Elucidation of the Mechanism of Bacterial Cytochrome c Thermostability by Protein
Grant-in-Aid for Scientific Research (B).
|Research Institution||The University of Tokyo|
KODAMA Tohru The Univ. of Tokyo, Fac. of Agriculture, Professor., 農学部, 教授 (30011901)
ISHII Masaharu The Univ. of Tokyo., Pept. of Agric. Chem., Assistant Professor, 農学部, 助手 (30193262)
|Project Fiscal Year
1989 – 1990
Completed(Fiscal Year 1990)
|Budget Amount *help
¥6,000,000 (Direct Cost : ¥6,000,000)
Fiscal Year 1990 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1989 : ¥5,100,000 (Direct Cost : ¥5,100,000)
|Keywords||Cytochrome c / Thermostability / シトクロムC / 耐熱化機構 / シトクロムc|
1. Clonimg of cytochrome c genes
(1) By using two probes which were made from cytochrome c_<552> protein sequence, cytochrome c_<552> gene from thermophilic bacterium was isolated and sequenced.
(2) By following the similar scheme, cytochrome c_<551> gene from Pseudomonas aeruginosa was isolated and sequenced.
2. Expression of cytochrome c genes
(1) Cytochrome c_<552-> gene without signal sequence was prepared. By using plasmid pKK223-3, Escherichia coli JM 109 was transformed by this gene. The transformant produced cytochrome c_<552-> and the cytochrome was purified from cell-free extract.
(2) Cytochrome c_<551> gene with signal sequence was prepared. By using plasmid pHA10, Pseudomonas aeruginosa PAO1161 was transformed by this gene. The transformant produced cytochrome c_<551>.
3. Changes in thermostability by amino acid replacement
(1) In the case of cytochrome c_<552>, the purpose was to get mutated proteins which shows decreased thermostability. The following substituted proteins were prepared (Ala26 to Lys, Lys30 to Ala, Ala26 to Lys and Lys30 to Ala, Asp37 to Gly)
(2) In the case of cytochrome c_<551>, the purpose was to get mutated proteins which shows increased thermostability. The following substituted proteins were prepared (Lys28 to Ala, Ala32 to Lys, and Gly39 to Asp).
(3) All of the mutated protein showed similar thermostability as those of original ones.
(1)シトクロムc_<552>の場合には耐熱性減少を目的とし、site directed mutagenesis の手法を用いて、アラニン26→リジン、リジン30→アラニン、アラニン26→リジン:リジン30→アラニンの二重変異、アスパラギン酸37→グリシンという変異シトクロムc_<552>蛋白質を調製した。
(2)シトクロムc_<551>の場合には耐熱性増加を目的とし、site directed mutagenesis の手法を用いて、リジン28→アラニン、アラニン32→リジン、グリシン39→アスパラギン酸という変異シトクロムc_<551>蛋白質を調製した。
Research Output (4results)