JP2980889B2 - Hepatocyte growth factor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to hepatocyte growth factor obtained by genetic recombination.

[0002]

2. Description of the Related Art The liver is the largest and most highly differentiated organ in a living body. It mainly has various important functions such as processing (metabolism), storage, detoxification, decomposition and excretion of various nutrients (sugars, proteins, lipids, vitamins, hormones, etc.). It is known to play a central role. Hepatic parenchymal cells responsible for these functions are placed under the control of various hormones in vivo, and in some cases, exhibit extremely vigorous proliferation. For example, if almost two thirds of a rat's liver is removed,
It is known that the size returns to the original size after about 10 days, and a treatment by partial hepatectomy and subsequent regeneration is also performed in humans with liver cancer. Many studies have been conducted on the mechanism of liver regeneration by proliferation of hepatocytes, and the presence of hepatocyte growth factor has been reported. In particular, some of the present inventors have found that hepatic parenchymal cell proliferating activity is extremely high in human fulminant hepatitis plasma.
(Biomed.Res., 6, 231 (1985) and Exp.Cell. Res. 16
6, 139 (1986)), and succeeded in purifying the active factor as a single protein for the first time in the world (JP-A-63-22526 and J. Clin. Invest., 81, 414 (198)).
8)).

[0003] This human hepatocyte growth factor (hereinafter abbreviated as "hHGF") has an estimated molecular weight of about 76000-92000 by SDS-PAGE under non-reducing conditions, and a molecular weight of 56000-65000 and Divided into two bands, 32000-35000. Nakamura et al. Reported a factor with similar activity derived from rat platelets (Biochem. Biophys. Res. Comm.
un., 122, 1450 (1984)). According to SDS-PAGE, the estimated molecular weight was about 27,000 (Proc. Natl. Ac).
ad. Sci. USA, 83, 6489 (1986)), which was subsequently purified as a single protein and reported to be a protein with a molecular weight of 82,000 consisting of two polypeptides with molecular weights of 69000 and 34000 (FEBS Letters, 224, 311 (1987)). Apart from these hHGFs and rat HGFs, no hepatocyte growth factor purified as a single protein has been reported so far. Regarding hHGFs and rat HGFs, the primary structure and the nucleotide sequence of the corresponding cDNA have not been determined. No reports have been made.

[0004]

In order to examine the detailed function of hHGF in the living body or the effect on liver regeneration in the case of liver damage in vitro, a large amount of hHGF is required, but a large amount of hHGF is required from the plasma of fulminant hepatitis patients. Purification of hHGF is not always easy in terms of man, time, and cost, and it is extremely difficult to stably extract only hHGF from plasma where the source of infection exists. For this reason, stable and large-scale purification of hHGF from plasma of fulminant hepatitis patients has not been performed.

[0005]

Means for Solving the Problems Accordingly, the present inventors have:
As a result of various investigations to obtain a large amount of hHGF by recombinant DNA technology, the gene encoding hHGF useful for this purpose was successfully cloned for the first time, and the present invention was completed.

[0006] That is, the gist of the present invention is to provide an hHG containing a signal sequence represented by the amino acid sequence shown in Figs.
F, hHGF represented by the sequence from the 30th glutamic acid residue (Glu) to the last serine residue (Ser) in the amino acid sequence represented by the amino acid sequence shown in FIGS. Glutamine at position 32 (Gl
h represented by the sequence from n) to the last serine residue (Ser)
Exists in HGF.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION In the following, the present invention will be described. The gene (cDNA) encoding hHGF of the present invention has, for example, a nucleotide sequence as shown in FIGS. In addition, other complementary base sequences were omitted from the base sequence, and only a single strand was described. From this gene, for example, hHGF having the amino acid sequence shown in FIGS. 1 and 2 can be expressed by recombinant DNA technology. At this time, the protein translated from the mRNA encoding hHGF contains a signal sequence, but when secreted from cells, the signal sequence is cleaved, resulting in glutamic acid residue (Glu) at position 30 or glutamine at position 32. residue
HHGF having the amino acid sequence after (Gln) is produced. A signal sequence of another protein can be used as the signal sequence. When mature hHGF without a signal sequence is expressed in a host cell, the hHGF-encoding gene has a base sequence from the 88th G or 94th C of the base sequence shown in FIGS. The gene may be used by connecting it to the ATG codon of the vector. Furthermore, in the present invention, modifications in which some amino acids or nucleic acids are removed, changed, or added within a range that does not impair the hepatic parenchymal cell growth promoting activity are also included in the present invention.

