Regular ArticleExpression and Coupling of Human Cytochrome P450 2E1 and NADPH–Cytochrome P450 Oxidoreductase in Dual Expression and Co-infection Systems with Baculovirus in Insect Cells☆
Abstract
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 inSpodoptera 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 PPolh, whereas hOR cDNA was expressed under the control of the P10promoter. 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 apparentKmandkcatforN-nitrosodimethylamine (NDMA) demethylase activity were 145 μMand 2.4 min−1, respectively. When Sf9 cells were co-infected with the dual-expression virus (v-h2E1-hOR) and human cytochrome b5recombinant virus (v-hb5), a 9-fold decrease in theKmof NDMA demethylase activity (16 μM) was observed in the membrane preparations, whereas thekcatwas 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 hb5effect on h2E1-catalyzed reactions in the native membranes, we co-infected Sf9 cells with v-h2E1, v-hOR, and v-hb5and 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 hb5decreased NADPH oxidation and H2O2formation by 72 and 80%, respectively, but increased product formation from NDMA 13-fold. These results suggest that hb5enhances 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 hb5for mechanistic studies.
References (0)
Cited by (19)
Recombinant baculovirus-based multiple protein expression platform for Drosophila S2 cell culture
2008, Journal of BiotechnologyA platform for selective and controllable expression of multiple foreign protein types was developed in insect cell culture. Based on the fact that baculovirus cannot replicate in nonpermissive Drosophila melanogaster Schneider line 2 (S2) cells, S2 cells that stably express human erythropoietin (hEPO) under the control of the S2-derived inducible metallothionein (MT) promoter were infected with three types of recombinant baculoviruses, each of which expressed a different fluorescent protein gene under the control of MT promoter. Addition of copper sulfate as an inducer to infected, stably transfected S2 cells resulted in simultaneous expression of hEPO and three fluorescent proteins. Expression profiles and levels of the three induced fluorescent proteins were similar in all single infected cells. Importantly, expression profiles and levels of hEPO were similar in both non-infected and infected cells, indicating that baculovirus expressed recombinant proteins do not adversely affect expression of host cell recombinant proteins. Expressions of the three fluorescent proteins were able to be selectively regulated by altering combination ratios of the three types of recombinant baculoviruses. Collectively, these data indicate that the baculovirus/stably transfected S2 cell system can be successfully used to express multiple foreign proteins in a controlled and selective manner without the burden of additional selection markers. Such a system would be expected to be attractive as a multiple protein expression platform for engineering metabolic or glycosylation pathways.
Dual expression of the HA protein of H5N2 avian influenza virus in a baculovirus system
2006, Journal of Virological MethodsBaculovirus/insect cell system is used widely for recombinant protein production. The hemagglutinin (HA) gene of H5N2 avian influenza virus (AIV) 1209 strain and the enhanced green fluorescent protein (EGFP) gene were cloned into pFastBac DUAL vector that has two promoters and cloning sites, allowing simultaneous expression of these two genes. The HA protein of AIV was fused with a hexahistidine (His6) tag for purification. The coexpression of EGFP allowed identification of the recombinant baculoviruses in Sf-9 insect cells, eliminating cumbersome and time-consuming assays. A recombinant baculovirus, Bac-HA, was generated by transfecting pBac-HA to bacmid inside DH10BAC Escherichia coli by site-specific transposition, followed by transfection into the Sf-9 cells. Fluorescence in the insect cells was observed from 3 days post-infection. The expressed HA protein was confirmed by Western blot using an anti-HA monoclonal antibody. Also, different detergents and incubation times on ice were tested. The two-stage extraction with Triton X-100 or Tween 20 and incubation on ice for 2 h exhibited high efficiency. Since purification of HA with ConA resin resulted in low protein recovery, lentil lectin affinity column was used and was useful for HA purification.
Expression of double foreign protein types following recombinant baculovirus infection of stably transfected Drosophila S2 cells
2004, Enzyme and Microbial TechnologyWe sought to develop a platform for simultaneous, regulatable expression of double foreign protein types in cell culture. Drosophila melanogaster Schneider line 2 (S2) insect cells that stably express human erythropoietin (hEPO) were infected with a recombinant baculovirus containing the green fluorescent protein (GFP) gene. Since baculovirus cannot replicate in nonpermissive S2 cells, baculovirus infection did not affect cell growth or viability. Expression of each foreign protein was under the control of the inducible metallothionein (MT) promoter. Addition of copper sulfate to infected, stably transfected cells resulted in simultaneous expression of both GFP and hEPO. Induced hEPO expression profile and levels were similar in both control and infected cells, indicating that baculovirus infection also did not affect expression of stably introduced foreign gene. GFP protein levels were regulated by the infection dose of recombinant baculovirus, while hEPO expression remained constant. hEPO levels were much higher (∼30-fold) than GFP, indicating plasmid-based introduced gene copies have higher expression than baculovirus-based introduced genes. These data suggest the baculovirus/stable S2 cell system can be used to produce a major target protein by plasmid-based stable transfection, and assistant proteins by recombinant baculovirus infection. Such a system appears to be very attractive as a multiple protein expression platform for engineering metabolic pathways in cell culture.
