论文部分内容阅读
结节性硬化症是一种在多器官发生良性肿瘤而表现出不同临床病症的常染色体显性疾病,由肿瘤抑制基因TSC1(tuberous sclerosis 1)和TSC2发生突变所致。这两个基因编码的蛋白形成TSC1/2复合物,在m TOR信号通路中整合上游生长信号,负调控细胞的合成代谢。该复合物的结构学研究有助于揭示其调控机理。利用Bac-to-Bac杆状病毒表达系统在昆虫细胞中表达来自裂殖酵母Schizosaccharomyces pombe(S.pombe)与人同源的TSC1和TSC2及其截短和突变蛋白。纯化结果表明,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1 000 k Da的多聚体;亲和纯化带Flag标签的TSC2,同时TSC2结合TSC1,可得到纯度高的TSC1/2复合物;TSC1需要其卷曲螺旋(coiled-coil)来稳定TSC2;TSC1 1-718 aa或1-799 aa和TSC2全长共表达有利于提高TSC2的表达量;TSC2的N-端和GAP结构域可以分开,TSC1和TSC2的N-端结合。总之,该研究阐释了裂殖酵母TSC1和TSC2的一些结构性质,提出的TSC1和TSC2表达纯化方法为其结晶研究奠定了重要基础。
Tuberous sclerosis is an autosomal dominant disease that manifests different clinical conditions in benign tumors of multiple organs and is caused by mutations in the tumor suppressor genes TSC1 and TSC2. The proteins encoded by these two genes form the TSC1 / 2 complex, which integrates upstream growth signals in the mTOR signaling pathway and negatively regulates the cellular anabolism. The structural study of the complex helps reveal its regulatory mechanism. Human-derived TSC1 and TSC2 from Schizosaccharomyces pombe (S. pombe) and their truncated and muteins were expressed in insect cells using a Bac-to-Bac baculovirus expression system. Purification results showed that the fission yeast TSC1 existed as a dimer. TSC1 bound to TSC2 and TSC1 / 2 complex to polymerize into multimer with molecular weight greater than 1000 kDa. Flag-tagged TSC2 was affinity-purified and TSC2 bound TSC1, TSC1 / 2 complex with high purity can be obtained; TSC1 needs its coiled-coil to stabilize TSC2; TSC1 1-718 aa or 1-799 aa and TSC2 co-expression for a long time can improve the expression of TSC2 ; N-terminal TSC2 and GAP domains can be separated, TSC1 and N-terminal TSC2 binding. In summary, this study elucidated some of the structural properties of the fission yeast TSC1 and TSC2, and proposed TSC1 and TSC2 expression and purification methods laid an important foundation for their crystallization studies.