OVER-EXPRESSION OF MEMBRANE-BOUND CYTOCHROME C-551 FROM THERMOPHILIC BACILLUS PS3 IN BACILLUS-STEAROTHERMOPHILUS K1041

Cytochrome c-551 is a lipoprotein of about 10 500 Da, found in thermop hilic Bacillus PS3 grown under air-limited conditions. An expression v ector was constructed from a structural gene of PS3 cytochrome c-551, syththetic oligonucleotide as a promoter for Bacillus stearothermophil us and a shuttle vector for Escherichia coli and B. stearothermophilus . The transformed cells of B. stearothermophilus K1041 expressed cytoc hrome 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 r ate and an increased synthesis of cytochrome oxidase (about twofold) o ccurred. Over-expressed (4-10-fold) cytochrome c-551 were purified, an d its properties were examined to know whether the protein is processe d as in PS3 cells grown under air-limited conditions. The molecular ma ss determination and treatment with Rhizopus lipase suggested that the same processes, cleavage of signal peptidase, blocking of the N-termi nal group and acylation of glycerol residue by two fatty acids, took p lace in the over-expression system. Fatty acylation seems useful for t he cytochrome c to be effectively oxidized.