title: Baculovirus expression of human P450 2E1 and cytochrome b5: spectral and catalytic properties and effect of b5 on the stoichiometry of P450 2E1-catalyzed reactions.
abstract: The baculovirus (BV)-insect cell expression system has been successfully used to express high levels of mammalian proteins. Here we report the baculovirus-mediated expression of human P450 2E1 and cytochrome b5 in Sf9 insect cells. The BglI-EcoRI fragment of the human P450 2E1 cDNA (Umeno et al., Biochemistry 27, 1988) was used for the expression of P450 2E1. Human cytochrome b5 cDNA was obtained by the polymerase chain reaction using human liver cDNA as template. Infection of Sf9 insect cells with a recombinant 2E1-BV resulted in the expression of a protein which comigrated with human liver P450 2E1 on SDS gels and cross-reacted with a polyclonal antibody against rat liver P450 2E1. Inclusion of hemin in the cell growth medium greatly enhanced the spectral activity of expressed 2E1. Expression levels of 1.5 nmol/mg cell lysate were obtained after 7 days of infection. However, maximal turnover numbers (min-1) were observed after 48 h of infection. The lambdamax of the CO:reduced CO difference spectrum of human P450 2E1 was found to be 451.1 nm. In the presence of exogenous hemin, BV-expressed human b5 showed a typical reduced-oxidized difference spectrum with lambdamax and lambdamin of 425 and 409 nm, respectively. With saturating levels of purified rat liver NADPH:P450 oxidoreductase and rat liver cytochrome b5 included in the incubations, expressed human P450 2E1 showed high catalytic activity for the metabolism of p-nitrophenol (PNP), ethoxycoumarin, N-nitrosodiethylamine, and N-nitrosodimethylamine (NDMA). The presence of b5 in the incubations increased the activities several-fold. The Km and kcat values for the N-demethylation of NDMA to HCHO in the presence of rat b5 by expressed 2E1 were 36.0 microM and 8.3 min-1, respectively. In the absence of extra added b5, the Km was increased sixfold and the kcat decreased fourfold. In the presence of extra added b5 expressed 2E1 showed a Km for PNP metabolism of 86 microM and a kcat of 7.8 min-1. Simultaneous infection of Sf9 insect cells with both 2E1 and human bE recombinant BV resulted in membrane fraction (2E1:b5) containing both proteins at a ratio of b5 to P450 of approximately 1.8:1. The Km and kcat values for NDMA demethylase activity by the 2E1:b5 membrane fraction were similar to those with exogenously added b5.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Drug Metabolism and Pharmacokinetics, Drug Safety, Sandoz Research Institute, Sandoz Pharmaceuticals Corporation, East Hanover, New Jersey 07936.
authors.forename: C J
authors.initials: CJ
authors.lastname: Patten
authors.forename: P
authors.initials: P
authors.lastname: Koch
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002384:Catalysis; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015786:Cytochromes b5 / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006427:Hemin / Q000494:pharmacology; D006801:Humans; D015227:Lipid Peroxidation; D008315:Malondialdehyde / Q000378:metabolism; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D013053:Spectrophotometry; D018411:Spodoptera / Q000378:metabolism; D013379:Substrate Specificity; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D017392:Thiobarbituric Acid Reactive Substances; D008315:Malondialdehyde; D009249:NADP; D006427:Hemin; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating