PMID: 9526119Jun 2, 1998Paper

Comparative structural and immunochemical characterization of recombinant and natural cytochrome p450scc (CYPXIAI)

Biochemistry. Biokhimii︠a︡
G I Lepesheva, S A Usanov

Abstract

Optimization of the conditions for heterologous expression of recombinant cytochrome P450scc in E. coli provided an expression level of about 420 nmoles of cytochrome P450scc per liter of bacterial culture. A new procedure for purification of recombinant protein in substrate-bound high-spin and substrate-free low-spin form is described. Highly purified electrophoretically homogeneous recombinant cytochrome P450scc contains 12.3 and 16.7 nmoles heme per mg protein for substrate-free and substrate-bound forms, respectively. The recombinant and natural cytochrome P450scc from bovine adrenocortical mitochondria were compared functionally and immunochemically. The dissociation constants for the complexes of cytochrome P450scc with cholesterol and adrenodoxin, the efficiency of enzymatic reduction in the reconstituted system (NADPH--adrenodoxin reductase--adrenodoxin), and cholesterol side-chain cleavage activity were determined. It was found that limited proteolysis of the recombinant cytochrome P450scc with trypsin forms two main fragments which are electrophoretically and immunochemically identical with the fragments F1 (29.8 kD) and F2 (26.6 kD) formed during proteolysis of bovine adrenocortical cytochrome P450scc. The quantitati...Continue Reading

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