A General Strategy for the Expression of Recombinant Human Cytochrome P450s inEscherichia coliUsing Bacterial Signal Peptides: Expression of CYP3A4, CYP2A6, and CYP2E1

Heterologous expression of unmodified recombinant human cytochrome P450 enzymes (P450s) inEscherichia colihas 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 inE. coli,by making NH2-terminal translational fusions to bacterial leader sequences. Using this approach, we initially tested two leader sequences,pelBandompA,fused to CYP3A4. These were compared with an expression construct producing a conventional NH2-terminally modified CYP3A4 (17α-3A4). Both leader constructs produced spectrally active, functional protein. Furthermore, theompA–3A4 fusion gave higher levels of expression, and a marked improvement in the recovery of active P450 in bacterial membrane fractions, when compared with 17α-3A4. We then tested theompAleader with CYP2A6 and CYP2E1, again comparing with the conventional (17α-) approach. As before, the leader construct produced active enzyme, and, for CYP2E1 at least, gave a higher level of expression than the 17α-construct. TheompAfusion strategy thus appears to represent a significant advance for the expression of P450s inE. coli,circumventing the previous need for individual optimization of P450 sequences for expression.