[0008] DNA of the gene encoding hHGF of the present invention
The fragment can be obtained, for example, by the following method. From plasma of fulminant hepatitis patients, for example, J. Clin. Invest, 81, 414 (198
Under reduced conditions, hHGF purified by the method described in 8) is cleaved at disulfide bonds and split into two polypeptides. A polypeptide having a molecular weight of 56000-65000 is defined as an H chain, and a polypeptide having a molecular weight of 32000-35000 is defined as an L chain. After carboxymethylation of the thiol group of the cysteine residue generated by reduction treatment of hHGF, H-chain and L-chain are separated by reversed-phase high-performance liquid chromatography, or hHGF is electrophoresed under reducing conditions, and the gel To H
After extracting each of the chains and the L chain, the amino-terminal amino acid sequences of both chains can be examined by, for example, analyzing with a gas phase protein sequencer manufactured by Applied Biosystems. In addition, hHGF itself or H
After separation of the chains, the appropriate proteolytic enzymes such as Achromobacter protease I (lysyl endopeptidase)
After separating the resulting peptide fragments by, for example, reversed-phase high-performance liquid chromatography, the amino acid sequence inside the polypeptide can be known by analyzing the amino acid sequence of each peptide in the same manner as described above. From these amino acid sequences, the DNA base sequence is deduced to select a sequence that is easy to produce an oligonucleotide, and the oligonucleotide,
For example, oligonucleotides such as those described in the examples below are synthesized and used as probes.

The cDNA library for screening the gene encoding hHGF includes human liver cDNA.
Libraries, spleen cDNA libraries, placental cDNA libraries, and the like can be used. These libraries are sold by Clonetech. Especially placenta cDNA
Libraries are preferred. In addition, a cDNA library may be prepared from a cell line expressing hHGF and tissue material according to a conventional method. Escherichia coli is infected with the λ phage having such cDNA incorporated therein by the method of Maniatis et al. (“Molecular Cloning”, Cold Spring Harbor Laboratory, pp. 56-73 (1982)) and cultured. A plaque hybridization method ("Molecular Cloning", Cold Spring Harbor Laboratory, pp. 320-328 (1982)) using the oligonucleotide formed from the nucleotide sequence deduced from a part of the amino acid sequence of hHGF to form a plaque as a probe Can easily have the same amino acid sequence as a part of the target amino acid sequence of hHGF and
Some different lambda phage clones having a nucleotide sequence corresponding to a region other than the probe of the amino acid sequence of the above can also be obtained.

Further, phages are grown from the plaques positive for screening by the method of Maniatis et al. ("Molecular Cloning", Cold Spring Harbor Laboratory, pp. 76-79 (1982)), and the phage is grown from the phage according to the glycerol gradient method. Is purified and cut with an appropriate restriction enzyme such as EcoRI.
8, plasmid vector such as pUC19 or M13mp
CD on single-stranded phage vectors such as 18, M13mp19
The NA was subcloned and the dideoxy method of Sanger et al. (Proc. Natl. Proc. Natl.
Acad. Sci. USA) 74 , 5463 (1977))
The nucleotide sequence of a DNA segment can be determined. By analyzing the nucleotide sequences of the obtained clones and integrating them, it is possible to obtain a gene corresponding to the entire amino acid sequence of hHGF shown in FIGS. 1 and 2 by a group of cDNA clones encoding a part of hHGF. it can.

[0011] The expression of the cDNA thus obtained can be carried out, for example, by ligating the DNAs in such a manner that their base sequence is in accordance with the amino acid sequence of hHGF.
This is a NA fragment, which is connected in phase with the translation initiation codon ATG downstream of the promoter of a plasmid such as pCDL-SRα296 to form a plasmid for protein expression, and is used in the host of animal cells transformed with the plasmid. It can be carried out. Next, the HGF of the present invention can be obtained by recovering the expressed protein according to a conventional method.

