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

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Abstract

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.

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    ☆

    This work was supported financially by the Biotechnology and Biological Sciences Research Council, the Department of Trade and Industry, the consortium of pharmaceutical companies within the LINK scheme—Astra, Glaxo–Wellcome, Janssen Pharmaceutica, Lilly, Novo Nordisk, Parke–Davis, Pfizer, Roche Products, Sanofi–Winthrop, Servier, Smith–Kline Beecham, Wyeth–Ayerst, and Zeneca—and the Imperial Cancer Research Fund (C.H.).

    2

    To whom correspondence should be addressed at the Biomedical Research Centre, University of Dundee, Level 5, Ninewells Hospital and Medical School, Dundee, Scotland, United Kingdom DD1 9SY. Fax: (+44) (0) 1382 669993. E-mail: [email protected].

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