title: Optimization of heme precursors for the expression of human cytochrome P450 2A13 and its co-expression with oxidoreductase in baculovirus/sf9 system.
abstract: Human cytochrome P450 2A13 (CYP2A13), mainly expressed in respiratory tract, is active towards numerous toxicants. To establish the metabolism in vitro, we expressed CYP2A13 and NADPH-CYP450 oxidoreductase (POR) in a baculovirus/sf9 system. Due to the deficiency of sf9 cells in heme incorporation, we investigated the effects of different heme precursors on the expression of CYP2A13, POR and their co-expression. The present results showed that both CYP2A13 and POR were presented the highest expression levels or activity with 0.2 mM δ-aminolaevulinic acid (5-ALA), 0.02 mM Fe(3+) and 0.5-1.0 μg/ml hemin. The combination of 0.2 mM 5-ALA and 0.02 mM Fe(3+) significantly improved CYP2A13 expression and content compared with heme precursors alone, so was POR activity. A multiplicity of infection (MOI) value of 5 pfu/cell for CYP2A13 baculovirus particles induced very high CYP2A13 expression. When co-infected with different POR MOI values, a viral ratio of 5 : 2 was associated with the highest CYP2A13 activity, whereas POR activity dose dependently increased with POR MOI. Furthermore, the expressed CYP2A13 in the optimized conduction could eliminate its substrate aflatoxin B1 at a significantly higher than those in other condition (P < 0.01). Our results provide an efficient approach for expressing functionally characterized, highly active and homogeneous CYP2A13 proteins.
authors: State Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, PR China.
authors: Hui-Yuan
authors: HY
authors: Lu
authors: Liang-Lin
authors: LL
authors: Qiu
authors: Xue-Jiao
authors: XJ
authors: Yang
authors: Xiao-Ming
authors: XM
authors: Zhang
authors: Zhan
authors: Z
authors: Zhang
authors: Shou-Lin
authors: SL
authors: Wang
mesh_terms: D016604:Aflatoxin B1 / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics*; D006418:Heme / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera; D014162:Transfection
chemical_list: D006418:Heme; D016604:Aflatoxin B1; D010088:Oxidoreductases; D001189:Aryl Hydrocarbon Hydroxylases; C414206:CYP2A13 protein, human; D009251:NADPH-Ferrihemoprotein Reductase
keywords: NADPH-CYP450 oxidoreductase; baculovirus; heme precursors; heterologous expression; human cytochrome P450 2A13; sf9 system