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Methods Mol Biol. 2018;1700:113-145. doi: 10.1007/978-1-4939-7454-2_8.

Biochemical Reconstitution and Characterization of Multicomponent Drug Efflux Transporters.

Author information

1
Laboratoire de Biologie Physico-chimique des ProtÕines Membranaires, UMR7099. IBPC, UniversitÕ Paris Diderot, 13, Rue Pierre et Marie Curie, 75005, Paris, France. martin.picard@ibpc.fr.
2
Department of Cell Biology and Biochemistry, Texas Tech University Health Science Center, 3601 4th Street, Lubbock, TX, 79430, USA.
3
Laboratoire de Cristallographie et RMN Biologiques, UMR 8015, CNRS, Université Paris Descartes, Faculté de Pharmacie de Paris, 4 Avenue de l'Observatoire, 75006, Paris, France.
4
Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK, 73019, USA.
5
Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK, 73019, USA. elenaz@ou.edu.

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

PMID:
29177829
DOI:
10.1007/978-1-4939-7454-2_8
[Indexed for MEDLINE]

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