| Project/Area Number |
02453129
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
応用微生物学・発酵学
|
|---|
| Research Institution | Kyoto University |
|---|
Principal Investigator |
KUMAGAI Hidehiko Kyoto Univ., Fac. Agric., Professor, 農学部, 教授 (70027192)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
YANO Toshihiro Kyoto Univ., Fac. Agric., Instructor, 農学部, 教務職員 (30135553)
SUZUKI Hideyuki Kyoto Univ., Fac. Agric., Research Associate, 農学部, 助手 (10202136)
YAMAMOTO Kenji Kyoto Univ., Fac. Agric., Assoc. Professor, 農学部, 助教授 (70109049)
|
|---|
| Project Period (FY) |
1990 – 1991
|
| Project Status |
Completed (Fiscal Year 1991)
|
| Budget Amount *help |
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1991: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1990: ¥4,600,000 (Direct Cost: ¥4,600,000)
|
|---|
| Keywords | glutathione / glutathione S-transferase / gamma-glutamyltranspeptidase / gamma-glutamyltransferase / detoxification / Escherichia coli / Issatchenkia orientalis / 大腸菌 / グルタチオン抱合 / クロ-ニング |
|---|
| Research Abstract |
(1) gamma-Glutamyltaranspeptidase (GGT) gene (ggt) was self-cloned in GGT-less E. coli mutant and this cloned strain showed low-temperature dependent expression of GGT as a wild strain. The results from Western and Northern blotting indicated that the expression controlled not at the processing but at the transcriptional step.
(2) The plasmid carrying ggt was transduced into a tola strain of E. coli. The transformant excreted GGT and the GGT was purified by a simple two-step method.
(3) The conditions were investigated for the preparation of crystals of GGT suitable for the x-ray analysis and the conditions to obtain large crystals were established.
(4) The mutation of Arg 571 to Ala in GGT caused accumulation of inactive pro-GGT in the periplasmic space of the host cell.
(5) GILitathione S-transferase (GST) cDNA from a yeast Issatchenkia orientalis was cloned into an E. coli strain and this transformant highly expressed GST. The nucleotide sequence of GST CDNA was determined and the amino acid sequence was deduced. The inducible formation of GST was elucidated to be controlled at the transcriptional step.
(6) The glutathione-conjugate was found to be metabolized to eysteine-conjugate by the action of carboxypeptidase-like enzyme in I. orientalis cells.
(7) GST cDNA was subcloned to a shuttle-vector and the reconstituted plasmid was transduced into Saccharomyces cerevisiae cells. The transformant showed 15 times higher GST activity than the host cells.
(8) The localization of GST at the surface of I. orientalis cells were indicated by cell-fractionation and immune-electromicroscope analysis.
|
