JPH06192297A - Factor for multiplying matured hepatic parenchyma cell - Google Patents

Abstract

PURPOSE:To obtain the novel multiplication factor separated and purified from the homogenate of blood cells or vital tissues with chromatography useful for liver-regenerating medicines used after the partial enucleation of a liver, hepatic disease-therapeutic medicines, carcinostatic agents, wound therapeutic medicines, diagnostic medicines for various kinds of diseases. CONSTITUTION:A single strand polypeptide having an estimated mol.wt. of 82000+ or -5000 dalton by SDS-PAGE under a non-reducing condition, an estimated mol.wt. of 92000+ or -5000 dalton by SDS-PAGE under a reducing condition, and multiplying primarily cultured parenchyma cells is separated and purified from the homogenate of blood cells or vital tissues by the use of at least one of ion exchange chromatography, pigment affinity chromatography, lectin affinity chromatography, heparin affinity chromatography, hydrophobic chromatography, adsorbing chromatography, reversed phase chromatography, etc., to provide the matured hepatic parenchyma cell-multiplying factor useful for liver regeneration-stimulating medicines, etc.

Description

Detailed Description of the Invention

[0001]

The present invention relates to mature hepatocyte growth factor II (hepatocyte growth) having a novel physicochemical form.
factor II; abbreviated as HGF (II)), and more specifically, to a peptide factor having a physiological activity that enables the culture of mature hepatocytes in vitro. The HGF (II) of the present invention is expected to be used as a research reagent for biological research of liver parenchymal cells and liver, and further as a drug for the liver such as a liver regeneration promoting reagent, a chronic hepatitis therapeutic drug, and a cirrhosis therapeutic drug. To be done. Also HG
F (II) has a proliferation-promoting action on endothelial cells and epithelial cells as well as a physiological action on hepatocytes, and also has a proliferation-inhibiting action and cytotoxic action on cancer cells and tumor cells. . From this, the HGF (II) of the present invention is expected to be used as a therapeutic drug for wounds and an anticancer agent.

[0002]

2. Description of the Related Art The liver has a wide variety of functions and is an organ that plays an indispensable and important role in maintaining homeostasis of a living body. The main functions of the liver are synthetic secretion of various plasma proteins, blood glucose regulation by gluconeogenesis and glycogen metabolism,
Urea synthesis, bile secretion, metabolism of various drugs, detoxification, etc. It is known that the mature liver parenchymal cells that make up the liver are responsible for the function of the liver. Also,
One of the great features of the liver is its remarkable ability to regenerate. For example, it is known that even if two-thirds of the rat liver is excised, it returns to the size in about 1 week to 10 days. It has been speculated that this liver regeneration function is carried by an unknown humoral factor (hepatotrophic factor), but its body has not been clarified for a long time.

The present inventors have found that the primary hepatocyte culture method is an effective means for studying this liver regeneration factor, and using this culture system, in the presence of a specific protein component contained in serum. In, for the first time to be able to proliferate hepatocytes, the specific serum components were separated and named hepatocyte growth factor (HGF) mature [Biochem. Biophys. Res. Commun., 122 (No.3), 145
0, 1984, JP-A-60-45534)]. Furthermore, the present inventors have succeeded in purifying HGF from thrombin-stimulated supernatant of mammalian platelets [FEBS LETTER, 224 (No. 2), 311.
(1987)], and clarified the various properties.

HGF has an estimated molecular weight of about 150,000 daltons by gel filtration, and is about 82,02 under non-reducing conditions by SDS-PAGE.
Under a reducing condition of 1,000 daltons, it is divided into two bands, an α chain of about 60,000 to 65,000 daltons and a β chain of about 30,000 to 35,000 daltons. That is, HGF is a heterodimer protein in which two types of polypeptides, an α chain and a β chain, are linked by a disulfide bond. It was also revealed that HGF is a protein that is unstable to heat and acid and has a strong affinity for heparin sepharose.

As a result of further studies, the present inventors have found that blood cells such as white blood cells such as mammalian platelets other than thrombin-stimulated platelet supernatant and plasma, liver, kidney, lung, placenta, brain and spleen. In various living tissue homogenates such as
It was found that there are substances that have extremely good activity on mature hepatocytes in vitro. Various investigations were carried out to purify and isolate the substance. As a result, the substance having the activity of proliferating mature hepatocytes contained in the above-mentioned living tissues was found to be a heterodimeric protein consisting of two polypeptide chains and SDS under reducing conditions. -PAGE revealed that there are two types of monomeric proteins showing a single band. Then, the heterodimer protein [(designated as HGF (I)]] was purified to a single form, and its N-terminal amino acid sequence and other properties were clarified (JP-A-2-2888).
No. 99). Further, based on the obtained amino acid sequence of β chain, gene cloning of HGF (I) was performed, and rat HGF cDNA was successfully cloned from rat liver [Proc. Natl. Acad. Sci. USA, 87, 3200 ( 1989)]]
Furthermore, HGF cDNA derived from human liver [Nature, 342 , (62
48), 440 (1989)] and human leukocyte-derived HGF cDNA
[Biochem. Biophy. Res Commu., 172 (1), 321 (1990)]
Was cloned to reveal the entire structure of HGF (I).