For the expression plasmid, it is desirable to construct a stable host-vector system for industrial production. For example, those described in Japanese Patent Application No. 1-115831 can be mentioned. Specifically, when a microorganism such as Escherichia coli or Bacillus subtilis is used as a host, it is preferably composed of a promoter, a ribosome binding sequence, an hHGF gene, a transcription termination factor, and a gene that controls the promoter. Examples of the promoter include tryptophan synthase operon (trp), lactose operon (lac), lipoprotein promoter (lpp), and the like.
Hybrid promoters such as tac (trp: lac), trc (trp: lac) and pac (phage: E. coli) may be used.
The hHGF gene preferably has a portion corresponding to the signal sequence removed, but hHGF can be obtained by removing the signal sequence from the produced preform even if the portion includes the portion corresponding to the signal sequence. As the plasmid to be used, a plasmid which becomes a high copy number in Escherichia coli or Bacillus subtilis, such as a pBR322-based plasmid or a pUB110-based plasmid, is desirable. Escherichia coli, Bacillus subtilis, and the like transformed by a conventional method can be used in a conventional medium for 15-42.
The culture may be performed at ℃. When yeast is used as a host, a yeast-derived promoter, such as pyruvate kinase (pYK),
HHG under the control of a sequence such as phosphoglycerokinase (pGK)
The F gene may be connected, introduced into yeast, and cultured at about 30 ° C.

However, considering that natural hHGF is a glycoprotein, animal cells are preferable as the host. In addition, when an animal cell is used as a host, the introduction of the hHGF gene containing a portion corresponding to the signal sequence is expected to have the advantage that hHGF excluding the signal sequence is secreted and produced. As the signal sequence, other than the original signal sequence of hHGF, a signal sequence of human serum albumin, interferon, human amylase, etc. may be used.In such a case, the DNA fragment encoding the original signal sequence may be used. May be substituted on the 5 'side with a DNA fragment having a base sequence corresponding to the signal sequence of (1). When an animal cell is used as a host, the promoter includes the SV40 late promoter, the promoter of the apolipoprotein E gene, the promoter of the apolipoprotein A1 gene,
Examples thereof include a heat shock protein gene promoter, a metallothionein gene promoter, an HSVTK promoter, an adenovirus promoter, and a retrovirus LTR. Among them, the SV40 promoter and the metallothionein gene promoter are preferable. Expression vectors include:
A polyadenylation site is included downstream of the hHGF gene.
Specific examples of the polyadenylation site include SV40 DNA, β
-Those derived from the globin gene or the metallothionein gene.

The polyadenylation site of the β-globin gene and the polyadenylation site of SV40 DNA may be linked. The expression vector may have a transformant selection marker. Even without a selectable marker in the expression vector, transformed animal cells can be selected by double transformation. Such selectable markers include the DHFR gene, which confers methotrexate resistance,
Herpes simplex virus (HSV) tk ^- gene, 3'-, which allows selection of transformed tk strains in HAT medium
Examples include the aminoglycoside 3'-phosphotransferase gene from Escherichia coli transposon Tn5 that confers resistance to the deoxystreptamine antibiotic G418, the bovine papillomavirus gene by overgrowth, and the aprt gene. Further, to select animal cells transformed with the expression vector by the double transformation method, a plasmid or other DNA containing the above-described gene serving as a selection marker is transformed together with the expression vector, and Transformed cells can be selected according to the phenotype described above by expression.

[0015] If the expression vector has a plasmid fragment having a replication origin derived from a bacterium such as Escherichia coli, cloning in bacteria is advantageously possible. Examples of such plasmid fragments include plasmid fragments such as pBR322, pBR327, and pML. Specific examples of the plasmid vector used for the expression vector include the SV40 early promoter, a splice sequence DNA derived from the rabbit β-globin gene, a polyadenylation site from the rabbit β-globin gene, and a polyadenylation site from the SV40 early region. PKCR containing the replication origin from pBR322 and the ampicillin resistance gene, the pBR322 portion of pKCR was replaced with pBR327, and the EcoR1 site present in exon 3 of the rabbit β-globin gene was replaced with the HindIII site. PKCR H2, pBPV MT1 containing the BPV gene and the metallothionein gene, and the like.

Animal cells transformed with the expression vector include CHO cells, COS cells, mouse L cells, and mouse C cells.
127 cells and mouse FM3A cells. The transfer of the expression vector into animal cells is performed by a transfection method, a microinjection method, etc. Among them, the calcium phosphate method is the most common. The culture of the animal cells transformed by the transfer can be performed by suspension culture or adherent culture by a conventional method. As a medium, MEM, RPMI 1640 and the like are generally used.

