Expression and purification of integral membrane metallopeptidase HtpX
Expression and purification of integral membrane metallopeptidase HtpX
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DOI：
10.1016/j.pep.2014.04.008
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发表时间：
2014-07-01
影响因子：
1.6
通讯作者：
Xavier Gomis-Rueth, F.
中科院分区：
文献类型：
--
作者：
Arolas, Joan L.;Garcia-Castellanos, Raquel;Xavier Gomis-Rueth, F.
关于整合膜（IM）肽酶的催化机制知之甚少。HtpX是一种IM金属肽酶，通过防止膜中错误折叠蛋白的积累，在蛋白质质量控制中发挥核心作用。在这里，我们报告的重组过表达和纯化的催化消融形式的HtpX从大肠杆菌。几个E.大肠杆菌菌株、表达载体、去污剂和纯化策略进行了测试，以获得纯的和良好折叠的蛋白质的最大产量。在E. coli BL 21（DE 3）细胞，使用连接C-末端His（8）-标签的pET衍生载体，使用辛基-β-D-葡糖苷从膜中提取，并在存在该去污剂的情况下在三个连续步骤中纯化至均匀：钴亲和、阴离子交换和尺寸排阻色谱。以毫克量生产HtpX为结构研究铺平了道路，这对于了解这种IM肽酶和相关家族成员的催化机制至关重要。(C)2014爱思唯尔公司All rights reserved.
Little is known about the catalytic mechanism of integral membrane (IM) peptidases. HtpX is an IM metallopeptidase that plays a central role in protein quality control by preventing the accumulation of misfolded proteins in the membrane. Here we report the recombinant overexpression and purification of a catalytically ablated form of HtpX from Escherichia coli. Several E. coli strains, expression vectors, detergents, and purification strategies were tested to achieve maximum yields of pure and well-folded protein. HtpX was successfully overexpressed in E. coli BL21(DE3) cells using a pET-derived vector attaching a C-terminal His(8)-tag, extracted from the membranes using octyl-beta-D-glucoside, and purified to homogeneity in the presence of this detergent in three consecutive steps: cobalt-affinity, anion-exchange, and size-exclusion chromatography. The production of HtpX in milligram amounts paves the way for structural studies, which will be essential to understand the catalytic mechanism of this IM peptidase and related family members. (C) 2014 Elsevier Inc. All rights reserved.