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 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.
Charbonnier
F
F
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
Köhler
T
T
Pechère
J C
JC
Ducruix
A
A
D000345:Affinity Labels; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002462:Cell Membrane; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000648:ultrastructure; D006639:Histidine; D006358:Hot Temperature; D002479:Inclusion Bodies; D026901:Membrane Transport Proteins; D017510:Protein Folding; D020673:Protein Renaturation; D011550:Pseudomonas aeruginosa / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D000345:Affinity Labels; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; C402263:OprM protein, Pseudomonas aeruginosa; D011994:Recombinant Proteins; D006639:Histidine