On the other hand, in SDS-PAGE under reducing conditions, HGF (I) was found to be mixed in the monomer protein showing a single band (named HGF (II)).
Only GF (II) could not be isolated, and the monomer protein HGF (II) itself was completely unknown.

[0007]

Therefore, the object of the present invention is to provide HGF (II), a method for producing the same, and HGF (I).
To provide a composition comprising I).

[0008]

[Means for Solving the Problems] The present inventors have conducted extensive studies to isolate HGF (II), and as a result,
The HGF (II) was successfully purified singly, the chemical structure was analyzed, the biological function was clarified, and the main body was clarified, thus completing the present invention. Came to.

The present invention is a protein which has the activity of proliferating mature hepatocytes in vitro, is unstable to heat and has a high affinity for heparin-sepharose, and has the following constitution. . That is, the present invention relates to a mature hepatocyte growth factor comprising a single-chain polypeptide having the following physicochemical properties, a composition containing at least the growth factor, and a method for producing the same. (1) Estimated molecular weight by SDS-PAGE under non-reducing conditions is 82,000 ± 5,000 daltons. (2) Estimated molecular weight by SDS-PAGE under reducing conditions is 92,000 ± 5,000 daltons.

The conventional method for preparing HGF is to use ion exchange chromatography, heparin affinity chromatography, dye affinity chromatography, and hydroxyapatite chromatography, using thrombin-stimulated supernatant of platelets or blood of a patient with fulminant hepatitis as a starting material. Was obtained after purification by [FEBS Letter, 224
(2), 311 (1987), and J. Clin. Invest., 8, 414 (1988).
See]. The HGF obtained by the above method is S
DS-PAGE has a molecular weight of 76,000 under non-reducing conditions.
It migrates as a single band of 92,000 daltons and has a molecular weight of 56,000-by SDS-PAGE under reducing conditions.
It has been considered to be a heterodimeric protein composed of two kinds of polypeptides by migrating in two bands of 69,000 and 32,000 to 35,000 daltons.

On the other hand, the present inventors have found that HGF active substances are widely contained in living tissues such as blood cells such as platelets and white blood cells, liver, kidney, lung, spleen and placenta. The homogenates of these living tissues were purified by a method combining ion exchange chromatography, dye affinity chromatography, lectin affinity chromatography, heparin chromatography, reverse phase chromatography, etc., and SD under non-reducing conditions was used.
An HGF active substance showing a single band of about 82,000 daltons was obtained by S-PAGE. This HGF active substance was shown to migrate as three bands of about 92,000 daltons, about 60,000 daltons and about 32,000 daltons by SDS-PAGE under reducing conditions. This result indicates that HGF active substances derived from various biological tissue homogenates including platelets contain two components, a heterodimer protein (named HGF (I)) and a monomer protein (named HGF (II)). It indicates that

The present inventors have found that HGF (II) HGF
Various studies were conducted on the method of separation from (I), and finally HGF (II) was successfully purified to homogeneity.
The main body of F (II) was clarified. Furthermore, the present inventors produced recombinant cells into which the HGF gene had been introduced, cultured the recombinant cells in a serum-free medium supplemented with a protein inhibitor, and secreted HGF (II) extracellularly. Homogeneous HGF (II) was obtained in a high yield from the culture solution by a method combining ion exchange chromatography, dye affinity chromatography, heparin affinity chromatography, reverse phase chromatography, hydrophobic chromatography, adsorption chromatography and the like. After that, the present invention was completed.

H according to the present invention produced as described above
GF (II) is characterized as a single-chain polypeptide protein having hepatocyte proliferative activity.

The method for producing HGF (II) of the present invention will be described in detail below. The HGF (II) of the present invention can be efficiently isolated from, for example, mammalian blood cells and homogenates of living tissues such as liver, lung, kidney, spleen and placenta. In particular, it can be obtained more efficiently and in high yield than a cell into which the HGF gene has been introduced by a gene recombination technique or a culture in which a cell line secreting HGF is cultured in the presence of a protease inhibitor. There is no particular limitation on the mammalian tissue or genetically modified cells or cell lines used as raw materials here, and examples thereof include humans, horses, cows, pigs, sheep,
Any one derived from rabbit, mouse, rat, monkey, etc. can be used.

