A GENERAL STRATEGY FOR THE EXPRESSION OF RECOMBINANT HUMAN CYTOCHROMEP450S IN ESCHERICHIA-COLI USING BACTERIAL SIGNAL PEPTIDES - EXPRESSION OF CYP3A4, CYP2A6, AND CYP2E1

Heterologous expression of unmodified recombinant human cytochrome P45 0 enzymes (P450s) in Escherichia coli has proved to be extremely diffi cult, To date, high-level expression has only been achieved after alte ring the 5'-end of the native cDNA, resulting in amino acid changes wi thin the P450 protein chain, We have devised a strategy whereby unmodi fied P450s can be expressed to high levels in E. coli, by making NH2-t erminal translational fusions to bacterial leader sequences, Using thi s approach, we initially tested two leader sequences, pelB and ompA, f used to CYP3A4. These were compared with an expression construct produ cing a conventional NH2-terminally modified CYP3A4 (17 alpha-3A4), Bot h leader constructs produced spectrally active, functional protein, Fu rthermore, the ompA-3A4 fusion gave higher levels of expression, and a marked improvement in the recovery of active P450 in bacterial membra ne fractions, when compared with 17 alpha-3A4, We then tested the ompA leader with CYP2A6 and CYP2E1, again comparing with the conventional (17 alpha-) approach, As before, the leader construct produced active enzyme, and, for CYP2E1 at least, gave a higher level of expression th an the 17 alpha-construct. The ompA fusion strategy thus appears to re present a significant advance for the expression of P450s in, coli, ci rcumventing the previous need for individual optimization of P450 sequ ences for expression. (C) 1997 Academic Press.