Bacterial Expression of Membrane-Associated Cytochrome P450s and Their Activity Assay in Nanodiscs.

Abstract

Eukaryotic membrane bound cytochrome P450s are expressed in bacterial systems to produce large yields of catalytically active protein for structure function studies. Recently, there have been several instances of expressing eukaryotic membrane bound CYPs in bacteria after making various modifications to both the N-terminus membrane binding domains of the protein and to noncontiguous F-G membrane binding loop that is also implicated in substrate binding. These modifications have been shown not to disturb the function of the protein of interest. The major factors that have been key to express the membrane bound cytochrome P450s in bacteria have been the following: (a) exon optimization (b) selection of the appropriate vector and host strain, and (c) growth and expression conditions with respect to temperature and speed of shaking the media flask. Herein, we describe methods to express and purify eukaryotic membrane bound cytochrome P450s. We also describe the measurement of the activity of the cytochrome P450 expressed by taking the example of cytochrome P450 2J2, the primary P450 expressed in the human heart and CYP725A4, the primary cytochrome P450 expressed in the first step of taxol synthesis. Additionally, we discuss the pros and cons of the different modifications done in order to express the membrane bound cytochrome P450s.