[0017] Separation and purification of the produced hHGF can be performed by column chromatography using heparin / sepharose, hydroxyapatite or the like, similarly to the purification from plasma of a fulminant hepatitis patient.

[0018]

The gene encoding hHGF according to the present invention can be introduced into an expression vector by a conventional method.
By using this as a template, hHGF or an hHGF-like substance or a fusion protein containing the same can be obtained. The obtained recombinant hHGF, hHGF-like substance or fusion protein containing hHGF may be used as a therapeutic agent for liver diseases such as a liver regeneration promoter, a liver function improving agent, a therapeutic agent for hepatitis or a cirrhosis inhibitor.

[0019]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the scope of the invention. Example 1 [1] Determination of partial amino acid sequence of hHGF and preparation of probe J. Clin. Invest., 81, 414 (19)
HHGF was purified according to the method described in 88). When subjected to SDS-PAGE, a slightly broad single band appeared at the position of molecular weight 76000-92000 under non-reducing conditions, and a slightly broad band with molecular weight of 56000-65000 and a molecular weight of 32000-35000 under reducing conditions. Two bands appeared. 50 μg of this purified hHGF was dissolved in 100 μl of 50 mM Tris-HCl buffer (pH 9) containing 5 molar urea, and Achromobacter protease I corresponding to 1/200 in molar ratio to hHGF was added thereto. In addition, the reaction was carried out at 37 ° C. for 6 hours. After the resulting peptide mixture is subjected to reductive carboxymethylation by a conventional method, J.
Each peptide was separated by reversed-phase high performance liquid chromatography using Bakerbond WP Octyl Column manufactured by T. Baker.

Amino acid sequence analysis was performed on the six peptides using a gas-phase protein sequencer (Applied Biosystems, Model 470A), and the sequences shown in Table 1 were found.

[Table 1]

[2] Screening of cDNA Encoding a Part of hHGF (1) Plaque Hybridization Screening Screening of 34-week-old human placenta-derived cDNA (Clontech) as a λ phage cDNA library Was performed according to Infect 1 million clones of phage into E. coli strain Y-1090 24.
NZY soft agar medium in a 5 cm x 24.5 cm petri dish [NZY medium; 1% NZ-amine, 0.5% yeast extract, 0.1%
5% sodium chloride, adjusted to pH 7.5 and added with 0.25% magnesium chloride, NZY soft agar medium; 0.7% in NZY medium
Agar droplets were added and the mixture was autoclaved, and five of them were cultured overnight at 42 ° C. at a ratio of 200,000 clones per plate.

Next, the λ phage clone in the medium was transferred onto Gene Screening Plus (DuPont), a commercially available nylon membrane, and plaque hybridization described below was performed. That is, the phage particles were transferred at a rate of two nylon membranes per petri dish, and the nylon membrane thus formed was washed with 0.1 M sodium hydroxide-
After 1.5M sodium chloride had been impregnated, leave it on paper for 2 minutes and remove water on a separately prepared dry filter paper. Then, apply 2xSSCP (2 times concentration of SSCP solution. Take the notation: 10 × SSCP; 1.2M sodium chloride,
The nylon membrane was allowed to stand on a filter paper impregnated with 150 mM sodium citrate, 130 mM potassium dihydrogen phosphate, 1 mM EDTA pH 7.2) -0.2 M Tris-hydrochloric acid (pH 7.4), air-dried on a dry filter paper, and then dried. The operation was repeated again. Nylon membrane treated in this way is 3 × SSC (20 times concentration SSC solution; 3M sodium chloride, 0.3M sodium citrate) −0.0.
After washing twice with 1% SDS at 60 ° C. for 15 minutes, 5 ml of a prehybridization solution [3 × SSC,
0.1% SDS, 10 × Denhardt's (50 times concentration of Denhardt's solution; 1% BSA (bovine serum albumin) 1% polyvinylpyrrolidone and 1% Ficoll 400), 20 μg / ml salmon sperm DNA]
At 65 ° C. for 3 hours.

