A general strategy for the expression of recombinant human cytochrome P450s in Escherichia coli using bacterial signal peptides: expression of CYP3A4, CYP2A6, and CYP2E1

Heterologous expression of unmodified recombinant human cytochrome P450 enzymes (P450s) in Escherichia coli has proved to be extremely difficult, To date, high-level expression has only been achieved after altering the 5'-end of the native cDNA, resulting in amino acid changes within the P450 protein chain, We have devised a strategy whereby unmodified P450s can be expressed to high levels in E. coli, by making NH2-terminal translational fusions to bacterial leader sequences, Using this approach, we initially tested two leader sequences, pelB and ompA, fused to CYP3A4. These were compared with an expression construct producing a conventional NH2-terminally modified CYP3A4 (17 alpha-3A4), Both leader constructs produced spectrally active, functional protein, Furthermore, the ompA-3A4 fusion gave higher levels of expression, and a marked improvement in the recovery of active P450 in bacterial membrane 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 than the 17 alpha-construct. The ompA fusion strategy thus appears to represent a significant advance for the expression of P450s in, coli, circumventing the previous need for individual optimization of P450 sequences for expression. (C) 1997 Academic Press.