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公开(公告)号：JPS63267276A
公开(公告)日：1988-11-04
申请号：JP31283686
申请日：1986-12-26
Applicant: AGENCY IND SCIENCE TECHN
Inventor： IWAKURA MASAHIRO , TSUDA KEISHIRO
Abstract: PURPOSE:To provide a novel recombinant plasmid pTP70-1 containing a gene obtained by modifying a dihydro-folate reductase gene originated from E.coli, having a specific DNA sequence and capable of facilitating the use of dihydro- folate reductase gene as a trimethoprim-resistance marker. CONSTITUTION:The novel recombinant plasmid pTP70-1 is stably replicable in E.coli, capable of imparting the host E.coli with trimethoprim resistance and ampicillin resistance, has a size of 4608 base pairs and is composed of a specific DNA sequence (omitted). E.coli K12C600 strain containing pTP70-1 has been deposited in the Fermentation Research Institute in the name of FERM P-9091.
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公开(公告)号：JPS63245680A
公开(公告)日：1988-10-12
申请号：JP7937887
申请日：1987-03-31
Applicant: AGENCY IND SCIENCE TECHN
Inventor： IWAKURA MASAHIRO , KOKUBU TOMOKUNI , FURUSAWA KIYOTAKA , OHASHI SHINICHI , TSUDA KEISHIRO
Abstract: PURPOSE:To readily give a fused protein measurable enzymatic activity by making up a novel recombined plasmid pGIF1 including a gene coding fused protein from dehydrofolate reductase and somastatin to solubilize the fused protein. CONSTITUTION:The recombinant plasmid pGIF1 is stably copied in E. coli and gives the host E. coli, resistance to trimethoprim and ampicillin. In the plasmid, the gene giving trimethoprim resistance is recombined by modifying a part of the 3'-terminal of dihydrofolate reductase in Bacillus subtilis to code the fused protein with 4789 pairs of bases. The recombinant plasmid pGIF1 is introduced into E. coli C600 strain and the transformed strain is deposited as FERM P-9301.
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公开(公告)号：JPS63102698A
公开(公告)日：1988-05-07
申请号：JP24925986
申请日：1986-10-20
Applicant: AGENCY IND SCIENCE TECHN
Inventor： IWAKURA MASAHIRO , KOKUBU TOMOKUNI , FURUSAWA KIYOTAKA , OHASHI SHINICHI , TSUDA KEISHIRO
IPC: C12N15/09 , C07K1/12 , C07K14/00 , C07K14/575 , C07K14/655 , C07K14/70 , C07K19/00 , C12N1/20 , C12N9/06 , C12N15/00 , C12P21/02 , C12R1/19
Abstract: PURPOSE:To make it possible to produce leucine enkephalin, by separating dehydrofolate reductase-leucine enkephalin fusion protein produced by E.coli integrated with a specific plasmid from the other substances in a medium, purifying and decomposing by cyanogen bromide method. CONSTITUTION:Dehydrofolate reductase-leucine enkephalin fusion protein is separated from a medium in which E.coli integrated with plasmid pBSFOLEK1 by ion exchange column chromatography and gel filtration chromatography and purified. Then the protein is decomposed by cyanogen bromide decomposition method and leucine enkephalin is separated and purified by reverse phase high performance liquid chromatography.
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公开(公告)号：JPS63102696A
公开(公告)日：1988-05-07
申请号：JP24926086
申请日：1986-10-20
Applicant: AGENCY IND SCIENCE TECHN
Inventor： IWAKURA MASAHIRO , KOKUBU TOMOKUNI , FURUSAWA KIYOTAKA , OHASHI SHINICHI , TSUDA KEISHIRO
IPC: C12N15/09 , C07K1/16 , C07K14/575 , C07K14/655 , C07K14/70 , C07K19/00 , C12N9/06 , C12N15/00 , C12P21/00 , C12P21/02 , C12R1/19
Abstract: PURPOSE:To obtain a dihydrofolate reductase-leucine enkephalin fusion protein which is protein produced by Escherichia coli integrated with a specific plasmid and a raw material for leucine enkephalin having nonhabitual morphine-like analgesic action. CONSTITUTION:Escherichia coli integrated with plasmid pBSFOLEK1 is cultivated to produce dihydrofolate reductase-leucine enkephalin fusion protein having an amino acid sequence shown by the fig. The cell is separated from the culture solution, removed, the culture solution is subjected to ion exchange chromatography and then the aimed fusion protein is separated and purified by using dihydrofolate reductase as a standard while passing through the solution gel filtration chromatography.
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