公开(公告)号：JPH01144979A

公开(公告)日：1989-06-07

申请号：JP30215787

申请日：1987-11-30

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  FURUSAWA KIYOTAKA ,  SAKAI TSUKASA ,  TANAKA YOSHIO

IPC: C12N1/21 ,  C12N15/00 ,  C12N15/09 ,  C12N15/53 ,  C12N15/62 ,  C12P21/00 ,  C12P21/02 ,  C12R1/19

Abstract: PURPOSE:To produce a substance containing a heterogenic product fused to a carboxy-terminal of dihydrofolic acid reductase(DHFR), in high efficiency, by modifying a plasmid pTP70-1 capable of easily utilizing a DHFR gene as a genetic marker. CONSTITUTION:A terminator region of rrnB gene (one of ribosomal RNA genes) known as an efficient terminator is introduced at the downstream side of a DHFR gene of plasmid pTP70-1 to obtain a plasmid pTP104-4 capable of efficiently producing a substance containing heterogenic product fused to the carboxy-terminal of DHFR. The plasmid has a size of 4,466 base pairs and can impart a host E.coli with trimethoprim resistance and ampicillin resistance.

公开(公告)号：JPH01144978A

公开(公告)日：1989-06-07

申请号：JP30215687

申请日：1987-11-30

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  KOKUBU TOMOKUNI ,  FURUSAWA KIYOTAKA ,  OHASHI SHINICHI ,  SAKAI TSUKASA ,  TANAKA YOSHIO

IPC: C12P21/02 ,  C07H21/04 ,  C12N1/20 ,  C12N1/21 ,  C12N15/00 ,  C12N15/09 ,  C12R1/19

Abstract: PURPOSE:To produce a peptide (GRF1-29) containing the factors from the 1st to the 29th of bovine growth hormone releasing factors, by using a specific recombinant plasmid pGRE2-15. CONSTITUTION:A recombinant plasmid is prepared by linking a GRF1-29 gene to the 3'-terminal of a dihydrofolic acid reductase (DHFR) gene using a plasmid vector pTP70-1 capable of fusing a heteropeptide to a carboxy-terminal or DHFR of E.coli. The plasmid is introduced into E.coli and expressed to enable the stable production and accumulation of a fused protein containing GRF1-29 bonded to the carboxy-terminal of DHFR in E.coli.

公开(公告)号：JPS63111465A

公开(公告)日：1988-05-16

申请号：JP25719086

申请日：1986-10-29

Applicant: AGENCY IND SCIENCE TECHN

Inventor： KOKUBU TOMOKUNI ,  IWAKURA MASAHIRO ,  OHASHI SHINICHI ,  TSUDA KEISHIRO

IPC: C12N15/02 ,  C12N15/00 ,  C12P21/00 ,  C12Q1/00 ,  G01N33/53 ,  G01N33/535 ,  G01N33/577

Abstract: PURPOSE:To improve measurement sensitivity and reproducibility by utilizing the fused protein genetically bonded with labeling enzyme and antigen by a gene manipulation as a labeling antibody. CONSTITUTION:The coliform bacilli contg. the plasmid having the gene coding the dihydro folic acid reductase-leucine enkephalline fused protein (hereafter expressed as DHFR-L-Enk) are prepd. by the gene manipulation and are subjected to fractionating, refining and purifying after liquid culture to obtain the DHFR-L-Enk exhibiting a uniform band in SDS electrophoresis. Said liquid is added together with an antirabbit leucine enkephalline (hereafter expressed as L-Enk) antibody to cause reaction, by which a soln. for measuring enzyme activity is obtd. A DHFR substrate soln. is then added thereto to initiate reaction. A change in the absorption at 340nm wavelength is measured by a spectrophotometer, by which L-Enk is measured.

