Overexpression, refolding, and purification of the histidine-tagged outer membrane efflux protein OprM of Pseudomonas aeruginosa
Overexpression, refolding, and purification of the histidine-tagged outer membrane efflux protein OprM of Pseudomonas aeruginosa
复制标题
DOI：
10.1006/prep.2001.1473
复制
发表时间：
2001-10-01
影响因子：
1.6
通讯作者：
Ducruix, A
中科院分区：
文献类型：
--
作者：
Charbonnier, F;Köhler, T;Ducruix, A
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. (C) 2001 Academic Press.
