Noguchi S, Yamazaki T, Yaginuma A, Sakamoto J, Sone N
Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Fukuoka-ken 820, Japan.
Biochim Biophys Acta. 1994 Dec 30;1188(3):302-10. doi: 10.1016/0005-2728(94)90049-3.
Cytochrome c-551 is a lipoprotein of about 10500 Da, found in thermophilic Bacillus PS3 grown under air-limited conditions. An expression vector was constructed from a structural gene of PS3 cytochrome c-551, synthetic oligonucleotide as a promoter for Bacillus stearothermophilus and a shuttle vector for Escherichia coli and B. stearothermophilus. The transformed cells of B. stearothermophilus K1041 expressed cytochrome c-551 as much as 5 nmol/mg membrane protein. The effects of over-expression on the host cells are analyzed; a slightly slower growth rate and an increased synthesis of cytochrome oxidase (about twofold) occurred. Over-expressed (4-10-fold) cytochrome c-551 were purified and its properties were examined to know whether the protein is processed as in PS3 cells grown under air-limited conditions. The molecular mass determination and treatment with Rhizopus lipase suggested that the same processes, cleavage of signal peptidase, blocking of the N-terminal group and acylation of glycerol residue by two fatty acids, took place in the over-expression system. Fatty acylation seems useful for the cytochrome c to be effectively oxidized.
细胞色素c-551是一种分子量约为10500道尔顿的脂蛋白，存在于在空气受限条件下生长的嗜热芽孢杆菌PS3中。利用PS3细胞色素c-551的结构基因、作为嗜热脂肪芽孢杆菌启动子的合成寡核苷酸以及用于大肠杆菌和嗜热脂肪芽孢杆菌的穿梭载体构建了表达载体。嗜热脂肪芽孢杆菌K1041的转化细胞表达的细胞色素c-551高达5 nmol/mg膜蛋白。分析了过表达对宿主细胞的影响；观察到生长速率略有减慢，细胞色素氧化酶的合成增加（约两倍）。对过表达（4-10倍）的细胞色素c-551进行了纯化，并检测了其性质，以了解该蛋白是否如在空气受限条件下生长的PS3细胞中那样进行加工。分子量测定和用根霉脂肪酶处理表明，在过表达系统中发生了相同的过程，即信号肽酶切割、N端基团封闭以及甘油残基被两个脂肪酸酰化。脂肪酸酰化似乎有助于细胞色素c被有效氧化。