Cytochrome b<inf>5</inf> reductase and cytochrome b<inf>5</inf> support the CYP2E1-mediated activation of nitrosamines in a recombinant Ames test
2003, Archives of Biochemistry and BiophysicsWith CYP2E1 in vitro both the first and the second electron of the catalytic cycle can come from cytochrome b5 via either NADPH–cytochrome P450 reductase or NADH–cytochrome b5 reductase, and the presence of cytochrome b5 stimulates CYP2E1 turnover both in vitro and in vivo. To determine whether electron input via the NADH-dependent pathway was similarly functional in whole cells and necessary for the stimulation by cytochrome b5, we constructed five plasmids designed to express human CYP2E1 in various combinations with cytochrome b5 reductase, cytochrome b5, and cytochrome P450 reductase. CYP2E1 activity in Salmonella typhimurium cells transformed with each plasmid was assessed by mutagenic reversion frequency in the presence of dimethylnitrosamine. A fivefold increase in reversion frequency when cytochrome b5 was coexpressed with P450 reductase was abolished by disruption of heme-binding in cytochrome b5 by site-directed mutagenesis (His68Ala), suggesting that electron transfer to cytochrome b5 was necessary for the stimulation. Addition of cytochrome b5 reductase to the cytochrome b5/P450 reductase coexpression plasmid did not further increase the stimulation by cytochrome b5, but b5 reductase could support CYP2E1 activity in the absence of P450 reductase at a level equivalent to that obtained with just CYP2E1 and P450 reductase. Neither cytochrome b5 reductase nor cytochrome b5 alone could support CYP2E1 activity. These results demonstrate that the cytochrome b5 reductase/cytochrome b5 pathway can support CYP2E1 activity in bacterial cells.
Cytochrome b<inf>5</inf> coexpression increases the CYP2E1-dependent mutagenicity of dialkylnitrosamines in methyltransferase-deficient strains of Salmonella typhimurium
2001, Mutation Research - Fundamental and Molecular Mechanisms of MutagenesisAddition of cytochrome b5 to recombinant cytochrome P450 2E1 systems has been shown to enhance the metabolism of dialkylnitrosamines in vitro. To determine if this effect could be observed with recombinant expression systems in vivo, we have constructed mutagenicity tester strains that coexpress full-length human cytochrome P450 2E1 (CYP2E1), rat cytochrome P450 reductase, and human cytochrome b5 in Salmonella typhimurium lacking ogt and ada methyltransferases (YG7104, ogt−; and YG7108, ogt−, ada−). These new recombinant strains exhibit a four- to five-fold greater mutagenic response to dimethylnitrosamine, diethylnitrosamine, and dipropylnitrosamine than strains that contain only CYP2E1 and reductase, and are over 100-fold more sensitive to nitrosamines than the parental strains in the presence of an exogenous activating system (S9 fraction). The four-fold increase in mutagenicity in the presence of cytochrome b5 was consistent with increasing alkyl chain length up to dibutylnitrosamine, which was poorly activated by CYP2E1. The greatest enhancement was obtained with a tricistronic construct in which the b5 cDNA preceded the P450 and reductase cDNAs; placing the b5 cDNA after the reductase cDNA was substantially less effective. These new, highly sensitive strains may prove useful in the detection of nitrosamine contamination of food and environmental samples.
Expression and characterization of canine cytochrome P450 2D15
1998, Archives of Biochemistry and BiophysicsCYP2D15 is the canine ortholog of human CYP2D6, the human CYP2D isoform involved in the metabolism of drugs such as antiarhythmics, adrenoceptor antagonists, and tricyclic antidepressants. Similar to human, canine CYP2D15 is expressed in the liver, with detectable levels in several other tissues. Three different CYP2D15 cDNA clones were obtained by RT-PCR from dog liver RNA. Two clones corresponded to variant full-length CYP2D15 cDNAs (termed CYP2D15 WT2 and CYP2D15 V1); the third was identified as a splicing variant missing exon 3 (termed CYP2D15 V2). Recombinant baculoviruses were constructed containing full-length cDNAs and used to express CYP2D15 WT2 and CYP2D15 V1 inSpodoptera frugiperda(Sf9) cells with expression levels of up to 0.14 nmol/mg cell protein. As with human CYP2D6, the recombinant CYP2D15 enzymes exhibited bufuralol 1′-hydroxylaseand dextromethorphanO-demethylase activities whencoexpressed with rabbit NADPH:P450 oxidoreductase. For bufuralol 1′-hydroxylase, apparentKmvalues were 4.9, 3.7, and 2.5 μM and theVmaxvalues were 0.14, 0.034, and 0.60 nmol/min/mg protein for dog liver microsomes, CYP2D15 WT2, and the variant CYP2D15 V1, respectively. For dextromethorphanO-demethylase, apparentKmvalues were 0.6, 0.6, and 2.0 μM and theVmaxvalues were 0.18, 0.034, and 0.057 nmol/min/mg protein for dog liver microsomes, CYP2D15 WT2, and the variant CYP2D15 V1, respectively. The human CYP2D6-specific inhibitor quinidine and the rat CYP2D1-specific inhibitor quinine were both shown to be inhibitors of bufuralol 1′-hydroxylase activity for dog liver microsomes, CYP2D15 WT2, and the CYP2D15 V1 variant with nearly equal potency. Thus, the dog expresses a CYP2D ortholog possessing enzymatic activities similar to human CYP2D6, but is affected by the inhibitors quinine and quinidine in a manner closer to that of rat CYP2D1.
- ☆
Supported by NIEHS Grant ES03938 and Center Grant ES05022.
- 2
Conducted in partial fulfillment of a requirement for a Ph.D. in the Graduate Program of Biochemistry and Molecular Biology, Rutgers University. Current address: Department of Pharmacology, New York Medical College, Valhalla, NY 10595.
- 3
To whom correspondence should be addressed.