TY - JOUR T1 - Coexpression of Cytochrome P4502A6 and Human NADPH-P450 Oxidoreductase in the Baculovirus System JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 399 LP - 405 VL - 25 IS - 4 AU - Liping Chen AU - Jeroen T. M. Buters AU - James P. Hardwick AU - Shingi Tamura AU - Bruce W. Penman AU - Frank J. Gonzalez AU - Charles L. Crespi Y1 - 1997/04/01 UR - http://dmd.aspetjournals.org/content/25/4/399.abstract N2 - 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 × 10−2) and a vOR of 10- to 20-fold less. This activity was ∼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. The American Society for Pharmacology and Experimental Therapeutics ER -