DNA region and a restriction enzyme map. Figure 2 (Figures 2-1 to 2-3) shows the h-HGF of the present invention.
It is a diagram showing the entire DNA base sequence of the gene. FIG. 3 shows p 1n )(Gp Bam-
3ca, phHG F Pst-Hind, ph)
(FIG. 4 is a schematic diagram of producing phHGF from G F Bgl-Dra. FIG. 4 is an elution pattern showing the results of purifying recombinant h-HGF using a mono S column according to Example 3. Figure 6 is an elution pattern showing the results of purifying recombinant h-HGF using a heparin-Sepharose CL-6B column according to Example 3.
FIG. 3 is a diagram showing the DNA synthesis activity of GF. (and above) ← − I-1I+1 Kux Ku- + Q Ku-

Claims (1)

[Scope of Claims] [1] (1) The molecular weight (by SDS-PAGE analysis) is in the range of 96,000 to 82,000, and is mainly between 92,000 and 84,000, and (2) by reduction treatment in the presence of mercaptoethanol. Asp106:Thr48:Ser51:Glu87:Gly76:Ala31:Val38:Met17, which is separated into the following subunit α and subunit β_1 or β_2, and (3) has the following amino acid composition based on Phe (22 mol). :Ile40:Leu50:Tyr36:Phe22:Lys58:His27:Arg52 A liver regeneration factor characterized by the following. Subunit α: (1) Molecular weight (according to SDS-PAGE analysis) is approximately 62
000; (2) has at least the following amino acid sequence; (i) [
[There is a gene sequence] (ii) [There is a gene sequence] (iii) [There is a gene sequence] (iv) [There is a gene sequence] (v) [There is a gene sequence] [There is a gene sequence] (vi) [Gene sequence is available] (3) It has the following amino acid composition based on Phe (18 moles): Cmc38.5:Asp87.5:Thr37.1:Ser32.7:Glu60.8:Gly43.0:Ala14. 9: Val13.3: Met10.4: Ile22.4: Leu23.7: Tyr23.5: Phe18.0: Lys41.4: His20.8: Arg36.8 Subunit β_1: (1) Molecular weight (SDS-PAGE analysis ) is approximately 34
000, (2) The sequence of the 25 amino acids from the N-terminus is as follows. [There is a gene sequence] (3) It has the following amino acid composition based on Phe (4 moles), Cmc12.2: Asp28.7: Thr12.3: Ser19.4: Glu28.1: Gly41.1: Ala15.3: Val22.0: Met3.2: Ile15.7: Leu26.1: Tyr14.6: Phe4.0: Lys18. 5: His9.2: Arg19.0 Subunit β_2: (1) Molecular weight (according to SDS-PAGE analysis) is approximately 32
000. (2) The 25 amino acid sequence from the N-terminus is as follows, and it matches that of β_1. [There is a gene sequence] (3) The following amino acid composition is based on Phe (4 moles). Has Cmc15.0:Asp32.4:Thr13.0:Ser21.0:GIu31.0:Gly44.2:Ala15.7:Val24.1:Met3.9:Ile17.8:Leu29.5:Tyr15.7: Phe4.0: Lys21.6: His10.3: Arg21.3 [2] Formula [Gene sequence available] [Gene sequence available] DNA base sequence of human liver regeneration factor. [3] The DNA base sequence of human liver regeneration factor expressed by the formula [Gene sequence available] [Gene sequence available]. [4] A plasmid for expressing a human liver regeneration factor, comprising the DNA base sequence according to claim [3]. [5] A transformant transformed with the plasmid according to claim [4]. [6] A method for producing a recombinant liver regeneration factor, which comprises culturing the transformant according to claim [5] and collecting the expressed liver regeneration factor. [7] A recombinant liver regeneration factor characterized by having an amino acid sequence represented by the formula [There is a gene sequence] [There is a gene sequence].