Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus
Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus
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DOI：
10.1107/s1744309106007779
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发表时间：
2006-04-01
影响因子：
0.9
通讯作者：
Ito, K
中科院分区：
文献类型：
--
作者：
Tsukazaki, T;Mori, H;Ito, K
嗜热热菌有一种多通路膜蛋白TSecDF，是大肠杆菌SecD和SecF的单链同源物，它们形成了一个跨位点相关复合体，这是有效的蛋白前体易位和膜蛋白整合所必需的。本文报道了TSecDF的克隆、在大肠杆菌中的表达、纯化和结晶。过量生产的TSecDF用十二烷基麦芽糖溶解，在聚乙二醇存在下进行色谱纯化和蒸汽扩散结晶。在x射线照射下，晶体的最大分辨率为4.2埃，表明它们属于P4(3)2(1)2空间群。通过微搅拌、激光照射和脱水等方法提高晶体的衍射质量，最终获得了3.74埃分辨率的完整数据集，并初步成功进行了单波长异常色散分析。这些结果为确定蛋白质转运机制中这一重要膜组分的三维结构提供了必要的信息。
Thermus thermophilus has a multi-path membrane protein, TSecDF, as a single-chain homologue of Escherichia coli SecD and SecF, which form a translocon-associated complex required for efficient preprotein translocation and membrane-protein integration. Here, the cloning, expression in E. coli, purification and crystallization of TSecDF are reported. Overproduced TSecDF was solubilized with dodecylmaltoside, chromatographically purified and crystallized by vapour diffusion in the presence of polyethylene glycol. The crystals yielded a maximum resolution of 4.2 angstrom upon X-ray irradiation, revealing that they belonged to space group P4(3)2(1)2. Attempts were made to improve the diffraction quality of the crystals by combinations of micro-stirring, laser-light irradiation and dehydration, which led to the eventual collection of complete data sets at 3.74 angstrom resolution and preliminary success in the single-wavelength anomalous dispersion analysis. These results provide information that is essential for the determination of the three-dimensional structure of this important membrane component of the protein-translocation machinery.