The HGF (II) of the present invention can be obtained together with HGF (I) by purifying it from a culture in which a homogenate of the above-mentioned living tissue or a protease inhibitor such as aprotinin or cetraxate is added. HGF (I)
And HGF (II) are purified by purifying the HGF (I) and (I
It can be carried out by a combination of various separation means utilizing the physical, chemical and immunological properties of I). Examples of particularly preferred purification means include salting out, ion exchange chromatography, immuno-antibody chromatography, dye affinity chromatography, metal chelate affinity chromatography, lectin affinity chromatography, molecular sieve chromatography, adsorption chromatography, hydrophobic chromatography. , Reverse phase chromatography, heparin affinity chromatography, chromatofocusing, and further electrophoresis,
A method combining an ultrafiltration method and a dialysis method can be exemplified.

For salting out, salts such as ammonium sulfate and sodium sulfate can be used. Further, as the carrier used for ion exchange chromatography, any of the carriers for various ordinary ion exchange chromatography used for separating proteins can be used. Specific examples thereof include CM-cellulose, CM-Sephadex, CM-Toyopearl, P-cellulose and S.
Cation exchange resins such as P-Toyopearl, Mono S, S-Sepharose FF, DEAE-Sepharose, DEA
E-Sephadex, Mono Q, Q-Sepharose FF,
Anion exchange resins such as DEAE-Toyopearl and QAE-Toyopearl are exemplified. Specific examples of dye affinity chromatography include Affigel Blue and Blue Tris Acrylic M, specific examples of lectin affinity chromatography include ConA Sepharose, and specific examples of adsorption chromatography include hydroxyapatite and HA-ultrogel. Specific examples of metal chelate affinity chromatography include chelating sepharose CL6BFF and TSK.
Specific examples of the gel AF chelate / Toyopearl include phenyl 5PW, phenyltoyopearl, butyl sepharose, butyl toyopearl, and ether toyopearl.

[0017]

HGF (II) of the present invention is a therapeutic agent for liver regeneration after partial hepatectomy by surgery and a therapeutic agent for liver diseases such as acute hepatitis, chronic hepatitis, transition to cirrhosis, and fulminant hepatitis. It is also useful as a pharmaceutical agent such as an anti-cancer agent and a therapeutic agent for wounds and as a diagnostic agent for each of the above diseases. Further H
By using GF (II), hepatocytes derived from various animals including humans can be extremely easily proliferated and maintained in vitro in the presence of the HGF (II). For basic research such as application to the liver and liver function, for studying the action of various hormones, peptides, or various drugs on hepatocytes, for screening tests for liver diseases, and for carcinogenicity tests and hepatitis virus. Is extremely useful as a host cell in in vitro culture.

Since the HGF (II) of the present invention is unstable to heat, it usually contains a surfactant such as Tween 80 as a stabilizer, mannitol, sorbitol, heparin, saccharides such as glucosaminoglucan, and glycerin. It is preferable to combine one or more of alcohols such as polyethylene glycol and ethylene glycol, amino acids such as glycine, arginine, alanine and aspartic acid, and proteins such as serum and albumin in combination.

When the HGF (II) of the present invention is used as a drug as described above, it is formulated into a lyophilized product or an injection with HGF (II) itself or a carrier known per se,
It is administered parenterally.

[0020]

EXAMPLES The present invention will be more specifically described below with reference to Reference Examples and Examples, but the present invention is not limited thereto.

Reference Example 1 (Measurement of HGF activity 1): [Nakamura, T., et al.
Proc. Natl. Acad. Sci. USA., 80, 7229-7233 (1983)] In situ collagen perfusion method [Tanaka, K., et al. J. Biochem. (TOKYO)
84 , 937-946 (1978), Toshikazu Nakamura, Primary hepatocyte experiment method, Academic Publishing Center pp 5-53 (1987)]. 5 rat hepatocytes obtained
% Bovine serum, 2 × 10 ^-9 M insulin and 2 × 10 ^-9 M dexamethasone were added to suspension in William E medium (Flow Laboratories), and the concentration of 1.25 × 10 ⁵ cells / well was applied to a 24-well multiplate. Sowed. 5% CO
After culturing in the presence of ₂ and 30% O ₂ and 65% N ₂ for 20 hours at 37 ° C., the medium was replaced with Williams E medium supplemented with 0.1 μg / ml aprotinin, and at the same time a predetermined amount of the sample was added. After 15 hours, 15 μCi / ml of ¹²⁵ I-deoxyuridine 10
μl / well was added. In the control group, 5 μg / ml of aphidicolin was added 15 minutes before the addition of ¹²⁵ I-deoxyuridine. Labeled with ¹²⁵ I, which was incubated for a further 6 hours. The cells were washed twice with PBS at pH 7.4 and then fixed with cold 10% trichloroacetic acid aqueous solution (TCA). The cells were solubilized with 0.5 ml of 1N aqueous sodium hydroxide solution per well, and the radioactivity thereof was measured by a gamma counter. Also, take a part of the sample after the radioactivity measurement and use the Lowry method (J.
ol. Chem., 193, 265, 1951). The amount of ¹²⁵ I taken up in hepatocytes when the test sample was added was calculated as the difference from the control, and this was converted to 1 mg of rat hepatocyte protein to calculate the DNA synthesis activity (cpm / mg protein). ) The HGF activity of the test sample was expressed by defining 1 unit as the activity corresponding to 50% of the DNA synthesis activity of hepatocytes when epidermal growth factor (EGF) 10 ng / ml was used in the same test.

