Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1994-12-23
1997-07-29
Jagannathan, Vasu
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4352523, 4353201, 530399, C12N 1518, C12N 121
Patent
active
056521200
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel gene coding human epidermal growth factor and a process for preparing the same. Specifically, the present invention relates to a novel human epidermal growth factor gene and a process for preparing the same employing a recombinant expression vector therefor.
2. Description of the Prior Art
It is known that human epidermal growth factor (hereinafter referred to as `hEGF`) is a polypeptide hormone consisting of 53 amino acids and 3 disulfide bridges[see: Cohen, S., J. Biol. Chem., 237:1555-1562(1962); Savage, C. R., Jr. et al., J. Biol. Chem., 248:7669-7672(1973); Savage, C. R., Jr. et al., J. Biol. Chem., 247:7612-7621 (1972)], and that hEGF plays an important role on the growth control in mammalian cells, inter alia epidermal and epithelial cells on molecular level[see: Sporn, M. B. et al., Nature (London), 313:745-747(1985); Sporn, M. B. et al., N. Engl. J. Med., 303:878-880(1980)] and the treatment of injury [see: Buckley, A. et al., Proc. Natl. Acad. Sci., USA, 82: 7340-7344(1985)]. Further, it has been reported that the hEGF can be applied in the treatment of a stomach ulcer, due to its ability to repress secretion of gastric acid into stomach[see: Gregory, H., J. Cell Sci. Suppl., 3: 11-17(1985)].
Under the circumstances, studies on the mass production of the hEGF has been actively carried out, since Starkey et al. reported the biochemical property of hEGF purified from human urine[see: Starkey, R. H. et al., Science, 189:800(1975); Cohen, S. et al., Proc. Natl. Acad. Sci., USA, 72:1317(1975)]. Several researchers have accomplished cloning of hEGF gene by the recombinant DNA technology in a successful manner[see: Smith, J. et al., Nucleic Acids Res., 10:4467-4482(1982); Urdea, M. S . et al., Proc. Natl. Acad. Sci., USA, 80:7461-7465(1983); Oka, T. et al., Proc. Natl. Acad. Sci., USA, 82:7212-7216(1985)]. The prior art, however, has not described methods of producing hEGF to the level sufficient for industrial application, due to its low activity and productivity.
Accordingly, studies on the elevation of activity and productivity in hEGF manufacture have been carried out. These studies have concentrated on the preparation of the nucleotide sequence of hEGF gene efficient for its massive production and the expression vector whose regulatory function is strengthened.
SUMMARY OF THE INVENTION
In accordance with the present invention, the inventors synthesized a novel hEGF gene and a novel expression vector therefor which expresses high levels of hEGF and developed a process for preparing hEGF therefrom.
A primary object of the invention is, therefore, to provide a novel hEGF gene which is designed and chemically synthesized for the purpose of producing high levels of hEGF in E. coli .
Another object of the invention is to provide a novel process for preparing hEGF from a recombinant expression vector comprising said hEGF gene and a regulatory sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
The above and the other objects and features of the present invention will become apparent from the following descriptions given in conjunction with the accompanying drawings, in which:
FIG. 1 depicts sequence of designed hEGF gene of the invention SEQ.ID.NO. 1 (DNA). and SEQ.ID.NO. 2(protein)
FIG. 2A depicts 10 oligonucleotides synthesized;
FIG. 2B depicts assembly pattern of synthesized 10 oligonucleotides of FIG. 2A;
FIG. 3 depicts construction strategy of pUE118;
FIG. 4 is a photograph showing agarose gel electrophoresis pattern of pUE118 carrying hEGF gene digested with restriction enzymes;
FIG. 5 depicts the Omp A leader-universal translation termination-Trp A transcription termination sequence;
FIG. 6 depicts construction strategy of pDT420;
FIG. 7 depicts construction strategy of pDE135;
FIG. 8 depicts construction strategy of pTC108;
FIG. 9 depicts construction strategy of pTC226;
FIG. 10 depicts construction strategy of pTE105;
FIG. 11A is a photograph showing SDS-PAGE pattern of hEGF expressed in E. coli JM101 ha
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Chung Ju Young
Jee Young Su
Koh Yeo Wook
Kwon Chang Hyuk
Lee Kang Moon
Daewoong Pharmaceutical Co., Ltd.
Jagannathan Vasu
Saoud Christine
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