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 Km and kcat for N-nitrosodimethylamine (NDMA) demethylase activity were 145 microM 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 Km of NDMA demethylase activity (16 microM) was observed in the membrane preparations, whereas the kcat 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 H202 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.
Laboratory for Cancer Research, College of Pharmacy, Rutgers University, Piscataway, New Jersey 08855-0789, USA.
M H
MH
Wang
C J
CJ
Patten
G Y
GY
Yang
S R
SR
Paranawithana
Y
Y
Tan
C S
CS
Yang
D015660:7-Alkoxycoumarin O-Dealkylase / Q000096:biosynthesis / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D019392:Cytochrome P-450 CYP2E1 / Q000096:biosynthesis* / Q000378:metabolism; D015786:Cytochromes b5 / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis* / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D018411:Spodoptera; D014162:Transfection / Q000379:methods*
D011994:Recombinant Proteins; D015786:Cytochromes b5; D015660:7-Alkoxycoumarin O-Dealkylase; D019392:Cytochrome P-450 CYP2E1; D009251:NADPH-Ferrihemoprotein Reductase