title: Coexpression of cytochrome P4502A6 and human NADPH-P450 oxidoreductase in the baculovirus system.
abstract: Heterologous expression using baculovirus vectors has become a popular method for the production of catalytically active cytochrome P450s (CYPs). We have systematically optimized the multiplicity of infection (MOI) for a coinfection approach for the coexpression of CYP2A6 (viral vector designated v2A6) and NADPH-P450 oxidoreductase (OR; viral vector designated vOR) using Sf9 insect cells. A 3000-fold range of MOI was examined in stationary culture and stirred suspension culture. Surprisingly, our results indicate that the best CYP2A6 catalytic activity (850-1300 pmol/ min/mg total lysate protein as measured by coumarin 7-hydroxylase activity) was obtained only when using a low MOI of v2A6 (1.5-3 x 10(-2)) and a vOR of 10- to 20-fold less. This activity was approximately 7- to 11-fold higher than the best activity obtained when infecting cells with v2A6 alone. At this level of coinfection, the P450 content ranged from 180 to 250 pmol/mg total lysate protein, and the NADPH cytochrome c reductase activity ranged from 350 to 520 nmol/min/mg total lysate protein. Increasing the MOI of both viruses to 50-fold higher resulted in lower overall activity with the optimum (250 pmol/min/mg total lysate protein) being seen earlier postinfection (60 vs. 72 hr). Increasing the MOI of vOR to levels comparable with those of v2A6, decreased coumarin 7-hydroxylase activity 14-fold. These results suggest that the best CYP2A6 catalytic activity depends on properly posttranslationally modified proteins accumulating in a right ratio as a result of primary, secondary, and possibly tertiary infection of both viruses. These results also suggest that high OR expression results in degradation of P450.
authors.affiliation: Gentest Corporation, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: L
authors.initials: L
authors.lastname: Chen
authors.forename: J T
authors.initials: JT
authors.lastname: Buters
authors.forename: J P
authors.initials: JP
authors.lastname: Hardwick
authors.forename: S
authors.initials: S
authors.lastname: Tamura
authors.forename: B W
authors.initials: BW
authors.lastname: Penman
authors.forename: F J
authors.initials: FJ
authors.lastname: Gonzalez
authors.forename: C L
authors.initials: CL
authors.lastname: Crespi
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D016367:Baculoviridae / Q000235:genetics*; D002384:Catalysis; D002460:Cell Line; D003001:Cloning, Molecular; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics*; D018411:Spodoptera
chemical_list: D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6; D009251:NADPH-Ferrihemoprotein Reductase