公开(公告)号：JP2000119300A

公开(公告)日：2000-04-25

申请号：JP28366998

申请日：1998-10-06

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO

IPC: C12N11/12 ,  C07K1/02 ,  C07K17/12

Abstract: PROBLEM TO BE SOLVED: To produce the subject protein capable of carrying out the complete reversion of the denaturation due to immobilization at one site of the carboxyl terminal and through a main chain by immobilizing the protein through the carboxyl group at the carboxyl terminal thereof. SOLUTION: This protein is represented by the formula NH2-R1-CO-NH-R2-CO-NH-Y (R1 and R2 are each an amino acid sequence; and Y is an immobilizing carrier) and immobilized on the immobilizing carrier at one site of the carboxyl terminal of a protein main chain. The protein is produced by binding a protein represented by the formula NH2-R1-COOH to the peptide represented by formula I (X is OH, an amino acid or an amino acid sequence), cyanating SH of the resultant fusion protein represented by formula II and subsequently reacting a cyano-containing protein represented by formula III with the immobilizing carrier represented by the formula NH2-Y. The fusion protein represented by formula II is prepared by binding, e.g. a gene encoding the protein represented by the formula NH2-R1-COOH to a gene encoding the peptide sequence represented by formula I, preparing a gene encoding the protein represented by formula II and then expressing the resultant gene in a host organism.

公开(公告)号：JPH05199866A

公开(公告)日：1993-08-10

申请号：JP25046091

申请日：1991-09-03

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO

IPC: C12N1/21 ,  C12N9/06 ,  C12N15/09 ,  C12N15/70 ,  C12R1/19

Abstract: PURPOSE:To efficiently produce a dihydrofolate reductase (DHFR) or its derivative. CONSTITUTION:Escherichia coli is cultured at a low temperature (

公开(公告)号：JPH02142480A

公开(公告)日：1990-05-31

申请号：JP29420388

申请日：1988-11-21

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  OHASHI SHINICHI ,  TANAKA YOSHIO

IPC: C12N1/21 ,  C07K14/00 ,  C07K14/575 ,  C07K14/60 ,  C07K19/00 ,  C12N15/09 ,  C12N15/16 ,  C12N15/62 ,  C12N15/70 ,  C12P21/02 ,  C12R1/19

Abstract: NEW MATERIAL:A plasmid pGRF44-22, stably replicative in Escherichia coli and capable of imparting trimethoprim and ampicillin tolerance to host Escherichia coli and encoding a fused protein of a derivative of a bovine growth hormone releasing factor having an amino acid sequence expressed by the formula to a dihydrofolate reductase. USE:For producing a bovine growth hormone releasing factor(GRF) and dihydrofolate reductase(DHFR). PREPARATION:For example, a DNA fragment prepared by cleaving a plasmid pMEK2 for producing leucine enkephalin with a restriction enzyme is linked to a DNA fragment obtained by cleaving a recombinant plasmid pGRF2-15 capable of coding DHFR and an amino acid sequence of the 1st to 29th residues of a bovine GRF using a T4-DNA ligase, inserted into Escherichia coli and cultured to select the resultant colony. Thereby, the objective pGRF44-22 is obtained.

公开(公告)号：JPH0279977A

公开(公告)日：1990-03-20

申请号：JP23124788

申请日：1988-09-14

Applicant: AGENCY IND SCIENCE TECHN

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

IPC: C12N15/09 ,  A61K38/22 ,  A61P23/00 ,  A61P25/04 ,  C07K1/16 ,  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 pENDC1 being a recombinant plasmid having 4676 pair of bases and capable of producing gamma-endorphin of morphine like peptide having high analgesic activity when Escherichia coli is transformed by the above-mentioned plasmid. CONSTITUTION:Gene coding gamma-endorphin expressed by the formula is synthesized and the synthesized gene is integrated into a plasmid pMEK2 to prepare a fused gene (pENDC1) of gamma-endorphin gene and dihydrofolic acid reducing enzyme (DHFR) gene, by which Escherichia coli is then transformed. DHFR-gamma-endorphin fused protein is produced by culturing the transformed Escherichia coli and then the fused protein is digested by a proteolytic enzyme to separate gamma- endorphin and the gamma-endorphin is purified.

公开(公告)号：JPH01144974A

公开(公告)日：1989-06-07

申请号：JP30215287

申请日：1987-11-30

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  SAKAI TSUKASA ,  TANAKA YOSHIO

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

Abstract: PURPOSE:To easily purify the title enzyme useful for the production of enzyme and medicine, in high purity, by culturing E.coli and purifying the cell-free extract of the cultured product with methotrexate-bonded affinity chromatography. CONSTITUTION:An E.coli containing plasmid pT64-1 is cultured and the cell-free extract of the cultured bacterial cell is treated with an ion-exchange column and purified by methotrexate-bonded affinity chromatography and anion exchange chromatography to obtain the objective enzyme.

