ISSN 1003-8280 CN 10-1522/R 中国疾病预防控制中心 主办
Chinese Journal of Vector Biology and Control >
Functional expression of NADPH-cytochrome P450 reductase of the house fly (Musca domestica) in Escherichia coli
Received date: 2015-11-12
Online published: 2016-04-20
Supported by
Supported by the Municipal Natural Science Foundation of Beijing of China (No. 5142014)
Objective House fly (Musca domestica) is a mechanical vector of many diseases which can cause severe consequences for human and animal health. Cytochrome P450 monooxygenases play an important role in resistance to many insecticides in the house fly. Establishment of functional expression system of NADPH-cytochrome P450 reductase will be helpful for the characterization of P450 mediated insecticide resistance. Methods In this study, we cloned the open reading frame of MdCPR gene into the expression vector pB508. The constructed plasmid MdCPR-pB508 was transformed into Escherichia coli (DH5α). The production of MdCPR protein in E. coli was induced by isopropyl β-thiogalactoside (IPTG). Results MdCPR was expressed in E. coli with an expected molecule weight of around 76×103. The E. coli-produced MdCPR protein was mainly present in the membrane fraction, showing the catalytic activity of NADPH-dependent reduction of cytochrome C. Conclusion The successful functional expression of MdCPR can facilitate further investigation of the role of various cytochrome P450s in insecticide resistance in house flies.
FENG Jing, TIAN Kai, HOU Xin, QIU Xing-hui . Functional expression of NADPH-cytochrome P450 reductase of the house fly (Musca domestica) in Escherichia coli[J]. Chinese Journal of Vector Biology and Control, 2016 , 27(2) : 121 -123 . DOI: 10.11853/j.issn.1003.8280.2016.02.007
[1] Scott JG, Liu N, Kristensen M, et al. A case for sequencing the genome of Musca domestica (Diptera:Muscidae)[J]. J Med Entomol,2009,46(2):175-182.
[2] Wang QM, Li M, Pan J, et al. Diversity and frequencies of genetic mutations involved in insecticide resistance in field populations of the house fly (Musca domestica L.) from China[J]. Pestic Biochem Physiol,2012,102(2):153-159.
[3] Feyereisen R. Insect CYP genes and P450 enzymes[M]//Gilbert L (Ed.). Insect Molecular Biology and Biochemistry. London:Elsevier B.V.,2012:236-316.
[4] Li XC, Berenbaum MR, Schuler MA. Plant allelochemicals differentially regulate Helicoverpa zea cytochrome P450 genes[J]. Insect Mol Biol,2002,11(4):343-351.
[5] Vermilion JL,Ballou DP,Massey V,et al. Separate roles for FMN and FAD in catalysis by liver microsomal NADPH-cytochrome P-450 reductase[J]. J Biol Chem,1981,256(1):266-277.
[6] Cheng J, Wan DF, Gu JR,et al. Establishment of a yeast system that stably expresses human cytochrome P450 reductase:application for the study of drug metabolism of cytochrome P450s in vitro[J]. Protein Expr Purif,2006,47(2):467-476.
[7] Horecker BL, Heppel LA. The reduction of cytochrome C by xanthine oxidase[J]. J Biol Chem,1949,178(2):683-690.
[8] Zhu F, Sams S, Moural T, et al. RNA interference of NADPH-cytochrome P450 reductase results in reduced insecticide resistance in the bed bug, Cimex lectularius[J]. PLoS One,2012,7(2):e31037.
[9] Liu D, Zhou XJ, Li M, et al. Characterization of NADPH-cytochrome P450 reductase gene from the cotton bollworm,Helicoverpa armigera[J]. Gene,2014,545(2):262-270.
[10] Koener JF, Cariño FA, Feyereisen R. The cDNA and deduced protein sequence of house fly NADPH-cytochrome P450 reductase[J]. Insect Biochem Mol Biol,1993,23(4):439-447.
[11] Andersen JF,Utermohlen JG,Feyereisen R. Expression of house fly CYP6A1 and NADPH-cytochrome P450 reductase in Escherichia coli and reconstitution of an insecticide-metabolizing P450 system[J]. Biochemistry,1994,33(8):2171-2177.
[12] Yang JN, An JF, Li M, et al. Characterization of chicken cytochrome P4501A4 and 1A5:inter-paralog comparisons of substrate preference and inhibitor selectivity[J]. Comp Biochem Physiol C Toxicol Pharmacol,2013,157(4):337-343.
[13] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem,1976,72(1/2):248-254.
[14] Guengerich F, Martin MV, Sohl CD, et al. Measurement of cytochrome P450 and NADPH-cytochrome P450 reductase[J]. Nat Protoc,2009,4(9):1245-1251.
[15] Riddick DS, Ding XX, Wolf CR, et al. NADPH-Cytochrome P450 oxidoreductase:roles in physiology, pharmacology, and toxicology[J]. Drug Metab Dispos,2013, 41(1):12-23.
[16] Sarapusit S, Pethuan S, Rongnoparut P. Mosquito NADPH-cytochrome P450 oxidoreductase:kinetics and role of phenylalanine amino acid substitutions at leu86 and leu219 in CYP6AA3-mediated deltamethrin metabolism[J]. Arch Insect Biochem Physiol,2010,73(4):232-244.
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