Pl. Holmans et al., THE HIGH-LEVEL EXPRESSION IN ESCHERICHIA-COLI OF THE MEMBRANE-BOUND FORM OF HUMAN AND RAT CYTOCHROME B(5) AND STUDIES ON THEIR MECHANISM OFFUNCTION, Archives of biochemistry and biophysics, 312(2), 1994, pp. 554-565
A T7 expression system is described for the high-level production in E
scherichia 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 sequ
ence for a four-member histidine domain at the amino-terminus of the r
ecombinant protein permitting the use of a nickel-chelate affinity col
umn for rapid purification of the detergent-solubilized hemoprotein. R
esults are presented demonstrating the ability of the purified recombi
nant b(5) proteins to stimulate the rate of oxidation of 17 alpha-hydr
oxypregnenolone to dehydroepiandrosterone, catalyzed by bovine P450 17
A, and to stimulate the 6 beta-hydroxylation of testosterone, catalyze
d by human P450 3A4. These P450-catalyzed reactions have been used to
compare the properties of different forms of b(5). Purified bg can ser
ve as a ''coupling protein'' as illustrated by its inhibition of NADPH
oxidation, catalyzed by a fusion protein containing the heme domain o
f P450 3A4 linked to rat NADPH-P450 reductase, and the associated inhi
bition of hydrogen peroxide formation. Kinetic studies show the format
ion of a complex of the flavoprotein, NADPH-P450 reductase, with b(5)
for the rapid transfer of electrons from NADPH. (C) 1994 Academic Pres
s, Inc.