Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.
When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system.
Barnes
H J
HJ
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
Arlotto
M P
MP
Waterman
M R
MR
D006907:17-alpha-Hydroxypregnenolone / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004247:DNA / Q000235:genetics; D003687:Dehydroepiandrosterone / Q000378:metabolism; D004926:Escherichia coli; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D011284:Pregnenolone / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013057:Spectrum Analysis; D013254:Steroid 17-alpha-Hydroxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D006907:17-alpha-Hydroxypregnenolone; D003687:Dehydroepiandrosterone; D011374:Progesterone; D011284:Pregnenolone; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D013254:Steroid 17-alpha-Hydroxylase