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Expression and purification of the recombinant membrane protein YidC: A case study for increased stability and solubility

机译:重组膜蛋白YidC的表达和纯化:提高稳定性和溶解度的案例研究

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摘要

YidC is an inner membrane protein from Escherichia coli and is an essential component in insertion, translocation and assembly of membrane proteins in the membranes. Previous purification attempts resulted in heavy aggregates and precipitated protein at later stages of purification. Here we present a rapid and straightforward stability screening strategy based on gel filtration chromatography, which requires as little as 10 mu g of protein and takes less than 15 min to perform. With this technique, we could rapidly screen several buffers in order to identify an optimum condition that stabilizes purified YidC. After optimization we could obtain several milligrams of purified YidC that could be easily prepared at high concentrations and that was stable for weeks at +4 degrees C. The isolated protein is thus well suited for structural studies. (C) 2008 Elsevier Inc. All rights reserved.
机译:YidC是大肠杆菌的内膜蛋白,是膜蛋白在膜中的插入,转运和组装中的重要组成部分。先前的纯化尝试在较晚的纯化阶段导致沉重的聚集体和沉淀的蛋白质。在这里,我们提出了一种基于凝胶过滤色谱的快速,直接的稳定性筛选策略,该策略仅需10μg蛋白质即可完成,所需时间不到15分钟。使用这种技术,我们可以快速筛选几种缓冲液,以鉴定稳定纯化的YidC的最佳条件。经过优化后,我们可以得到几毫克纯化的YidC,可以很容易地以高浓度制备它们,并且在+4摄氏度下可以稳定数周。因此,分离出的蛋白质非常适合结构研究。 (C)2008 Elsevier Inc.保留所有权利。

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