Example 1 Preparation of liver extract 100 rats (strain SD; body weight 200 to 300 g) were intraperitoneally administered with 10 ml / kg of 20% carbon tetrachloride solution dissolved in salad oil (2 ml / carbon tetrachloride). (kg administration), and after 30 hours, the liver was removed. The removed liver is 0.15M NaCl, 1 mM PMSF, 1 mM
Buffer solution A containing monoiodoacetic acid and 1 mM EDTA A [50 mM Tr
is-HCl (pH8.5), 10mM Hepes, 2mM CaCl 2, 0.01% Twe
en 80] Crushed with a Waring blender in 4 liters. After crushing, 10,000 rpm with a cooling centrifuge (Hitachi 20PR-52)
The precipitate was removed by centrifugation for a minute. Filter the supernatant with filter paper,
The filtrate was obtained as a liver extract.

Cation Exchange Chromatography About 4 liters of the liver extract was dialyzed against buffer A containing about 4 volumes of 0.15M NaCl three times for 2 hours or more, and then equilibrated with buffer A containing 0.15M NaCl. S-Sepharose FF (Pharmacia) column (size, inner diameter 11.3 cm x height 10 c
m) was added. After washing with buffer A containing 0.15M NaCl,
Elution was performed with a linear gradient of 0.15 M to 1.0 M NaCl (total volume: 6 l). The elution fraction was converted to H by the method shown in Reference Example 1.
The GF activity was measured and the active fractions were collected and S-Sepharose F
This was the F eluate. The elution pattern is shown in FIG.

Dye Affinity Chromatography The S-Sepharose FF eluate was adjusted to pH 7.5 with 1N HCl, diluted with distilled water containing the same amount of 0.01% Tween 80, and then used as buffer B [20 mM Tris-HCl (pH 7. 5) was added to a Blue-Trisacryl M column (manufactured by IBF, column size: inner diameter 2.6 cm x height 13.5 cm) equilibrated with 0.01% Tween 80]. After washing with buffer B, elution was performed with a linear concentration gradient of arginine from 0 to 0.5 M (total amount: 350 ml). The elution pattern is shown in FIG. Fractions having high HGF activity were collected and used as Blue-Trisacryl M eluate.

Heparin Affinity Chromatography Blue-Trisacryl M eluate was mixed with 9 volumes of buffer C [10 mM Tris
-HCl (pH 7.5), 0.01% Tween 80] and then 0.3M Na
Heparin Sepharose C equilibrated with buffer C containing Cl
L-6B column (Pharmacia, column size: inner diameter)
1.6 cm × height 7 cm). After washing with buffer C containing 0.3M NaCl, 0.3M to 2.0M NaCl linear concentration gradient (total amount
(150 ml). The elution pattern is shown in FIG. H
Fractions with high GF activity were collected and used as a heparin-sepharose eluate.

Hydrophobic Chromatography Solvent A [20 mM phosphate buffer (pH 7.5), 4 M NaCl] and solvent B
Phenyl 5PW column (manufactured by Tosoh Corporation, column size: inner diameter 7.5 mm x height 7.5 c) equilibrated with a 2: 1 mixture of [20 mM phosphate buffer (pH 7.5) and 50% ethylene glycol].
Heparin-Sepharose eluate was added to m). Elution was performed by continuously changing the composition ratio of the solvents A and B from 2: 1 to 0: 1. The elution pattern is shown in FIG.
The fraction showing HGF activity eluted around 45 minutes was homogeneous HG.
It was F (I) and the HGF active fraction eluted around 85 minutes was a fraction containing HGF (II). However, as shown in FIG. 6, as a result of analysis by SDS-PAGE under reducing, this H
Contamination of HGF (I) was still observed in the GF (II) fraction. To collect the HGF (II) fraction, the procedure from was repeated (for hydrophobic chromatography, the elution conditions were changed), and from a total of about 500 rat livers,
The HGF (II) fraction was collected.

