title: Overexpression, Membrane Preparation, and Purification of a Typical Multidrug ABC Transporter BmrA.
abstract: The production and purification is normally the first step in any biophysical or biochemical study of a new target protein. For membrane proteins, due to their generally low expression levels and hydrophobic properties this is often a major hurdle. Some multidrug transporters are members of one of the largest families of membrane proteins, the ABC ("ATP-binding cassette"), and are responsible for the uptake and export of a wide variety of molecules. This can lead to resistance when those molecules are antibiotics or chemotherapy drugs. To better understand their role in multidrug resistance pure and active protein is required. Here we outline a protocol to produce a highly pure and functionally active multidrug transporter BmrA that is suitable for use in biophysical and biochemical studies. We show that BmrA can be heterologously overexpressed in huge amount in E. coli and extracted from the membrane in a functionally active form.
authors.affiliation: UMR5086 CNRS/Université Claude Bernard Lyon 1, MMSB-IBCP, 7 passage du vercors, 69367, Lyon Cedex 7, France.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Wiseman
authors.affiliation: UMR5086 CNRS/Université Claude Bernard Lyon 1, MMSB-IBCP, 7 passage du vercors, 69367, Lyon Cedex 7, France. jean-michel.jault@ibcp.fr.
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Jault
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D018432:Drug Resistance, Multiple; D015870:Gene Expression; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D011994:Recombinant Proteins
keywords: ATP-binding cassette; Membrane protein; Multidrug resistance; Purification; Transporter