TSC1和TSC2蛋白的表达、纯化和性质探讨

发布时间：2018-03-20 14:14

本文选题：TSC1　切入点：TSC2　出处：《上海交通大学》2015年硕士论文　论文类型：学位论文

【摘要】：结节性硬化症(Tuberous Sclerosis Complex,TSC)是一种在多器官发生良性肿瘤的常染色体显性疾病。该疾病表现出多种临床病症,如癫痫、自闭症、肾血管平滑肌瘤等。结节性硬化症由肿瘤抑制基因TSC1和TSC2发生突变所致,不同突变导致的病症和严重程度都不同。这两个基因分别编码的TSC1和TSC2蛋白形成TSC1/2复合物,在mTOR信号通路中起到负调控的作用。TSC1/2复合能够整合上游的氨基酸、生长因子、能量、低氧和细胞因子等生长信号,转化为TSC1和TSC2不同位点的磷酸化状态。TSC2的GTP酶激活蛋白(GAP)结构域活化Rheb(Ras homolog enriched in brain GTPase),从而抑制mTORC1(哺乳动物雷帕霉素靶蛋白复合物1,mammalian target of rapamycin complex 1)的活性,降低细胞的合成代谢水平。该复合物的结构学研究有助于揭示其调控机理。本研究表达和纯化人和裂殖酵母Schizosaccharomyces pombe(S.pombe)的TSC1和TSC2蛋白,进行结晶尝试。我们利用大肠杆菌Escherichia coli(E.coli)表达TSC1的coiled coil结构域和TSC2的GAP结构域,裂殖酵母TSC1 540-675 aa的MBP(麦芽糖结合蛋白)融合蛋白表达较好,但聚合度高;TSC2完整的GAP结构域沉淀,人TSC2 1516-1708 aa可溶,但与人Rheb共点晶不结晶。用昆虫细胞Sf9共表达裂殖酵母TSC1和TSC2蛋白,TSC1全长(TSC1 FL)和TSC2全长(TSC2 FL)表达量低;TSC1的C端截短,如1-799 aa、1-718 aa表达量较高;TSC2的C端截短表达量不如TSC2 FL;Anti-Flag亲和纯化Flag-TSC2同时拉出His-TSC1,可得到纯度高的TSC1/2复合物,解决了TSC1表达量高于TSC2的问题;TSC1需要一定长度的coiled coil来稳定TSC2,部分C端截短的TSC1 1-718 aa或1-799 aa和TSC2FL共表达有利于提高TSC2的表达量。裂殖酵母TSC1以二聚体的形式存在;TSC1结合TSC2,TSC1/2复合物会聚合成分子量大于1 MDa的多聚体;TSC2的N端和GAP结构域可以分开,TSC1和TSC2的N端结合。Expi293F表达系统适合人TSC1 FL和TSC2 FL的表达,但表达量较低,截短蛋白的表达情况不如全长。总之,本研究对TSC1和TSC2的表达和纯化进行了深入的探索,阐释了蛋白的一些结构性质,研究结果和提出的方法为TSC1和TSC2的结晶研究奠定了重要基础。
[Abstract]:Tuberous Sclerosis complex (Sclerosis) is an autosomal dominant disease that develops benign tumors in multiple organs. Renal angiomyoma, etc. Nodular sclerosis is caused by mutations in tumor suppressor gene TSC1 and TSC2, and the severity and severity of these mutations are different. The TSC1 and TSC2 proteins encoded by these two genes form TSC1/2 complex, respectively. TSC 1 / 2 complex that plays a negative role in the mTOR signaling pathway can integrate upstream growth signals such as amino acids, growth factors, energy, hypoxia and cytokines. The domain of Rheb(Ras homolog enriched in brain GTPase, which was transformed into phosphorylated state of TSC1 and TSC2, inhibited the activity of Rheb(Ras homolog enriched in brain GTPase1 (mammalian rapamycin target protein complex 1 of rapamycin complex 1). The structure of the complex was helpful to reveal its regulatory mechanism. In this study, TSC1 and TSC2 proteins of human and merozoite yeast Schizosaccharomyces pombeia S.pombe. were expressed and purified. We expressed coiled coil domain of TSC1 and GAP domain of TSC2 using E. coli Escherichia coli. The fusion protein of TSC1 540-675 AA was expressed well, but the whole GAP domain of TSC2 was precipitated with high degree of polymerization. Human TSC2 1516-1708 AA was soluble, but it was not crystallized in cosite with human Rheb. The C terminal truncation of TSC1 and TSC2 protein (TSC1 FL1) and TSC2 (TSC2 FL1) were expressed in insect cell Sf9. For example, the C-terminal truncated expression of 1-799aaaAU 1-718aa is lower than that of TSC2 FL-Anti-Flag affinity purification Flag-TSC2, and His-TSC1 can be obtained with high purity TSC1/2 complex. It solves the problem that the expression of TSC1 is higher than that of TSC2. TSC1 requires a certain length of coiled coil to stabilize TSC2. The coexpression of TSC1 1-718aa or 1-799aa or 1-799aa at some C-terminal can increase the expression of TSC2. TSC1 exists in the form of dimer. Combining TSC1 with TSC1 / 2 complexes to synthesize the N-terminal and GAP domains of the polymer TSC2 with a molecular weight of more than 1 MDa, the N-terminal binding system of TSC1 and the N-terminal binding of TSC2. Expi293F expression system is suitable for the expression of human TSC1 FL and TSC2 FL. However, the expression of truncated protein was lower than that of full-length protein. In a word, the expression and purification of TSC1 and TSC2 were deeply explored, and some structural properties of the protein were explained. The results and the proposed methods lay an important foundation for the crystallization of TSC1 and TSC2.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R596.1

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