High-level expression in Escherichia coli of enzymatically active fusion proteins containing the domains of mammalian cytochromes P450 and NADPH-P450 reductase flavoprotein.
This report describes the properties of two mammalian cytochromes P450 that have been expressed at high levels in Escherichia coli as enzymatically active fusion proteins containing the flavoprotein domain of rat NADPH-cytochrome P450 reductase (EC 1.6.2.4). Fusion proteins were prepared by engineering the cDNAs for the steroid-metabolizing bovine adrenal P450 17A with the cDNA for rat liver NADPH-P450 reductase with the introduction of a Ser-Thr linker to give a protein we have named rF450[mBov17A/mRatOR]L1. Similarly, the cDNA for the omega-hydroxylase of rat liver (P450 4A1) was linked with the cDNA for rat liver NADPH-P450 reductase to give rF450[mRat4A1/mRatOR]L1. A procedure involving disruption of transformed E. coli by sonication, isolation of membranes by differential centrifugation, solubilization with detergent, and affinity chromatography provided significant amounts of purified fusion proteins of approximately 118 kDa. The purified fusion proteins had turnover numbers for the metabolism of steroids (rF450[mBov17A/mRatOR]L1) or fatty acids (rF450[mRat4A1/mRatOR]L1) ranging from 10/min to 30/min in the absence of added phospholipid. Addition of purified rat liver cytochrome b5 stimulated the 17,20-lyase reaction for the conversion of 17-hydroxypregnenolone to dehydroepiandrosterone, and addition of purified rat NADPH-cytochrome P450 reductase enhanced the formation of omega--1 metabolites from lauric and arachidonic acids. NADPH oxidation was tightly coupled to substrate hydroxylation with the purified fusion proteins.
Fisher
C W
CW
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
Shet
M S
MS
Caudle
D L
DL
Martin-Wixtrom
C A
CA
Estabrook
R W
RW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D011284:Pregnenolone / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D011374:Progesterone; D011284:Pregnenolone; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase