公开(公告)号：JPH1045796A

公开(公告)日：1998-02-17

申请号：JP20371296

申请日：1996-08-01

Applicant: AGENCY IND SCIENCE TECHN ,  EISAI CO LTD

Inventor： IWAKURA MASAHIRO ,  TAKENAWA TATSUYUKI ,  ODA YOSHIYA ,  ISHIHAMA YASUSHI

IPC: C12N15/09 ,  C07H21/04 ,  C07K1/12 ,  C12N1/21 ,  C12P21/02 ,  C12R1/19

Abstract: PROBLEM TO BE SOLVED: To raise the efficiency in the protein engineering field, especially a method for site-specific fragmentation of a protein. SOLUTION: A lysine-cysteine residue is introduced into a peptide and the cysteine is then cyanogenated. When the peptide is subsequently treated with a weak alkali, ε-amino group of the lysine acts as a nucleophilic group to attack carbonyl carbon on the peptide bond with the lysine to the cyanocysteine. Thereby, cleavage of the peptide bond of the lysine-cyanocysteine is efficiently caused.

公开(公告)号：JPH0638786A

公开(公告)日：1994-02-15

申请号：JP21813091

申请日：1991-05-21

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 provide a new manifestation vector useful for the production of a protein containing a heterogenic product fused to the carboxy-terminal of DHFR. CONSTITUTION:The manifestation vector pTP104-4 having the DNA sequence of formula. The vector can be prepared by introducing the terminator region of rrnB gene to the downstream of the DHFR gene of pTP70-1.

公开(公告)号：JPH05146291A

公开(公告)日：1993-06-15

申请号：JP33623691

申请日：1991-11-26

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  KOKUBU TOMOKUNI ,  OHASHI SHINICHI

IPC: C12N9/06 ,  C12N11/00 ,  C12N15/09 ,  C12N15/53 ,  C12R1/19

Abstract: PURPOSE:To obtain the subject enzyme capable of suppressing inactivation of the enzyme and providing a homogenous immobilized enzyme having high activity by transducing an amino acid sequence containing plural amino acid residues having cysteine residue as the carboxyl terminal into the carboxyl terminal side of an enzymic protein. CONSTITUTION:A genetic DNA capable of coding a dihydrofolate reductase is used to replace the sequence part capable of coding the carboxyl terminal side of this enzymic protein with a chemical synthetic DNA. Thereby, a genetic DNA capable of coding a modified enzymic protein in which a short amino acid sequence is transduced so that the carboxyl terminal may be the cysteine residue is prepared. The resultant genetic DNA is then integrated into a vector such as a plasmid for expression to construct an expression vector, which is subsequently transduced into a host such as Escherichia coli and expressed. The obtained modified enzymic protein can be separated and purified from the host microbial cell to afford a new dihydrofolate reductase, having an amino acid sequence expressed by the formula, capable of efficiently producing a homogenous immobilized enzyme having high activity and utilizable for producing various useful substances.

公开(公告)号：JPH04117286A

公开(公告)日：1992-04-17

申请号：JP12320390

申请日：1990-05-15

Applicant: AGENCY IND SCIENCE TECHN ,  HITACHI CHEMICAL CO LTD

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

IPC: C12N15/09 ,  A61K31/00 ,  A61K38/00 ,  A61K38/44 ,  A61P37/08 ,  C07K1/12 ,  C07K1/22 ,  C07K7/06 ,  C07K14/00 ,  C07K19/00 ,  C12N1/21 ,  C12N9/02 ,  C12N15/12 ,  C12N15/53 ,  C12N15/62 ,  C12P21/02 ,  C12R1/19 ,  C12R1/91

Abstract: NEW MATERIAL:A recombinant plasmid containing a base sequence encoding dihydrofolate reductase-antiallergic peptide polymer fused protein (II). EXAMPLE:A recombinant plasmid wherein an antiallergic pentapeptide, a fused protein (II) of dimer contains a base sequence encoding an amino acid sequence shown by the [formula (underline shows amino acids existing between dihydrofolate reductase and antiallergic pentapeptide). USE:Production of DSDGK. PREPARATION:A DNA encoding DSDGK polymer obtained by chemically synthesizing a single strand DNA and annealing both chains is inserted into a vector plasmid capable of manifesting DHFR polymer.

公开(公告)号：JPH02258799A

公开(公告)日：1990-10-19

申请号：JP7946289

申请日：1989-03-30

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  OBARA KAZUHIKO ,  KOKUBU TOMOKUNI ,  OHASHI SHINICHI

IPC: C12P21/02 ,  A61K38/04 ,  C07K1/20 ,  C07K1/22 ,  C07K14/00 ,  C07K14/575 ,  C07K14/60 ,  C07K19/00 ,  C12N15/09 ,  C12N15/18 ,  C12N15/62 ,  C12R1/19

Abstract: NEW MATERIAL:A dihydrofolate reductase-growth hormone-releasing factor derivative fused protein produced by Escherichia coli containing recombinant pGRF2-15. USE:An agent for fermentation, stock raising and drugs having bovine growth hormone-releasing factor and dihydrofolic acid reducing activity. PREPARATION:For example, recombinant plasmid pGRF2-15 containing a base sequence to code amino acid sequence of fused protein comprising dihydrofolate reductase-growth hormone-releasing factor shown by the formula is inserted into Escherichia coil, transformed, the transformant is cultured in a medium, the cell is ground, centrifuged, a fraction of supernatant liquid is collected and treated with streptomycin sulfate and ammonium sulfate to give an enzyme solution. The solution is purifying by using mesotrixate bonded affinity column chromatography to give growth hormone-releasing factor derivative.

