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

Arch Biochem Biophys. 1997 Sep 15;345(2):342-54. doi: 10.1006/abbi.1997.0265.


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 (17alpha-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 17alpha-3A4. We then tested the ompA leader with CYP2A6 and CYP2E1, again comparing with the conventional (17alpha-) approach. As before, the leader construct produced active enzyme, and, for CYP2E1 at least, gave a higher level of expression than the 17alpha-construct. The ompA fusion strategy thus appears to represent a significant advance for the expression of P450s in E. coli, circumventing the previous need for individual optimization of P450 sequences for expression.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminolevulinic Acid / metabolism
  • Aryl Hydrocarbon Hydroxylases*
  • Bacterial Outer Membrane Proteins / genetics
  • Cloning, Molecular / methods
  • Cytochrome P-450 CYP2A6
  • Cytochrome P-450 CYP2E1 / biosynthesis*
  • Cytochrome P-450 CYP2E1 / genetics
  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 Enzyme System / biosynthesis*
  • Cytochrome P-450 Enzyme System / genetics
  • Escherichia coli / genetics
  • Genetic Vectors
  • Humans
  • Mixed Function Oxygenases / biosynthesis*
  • Mixed Function Oxygenases / genetics
  • Plasmids
  • Polysaccharide-Lyases / genetics
  • Protein Sorting Signals / genetics
  • Recombinant Proteins / biosynthesis*


  • Bacterial Outer Membrane Proteins
  • Protein Sorting Signals
  • Recombinant Proteins
  • Aminolevulinic Acid
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • Cytochrome P-450 CYP2E1
  • Aryl Hydrocarbon Hydroxylases
  • CYP2A6 protein, human
  • CYP3A protein, human
  • Cytochrome P-450 CYP2A6
  • Cytochrome P-450 CYP3A
  • CYP3A4 protein, human
  • Polysaccharide-Lyases
  • pectin lyase B