Effects of heme precursors on CYP1A2 and POR expression in the baculovirus/ Spodoptera frugiperda system

Huiyuan Lu,Jun Ma,Nian Liu,Shoulin Wang

doi:10.1016/s1674-8301(10)60034-6

Abstract

Objective CYP1A2 and NADPH-CYP450 oxidoreductase (POR) were expressed in the baculovirus/ Spodoptera frugiperda (sf9) system. The aim of this study was to investigate the effects of heme precursors on the expression of CYP1A2 and POR. Methods The heme precursors [δ-Aminolaevulinic Acid (5-ALA), Fe 3+ and hemin] were introduced into the system to evaluate their effects on the expression of CYP1A2, POR and their co-expression. All the proteins were identified using immunoblotting, CO-difference spectroscopy, or cytochrome c assay. Results In the present study, functional CYP1A2 and POR were successfully expressed in the baculovirus/ sf9 system, and both of them showed high activities. Co-addition of 5-ALA and Fe 3+ significantly improved expression of CYP1A2 by about 50% compared with the addition of 5-ALA, Fe 3+ or hemin alone. Either co-addition of 5-ALA and Fe 3+ or addition of 5-ALA or Fe 3+ alone improved the POR expression level 2 fold and its activity 7–10 fold compared with control (no addition). However, unlike CYP1A2, there was no difference between the co-addition and addition of these heme precursors alone. Different ratios of BvCYP1A2 to BvPOR also affected the co-expression of CYP1A2 and POR, with a 3:1 ratio of BvCYP1A2/BvPOR significantly increasing their co-expression. Surprisingly, the addition of 0.1 mM 5-ALA or Fe 3+ alone, but not their co-addition, could significantly improve the CYP1A2 and POR co-expression ( P < 0.05). Conclusion 5-ALA and Fe 3+ increased the expression of CYP1A2 and POR in a baculovirus/ sf9 system, but the pattern of their expression was different between their expression alone and co-expression.

Highlights

Cytochrome P450 (CYP450) monooxgenases, make up 70%-80% of all phaseIxenobiotic-metabolizing enzymes
The recombinant plasmid pFastBacTM1-CYP1A2 digested with KpnI/XbaIgenerated a 1.5 kb fragment and a 4.7 kb fragment, while pFastBacTM1POR digested with BamHI/ KpnIgenerated a 2.0 kb fragment and a 4.7 kb fragment (Fig.1B)
The results showed that AcBacmid-CYP1A2 generated a 1.5 kb fragment, and Ac-BacmidPOR generated a 4.3 kb fragment

Summary

Introduction

Cytochrome P450 (CYP450) monooxgenases, make up 70%-80% of all phaseIxenobiotic-metabolizing enzymes. They can metabolize a large number of endogenous compounds and are involve in many cellular functions[1]. CYP450 oxidoreductase (POR) is a single protein which transfers electrons from NADPH to CYP450s in the endoplasmic reticulum[5,6]. It is usually regarded as the partner of the CYP450s in the metabolism of xenobiotics and endogenous compounds. Spodoptera frugiperda (sf9) insect cells are very important for the efficient expression of CYP450 because they have similar modes for post-transcription and posttranslation of proteins to mammalian cells. The addition of a heme precursor is necessary to compensate the baculovirus/sf system in order to maximize the expression of the functional CYP450s

Methods

Results

Conclusion