Repeated Hydrophobic Chromatography A phenyl 5PW column (manufactured by Tosoh Corp., column size: inner diameter 7.5 mm × height 7.5 cm) equilibrated with solvent A was doubled to the HGF (II) fraction collected in the section above. A solution containing an amount of solvent A was added. The solvent A is flown and the column is washed, then the solvent A
The elution operation was performed by continuously changing the composition ratio of B and B from 1: 0 to 0: 1. The elution pattern is shown in FIG.
Shown in. HGF contained in the HGF (II) fraction
(I) was eluted first, followed by HGF (II).
Fractions without HGF (I) contamination were determined by SDS-PAGE, and fractions containing only HGF (II) were collected. The amount of purified HGF (II) obtained was 95 μg.

Analysis of physicochemical properties of HGF (II) (i) Regarding HGF (II) obtained in the terms of SDS-PAGE and
SDS-PAGE analysis was performed according to the method of Laemmli. The result is shown in FIG. HGF obtained in the section
(II) has a molecular weight of 82,000 ± 5,000 under non-reducing conditions.
It shows 0 Dalton and has a molecular weight of 72,000 even under reducing conditions.
Shows only one band at 0 ± 5,000 daltons, H
It was shown that GF (II) is a protein consisting of a single polypeptide chain. The amount of protein added per well was about 200 ng, and staining was performed by the silver staining method (manufactured by Wako Pure Chemical Industries, Ltd.).

(Ii) Physiological activity Using HGF (II) purified to homogeneity and HGF (I) obtained in the section above in the section of the biological activity, each rat was tested according to the method shown in Reference Example 1. Primary culture hepatocyte proliferation promoting activity was measured. The homogeneous HGF (II) activity was 350,000 ± 100,000 units / mg, and the HGF obtained in
The activity of (I) showed a hepatocyte proliferation promoting activity equivalent to (300,000 ± 100,000 units / mg). The protein has an absorbance of 14 O.S. at 214 nm. D. At that time, the protein concentration was calculated as 1 mg / ml.

(Iii) HGF (II) purified in the section of partial amino acid sequence was evaporated to dryness and then 5M.
A 1M Tris-HCl (pH 8.1) buffer solution containing guanidine hydrochloride and 0.2% EDTA was added and redissolved. After the dissolved oxygen was blown off with nitrogen gas, 2-mercaptoethanol was reduced at 50 ° C. for 15 hours. After the reduction, an equal amount of monoiodoacetic acid was added and reacted at room temperature for 1 hour to carboxymethylate the cysteine residue. Reversed phase HPLC [μBondasphere 5μ
C4-300A, 3.9 mm x 15 cm (Waters Co.)] for desalting. Elution was performed by a continuous concentration gradient from water containing 0.1% TFA to a mixture of 20% water-80% [acetonitrile: isopropanol (1: 1)] containing 0.1% TFA.
As described above, the reduced-carboxymethylated HGF (I
I) was obtained. A portion of this was taken and dried under reduced pressure, and the resulting reduced-carboxymethylated HGF (II) was added to 200 μl of 0.1.
Redissolve in 1M Tris-HCl buffer (pH 9.5), add 1 μg of lysyl endopeptidase (Wako Pure Chemical Industries) from Achromobacter litikas, react at 37 ° C for 5 hours, and use HGF.
A partially digested product of (II) was obtained. This partial digest is reverse phase H
It was applied to PLC [μBondasphere 5μ C18-300A (Waters)] and peptide mapping was performed. Elution is 0.
Water containing 1% TFA to 20% water containing 0.1% TFA-80
% [Acetonitrile: isopropanol (1: 1)] mixed solution. Obtained HGF
The peptide map of (II) is shown in FIG. 7. Next, the peptide fragment of FIG. 7 was collected and its amino acid sequence was analyzed using a gas phase protein sequencer (Applied Biosystems 477A). The amino acid sequence of the peptide fragment thus obtained was HGF (I) cDNA.
Amino acid sequence deduced from the sequence [Proc. Natl. Acad. S
ci. USA, 87, 3200 (1989)]. That is, it was shown that HGF (II) is a single chain protein which is a precursor of HGF (I). Particularly, the amino acid sequence of the peptide marked with * in FIG. 7 is X-Leu-Arg-Val-Val-Asn-Gl.
It was y-Ile-, and was a peptide containing cleavage sites (between Arg-Val) of the α chain and β chain of HGF (I). Obtaining this peptide clearly shows that the protein before HGF (I) is cleaved into α chain and β chain is HGF (II). From the above results, as a mechanism of HGF production, first, HGF mRNA is translated, pre-HGF protein is produced, and then a secretory signal is cleaved to form single-chain HGF (II), which is stored in the endoplasmic reticulum. HGF
(II) is in the endoplasmic reticulum or extracellularly, and it is presumed that HGF (I) is cleaved into double chains by the action of protease. It was revealed for the first time by the present invention that single-chain HGF is present in natural tissues and retains biological activity.

