Overexpression, refolding, and purification of the histidine-tagged outer membrane efflux protein OprM of Pseudomonas aeruginosa |
| |
Authors: | Charbonnier F Köhler T Pechère J C Ducruix A |
| |
Institution: | Laboratoire de Cristallographie et RMN Biologiques, Faculté de Pharmacie, Centre National de la Recherche Scientifique, UMR 8015, 4 avenue de l'observatoire, Paris Cedex 06, 75270, France. fcharbo@pharmacie.univ-paris5.fr |
| |
Abstract: | This paper describes the overproduction and purification of the C-terminus polyhistidine-tagged outer membrane protein OprM, which is a part of the MexA-MexB-OprM active efflux system of Pseudomonas aeruginosa. Renaturation of 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 was purified by ion-exchange and nickel-affinity chromatography. The final yield was 6 mg of pure histidine-tagged OprM per liter of E. coli culture. Renaturation was monitored by the effects of heating prior to SDS-PAGE, using a typical and exclusive property of outer membrane proteins. Immunoblotting 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 histidine-tagged OprM protein restored antibiotic susceptibilities to wild-type levels, demonstrating functionality of recombinant OprM. |
| |
Keywords: | |
本文献已被 PubMed 等数据库收录! |
|