Next, peptide 4 in Table 1, ie, Asn-M
et-Glu-Asp-Leu-His-Arg-His-Ile-Phe-Trp-Glu-Pro-Asp
Asn-Met-Glu-Asp-Leu-His of -Ala-Ser-Lys and
Synthetic oligonucleotides were prepared based on His-Ile-Phe-Trp-Glu-Pro. That is, 17 bases in the order of the aforementioned amino acid sequence
64 types of TH 23 (5'-TGT / C / A / GAA / GA / GTCT / CT
-CCATA / GTT-3 '), 17 bases 24 types of TH 24 (5'-GG
-T / CTCCCAA / GAAA / G / TATA / GTG-3 ') was prepared. The 5 'end of these was reacted with a polynucleotide kinase according to a conventional method using a reaction solution [50 mM Tris-HCl pH 7.6.
After ³² P-labeled magnesium 10mM chloride, 10mM mercaptoethanol, 100 [mu] M (gamma ³² P) ATP, a substrate DNA] in, except for the excess mononucleotides over DEAE cellulose column according to a conventional method. The result is ³²
Hybridization solution containing P-labeled synthetic oligonucleotide probe [3 × SSC, 10 × Denhardt's, 50 μg / ml
Salmon sperm DNA, 1M sodium chloride, 1% SDS, 250μg / ml salmon sperm DNA, 100,000 cpm / ml per synthetic probe ³² P
In the labeled probe DNA], A or T is replaced with 2 ° C. and G or C is replaced with 4 ° C. according to the probe, and the total temperature of all the bases is replaced.
(TH23) The mixture was kept at 46 ° C (TH24) for 36 hours. Then, remove the nylon membrane, wash twice in a 4 × SSC solution at room temperature for 30 minutes, wash twice with a 4 × SSC solution at the same temperature as for hybridization for 30 minutes, and then in a 2 × SSC solution for 15 minutes at room temperature. Washed twice and autoradiographed.

There were 6 pairs of nylon membranes whose signals on autoradiography matched each other. To isolate clones corresponding to the signals obtained, plaques on soft agar medium corresponding to these signals were punched out with a glass tube, and 1 ml of T in the presence of 50 μl chloroform.
Phage particles were extracted overnight in MG buffer [50 mM Tris-HCl pH 7.5, 100 mM sodium chloride, 10 mM magnesium chloride and 0.01% gelatin], infected again with E. coli strain Y-1090, and cultured in a 9 cm Petri dish in an appropriate amount. Plaque hybridization was performed as described above. By repeating this series of operations, each clone corresponding to the signal could be isolated. As a result, six independent clones were obtained. Two of them, namely λ hHGF
For 21 and λhHGF502, the nucleotide sequences of the contained cDNA were analyzed.

(2) Subcloning of cDNA fragment and determination of nucleotide sequence DNA was extracted from these λ phage clones as follows, and subcloned into plasmid vectors pUC18 and pUC19 and single-stranded phage vectors M13mp18 and M13mp19. That is, 200 ml of N in a 500 ml Erlenmeyer flask
In a ZY medium, 2 × 10 ⁷ pfu of λ phage clone (pfu; plaque forming unit) suspended in 200 μl of TMG solution and 40 μl of E. coli Y-1090 strain 2 × 10 ⁸ were suspended at 37 ° C.
Infected by placing for 15 minutes. After 15 minutes 1 ml 1 more
M calcium chloride was added, and the cells were cultured overnight, approximately 14 hours. Next, add 2ml of chloroform and leave for 10 minutes,
Add 15.8 g of sodium chloride and dissolve.
Rotor RPR 9-2 with Hitachi cooling centrifuge SCR20BB
The mixture was centrifuged at 6,000 rpm for 20 minutes, and 20 g of polyethylene glycol 6000 was added to the supernatant, which was sufficiently dissolved.

