Construction of a human cytochrome P450 1A1: rat NADPH-cytochrome P450 reductase fusion protein cDNA and expression in Escherichia coli, purification, and catalytic properties of the enzyme in bacterial cells and after purification

Arch Biochem Biophys. 1996 Jun 1;330(1):48-58. doi: 10.1006/abbi.1996.0224.


A plasmid (pCW) was modified to code for a fusion protein consisting of the complete sequence of human cytochrome P450 (P450) 1A1 (with only the second amino acid changed) in the N-terminal portion connected by a Ser-Thr linker to the portion of rat NADPH-P450 reductase beginning at amino acid 57. This plasmid was used to express the fusion protein in Escherichia coli DH5alpha cells and the protein was purified from detergent-solubilized bacterial membranes using DEAE and 2',5'-ADP agarose chromatography. The purified fusion protein catalyzed benzo[a]pyrene 3-hydroxylation, 7-ethoxyresorufin O-deethylation, and zoxazolamine 6-hydroxylation. Catalytic activity was not increased in the presence of added NADPH-P450 reductase, cytochrome b5, or phospholipid. The fusion protein could also transfer electrons to cytochromes c and b5 but not P450 lA2. The same oxidation products of benzo[a]pyrene were formed with the purified fusion protein and the fusion protein functioning in bacterial cells. The catalytic activity of the human P450 1A1 fusion protein toward several substrates is markedly less than that of a similar fusion protein constructed with rat P450 1A1, in line with the reported differences in catalytic activities of the rat and human P450 1A1 enzymes. The purified fusion protein also oxidized (+)- and (-)-benzo[a]pyrene 7,8-dihydrodiols and eight aryl and heterocyclic amines to genotoxic products, in the absence of added NADPH-P450 reductase. The demonstration of catalytic activities of the human fusion protein within bacterial cells suggests the prospect of utilizing such cellular systems for production of human P450 metabolites.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Carcinogens / metabolism
  • Carcinogens / pharmacology
  • Chromatography, Affinity
  • Chromatography, DEAE-Cellulose
  • Cloning, Molecular
  • Cytochrome P-450 Enzyme System / biosynthesis
  • Cytochrome P-450 Enzyme System / isolation & purification*
  • Cytochrome P-450 Enzyme System / metabolism*
  • DNA Primers
  • Durapatite
  • Escherichia coli
  • Humans
  • Microbial Sensitivity Tests
  • Molecular Sequence Data
  • Molecular Weight
  • Mutagens / metabolism
  • Mutagens / pharmacology
  • NADPH-Ferrihemoprotein Reductase / biosynthesis
  • NADPH-Ferrihemoprotein Reductase / isolation & purification*
  • NADPH-Ferrihemoprotein Reductase / metabolism*
  • Plasmids
  • Polymerase Chain Reaction
  • Prodrugs / metabolism
  • Rats
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Restriction Mapping
  • Salmonella typhimurium / drug effects
  • Substrate Specificity


  • Carcinogens
  • DNA Primers
  • Mutagens
  • Prodrugs
  • Recombinant Fusion Proteins
  • Cytochrome P-450 Enzyme System
  • Durapatite
  • NADPH-Ferrihemoprotein Reductase