Purification of a Multidrug Resistance Transporter for Crystallization Studies.
Purification of a Multidrug Resistance Transporter for Crystallization Studies.
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DOI：
10.3390/antibiotics4010113
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发表时间：
2015-03-05
期刊：
影响因子：
--
通讯作者：
Law CJ
中科院分区：
文献类型：
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作者：
Alegre KO;Law CJ
整合膜蛋白的结晶是一个具有挑战性的领域，并且已经投入了大量的努力来优化所需的过表达和纯化步骤，以获得用于结晶学研究的毫克量的纯的、稳定的、单分散的蛋白质样品。我们目前的工作涉及大肠杆菌多药耐药转运蛋白MdtM的结构和功能特性，主要易化超家族（MFS）的成员。在这里，我们提出了一个协议MdtM的分离，以增加重组蛋白的产量毫克的数量，必要的追求使用X-射线晶体学的结构研究。MdtM的纯化通过引入延长的His-标签来增强，随后通过鉴定和随后去除伴侣蛋白污染。对于MdtM的结晶试验，使用尺寸排阻色谱法的洗涤剂筛选确定癸基麦芽糖苷（DM）是最短链的洗涤剂，其将蛋白质保持在稳定的单分散状态。使用悬滴扩散法用市售结晶筛进行的MdtM结晶试验在几种不同条件下产生3D蛋白质晶体。我们认为，这里描述的纯化协议可用于生产高品质的蛋白质的其他多药外排成员的MFS，一个无处不在的，生理和临床上重要的一类膜转运蛋白。
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.