Research On Expression, Purification And Protein Feature Of TSC1 And TSC2

Tuberous sclerosis complex(TSC) is an autosomal dominated disease characterized with benign tumors developed in multiple organs. This disease has various clinical manifestations, such as epilepsy, autism and renal angiomyolipoma. The occurrence of TSC results from the mutations of two tumor suppressor gene, TSC1 and TSC2. The manifestation and severity of TSC are related to the mutant conditions. These two genes respectively encode two proteins forming TSC1/2 complex, which is able to integrate a variety of growth related signals upstream mTOR pathway. The growth signals contain amino acid, growth factor, energy, hypoxia and cytokines. These signals are shifted to the multiple-site phosphorylation of TSC1 and TSC2. The GAP(GTPase activating protein) domain of TSC2 activates Rheb(Ras homolog enriched in brain GTPase), resulting the inhibition of mTORC1(mammalian target of rapamycin complex 1). Therefore, TSC1/2 complex negatively regulates the anabolism of cells. The 3D structure of TSC1/2 complex will contribute to revealing its regulation mechanism.We conducted the expression and purification of TSC1 and TSC2 proteins from Schizosaccharomyces pombe(S. pombe) and Homo Sapiens for their crystallization. We used Escherichia coli(E.coli) to express the TSC1 coiled coil domain and TSC2 GAP domain. The maltose-binding protein(MBP) fusion S. pombe TSC1 540-675 aa expressed well but aggregated; the TSC2 GAP domain precipitated; Human TSC2 1516-1708 aa was soluble but didn't co-crystalize with Rheb. We used insect cell Sf9 to express S. pombe TSC1 and TSC2. TSC1 FL(TSC1 full-length) and TSC2 FL(TSC2 full-length) had a low expression level; the C terminal truncated S. pombe TSC1, like 1-718 aa and 1-799 aa, expressed well; the expression of C terminal truncated S. pombe TSC2 was less than TSC2 FL; Anti-Flag affinity purification of Flag-TSC2 binding to His-TSC1 yielded considerably pure TSC1/2 complex, which solved the imbalance expression amount of TSC1 and TSC2; TSC1 stabilize TSC2 with the help of its coiled coil region; the co-expression of TSC1 1-718 aa or 1-799 aa with the TSC2 FL benefits the expression of TSC2. The purification results indicated that S. pombe TSC1 existed in the form of dimer; TSC1 bond to TSC2 and the TSC1/2 complex aggregated into a multimer whose molecular weight was over 1 MDa; the N-terminal and GAP domain of TSC2 could be divided, and TSC1 bond to the N-terminal of TSC2. The Expi293 F expression system was fit for the expression of Human TSC1 FL and TSC2 FL but in a low expression level. However, the expression of truncated Human TSC1 and TSC2 was more terrible than their FL. In conclusion, we made an intensive study of expression and purification of TSC1 and TSC2, which had elucidated some structural features of TSC1 and TSC2. Moreover, the research results and developed expression methods of TSC1 and TSC2 lay a solid foundation for their crystallization.