This was centrifuged at 6000 rpm for 20 minutes in a rotor RPR 9-2 in a Hitachi cooling centrifuge SCR 20BB to precipitate 6 ml.
A buffer solution (0.5% NP 40, 36 mM calcium chloride, 30 mM Tris-HCl pH 7.5 50 mM magnesium chloride, 125 mM potassium chloride, 0.5 mM EDTA, 0.25% deoxycholic acid, 0.6 mM
M mercaptoethanol] and add 100 μl of 10
mg / ml of deoxyribonuclease I and 10 μl of 10 mg / ml
E. coli-derived nucleic acid was degraded by adding ml of ribonuclease A and keeping the mixture at 30 ° C for 30 minutes. Thereafter, an equal volume of chloroform was added to the above reaction solution, and the mixture was stirred well, and then centrifuged at 3,000 rpm for 10 minutes with a Tommy centrifuge LC-06 and a rotor TS-7 to obtain a supernatant. Meanwhile, Hitachi Ultracentrifuge Rotor RPS
40% glycerol solution (0.5% NP 40, 30) in a 40T centrifuge tube
mM Tris-HCl pH 7.5, 125 mM potassium chloride, 0.5 mM
1 ml of EDTA, 0.6 mM mercaptoethanol, 10% glycerol], and 3 ml of 10% glycerol solution [0.5% NP40, 30 mM Tris-HCl pH 7.5, 125 mM
Potassium chloride, 0.5mM EDTA, 0.6mM mercaptoethanol, 40% glycerol] were prepared, and the nuclease-treated phage suspension was layered on top of it, and the Hitachi Ultracentrifuge 70P72, rotor RPS 35000 at 40T
Centrifuged for 1 hour at rotation.

After centrifugation, the phage particles falling down as a precipitate were washed with 0.4 ml of 40 mM Tris-HCl pH 7.5, 10 mM EDTA, 2% S
Suspend in DS and add 4 μL of 10 mg / ml proteinase K
The temperature was kept at 55 ° C for 1 hour. Thereafter, the solution was transferred to an Eppendorf tube, and the phage DNA was extracted with an equal amount of phenol / chloroform, followed by ethanol precipitation to obtain 200 μg of the target phage DNA.
The phage DNA was digested with a restriction enzyme EcoRI according to a conventional method, and analyzed by agarose electrophoresis. As a result, three EcoRIs of 0.2 kb, 0.85 kb and 0.72 kb from clone λ hHGF 21 were obtained.
A fragment was obtained. On the other hand, the insert cDN
The desired cDN is obtained by recovering the A fragment according to a conventional method.
A fragment was obtained. A reaction mixture of 10 μl of plasmid vector pUC18, pUC19 and single-stranded phage vector M13mp18, M13mp19 200 ng of 100 ng of these cDNA fragments previously digested with restriction enzyme EcoRI in a usual manner
[66 mM Tris-HCl pH 7.6, 6.6 mM magnesium chloride, 1
In the 0 mM dithiothreitol, 66 μM ATP, substrate DNA], the HGF protein has a partial sequence at the EcoRI insertion site by transforming the host Escherichia coli suitable for each vector with the unit T4 DNA ligase according to a standard method. A subclone was obtained.

The nucleotide sequence of the obtained cDNA subclone was determined by the dideoxy method of Sanger et al. Primers corresponding to commercially available M13 phage vectors were used. As a result, clone λ with the longest cDNA
When the nucleotide sequence of hHGF 21 is translated into amino acids, FIGS.
As shown in Fig. 7, it was found that the clone contained at least a part of the amino acid sequence of a region different from the amino acid sequence used for the probe design among the amino acid sequences already clarified. It was found to be a cDNA containing the region of Also, λ hHG
Of clone λ hHGF502 containing a cDNA fragment not found in F21
As a result of analyzing the nucleotide sequence of cDNA according to the Sanger method, clone λ hHGF502 has the same nucleotide sequence as clone λ hHGF 21 and is the third EcoRI counted from the 5 ′ upstream from the vicinity of restriction site NcoI shown in FIGS. 0.8 kb shared to the vicinity of the cleavage site and not 3 'λ hHGF 21
It was found to have a base sequence of 0.7 kb. λ hHGF 502
It was found that among the base sequences of, there was a base sequence corresponding to the already analyzed amino acid sequence among the base sequences not having λhHGF21. It was found that when the nucleotide sequences of these two clones were joined together in a partially overlapping manner, the entire amino acid sequence of hHGF was covered.

[Brief description of the drawings]

FIG. 1 shows the first amino acid sequence of the hHGF of the present invention.
The 360th amino acid (Ser) from the 360th amino acid (Met)
FIG. The underline in the figure indicates the amino acid sequence that has been already clarified.

FIG. 2 shows 36th amino acid sequence of the entire amino acid sequence of hHGF of the present invention.
From the 1st amino acid (Glu) to the 728th amino acid (Se
It is a figure showing the arrangement | sequence to r). The underline in the figure is the same as in FIG.

