Arolas Joan L, García-Castellanos Raquel, Goulas Theodoros, Akiyama Yoshinori, Gomis-Rüth F Xavier
Proteolysis Lab, Department of Structural Biology, Molecular Biology Institute of Barcelona, CSIC, Barcelona Science Park, E-08028 Barcelona, Spain.
Proteolysis Lab, Department of Structural Biology, Molecular Biology Institute of Barcelona, CSIC, Barcelona Science Park, E-08028 Barcelona, Spain.
Protein Expr Purif. 2014 Jul;99:113-8. doi: 10.1016/j.pep.2014.04.008. Epub 2014 Apr 24.
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 His8-tag, extracted from the membranes using octyl-β-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.
关于整合膜（IM）肽酶的催化机制，人们了解甚少。HtpX是一种IM金属肽酶，通过防止错误折叠的蛋白质在膜中积累，在蛋白质质量控制中发挥核心作用。在此，我们报告了从大肠杆菌中重组过表达和纯化催化失活形式的HtpX的过程。我们测试了几种大肠杆菌菌株、表达载体、去污剂和纯化策略，以实现纯的且折叠良好的蛋白质的最大产量。使用连接C末端His8标签的pET衍生载体，HtpX在大肠杆菌BL21(DE3)细胞中成功过表达，使用辛基-β-D-葡萄糖苷从膜中提取，并在此去污剂存在下通过连续三步：钴亲和、阴离子交换和尺寸排阻色谱法纯化至同质。毫克量的HtpX的产生为结构研究铺平了道路，这对于理解这种IM肽酶和相关家族成员的催化机制至关重要。
