title: Purification of a Multidrug Resistance Transporter for Crystallization Studies.
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.
authors.affiliation: Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK. k.alegre@qub.ac.uk.
authors.forename: Kamela O
authors.initials: KO
authors.lastname: Alegre
authors.affiliation: Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK. c.law@qub.ac.uk.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Law
keywords: antibiotics; antiporter; chromatography; crystals; membrane protein; transport