Bacterial expression, purification, and characterization of human cytochrome P450 3A4 without N-terminal modifications

Abstract

In this communication we reported a bacterial system that over-expressed full-length wild-type (WT) human CYP3A4 in Escherichia coli (E. coli) at a level of 495 nmol/L culture. This level of expression was achieved by cloning the cDNA sequence of CYP3A4 WT to a pLW01-P450 vector and co-expressing it with chaperones GroEL/ES in bacterial C41(DE3) cells. Aided with a C-terminal His₅-tag, the expressed CYP3A4 WT was purified to homogeneity with a specific content of 14.3 ± 2.0 nmole P450/mg protein using a single Ni-Penta agarose column. Like the N-terminal modified form (CYP3A4-NF14), CYP3A4 WT binds substrate testosterone with a typical sigmoidal feature at slightly higher affinity. Functional characterization revealed that CYP3A4 WT exhibited lower testosterone 6β-hydroxylase activities than CYP3A4-NF14 in reconstituted phospholipid systems. In addition, it was found that the 6β-hydroxylase activity of CYP3A4 WT was less dependent on excess cytochrome P450 oxidoreductase (POR), compared with CYP3A4-NF14. These results suggest that the N-terminal membrane anchor of CYP3A4 WT enhances its interactions with POR and marginally increases testosterone binding.