Development of bacterial expression system with high yield of CYP3A7, a human fetus-specific form of cytochrome P450

In an E. coli expression system for human cytochrome P450 3A7 (CYP3A7), hol o-CYP3A7 was not expressed as judged by GO-difference spectra, although apo -GYP3A7 was clearly detected by Western blot analysis. Unlike CYP3A7, CYP3A 4 was expressed efficiently as a hemoprotein in E. coli transformed with a CYP3A4 expression plasmid. To achieve the high yield of the holo-CYP3A7 in E. coli, we examined a causal residue(s) preventing the expression of the h olo-CYP3A7 using the chimeric gene of CYP3A4 with CYP3A7. It was found that the region between residues 405 and 503 of CYP3A7 was responsible for the prevention of the holo-CYP3A7 expression in E. coli. Among amino acids exam ined, substitution of Thr at position 485 in CYP3A7 with Pro, which is at t he corresponding position of CYP3A4, resulted in an increase in the amount of holo-CYP3A7. The Thr residue was adjacent to the heme-binding region of CYP3A7. Thus, it appeared that the incorporation of heme into CYP3A7 was po ssibly affected by this particular amino acid residue. Moreover, holo-CYP3A 7 was expressed efficiently when CYP3A7 was co-expressed with molecular cha perone GroEL, known to assist the correct folding of unfolded proteins. Deh ydroepiandrosterone 16 alpha-hydroxylation was catalyzed by CYP3A7 expresse d in the presence of GroEL. (C) 2000 Academic Press.