Prokaryotic Expression And Functional Characterization Of CYP6G4 From Housefly(Musca Domestica)

House fly (Musca domestica L.) is a mechanical vector of many diseases which can cause severe consequences for human and animal health. In this study, the protein of CYP6G4 and CPR is heterogeneous expressed successfully in E. coli each. The metabolic activities of CYP6G4 to seven pyrethroids and three coumarins are determined by HPLC using recombinant enzyme system in vitro, which indicates the potential resistance to insecticide. Main results obtained as follows:(1)CYP6G4 and CPR are expressed in the membrane fraction of E. coli with an expected molecule weight of 59×103Da and 76×103Da respectively. The harvest of CYP6G4 protein is 0.37±0.02 nmol/mg protein as estimated by reduced CO difference spectrum, and CPR protein which is 0.47±0.05 nmol/mg protein has a catalytic activity of NADPH-dependent reduction of cytochrome C.(2)CYP6G4/CPR reconstitution system exhibits metabolic activities to tetramethrin, coumarin and imperatorin by HPLC detection. Rates are enhanced by cytochrome b5 with kinetic parameters of km=94.25±8.65μM and Vmax=686.8 peak area/min/pmol P450, in contrast, km=36.37±3.62μM and Vmax=536.5 peak area/min/pmol P450 without cytochrome b5 in reaction. HPLC-Mass spectrometry analysis identifies hydroxyl-tetramethrin as main metabolite.