FIG. 3 is a view showing the sequence from the first base to the 780th base in the entire base sequence of the cDNA containing the gene encoding hHGF of the present invention obtained in Example 1. The main restriction enzyme recognition sites are also shown in the figure. The underline indicates a portion corresponding to the amino acid sequence that has already been revealed.

FIG. 4 is a view showing the sequence from the 781st base to the 1620th base in the entire base sequence of the cDNA containing the gene encoding hHGF of the present invention obtained in Example 1. The main restriction enzyme recognition sites are also shown in the figure. The underline in the figure is the same as in FIG. 3, and the double underline represents the base sequence corresponding to the probe used to obtain the first clone.

FIG. 5 is a view showing a sequence from the 1621st base to the 2187th base in the entire base sequence of the cDNA containing the gene encoding hHGF of the present invention obtained in Example 1. The main restriction enzyme recognition sites are also shown in the figure. The underline is the same as FIG.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12N 15/09 ZNA C12N 5/00 B C12P 21/02 15/00 ZNAA (C12N 1/21 C12R 1:19) (C12N 5 / 10 C12R 1:91) (C12N 15/09 ZNA C12R 1:91) (C12P 21/02 C12R 1:19) (C12P 21/02 C12R 1:91) (72) Inventor Kazunobu Takahashi Midori, Yokohama, Kanagawa 1000 Kamo-shida-ku, Mitsubishi Research Institute, Ltd. (56) References JP-A-6-153946 (JP, A) JP-A-63-22526 (JP, A) Clin. Invest. , Vol. 81 (1988) p. 414-419 (58) Fields investigated (Int. Cl. ⁶ , DB name) C07K 14/52 C12N 15/09 ZNA BIOSIS (DIALOG) GenBank / EMBL / DDBJ (GENETYX) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. The following amino acid sequence: Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu 1 5 10 15 Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln 20 25 30 Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr 35 40 45 Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val 50 55 60 Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu 65 70 75 80 Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys 85 90 95 Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe 100 105 110 Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys 115 120 125 Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys 130 135 140 Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His 145 150 155 160 Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr 165 170 175 Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser 180 185 190 Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu 195 200 205 Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp 210 215 220 His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro 225 230 235 240 His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp 245 250 255 Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr 260 265 270 Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys 275 280 285 Ala Asp Asn Thr Met Asn Asp Thr Asp Val Pro Leu Glu Thr Thr Glu 290 295 300 Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile 305 310 315 320 Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu 325 330 335 His Asp Met Thr Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn 340 345 350 Tyr Cys Arg Asn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr 355 360 365 Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp 370 375 380 Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met 385 390 395 400 Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp 405 410 415 Lys Asn Met Glu Asp Leu His Arg His Ile Phe Trp Glu Pro Asp Ala 420 425 430 Ser Lys Leu Asn Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His 435 440 445 Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys 450 455 460 Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu 465 470 475 480 Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val 485 490 495 Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp Met Val Ser Leu Arg 500 505 510 Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp 515 520 525 Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr 530 535 540 540 Glu Ala Trp Leu Gly Ile His Asp Val His Gly Arg Gly Asp Glu Lys 545 550 555 560 Cys Lys Gln Val Leu Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly 565 570 575 Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp 580 585 590 Phe Val Ser Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu 595 600 605 Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn 610 615 620 Tyr Asp Gly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly Asn Glu 625 630 635 640 Lys Cys Ser Gln His His Arg Gly Lys Val Thr Leu Asn Glu Ser Glu 645 650 655 Ile Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp 660 665 670 Tyr Gly Gly Pro Leu Val Cys Glu Gln His Lys Met Arg Met Val Leu 675 680 685 Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly 690 695 the hepatocyte growth factor comprising a signal sequence represented by 700 Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile 705 710 715 720 Leu Thr Tyr Lys Val Pro Gln Ser 725
Fraud and mitigating risk signal sequence which is characterized by being represented by the sequence from 30th glutamic acid to the 728 th serine containing
Manai hepatocyte growth factor. 2. It is represented by the amino acid sequence according to claim 1.
Hepatocyte growth factor containing a signal sequence
A hepatocyte growth factor not containing a signal sequence, which is represented by a sequence from glutamine to serine at position 728.