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

Expression of the membrane-bound form of rabbit cytochrome b(5) in Escheric hia coli has been significantly improved through the use of the T7 expressi on vector pLW01 (A. Bridges, L. Gruenke, Y.-T. Chang, I. Vakser, G. Loew, a nd L. Waskell, 1998, J. Biol. Chem. 273, 17036-17049) in conjunction with s train 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 m g/liter of culture and reached peak levels as high as 1100 mg/liter when hi gher antibiotic concentrations were used. Maximal levels were obtained from cultures when expression was induced with 10 mu M 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 deterg ent-solubilized bacterial membranes using anion-exchange chromatography on DEAE-Sepharose followed by size-exclusion chromatography on Superdex-75. Pu rification of cytochrome b(5) from a 500-ml culture yielded 121 mg of prote in which had a specific content of 50 nmol of heme per milligram of protein with an overall recovery of 35%. The final cytochrome bs was free of any d etectable contaminants when analyzed by SDS-PAGE. (C) 2000 Academic Press.