Abstract
Efflux pumps are the major determinants in bacterial multidrug resistance. In Gram-negative bacteria, efflux transporters are organized as macromolecular tripartite machineries that span the two-membrane cell envelope of the bacterium. Biochemical data on purified proteins are essential to draw a mechanistic picture of this highly dynamical, multicomponent, efflux system. We describe protocols for the reconstitution and the in vitro study of transporters belonging to RND and ABC superfamilies: the AcrAB-TolC and MacAB-TolC efflux systems from Escherichia coli and the MexAB-OprM efflux pump from Pseudomonas aeruginosa.
Keywords:
Membrane protein purification; Proteoliposomes; Transport kinetics.
MeSH terms
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ATP-Binding Cassette Transporters / metabolism
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Bacterial Outer Membrane Proteins / metabolism
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Bacterial Proteins / isolation & purification
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Bacterial Proteins / metabolism
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Carrier Proteins / metabolism
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Drug Resistance, Multiple, Bacterial
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Escherichia coli / metabolism*
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Escherichia coli Proteins / metabolism
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Membrane Transport Proteins / isolation & purification*
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Membrane Transport Proteins / metabolism
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Pseudomonas aeruginosa / metabolism*
Substances
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ATP-Binding Cassette Transporters
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AcrAB-TolC protein, E coli
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Bacterial Outer Membrane Proteins
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Bacterial Proteins
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Carrier Proteins
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Escherichia coli Proteins
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MacAB protein, E coli
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Membrane Transport Proteins
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MexA protein, Pseudomonas aeruginosa
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MexB protein, Pseudomonas aeruginosa
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OprM protein, Pseudomonas aeruginosa