Chen Hanyu, Wang Huilin, Sun Tao, Tian Shuangliang, Lin Donghai, Guo Chenyun
Department of Chemical Biology, College of Chemistry and Chemical Engineering, The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen 361005, China.
Department of Chemical Biology, College of Chemistry and Chemical Engineering, The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen 361005, China.
Protein Expr Purif. 2016 Jul;123:51-9. doi: 10.1016/j.pep.2016.03.009. Epub 2016 Mar 25.
The ESX-1 secretion system of Mycobacterium tuberculosis is required for the virulence of tubercle bacillus. EspI, the ESX-1 secretion-associated protein in Mycobacterium tuberculosis (MtEspI), is involved in repressing the activity of ESX-1-mediated secretion when the cellular ATP level is low. The ATP binding domain of MtEspI plays a crucial role in this regulatory process. However, further structural and functional studies of MtEspI are hindered due to the bottleneck of obtaining stable and pure recombinant protein. In this study, we systematically analyzed the structure and function of MtEspI using bioinformatics tools and tried various expression constructs to recombinantly express full-length and truncated MtEspI ATP binding domain. Finally, we prepared pure and stable MtEspI ATP binding domain, MtEspI415-493, in Escherichia coli by fusion expression and purification with dual tag, Glutathione S-transferase (GST) tag and (His)6 tag. (31)P NMR titration assay indicated that MtEspI415-493 possessed a moderate affinity (∼μM) for ATP and the residue K425 was located at the binding site. The protocol described here may provide a train of thought for recombinant preparation of other ESX-1 secretion-associated proteins.
结核分枝杆菌的ESX-1分泌系统是结核杆菌毒力所必需的。EspI是结核分枝杆菌中与ESX-1分泌相关的蛋白（MtEspI），当细胞ATP水平较低时，它参与抑制ESX-1介导的分泌活性。MtEspI的ATP结合结构域在这一调节过程中起关键作用。然而，由于获得稳定和纯的重组蛋白存在瓶颈，对MtEspI的进一步结构和功能研究受到阻碍。在本研究中，我们使用生物信息学工具系统地分析了MtEspI的结构和功能，并尝试了各种表达构建体来重组表达全长和截短的MtEspI ATP结合结构域。最后，我们通过谷胱甘肽S-转移酶（GST）标签和（His）6标签的双标签融合表达和纯化，在大肠杆菌中制备了纯的和稳定的MtEspI ATP结合结构域MtEspI415-493。（31）P NMR滴定分析表明，MtEspI415-493对ATP具有中等亲和力（~μM），且残基K425位于结合位点。本文所述方案可能为其他ESX-1分泌相关蛋白的重组制备提供思路。
