Recombinant protein and insecticide metabolism
The CYP6P4-236M variant, and its redox partner cytochrome P450 reductase
gene (CPR), were expressed in E. coli as per standard
protocols [32]
(Supplementary materials Appendix 2). Initial efforts to generate
recombinant CYP6AA1 in an E. coli system with optimised
codon usage failed, and we therefore used an Sf9-baculovirus-based
expression system. Since P450 catalytic activity is dependent on
electrons supplied by NADPH via CPR, insecticide metabolism was assayed
with cell pellets of CYP6P4/CPR or CYP6AA1/CPR in the presence or
absence of NADPH. The depletion of the substrate and the appearance of
metabolites were monitored by reverse-phase HPLC (Supplementary
materials Appendix 3).