结核分枝杆菌DNA糖基化酶TagA的表达、纯化及晶体学研究  

Expression,Purification and Crystallographic Study of DNA Glycosylase TagA of Mycobacterium tuberculosis

作　　者：刘作隆 祁祺[1] 战博闻 耿璐 李继喜[1] LIU Zuolong;QI Qi;ZHAN Bowen;GENG Lu;LI Jixi(School of Life Sciences,Fudan University,Shanghai 200438,China)

机构地区：[1]复旦大学生命科学学院,上海200438

出　　处：《激光生物学报》2021年第4期325-332,共8页Acta Laser Biology Sinica

基　　金：国家重点研发计划重点专项项目(2016YFA0500600);国家自然科学基金面上项目(82071782)。

摘　　要：结核病的病原体结核分枝杆菌是最致命的传染病病原体之一,然而2019年新冠疫情对结核病防治工作造成了沉重打击,因此对结核分枝杆菌进行深入研究有着重要的意义。基因组学的研究表明,结核分枝杆菌来源Ⅰ型3-甲基腺嘌呤DNA糖基化酶TagA(MtbTagA)可识别和切除受损烷基化碱基,以此维持基因组的正常复制、转录和翻译,但其结合底物的类型、催化机制以及结构基础尚不清楚。在大肠杆菌中异源表达MtbTagA,通过镍介质亲和层析、离子交换层析以及凝胶过滤层析的方法获得高纯度的蛋白。动态光散射(DLS)试验发现MtbTagA蛋白以单体分布于溶液中,可与底物次黄嘌呤(Hx)和3-甲基腺嘌呤(3MA)结合,并改变其聚集状态。进一步结晶筛选优化出片状晶体,通过X光衍射获得分辨率为7Å的衍射数据。该研究为进一步探究DNA糖基化酶蛋白的生化性质和结构性质提供了参考。Mycobacterium tuberculosis, the pathogen of tuberculosis, is one of the deadliest pathogens of infectious diseases, however the epidemic situation of COVID-19 in 2019 dealt a heavy blow to the prevention and treatment of tuberculosis. Therefore, the in-depth study of Mycobacterium tuberculosis is of great significance. The study of genomics shows that DNA-3-methyladenine glycosylase I TagA from Mycobacterium tuberculosis(Mtb Tag A) recognizes and removes damaged alkylated bases in order to maintain normal replication, transcription and translation of the genome, whereas the type of binding substrate, catalytic mechanism and structural basis are not clear. In this study, Mtb TagA was heterologous expressed in E. coli, and the high purity protein was obtained by nickel medium affinity chromatography, anion exchange chromatography and gel filtration chromatography. Dynamic light scattering(DLS) experiments showed that Mtb TagA protein was distributed as a monomer in solution and could bind to substrate 3-methyladenine(3 MA) and hypoxanthine(Hx) and change its aggregation state. The flake crystals were selected and optimized by further crystallization, and the diffraction data with a resolution of 7 ? were obtained. It provides a reference for further study on the biochemical properties and structure of DNA glycosylase protein.

关 键 词：DNA糖基化酶 TagA 结核分枝杆菌 动态光散射 结晶 

分 类 号：Q71[生物学—分子生物学]