公开(公告)号：JPS6269990A

公开(公告)日：1987-03-31

申请号：JP21081385

申请日：1985-09-24

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  TSUDA KEISHIRO

IPC: C12N15/09 ,  C12N1/20 ,  C12N15/70 ,  C12R1/19

Abstract: PURPOSE:To produce dihydrofolic acid reductase, by producing recombinant plamid PTA64-1 which is stably replicated with Escherichia coli and provides host Escherichia coli with trimethoprim resistance and ampicillin resistance. CONSTITUTION:A promoter sequence existing in a smaller one of two DNA fragments obtained by cleaving plasmid pTP52-1 with restriction enzyme PstI and C1aI is related to highly efficient manifestation of structural gene of dehydrofolic acid reductase. This DNA fragment is bonded to a larger one of two DNA fragments obtained by clevage of plasmid pTP63-21 with PstI and C1aI so that pTP64-1 can be replicated from the plasmid pTP63-21 and plasmid pTP52-1.

公开(公告)号：JPH02299589A

公开(公告)日：1990-12-11

申请号：JP11744289

申请日：1989-05-12

Applicant: AGENCY IND SCIENCE TECHN ,  HITACHI CHEMICAL CO LTD

Inventor： IWAKURA MASAHIRO ,  OHASHI SHINICHI ,  TANAKA YOSHIO ,  BABA KENZO ,  IZUTSU HIROSHI ,  OBARA KAZUHIKO

IPC: C12N15/09 ,  C07K1/22 ,  C07K7/06 ,  C07K14/00 ,  C07K19/00 ,  C12N1/21 ,  C12N15/62 ,  C12P21/02 ,  C12P21/06 ,  C12R1/19

Abstract: PURPOSE:To produce DHFR-DSDGK fused protein in a mass by transforming an E.coli with a recombinant plasmid containing a base sequence coding a DHFR-DSDGK fused protein. CONSTITUTION:An E.coli is transformed with a recombinant plasmid containing a base sequence coding dihydrofolic acid reductase-anti-allergic pentapeptide (DHFR-DSDGK) fused protein. The transformed E.coli is cultured and the objective DHFR-DSDGK fused protein is separated from the cultured bacterial cell. The separation of the DHFR-DSDGK fused protein is preferably carried out by purifying a cell-free extract of the cultured cell by successively treating the extract by an ion exchange column, a methotrexate-bonded affinity column chromatography and an anion exchange column chromatography.

公开(公告)号：JPH0279976A

公开(公告)日：1990-03-20

申请号：JP23124688

申请日：1988-09-14

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  SAKAI TSUKASA ,  TANAKA YOSHIO

IPC: C12N15/09 ,  A61K38/22 ,  A61P23/00 ,  A61P25/04 ,  C07K1/14 ,  C07K1/22 ,  C07K14/00 ,  C07K14/575 ,  C07K14/655 ,  C07K14/675 ,  C07K19/00 ,  C12N1/20 ,  C12N1/21 ,  C12N15/16 ,  C12N15/62 ,  C12P21/02 ,  C12R1/19

Abstract: PURPOSE:To obtain a recombinant plasmid pENDA1 being a recombinant plasmid having 4673 pair of bases and capable of producing alpha-endorphin of morphine like peptide having high analgesic activity when Escherichia coli is transformed by the above-mentioned plasmid. CONSTITUTION:Gene coding alpha-endorphin expressed by the formula is synthesized and the synthesized gene is integrated into a plasmid pMEK2 to prepare a fused gene (pENA1) of alpha-endorphin gene and dihydrofolic acid reducing enzyme gene, by which Escherichia coli is then transformed. Dihydrofolic acid reducing enzyme-alpha-endorphin fused protein is produced by the transformed Escherichia coli and then the fused protein is digested with a proteolytic enzyme to separate alpha-endorphin and then alpha-endorphin is purified.

公开(公告)号：JPH01252289A

公开(公告)日：1989-10-06

申请号：JP7968088

申请日：1988-03-31

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  OHASHI SHINICHI ,  SAKAI TSUKASA ,  TANAKA YOSHIO

IPC: C12N15/09 ,  A61K38/22 ,  A61P23/00 ,  A61P25/04 ,  C07K1/14 ,  C07K1/22 ,  C07K14/00 ,  C07K14/575 ,  C07K14/655 ,  C07K14/70 ,  C07K19/00 ,  C12N1/20 ,  C12N1/21 ,  C12N9/06 ,  C12P21/02 ,  C12R1/19

