Over-expression of membrane-bound cytochrome c-551 from thermophilic Bacillus PS3 in Bacillus stearothermophilus K1041.
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.
Noguchi
S
S
Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Fukuoka-ken 820, Japan.
Yamazaki
T
T
Yaginuma
A
A
Sakamoto
J
J
Sone
N
N
D000215:Acylation; D001426:Bacterial Proteins; D001483:Base Sequence; D003574:Cytochrome c Group / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D008049:Lipase; D008969:Molecular Sequence Data
D001426:Bacterial Proteins; D003574:Cytochrome c Group; C017167:cytochrome C(551); D008049:Lipase