公开(公告)号：JPH02142481A

公开(公告)日：1990-05-31

申请号：JP29551988

申请日：1988-11-22

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO

IPC: C12N9/06 ,  C12N1/21 ,  C12N15/09 ,  C12N15/16 ,  C12N15/53 ,  C12N15/62 ,  C12N15/70 ,  C12R1/19

Abstract: NEW MATERIAL:A recombinant plasmid pDDM1 having a DNA sequence expressed by the formula. USE:For producing dihydrofolate reductase. PREPARATION:For example, a plasmid pTP64-1 is cleaved with restriction enzymes BclI and BglII and then subjected to treatment with an alkaline phosphatase and phenol to denature and remove coexisting enzymic proteins. The resultant DNA is subsequently precipitated with ethanol and linked to a plasmid pTP70-1 cleaved with the restriction enzyme BglII using a T4-DNA ligase, inserted into Escherichia coli and the obtained transformant is then cultured to select a strain capable of producing dimer of dihydrofolate reductase(DHFR) from the formed colony. Thereby, the objective recombinant plasmid pDDM1 is obtained.

公开(公告)号：JPH02138981A

公开(公告)日：1990-05-28

申请号：JP29338988

申请日：1988-11-19

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  OHASHI SHINICHI ,  TANAKA YOSHIO

IPC: C12N15/09 ,  C07K14/00 ,  C07K14/575 ,  C07K14/60 ,  C07K19/00 ,  C12N1/21 ,  C12N15/18 ,  C12N15/62 ,  C12N15/70 ,  C12P21/02 ,  C12R1/19

Abstract: PURPOSE:To mass-produce a GRF1-29 derivative by forming a recombinant plasmid pSG1-12 having a DNA sequence coding a peptide fragment of GRF with a fused protein of dihydrofolate reductase. CONSTITUTION:A recombinant plasmid pSG1-12, stably replicative in Escherichia coli, capable of imparting tolerance to trimethoprim and ampicillin to Escherichia coli which is a host, coding a fused protein of a peptide fragment of a growth hormone releasing factor having a specific amino acid sequence and a dihydrofolate reductase and forming a recombinant plasmid and having a specific DNA sequence is formed. The recombinant plasmid encodes DHFR- GRFM and Escherichia coli having the pSG1-12 is capable of accumulating and producing the DHFR-GRFM in a large amount.

公开(公告)号：JPH01252290A

公开(公告)日：1989-10-06

申请号：JP7968188

申请日：1988-03-31

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  KOKUBU TOMOKUNI ,  FURUSAWA KIYOTAKA ,  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 reductaseleucine enkephalin fused protein, by manifesting a fused gene in which a leucine enkephalin gene is included in a plasmid vector pTP 104-4 with a colon bacillus. CONSTITUTION:A newly recombined plasmid pLEK 1, 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 4207 basic pairs and having a bonded structure of chemically synthesized DNA of 34 basic pairs containing conformations coding BamHI of pTP 104-4, 4173 basic pairs DNA fragments of large side obtained by Sa I abscission and a leucine enkephalin, is produced. A colon bacillus introduced of the plasmid pLEK 1 (FERM BP-1818) produces a large amount of dihydrofolic acid reductase-leucine enkephalin fused protein.

公开(公告)号：JPH01252287A

公开(公告)日：1989-10-06

申请号：JP7967888

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 fused protein, by manifesting a fused gene in which an angiotensin I gene is included in a recombined plasmid pLEK1 with a colon bacillus. CONSTITUTION:A newly recombined plasmid pANG1-23, 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 4222 basic pairs and having a replaced structure with a conformation of 28 basic pairs containing a conformation coding leucine enkephaline between BamHI site and MluI site in pLEK1 to a chemically synthesized DNA of 43 basic pairs containing a conformation coding angiotensin I, is produced. A colon bacillus introduced of pANG1-23 produces a large amount of dihydrofolic acid reductase- angiotensin fused protein.

公开(公告)号：JPH01144977A

公开(公告)日：1989-06-07

申请号：JP30215487

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: C07K14/575 ,  C07H21/04 ,  C07K14/00 ,  C07K14/195 ,  C07K14/41 ,  C07K14/655 ,  C07K19/00 ,  C12N1/20 ,  C12N1/21 ,  C12N15/00 ,  C12N15/09 ,  C12P21/02 ,  C12R1/19

Abstract: NEW MATERIAL:Recombinant plasmid pTPGIF2 having a size of 4,660 base pairs and a structure obtained by inserting a DNA having a size of 52 base pairs and containing a sequence coding somatostatin into a BamHI incision site of a plasmid vector pTP70-1 capable of fusing a heteropeptide to a carboxy- terminal of a dihydrofolic acid reductase gene of E.coli. USE:Production of fused protein containing somatostatin. PREPARATION:A somatostatin gene is integrated into a plasmid vector pTP70-1. pTPGIF2 is kept in stable state when introduced into E.coli. C600 strain. The E.coli. C600 strain containing pTPGIF2 is deposited in Fermentation Research Institute as FERM BP-1577.