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.
Highlights
Despite intense efforts put into the structural characterization of membrane proteins, their structures still represent only a small fraction of the total protein structures available
The relative scarcity of membrane protein structures can be attributed to hurdles in overexpression and purification, resulting in failure to obtain the milligram amounts of pure, stable protein needed for protein crystallography [2]
The major facilitator superfamily (MFS) is the largest family of secondary active membrane transporter proteins, accounting for up to 1%–2% of prokaryotic genomes [3], with members that function in a diversity of transport roles in both prokaryotes and eukaryotes; these include nutrient, ion and even antibiotic uptake, and multidrug efflux [4]
Summary
Despite intense efforts put into the structural characterization of membrane proteins, their structures still represent only a small fraction of the total protein structures available (www.pdb.org). 3D structural data exist for only 14 unique members of the MFS; 11 of bacterial origin [5,6,7,8,9,10,11,12,13,14,15], one fungal [16], one plant [17] and one human [18] Information derived from these structures has provided key insights into their mechanism and how they access, transport and release substrates. While one of these transporter structures is of EmrD, a multidrug efflux representative of the superfamily [7], the intermediate resolution of the structural model, combined with the absence of any bound antimicrobial substrate in the model, has prevented a more comprehensive understanding of the complete mechanism of multidrug efflux by this family of proteins. MdtM prepared using the modified protocol produced 3D protein crystals in DDM, but not in UDM or DM, under a number of different conditions
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