Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1992-03-11
1994-07-12
Hill, Jr., Robert J.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4352401, 4353201, 536 2351, 935 13, 935 70, C12P 2102
Patent
active
053288360
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to plasmids containing DNA encoding the amino acid sequence of a novel glycoprotein derived from human fibroblasts ( it is designated as TCF-II hereafter), transformed cells with the plasmids and a production method for a biologically active substance using the transformed cells.
TCF-II in the present invention is useful for pharmaceutical products as a hepatocyte growth factor, a tumor cytotoxic factor, etc. or a biochemical or pharmacological reagent.
BACKGROUND OF THE INVENTION
Interferon- .beta. is a representative factor as the biologically active factor, for example, the tumor cytotoxic factor which is produced by human-derived fibroblasts. This is a glycoprotein which is secreted by the fibroblasts, when the cells after cultivation are harvested and stimulated by polyI-poly C or Sendai virus. It has been clarified that the protein has various physiological activities besides its anti-virus or anti-tumor activity. A fibroblast-derived tumor cytotoxic glycoprotein designated as CBF is disclosed in Japanese Patent Laid-Open No. 58-146293. A tumor growth inhibitory factor (INF) with molecular. weight of 35,000.about.45,000, which is purified from the culture broth of fibroblasts derived from human tissue, is disclosed in Japanese Patent Laid-Open No. 61-33120. Furthermore, a tumor necrosis factor-like substance which is purified from the culture broth of fibroblasts, a fibroblast-derived necrosis factor, FNF, and a biologically active substance with cytotoxic activity, which is produced by animal-derived fibroblasts and has a molecular weight of 40,000 to 60,000 and an isoelectric point value of 5.0.+-.0.5, are disclosed in Japanese Patent Laid-Open No. 61-56131, Japanese Patent Laid-Open No. 61-1872, and Japanese Patent Laid-Open No. 62-103021, respectively. In addition, all the primary amino acid sequence and cDNA encoding the amino acid sequence of a tumor cytotoxic factor, which is obtained from the culture broth of human-derived fibroblasts, with a molecular weight of 36,000.+-.1,000 and an isoelectric point value more than 10.5 are disclosed in Japanese Patent Laid Open No. 64-10998.
DISCLOSURE OF THE INVENTION
The present inventors investigated biologically active substances which are present in culture broth of human-derived fibroblasts and found a glycoprotein with various biological activities, which is different in molecular weight, isoelectric point value, etc. from previously reported substances. A patent application directed to the novel glycoprotein has been filed (PCT/JP 90/00314; International patent published, WO 90/10651; published date, Sep. 20, 1990).
This novel glycoprotein (TCF-II) derived from human fibroblasts is a glycoprotein which is characterized by the following physicochemical properties.
a. Molecular weight (Mw): On SDS-polyacrylamide gel electrophoresis, it shows bands with Mw 74,000.+-.2,000 and 78,000.+-.2,000 under non-reducing conditions, and also shows a common band A with Mw 52,000.+-.2,000 and band B with Mw 30,000.+-.2,000 and band C with Mw 26,000.+-.2,000 under reducing conditions.
b. Isoelectric point value: 7.4 to 8.6.
c. Heat stability: Stable in the heating at 60.degree. C. for 10 min.
d. pH stability: Stable in the range of pH 6 to 9.
e. Glycoprotein: It is adsorbed to a Concanavalin A (Con A)-Sepharose column.
f. Biological activity: It inhibits the growth of KB cells, HeLa cells and L929-C18 cells, but not IMR-90 cells.
g. Reactivity to antibodies: Its cytotoxic activity is not neutralized by anti-T NF, anti-lymphotoxin, and anti-interferon- .beta. antibodies.
Furthermore, preferably TCF-II in the present invention has the following N-terminal amino acid sequence and amino acid composition.
h. N-terminal amino acid sequence: The above mentioned band B and band C are subchains of band A, respectively. The N-terminus of band A is blocked. Band B and band C have the same N-terminal amino acid sequence as follows; ##STR1## where X means an unidentified amino acid.
i. Amino acid composition: When it
REFERENCES:
Biochem Biophys Res Comm 172(1): 321-327 Oct. 5, 1990 T. Seki et al. "Isolation & Expression . . . ".
S. Sone et al. Biotherapy 1: 233-243 1989, "Effector Mechamism . . . ".
Miyazawa et al., "Molecular Cloning and Sequence Analysis of cDNA for Human Hepatocyte Growth Factor," Biochemical Biophysical Research Communications, 170, pp. 967-973 (1989).
Higashio Kanji
Nagao Masaya
Oogaki Fumiko
Shima Nobuyuki
Takaoka Hiroaki
Hill Jr. Robert J.
Snow Brand Milk Products Co. Ltd.
Spector L.
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