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结核分枝杆菌Pup-蛋白酶体系统蛋白的表达纯化研究
The Research on Mycobacterium Tuberculosis Prokaryotic Ubiquitin-like Protein (Pup)-Proteasome System’s Protein Expression and Purification
【作者】 汪春军；
【作者基本信息】 北京林业大学 ， 生物物理学， 2011， 硕士
【摘要】 结核分枝杆菌(Mycobacterium tuberculosis Mtb)中类泛素蛋白Pup (Prokaryotic ubiquitin-like protein)-蛋白酶体系统是胞内蛋白降解重要机制,在去酰胺酶Dop(Deamidase of Pup)和连接酶PafA(Proteasome accessory factor A)、ATP酶Mpa(Mycobacterial proteasome ATPase)等辅助因子的作用下,Pup可以共价标记多种功能蛋白,并介导被标记蛋白通过蛋白酶体降解,靶蛋白涉及物质中间代谢、信号通路、毒性和抗毒性因子、细胞壁和细胞膜组份等多个方面,并且与结核分枝杆菌的致病性相关,被认为是新的结核病治疗药物靶点,因此本领域研究具有深远的意义。本文以Mtb为研究对象,优化并克隆出pup基因,同时克隆出dop、pafA和底物蛋白fabD基因,将这些基因加上用于纯化的标签构入表达载体,得到pGEX-2T-his6-pup, pET21cc-dop-his6, pET21cc-fabD-his6, pET21cc-pafA-his6, pBAD-pafA-his6重组表达质粒。通过重组蛋白技术,在大肠杆菌BL21中诱导表达出GST-His6-Pup、Dop-His6、FabD-His6和PafA-His6蛋白,并分别通过常规和变复性等方法纯化出这些蛋白,用蛋白免疫印迹等技术鉴定了这些蛋白,为建立Pup-蛋白酶体体外蛋白降解系统提供了蛋白。合成Pup的N端十肽免疫兔,得到能识别Pup蛋白且有较高效价的Pup多克隆抗体。对Mtb的非致病同源菌耻垢分枝杆菌(Mycobacterium smegmatis, Msm)的dop基因敲除进行了初步探索,为进一步研究Pup-蛋白酶体系统参与的细胞调控功能做准备。
【Abstract】 Prokaryotic ubiquitin-like protein (Pup)-proteasome system, the essential mechanism for prokaryotic cellular protein degradation in Mycobacterium tuberculosis(Mtb).With the help of accessory factors, Dop(Deamidase of Pup), PafA(Proteasome accessory factor) and Mpa(Myco-bacterial proteasome ATPase), Pup covalently linked to the Lysε-NH2 in the target proteins and mediated the target protein degradation through the proteasome. The discovery of Pup-proteas-ome system revealed a novel mechanism of prokaryotic protein degradation, which is involved essential physiological function including the intermediary metabolism, information pathway, detoxification/virulence, cell wall and cell membrane formation and so on. Disruption of Pup-proteasome system can suppress the pathogenicity of Mtb. Therefore it is regarded as the new therapeutic target for tuberculosis. In the present paper, the progress in the study on mechanism and function of Pup-proteasome system is reviewed.In this study, the genes of pup, dop, pafA and fabD are cloned from Mtb with gst or his6 tag for purification. With recombinant plasmids pGEX-2T-his6-pup, pET21cc-fabD-his6, pET21cc-dop-his6, pET21 cc-pafA-his6 and pBAD-pafA-his6, proteins are expressed and purified from E.coli (BL21) by nature or denature protein purification methods. These proteins are identified by western blot. Preparation of polyclonal antibody against Pup and preliminary study on dop knockout in Mycobacterium smegmatis can give us more knowledge and insight into Pup-Proteasome system and prokaryotic celluar regulatory mechanism.