Expression and coupling of human cytochrome P450 2E1 and NADPH- cytochrome P450 oxidoreductase in dual expression and co-infection systems with baculovirus in insect cells

In order to study the interaction between human cytochrome P450 2E1 (h2E1) and NADPH-P450 oxidoreductase (hOR) in a native membrane environment, we used two approaches to express both h2E1 and hOR in a baculovirus expression system. For a dual-expression system, h2E1 and hOR were coexpressed in Spodoptera frugiperda (Sf9) insect cells using a recombinant baculovirus carrying both h2E1 and hOR cDNAs (v-h2E1-hOR). The h2E1 cDNA was expressed under the control of the polyhedrin promoter P(Polh), whereas hOR cDNA was expressed under the control of the P₁₀, promoter. The expressed enzymes were catalytically active in the cell membrane preparations. The estimated molar expression ratio of h2E1 to hOR in the membranes was 1:5. The apparent K(m) and k(cat) for N-nitrosodimethylamine (NDMA) demethylase activity were 145 μM and 2.4 min^-1, respectively. When Sf9 cells were co- infected with the dual-expression virus (v-h2E1-hOR) and human cytochrome b₅ recombinant virus (v-hb₅), a 9-fold decrease in the K(m) of NDMA demethylase activity (16 μM) was observed in the membrane preparations, whereas the k(cat) was increased to 4 min^-1. In the second approach, recombinant viruses of h2E1 and hOR (v-h2E1 and v-hOR) were used to co-infect the Sf9 cells. In this double-expression system, with a fixed amount of v-h2E1, the expression of h2E1 in the Sf9 cells decreased as the amount of v-hOR increased. Western blot analysis of the membrane preparations showed that the level of hOR increased, but the level of h2E1 decreased with increasing amounts of v-hOR. A corresponding decrease in h2E1 mRNA, however, was not observed. In the presence of human cytochrome b₅ (hb₅), the optimal h2E1:hOR molar ratio for h2E1 catalytic activity was 1:1. In order to further investigate the hb₅ effect on h2E1-catalyzed reactions in the native membranes, we co-infected Sf9 cells with v-h2E1, v-hOR, and v-hb₅ and obtained a membrane preparation containing h2E1, hOR, and hb₅. Stoichiometric analysis with membrane preparations from double-infection and triple-infection systems revealed that the presence of hb₅ decreased NADPH oxidation and H₂O₂ formation by 72 and 80%, respectively, but increased product formation from NDMA 13-fold. These results suggest that hb₅ enhances the coupling between h2E1 and hOR for product formation. These studies also demonstrate that the baculovirus-insect cell system can produce high levels of expression of functional h2E1, hOR, and hb₅ for mechanistic studies.