Example 2 HGF (II) by gene recombination method Expression plasmid pEVSSV (dLeHGF, incorporated by human leukocyte-derived HGF cDNA)
No. 00) was introduced into a DHFR-deficient Chinese hamster ovary cell line (CHO ^dhfr- ) cell by the method of Wigler et al. That is, 30 μg of pEVSS
The V (dLeHGF) plasmid was dissolved in 240 μl of 0.5 M calcium chloride solution, and 2 × HEPES buffer [280 mM N
20 mM HEPES containing aCl and 1.5 mM sodium phosphate (pH 7.
1)] was added with stirring. The mixture was stirred at room temperature for 30 minutes to form a coprecipitate of plasmid DNA and calcium phosphate. The cells were cultured in an α-MEM medium containing 10% fetal bovine serum and 1% glutamine at 37 ° C for 24 hours. CHO ^dhfr-
The co-precipitate was added to the cells and left at room temperature for 20 minutes. After further culturing at 37 ° C. for 4 hours, the medium was removed, 1 × HEPES buffer containing 1.5% glycerin was added, and the mixture was allowed to stand at room temperature for 5 minutes. Nucleoside-free α-containing 10% dialyzed fetal serum, 1% glutamine and 50 nM methotrexate.
After washing the cells with MEM medium, the cells were cultured at 37 ° C. for 7 days to obtain transformed cells. After subculture of the transformed cells in the above medium, the concentration of methotrexate in the medium was adjusted to 100 nM, 250 nM, 500 nM, 750 in order to obtain a stable HGF high-producing strain.
Subculture was carried out while sequentially increasing nM, 1 μM, and 2 μM. The resulting HGF-producing transformant cells were clonally selected to select HGF-producing strain 515C. Using a roller bottle, the selected 515C strain was cultured in a nucleoside-free α-MEM medium containing 10% fetal bovine serum (Gibco), 1% glutamine and 2 μM methotrexate (Flow Laboratories) at 37 ° C. under 5% CO _2. Cultured in
The cells were cultured until they grew to a conflict. Then, the culture solution was extracted, the cells were washed twice with PBS, and 1% glutamine, 500 μM methotrexate and a protease inhibitor were added. Α-MEM medium containing 400 units / ml of aprotinin (without nucleoside) was added, and the mixture was incubated at 37 ° C for 5
Cultured under% CO ₂ . The culture supernatant was collected by exchanging the culture supernatant daily. About 500 ml of the obtained culture supernatant
The recombinant HGF (II) was purified by a three-step chromatographic procedure.

(1) Cation exchange chromatography PM was added to a serum-free medium containing a protease inhibitor of 515C strain.
SF and Tween 80 were added to a final concentration of 1 mM and 0.01%, respectively, and insoluble matters were removed by filtration with a filter. 1/20 volume of 1M Tris-HCl (p
H8.5) Add buffer and buffer D [150mM NaCl, 1mM PMSF
, 50mM Tris / HCl (pH8.5) containing 0.01% Tween 80]
Was added to S-Sepharose FF (manufactured by Pharmacia, column size: inner diameter 1.6 cm × height 5 cm).
After washing the column with buffer D, buffer E [400 mM NaCl
, 50 mM Tris-HCl (pH 8.5) containing 1 mM PMSF, 0.01% Tween 80], and then the buffer F [1M NaC]
50 mM Tris-HCl (pH 8.5) containing 1 mM PMSF and 0.01% Tween 80] was flowed down to elute the fraction containing HGF (II). The elution chromatogram pattern is shown in FIG.
Fractions eluted with buffer F were collected and used as a recombinant S-Sepharose eluate.

(2) Affinity Chromatography The recombinant S-Sepharose eluate was adjusted to pH 7.5 with 1N hydrochloric acid and then diluted with distilled water containing 3 volumes of 0.01% Tween 80 and 1 mM PMSF. Buffer G [0.3M NaCl, 1mM PMSF, 0.01
10 mM Tris / HCl (pH 7.5) containing 10% Tween 80], and the above diluent was added to heparin Sepharose CL-6B (Pharmacia, column size: inner diameter 1 cm x height 5 cm), and then the buffer solution was added. After thoroughly washing the column with G, the NaCl solution in buffer G was eluted by a concentration gradient method in which the concentration was continuously changed from 0.3 M to 2.0 M (total volume 40 ml), and HGF (I
I) was eluted. Fractions having HGF activity were collected and used as a recombinant heparin eluate. The chromatographic pattern is shown in FIG.

