Soluble and membrane-bound Drosophila melanogaster CYP6G1 expressed in Escherichia coli: Purification, activity, and binding properties toward multiple pesticides
Soluble and membrane-bound Drosophila melanogaster CYP6G1 expressed in Escherichia coli: Purification, activity, and binding properties toward multiple pesticides
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DOI：
10.1016/j.ibmb.2013.02.003
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发表时间：
2013-05-01
影响因子：
3.8
通讯作者：
Oakeshott, John G.
中科院分区：
文献类型：
--
作者：
Cheesman, Matthew J.;Traylor, Matthew. J.;Oakeshott, John G.
细胞色素P450和细胞色素P6G1参与了果蝇对多种杀虫剂的抗性。虽然体内和体外研究已经对该酶的不同功能提供了深入的了解，但由于需要重组酶的来源，对分离的CYP6G1酶的直接研究是不可能的。在本研究中，从黑腹果蝇中分离了Cyp6g1基因，并对其进行了重组，使其在大肠杆菌中获得了异源表达。500mLP450全酶活力约为460nmoL L-1。重组酶主要位于细菌胞浆中。一种使用镍螯合亲和层析的两步纯化方案，然后在羟基磷灰石柱上去除洗涤剂，从可溶性和膜组分中产生基本上均一的酶。重组CYP6G1具有对硝基苯甲醚O-脱烷基活性，但对其他11种典型的P450标记底物无活性。底物诱导结合光谱和抑制对-硝基苯甲醚O-脱烷基的IC50值分别为DDT、吡虫啉、敌敌畏、马拉硫磷、硫丹、敌敌畏、双环胺、氟虫隆和西维因等多种农药的底物诱导结合光谱和IC50值，支持了以往对果蝇体内和体外的研究，表明该酶参与了昆虫对多种农药的抗性。(C)2013爱思唯尔有限公司。保留所有权利。
Cytochrome P450 CYP6G1 has been implicated in the resistance of Drosophila melanogaster to numerous pesticides. While in vivo and in vitro studies have provided insight to the diverse functions of this enzyme, direct studies on the isolated CYP6G1 enzyme have not been possible due to the need for a source of recombinant enzyme. In the current study, the Cyp6g1 gene was isolated from D. melanogaster and re-engineered for heterologous expression in Escherichia coli. Approximately 460 nmol L-1 of P450 holoenzyme were obtained in 500 mL cultures. The recombinant enzyme was located predominantly within the bacterial cytosol. A two-step purification protocol using Ni-chelate affinity chromatography followed by removal of detergent on a hydroxyapatite column produced essentially homogenous enzyme from both soluble and membrane fractions. Recombinant CYP6G1 exhibited p-nitroanisole O-dealkylation activity but was not active against eleven other typical P450 marker substrates. Substrate-induced binding spectra and IC50 values for inhibition of p-nitroanisole O-dealkylation were obtained for a wide selection of pesticides, namely DDT, imidacloprid, chlorfenvinphos, malathion, endosulfan, dieldrin, dicyclanil, lufenuron and carbaryl, supporting previous in vivo and in vitro studies on Drosophila that have suggested that the enzyme is involved in multi-pesticide resistance in insects. (C) 2013 Elsevier Ltd. All rights reserved.