High-level expression in Escherichia coli and purification of the membrane-bound form of cytochrome b(5)

Protein Expr Purif. 2000 Jun;19(1):173-8. doi: 10.1006/prep.2000.1228.


Expression of the membrane-bound form of rabbit cytochrome b(5) in Escherichia coli has been significantly improved through the use of the T7 expression vector pLW01 (A. Bridges, L. Gruenke, Y.-T. Chang, I. Vakser, G. Loew, and L. Waskell, 1998, J. Biol. Chem. 273, 17036-17049) in conjunction with strain C41(DE3) (B. Miroux and J. Walker, 1996, J. Mol. Biol. 260, 289-298). Cell cultures expressing the cytochrome b(5) contained an average of 820 mg/liter of culture and reached peak levels as high as 1100 mg/liter when higher antibiotic concentrations were used. Maximal levels were obtained from cultures when expression was induced with 10 microM IPTG. Approximately 90% of the cytochrome b(5) was expressed as apoprotein which was reconstituted by addition of exogenous heme. The cytochrome b(5) was purified from detergent-solubilized bacterial membranes using anion-exchange chromatography on DEAE-Sepharose followed by size-exclusion chromatography on Superdex-75. Purification of cytochrome b(5) from a 500-ml culture yielded 121 mg of protein which had a specific content of 50 nmol of heme per milligram of protein with an overall recovery of 35%. The final cytochrome b(5) was free of any detectable contaminants when analyzed by SDS-PAGE.

Publication types

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

MeSH terms

  • Animals
  • Chromatography, Agarose
  • Cytochromes b5 / genetics
  • Cytochromes b5 / isolation & purification*
  • Cytochromes b5 / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Intracellular Membranes / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / isolation & purification*
  • Membrane Proteins / metabolism
  • Rabbits
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism


  • Membrane Proteins
  • Recombinant Proteins
  • Cytochromes b5