How to successfully express insect microsomal P450 using a baculovirus system

Cytochrome P450 enzymes (P450s) play a key role in the metabolism of a diverse array of exogenous compounds. In insects they are often implicated in the phase I metabolism of many insecticides, which can lead to the emergence of resistance. Studies of such P450s have frequently employed heterologous expression in insect cell lines for in vitro functional characterisation. However, identifying optimal conditions for insect P450s expression is challenging. Here, we address this challenge by developing an improved protocol for insect P450s expression in insect cell lines employing two new concepts: CPR Balance and P450 Point. By adjusting the viral titer of CPR/P450, stable expression of microsomal P450 in the cellular system was achieved; By establishing time gradients for viral infection of H5 cells, the optimal time point for P450 harvest was determined. These two improvements enhanced the operability of P450 in vitro expression and increased the content of active P450. Using three P450s from agricultural pest silverleaf whitefly, Bemisia tabaci, we demonstrated that the protocol is efficient and stable in expressing active P450 proteins in vitro and is applicable to multiple CYPs from different subfamilies. Additionally, a detailed step-by-step protocol is included in the Appendix. Our work provides an improved reference for eukaryotic expression of P450s from insects and will inform future in vitro expression work using the bac-to-bac system.