Abstract: PURPOSE:To obtain a large amount of dihydrofolic acid reductase-methionine enkephalin fused protein, by manifesting a fused gene in which a methionine enkephalin gene is included in a plasmid vector pTP 70-1 with a colon bacillus. CONSTITUTION:A newly recombined plasmid pMEK 2, which is replicated in stable with a colon bacillus, imparting resistance to trimethoprim and resistance to ampicillin to a colon bacillus as a host, having a size of 4640 basic pairs and having a bonded structure of chemically synthesized DNA of 32 basic pairs containing a conformation coding methionine enkephalin at BamHI site of pTP 70-1, is produced. A colon bacillus introduced of the plasmid pMEK 2 (FERM BP-1816) produces a large amount of dihydrofolic acid reductase- methionine enkephalin fused protein.

公开(公告)号：JPH01252288A

公开(公告)日：1989-10-06

申请号：JP7967988

申请日：1988-03-31

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  OHASHI SHINICHI ,  SAKAI TSUKASA ,  TANAKA YOSHIO

IPC: C12N15/09 ,  C07K1/14 ,  C07K1/22 ,  C07K7/14 ,  C07K14/00 ,  C07K19/00 ,  C12N1/20 ,  C12N1/21 ,  C12N9/06 ,  C12P21/02 ,  C12R1/19

Abstract: PURPOSE:To obtain a large amount of dihydrofolic acid reductase-angiotensin II fused protein, by manifesting a fused gene in which an angiotensin II gene is included in a plasmid vector pTP 70-1 with a colon bacillus. CONSTITUTION:A newly recombined plasmid pANG2-11, which is replicated in stable with a colon bacillus, imparting resistance to trimethoprim and resistance to ampicillin to a colon bacillus as a host, having a size of 4651 basic pairs and having a bonded structure of chemically synthesized DNA of 43 basic pairs containing a conformation coding angiotensin II at BamHI site of pTP 70-1, is produced. A colon bacillus introduced of the plasmid pANG2-11 (FERM BP-1815) produces a large amount of dihydrofolic acid reductase-angiotensin II fused protein.

公开(公告)号：JPH01144992A

公开(公告)日：1989-06-07

申请号：JP30215387

申请日：1987-11-30

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO

IPC: C12N1/20 ,  C12N9/06 ,  C12N15/09 ,  C12P21/00 ,  C12P21/02 ,  C12R1/19

Abstract: PURPOSE:To produce a protein composed of a heterogene fused with dihydrofolic acid reductase, in high efficency, by introducing a heterogene into a specific recombinant plasmid. CONSTITUTION:A dihydrofolic acid redcuctase (DHFR) gene originated from E.coli is modified to attain trimethoprim-resistance and ampicillin-resistance. A heterogene is introduced to the 3'-terminal of the modified DHFR gene in a recombinant plasmid pT70-1 containing the modified gene in such a manner as to match the reading frame of the genetic code. A protein composed of a heterogene fused with DHFR can be produced in high efficiency as a protein having a DHFR activity.

公开(公告)号：JPS6471497A

公开(公告)日：1989-03-16

申请号：JP22612787

申请日：1987-09-09

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  OHASHI SHINICHI ,  SAKAI TSUKASA ,  TANAKA YOSHIO

IPC: C12N15/09 ,  C07K1/12 ,  C07K7/06 ,  C07K7/18 ,  C07K19/00 ,  C12N1/21 ,  C12P21/02 ,  C12R1/19

Abstract: PURPOSE:To obtain bradykinin having hypotensive action, by separating bacterial cell from E.coli C600 strain containing pBLAK1, treating the cell with cyanogen bromide and separating the objective compound by high-performance liquid chromatography. CONSTITUTION:E.coli C600 strain containing recombinant plasmid pBLAK1 is cultured in a medium, the bacterial cells are collected after cultivation and centrifuged to obtain a protein composed of dihydrofolic acid reductase-reductase fused to bradykinin. The protein is mixed and treated with cyanogen bromide and subjected to high-performance liquid chromatography to obtain bradykinin useful in the fields of medical and pharmaceutical industries, etc.