(3) First hydrophobic chromatography buffer H [20 mM sodium phosphate (pH 7.0) containing 4 M NaCl]
The recombinant heparin eluate was added to a phenyl 5PW column (manufactured by Tosoh Corp., column size: 0.75 cm inner diameter × 7.5 cm height) equilibrated with 1. and the column was washed with buffer solution H. Elution was performed with buffer H and buffer L (20 mM containing 50% ethylene glycol
It was carried out by a concentration gradient method in which the mixing ratio with sodium phosphate (pH 7.0)] was continuously changed. Figure 10 shows the elution pattern.
Shown in. HGF activity of each elution fraction was measured and
For fractions with GF activity, SDS-P
AGE to perform recombinant HGF (I) and recombinant HGF
The content of (II) was measured. Fractions mainly containing recombinant HGF (II) were collected and used as a hydrophobic column eluate. A slight amount of recombinant HGF (I) was still observed in this eluate. The hydrophobic column eluate H was added for dilution.

(4) Second hydrophobic chromatography The above-mentioned diluted hydrophobic column eluate was added to a phenyl 5PW column (same as (3)) equilibrated with buffer H. After washing the column with buffer solution H, elution was performed by a concentration gradient method in which the mixing ratio of buffer solution H and buffer solution I was continuously changed. The elution chromatography pattern is shown in FIG. Figure 11
The homologous recombinant HG was collected by collecting the fractions indicated by the arrow in the inside.
F (II) [human leukocyte-derived 5 amino acid deletion type HGF
(II)] was obtained. The yield of recombinant HGF (II) is about 75.
At μg, the activity recovery was 15%.

(5) SDS-Acrylamide Gel Electrophoresis The purified recombinant HGF (II) of human leukocyte-derived 5 amino acids obtained in section (4) was subjected to SDS-acrylamide gel (Tefco 01025) electrophoresis. went.
The protein was stained by the silver staining method (manufactured by Wako Pure Chemical Industries, Ltd.). The result is shown in FIG. Purified recombinant H
GF (II) has an estimated molecular weight of 82,000 under non-reducing conditions.
± 5,000 Daltons, and estimated molecular weight 92,000 ± under reducing conditions with 2-mercaptoethanol
A single band of 5,000 daltons was shown. Recombinant HG
Bands derived from the α and β chains of F (I) and bands of other impurities were not detected.

(6) Measurement of physiological activity Regarding purified recombinant HGF (II) of 5 amino acid deletion type derived from human leukocytes obtained in the item (4), proliferation of rat primary culture hepatocytes in accordance with the method shown in Reference Example 1. The accelerating activity was measured. As a result, the specific activity of the recombinant HGF (II) was 270,000 ± 80,000 units / mg. still,
The absorbance at 214 nm is 14O. D. At that time, the protein concentration was calculated as 1 mg / ml.

Formulation Example 1 mg of purified HGF (II) was taken to obtain 0.8766 g of Na.
Cl, 10 mg of surfactant, Tween 80 and 100 mg of human serum albumin were added, and the total volume was aseptically adjusted to 100 ml with 0.02M phosphate buffer at pH 7.0, and 1 ml was aseptically dispensed into ampoules. To close.

[Brief description of drawings]

FIG. 1 is a diagram showing the relationship between the fraction of S-Sepharose eluate of liver extract, the absorbance of eluate components, and their HGF activities.

FIG. 2 is a diagram showing the relationship between the fraction of the Blue Trisacryl M eluate of the liver extract, the absorbance of the eluted components, and their HGF activities.

FIG. 3 is a diagram showing a relation between a fraction of a heparin-sepharose eluate of a liver extract, absorbance of eluate components, and their HGF activities.

FIG. 4 is a diagram showing the relationship between the fraction of the phenyl 5PW eluate of the liver extract, the absorbance of the eluted components, and their HGF activities.

FIG. 5: Phenyl 5PW with different elution conditions of liver extract
It is a diagram which shows the elution pattern by.

FIG. 6 shows SDS-polyacrylamide electrophoresis patterns of HGF (II) and purified HGF (II) under reducing and non-reducing conditions.

FIG. 7 is a diagram showing a peptide map of purified HGF (II).

FIG. 8: Fraction of S-Sepharose eluate of recombinant HGF (II) and absorbance of eluted components and their D
It is a diagram which shows the relationship with NA synthetic activity.

FIG. 9 is a diagram showing the relationship between the fraction of recombinant HGF (II) heparin-sepharose eluate, the absorbance of the eluted components, and their DNA synthesis activity.

FIG. 10: First round phenyl 5P of recombinant HGF (II)
FIG. 3 is a diagram showing the relationship between the fraction of W eluate, the absorbance of eluate components, and their DNA synthesis activity.

FIG. 11: Second round phenyl 5P of recombinant HGF (II)
FIG. 3 is a diagram showing the relationship between the fraction of W eluate, the absorbance of eluate components, and their DNA synthesis activity.

FIG. 12: Purified recombinant HGF (II) and recombinant purified H
3 shows SDS-polyacrylamide electrophoresis patterns of GF (II) under reducing and non-reducing conditions.

