Overexpression, refolding, and purification of the histidine-tagged outer membrane efflux protein OprM of Pseudomonas aeruginosa

This paper describes the overproduction and purification of the C-terminus polyhistidine-tagged outer membrane protein OprM, which is a part of the Me xA-MexB-OprM active efflux system of Pseudomonas aeruginosa. Renaturation o f the protein from inclusion bodies of Escherichia coli was achieved using guanidine-HCl as denaturing agent and n-octylpolyoxyethylene (C8POE) and n- octyltetraoxyethylene (C8E4) as nonionic detergents. The refolded protein w as purified by ion-exchange and nickel-affinity chromatography. The final y ield was 6 mg of pure histidine-tagged OprM per liter of E. coli culture. R enaturation was monitored by the effects of heating prior to SDS-PAGE, usin g a typical and exclusive property of outer membrane proteins. Immunoblotti ng revealed that the recombinant protein is addressed to the outer membrane of E. coli, after maturation by excision of its N-terminal signal sequence . Complementation of an oprM deletion mutant with the plasmid encoded histi dine-tagged OprM protein restored antibiotic susceptibilities to wild-type levels, demonstrating functionality of recombinant OprM. (C) 2001 Academic Press.