The high-level expression in Escherichia coli of the membrane-bound form of human and rat cytochrome b5 and studies on their mechanism of function

Arch Biochem Biophys. 1994 Aug 1;312(2):554-65. doi: 10.1006/abbi.1994.1345.


A T7 expression system is described for the high-level production in Escherichia coli of the membrane-bound form of human and rat cytochrome b5. The cDNAs of b5 have been engineered to contain a coding sequence for a four-member histidine domain at the amino-terminus of the recombinant protein permitting the use of a nickel-chelate affinity column for rapid purification of the detergent-solubilized hemoprotein. Results are presented demonstrating the ability of the purified recombinant b5 proteins to stimulate the rate of oxidation of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone, catalyzed by bovine P450 17A, and to stimulate the 6 beta-hydroxylation of testosterone, catalyzed by human P450 3A4. These P450-catalyzed reactions have been used to compare the properties of different forms of b5. Purified b5 can serve as a "coupling protein" as illustrated by its inhibition of NADPH oxidation, catalyzed by a fusion protein containing the heme domain of P450 3A4 linked to rat NADPH-P450 reductase, and the associated inhibition of hydrogen peroxide formation. Kinetic studies show the formation of a complex of the flavoprotein, NADPH-P450 reductase, with b5 for the rapid transfer of electrons from NADPH.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism
  • Cytochromes b5 / genetics*
  • Cytochromes b5 / metabolism*
  • Escherichia coli / genetics
  • Humans
  • Hydrogen Peroxide / metabolism
  • Membrane Proteins / metabolism
  • Molecular Sequence Data
  • NADP / metabolism
  • NADPH-Ferrihemoprotein Reductase
  • Oxidation-Reduction
  • Oxidoreductases / metabolism
  • Progesterone / analogs & derivatives
  • Progesterone / metabolism
  • Protein Binding
  • Protein Engineering
  • Rats
  • Recombinant Proteins / metabolism
  • Spectrophotometry
  • Testosterone / metabolism


  • Membrane Proteins
  • Recombinant Proteins
  • Testosterone
  • Progesterone
  • NADP
  • Cytochromes b5
  • Cytochrome P-450 Enzyme System
  • Hydrogen Peroxide
  • Oxidoreductases
  • NADPH-Ferrihemoprotein Reductase