Elucidation of the Mechanism of Bacterial Cytochrome c Thermostability by Protein

1.细胞色素c基因的克隆（1）以细胞色素c蛋白序列为探针<552>，对嗜热菌细胞色素c<552>基因进行了克隆和测序。(2)按照相似的方案，对铜绿假单胞菌细胞色素c<551>基因进行了分离和测序.细胞色素c基因的表达（1）<552->制备无信号肽的细胞色素c基因。用质粒pKK 223 -3转化大肠杆菌JM 109。该菌产生细胞色素c_1<552->，并从无细胞提取物中纯化细胞色素c_1。(2)制备<551>了带有信号序列的细胞色素c基因。利用质粒pHA 10转化铜绿假单胞菌PAO 1161。该菌产生细胞色素<551>c_3。氨基酸置换引起的热稳定性的变化（1）对于细胞色素c_<552>，目的是获得热稳定性降低的突变蛋白。制备了以下取代蛋白（Ala 26至Lys，Lys 30至Ala，Ala 26至Lys和Lys 30至Ala，Asp 37至Gly）（2）在细胞色素c的情况下<551>，目的是获得表现出增加的热稳定性的突变蛋白。制备以下取代的蛋白质（Lys 28至Ala、Ala 32至Lys和Gly 39至Asp）。(3)所有突变蛋白均表现出与原始蛋白相似的热稳定性。

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.