Elucidation of the Mechanism of Bacterial Cytochrome c Thermostability by Protein

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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.