THE HIGH-LEVEL EXPRESSION IN ESCHERICHIA-COLI OF THE MEMBRANE-BOUND FORM OF HUMAN AND RAT CYTOCHROME B(5) AND STUDIES ON THEIR MECHANISM OF FUNCTION

A T7 expression system is described for the high-level production in Escherichia coli of the membrane-bound form of human and rat cytochrome b(5). The cDNAs of b(5) have been engineered to contain a coding sequence for a four-member histidine domain at the amino-terminus of the recombinant protein permitting the use of a nickel-chelate affinity column for rapid purification of the detergent-solubilized hemoprotein. Results are presented demonstrating the ability of the purified recombinant b(5) proteins to stimulate the rate of oxidation of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone, catalyzed by bovine P450 17A, and to stimulate the 6 beta-hydroxylation of testosterone, catalyzed by human P450 3A4. These P450-catalyzed reactions have been used to compare the properties of different forms of b(5). Purified bg can serve as a ''coupling protein'' as illustrated by its inhibition of NADPH oxidation, catalyzed by a fusion protein containing the heme domain of P450 3A4 linked to rat NADPH-P450 reductase, and the associated inhibition of hydrogen peroxide formation. Kinetic studies show the formation of a complex of the flavoprotein, NADPH-P450 reductase, with b(5) for the rapid transfer of electrons from NADPH. (C) 1994 Academic Press, Inc.