Expression, Purification and Characterization of the Escherichia coli Integral Membrane Protein YajC
Expression, Purification and Characterization of the Escherichia coli Integral Membrane Protein YajC
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DOI：
10.2174/092986611795222713
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发表时间：
2011-06-01
影响因子：
1.6
通讯作者：
Wei, Yinan
中科院分区：
文献类型：
--
作者：
Fang, Jun;Wei, Yinan
大肠杆菌YajC是一种具有单个跨膜螺旋的小型整合膜蛋白。yajC基因是secD操纵子的一部分，该蛋白在SecDF - YajC复合物中被鉴定出来。然而，YajC的确切功能仍然是个谜。虽然其功能通常在SecDF - YajC复合物的背景下被讨论，但研究表明，对于那些功能而言，SecD/F而非YajC是必不可少的。最近，YajC被确定为与AcrB共结晶的神秘蛋白。为了进一步研究YajC的结构，我们在洗涤剂溶解状态下表达并纯化了该蛋白。该蛋白呈现出包含混合的α/β二级结构的折叠结构，这与结构预测相符。通过使用信号半胱氨酸突变和巯基特异性探针，我们发现YajC的C末端在细胞质中，而YajC的N末端埋在膜内。此外，我们表达并纯化了YajC的一个截短片段，它对应于C末端细胞质结构域（YajC(CT)）。YajC(CT)形成了一个富含β链的紧凑结构，并以三聚体形式存在。
Escherichia coli YajC is a small integral membrane protein with a single transmembrane helix. The gene yajC is part of the secD operon and the protein is identified in the SecDF-YajC complex. However, the exact function of YajC remains a mystery. While its function is usually discussed in the context of the SecDF-YajC complex, studies have shown that SecD/F, rather than YajC, are essential for those functions. Recently YajC is identified as the mysterious protein that co-crystallized with AcrB. To further investigate the structure of YajC, we expressed and purified the protein in a detergent solubilized state. The protein assumed a folded structure containing mixed alpha/beta secondary structures, consistent with the structural prediction. Using signal Cys mutations and thiol-specific probes, we found the C-terminus of YajC was cytoplasmic, while the N-terminus of YajC was buried in the membrane. In addition, we expressed and purified a truncated fragment of YajC that corresponded to the C-terminal cytoplasmic domain (YajC(CT)). YajC(CT) formed a compact structure rich in beta-strands and existed as a trimer.