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Antibiotics 2015, 4(1), 113-135; doi:10.3390/antibiotics4010113

Purification of a Multidrug Resistance Transporter for Crystallization Studies

Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK
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Author to whom correspondence should be addressed.
Academic Editor: Helen I. Zgurskaya
Received: 22 January 2015 / Revised: 23 February 2015 / Accepted: 25 February 2015 / Published: 5 March 2015
(This article belongs to the Special Issue Multi-drug Efflux and Drug Permeation)
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Abstract

Crystallization of integral membrane proteins is a challenging field and much effort has been invested in optimizing the overexpression and purification steps needed to obtain milligram amounts of pure, stable, monodisperse protein sample for crystallography studies. Our current work involves the structural and functional characterization of the Escherichia coli multidrug resistance transporter MdtM, a member of the major facilitator superfamily (MFS). Here we present a protocol for isolation of MdtM to increase yields of recombinant protein to the milligram quantities necessary for pursuit of structural studies using X-ray crystallography. Purification of MdtM was enhanced by introduction of an elongated His-tag, followed by identification and subsequent removal of chaperonin contamination. For crystallization trials of MdtM, detergent screening using size exclusion chromatography determined that decylmaltoside (DM) was the shortest-chain detergent that maintained the protein in a stable, monodispersed state. Crystallization trials of MdtM performed using the hanging-drop diffusion method with commercially available crystallization screens yielded 3D protein crystals under several different conditions. We contend that the purification protocol described here may be employed for production of high-quality protein of other multidrug efflux members of the MFS, a ubiquitous, physiologically and clinically important class of membrane transporters. View Full-Text
Keywords: antiporter; antibiotics; crystals; membrane protein; transport; chromatography antiporter; antibiotics; crystals; membrane protein; transport; chromatography
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Alegre, K.O.; Law, C.J. Purification of a Multidrug Resistance Transporter for Crystallization Studies. Antibiotics 2015, 4, 113-135.

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