公开(公告)号：JPS6486871A

公开(公告)日：1989-03-31

申请号：JP24701787

申请日：1987-09-30

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO

IPC: C12N9/06 ,  C12N15/09 ,  C12P21/00

Abstract: PURPOSE:To obtain a completely homogeneous enzymic sample with excellent reproducibility, by carrying out special treatment, such as crushing of cultivated microbial cells, treatment with streptomycin sulfate and fractionation with ammonium sulfate. CONSTITUTION:Cultivated microbial cells are crushed and centrifuged to provide a supernatant, which is then treated with streptomycin sulfate, fractionated with ammonium sulfate and subjected to operation of butyl Toyopearl(R) column chromatography, gel filtration chromatography and DEAE-Toyopearl(R) chromatography. Escherichia coli transformed with a recombinant plasmid pBSFOL14-1 containing Bacillus subtilis dihydrofolic reductase gene integrated therein is used as the cultivated microbial cells to purify the Bacillus subtilis dihydrofolate reductase. If Escherichia coli transformed with a recombinant plasmid pBSFOLEK1 is used, dihydrofolic reductase-leucine enkephalin fused protein can be purified.

公开(公告)号：JPS6438099A

公开(公告)日：1989-02-08

申请号：JP8540687

申请日：1987-04-07

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  TSUDA KEISHIRO

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

Abstract: NEW MATERIAL:A compound, produced by Escherichia coli C600 strain containing a recombinant plasmid pBLAK1 and having an amino acid sequence expressed by the formula. USE:A hypotensive agent (vasodilator) and intestinal contracting agent. PREPARATION:For example, Escherichia coli C6,000 strain containing a recombinant plasmid pBLAK1 is cultivated in a culture medium and centrifuging is carried out to collect microbial cells, which are then suspended in a buffer solution at pH 7.0. The cells are subsequently crushed by ultrasonic crushing and centrifuged to afford a supernatant. The resultant cell-free extract solution of the cultivated cells is then purified by means of ion exchange column chromatography and succeeding gel filtration column chromatography using a dihydrofolate reductase activity as a measure to provide the aimed protein expressed by the formula.

公开(公告)号：JPS63258597A

公开(公告)日：1988-10-26

申请号：JP9288187

申请日：1987-04-15

Applicant: AGENCY IND SCIENCE TECHN

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

IPC: C12N15/09 ,  C07K1/16 ,  C07K14/00 ,  C07K14/575 ,  C07K14/655 ,  C07K19/00 ,  C12N9/06 ,  C12N13/00 ,  C12P21/02 ,  C12R1/19

Abstract: PURPOSE:To enable purification of the titled protein from a culture liquid, by passing a cell-free extract liquid of cultured microbial cells through an ion exchange column and gel-filtration column. CONSTITUTION:Microbial cells are collected from cultured product of E.coli C600 (FERM P-9301) containing recombinant plasmid pGIF1 and are disintegrated by ultrasonic radiation. The disintegrated product is subjected to centrifugal separation and the obtained supernatant liquid is purified by ion exchange chromatography and subsequent gel-filtration chromatography. The titled protein having amino acid sequence of formula, i.e. fused protein of dihydrofolic acid reductase and somatostatin is produced by the process.

公开(公告)号：JPS62115287A

公开(公告)日：1987-05-26

申请号：JP25566085

申请日：1985-11-14

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  FURUSAWA KIYOTAKA ,  OHASHI SHINICHI ,  TSUDA KEISHIRO

IPC: C12N15/09 ,  C07H21/04 ,  C07K14/60 ,  C12N1/20 ,  C12N1/21 ,  C12P21/02 ,  C12R1/19

Abstract: PURPOSE:A recombinant plasmid containing a gene to code 1st-29th peptide fragments among bovine growth hormone release factor peptide having secretion adjusting activity of growth hormone. CONSTITUTION:Novel recombinant plasmid pGRF29-28 consisting of 5406 pairs of bases having DNA sequence partially shown by formula II, a recombinant plasmid which can provide a host with ampicillin resistance, codes a peptide wherein a peptide sequence consisting of 6 amino acids of methionine-isoleucine- isoleucine-glutamic acid-glycine-alginine is fused to an amino end side of 1st-29th peptide fragment among bovine growth hormone release factor peptide by cleavage with restriction enzyme BclI and can free a DNA sequence consisting of 107 pairs of bases shown by formula I.

公开(公告)号：JPS61271990A

公开(公告)日：1986-12-02

申请号：JP11369285

申请日：1985-05-27

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  FURUSAWA KIYOTAKA ,  TSUDA KEISHIRO

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

Abstract: PURPOSE:A DNA sequence, consisting of a specific base sequence and containing a gene of an enzyme capable of catalyzing a reaction for reducing dihydrofolic acid and producing tetrahydrofolic acid. CONSTITUTION:A DNA sequence expressed by the formula. The DNA sequence consists of a part coding dihydrofolic acid reductase protein of E. coli (sequence A at the 1- - 477-position), part on the upstream side thereof (sequence B at the -56- - -1-position) and part on the downstream side thereof (sequence C consisting of TAA at the 478- - 480-translation stopping number). The sequences (A) and (C) are the same as the sequence derived from a chromosome DNA of E. coli, and the sequence (B) is derived from a chemically synthesized DNA. The sequence (B) consists of a cleavage site of a restriction enzyme, a site of translation stopping group corresponding to the reading frame of three gene translations and ribosome linking sites for E. coli (DNA sequence of AGGA) and for B. subtilis (DNA sequence of AAAGGAGG).