Overexpression and purification of the three components of the Enterobacter aerogenes AcrA-AcrB-TolC multidrug efflux pump.
The tripartite AcrA-AcrB-TolC system is the major efflux pump of the nosocomial pathogen Enterobacter aerogenes. AcrA is a trimeric periplasmic lipoprotein anchored in the inner membrane, AcrB is an inner membrane transporter and TolC is a trimeric outer membrane channel. In order to reconstitute the AcrA-AcrB-TolC system of E. aerogenes in artificial membranes, we overexpressed and purified the three proteins. The E. aerogenes acrA, acrB and tolC open reading frames were individually inserted in the expression vector pET24a(+), in frame with a sequence coding a C-terminal hexahistidine tag to allow purification by INAC (Immobilized Nickel Affinity Chromatography). The mature AcrA-6His was overproduced in a soluble form in the cytoplasm of Escherichia coli BL21(DE3). AcrA-6His was purified under native conditions in two steps using INAC and gel permeation chromatography. We obtained about 25 mg of 97% pure AcrA-6His per liter of culture. AcrB-6His was solubilized from the membrane fraction of E. coli C43(DE3) in 300 mM NaCl, 5% Triton X-100 and purified in one step by INAC. The AcrB-6His enriched fraction was eluted with 100 mM imidazole. The final yield was 1-2 mg of 95% pure AcrB-6His per liter of culture. The membrane fraction of E. coli BL21(DE3)pLysS containing TolC-6His was first treated with 2% Triton X-100, 30 mM MgCl(2) to solubilize the inner membrane proteins. After ultracentrifugation, the pellet was treated with 5% Triton X-100, 5 mM EDTA to solubilize the outer membrane proteins. Approximately 5 mg of 95% pure TolC-6His trimers per liter of culture was purified by INAC.
Masi
Muriel
M
EA2197, INSERM CJF9606, Enveloppe bactérienne, perméabilité et antibiotiques, Faculté de Médecine-Timone, 27 Bd Jean Moulin, 13385 cedex 05, Marseille, France. mmasi@caramail.com
Pagès
Jean-Marie
JM
Pradel
Elizabeth
E
D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D002846:Chromatography, Affinity; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D021902:Enterobacter aerogenes / Q000378:metabolism*; D013058:Mass Spectrometry; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D010957:Plasmids; D012995:Solubility
D001426:Bacterial Proteins; D017931:DNA Primers; D004364:Pharmaceutical Preparations