title: Soluble and membrane-bound Drosophila melanogaster CYP6G1 expressed in Escherichia coli: purification, activity, and binding properties toward multiple pesticides.
abstract: 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.
authors.affiliation: CSIRO Ecosystem Sciences, GPO Box 1700, Canberra, Australian Capital Territory 2601, Australia. m.cheesman@uq.edu.au
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Cheesman
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Traylor
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Hilton
authors.forename: Katelyn E
authors.initials: KE
authors.lastname: Richards
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Taylor
authors.forename: Phillip J
authors.initials: PJ
authors.lastname: Daborn
authors.forename: Robyn J
authors.initials: RJ
authors.lastname: Russell
authors.forename: Elizabeth M J
authors.initials: EM
authors.lastname: Gillam
authors.forename: John G
authors.initials: JG
authors.lastname: Oakeshott
mesh_terms: D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004331:Drosophila melanogaster / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005786:Gene Expression Regulation; D007305:Insecticide Resistance; D007306:Insecticides / Q000378:metabolism / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013057:Spectrum Analysis
chemical_list: D029721:Drosophila Proteins; D007306:Insecticides; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; C446928:Cyp6g1 protein, Drosophila