Expression of a catalytically active human cytochrome P-4502E1 in Escherichia coli.
The cDNA coding for human cytochrome P-4502E1 has been expressed in Escherichia coli by placing it under the control of the isopropylthiogalactoside inducible trc promoter. Production of P4502E1 was demonstrated by immunoblots and by catalytic activity with dimethylnitrosamine as substrate. Modifications which favor expression in E. coli were made within the first seven codons. This resulted in approx. a 2- to 2.5-fold increase in P4502E1 isolated from the bacterial membranes as detected by immunoblots and catalytic activity. CO-reduced difference spectra of the modified P4502E1 revealed a peak at 452 nm, which is characteristic of hepatic P4502E1, and a molecular weight of 54 kDa. A partially purified preparation of recombinant P450 protein was active with dimethylnitrosamine, a substrate specific for this isozyme, when reconstituted with purified rat liver NADPH-cytochrome P-450 oxidoreductase. This activity was enhanced in the presence of cytochrome b5 and inhibited by the addition of antibody to the P4502E1 purified from pyrazole-treated rats. E. coli were capable of oxidizing p-nitrophenol when transformed with vector containing the human P4502E1 cDNA but not with vector alone. This in vivo metabolism of p-nitrophenol was increased 2-fold when the modified P4502E1 cDNA was used, which corresponds to the increase in P4502E1 content. Expression of human P4502E1 in E. coli appears to be an attractive system for producing large amounts of this isozyme, and for studies on the toxicological properties and structure-function relationship of the human P4502E1.
Winters
D K
DK
Department of Biochemistry, Mount Sinai School of Medicine, New York, NY 10029.
Cederbaum
A I
AI
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis*
D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System