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 (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.
Pritchard
M P
MP
Biomedical Research Centre, University of Dundee, Scotland, United Kingdom.
Ossetian
R
R
Li
D N
DN
Henderson
C J
CJ
Burchell
B
B
Wolf
C R
CR
Friedberg
T
T
D000622:Aminolevulinic Acid / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D065637:Cytochrome P-450 CYP2A6; D019392:Cytochrome P-450 CYP2E1 / Q000096:biosynthesis* / Q000235:genetics; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D011133:Polysaccharide-Lyases / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*
D001425:Bacterial Outer Membrane Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000622:Aminolevulinic Acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; C104464:CYP3A protein, human; D065637:Cytochrome P-450 CYP2A6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D011133:Polysaccharide-Lyases; C074124:pectin lyase B