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[Procedure amendment]

[Submission date] March 6, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0018

[Correction method] Change

[Correction content]

Since HGF (II) of the present invention is unstable to heat, it is usually used as a stabilizer in Tween 80.
Surfactants such as, mannitol, sorbitol, heparin, sugars such as glycosaminoglucan, glycerin,
Polyethylene glycol, alcohols such as ethylene glycol, glycine, arginine, alanine, amino acids such as aspartic acid, and is preferably blended in combination one kind or two or more of proteins such as blood Kiyoshia albumin.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0028

[Correction method] Change

[Correction content]

Analysis of Physicochemical Properties of HGF (II) (i) SDS-PAGE The HGF (II) obtained in the section of SDS-PAGE and in the section of SDS-PAGE was analyzed according to the method of Laemmli. The result is shown in FIG. HG obtained in section
F (II) has a molecular weight of 82,000 ± 5,0 under non-reducing conditions.
It has a molecular weight of 92.0 even under reducing conditions.
00 ± 5,000 Dalton showing only one band,
It was shown that HGF (II) is a protein consisting of a single polypeptide chain. The amount of protein added per well was about 200 ng, and staining was performed by the silver staining method (manufactured by Wako Pure Chemical Industries, Ltd.).

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0034

[Correction method] Change

[Correction content]

(3) First hydrophobic chromatography Phenyl 5PW column (manufactured by Tosoh Corporation, column size: 0.75 cm inner diameter) equilibrated with buffer H [20 mM sodium phosphate (pH 7.0) containing 4M NaCl] Recombinant heparin eluate was added to the (height x 7.5 cm), and the column was washed with buffer solution H. Elution is with buffer H and buffer L
This was performed by the concentration gradient method in which the mixing ratio with (20 mM sodium phosphate (pH 7.0) containing 50% ethylene glycol) was continuously changed.
Shown in. HGF activity of each elution fraction was measured and
For fractions with GF activity, SDS-P
AGE to perform recombinant HGF (I) and recombinant HGF
The content of (II) was measured. Fractions mainly containing recombinant HGF (II) were collected and used as a hydrophobic column eluate. A slight amount of recombinant HGF (I) was still observed in this eluate. Buffer H was added to the eluate of the hydrophobic column to dilute it.

[Procedure amendment]

[Submission date] September 8, 1993

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be amended] Title of invention

[Correction method] Change

[Correction content]

Title: Mature hepatocyte growth factor

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number FI technical display location // (C12P 21/00 C12R 1:91) (72) Inventor Norihide Shimizu Katada, Otsu, Shiga Prefecture 1-1-1 Toyobo Co., Ltd. Pharmaceutical Research Center

Claims (5)

[Claims] 1. A molecular weight estimated by SDS-PAGE under non-reducing conditions is 82,000 ± 5,000 daltons, a molecular weight estimated by SDS-PAGE under reducing conditions is 92,000 ± 5,000 daltons, A mature hepatocyte growth factor (II), which is a single-chain polypeptide that proliferates primary cultured hepatocytes. 2. A homogenate of blood cells or biological tissues, selected from ion exchange chromatography, dye affinity chromatography, lectin affinity chromatography, heparin affinity chromatography, hydrophobic chromatography, adsorption chromatography, and reverse phase chromatography. The estimated molecular weight by SDS-PAGE under non-reducing conditions was at least 82
000 ± 5,000 daltons, SD under reducing conditions
Estimated molecular weight by S-PAGE is 92,000 ± 5,0
A method for producing mature hepatocyte growth factor (II), which comprises purification of a single-chain polypeptide that is 00 daltons and grows primary cultured hepatocytes. 3. A transformant transformed with a recombinant expression vector capable of expressing a nucleotide sequence encoding mature hepatocyte growth factor is cultured in the presence of a protease inhibitor, and the culture is subjected to non-reducing conditions. Lower SDS-PAG
The molecular weight estimated by E is 82,000 ± 5,000 daltons, the molecular weight estimated by SDS-PAGE under reducing conditions is 92,000 ± 5,000 daltons, and a single-chain polyproliferating primary cultured hepatocyte is obtained. A method for producing mature hepatocyte growth factor (II), which comprises collecting and purifying a peptide. 4. The method for producing mature hepatocyte growth factor (II) according to claim 3, wherein the purification is performed in the presence of a protease inhibitor. 5. The estimated molecular weight by SDS-PAGE under non-reducing conditions is 82,000 ± 5,000 daltons, and the estimated molecular weight by SDS-PAGE under reducing conditions is 92,000 ± 5,000 daltons. A mature hepatocyte growth factor (II) composition, comprising a mature hepatocyte growth factor (II), which is a single-chain polypeptide that grows primary cultured hepatocytes, and a stabilizer.