Research on Expression， Purification and Protein Feature of chizosaccharomyces pombe TSC1 and TSC2

Abstract: Tuberous sclerosis complex (TSC) is an autosomal dominated disease characterized with benign tumors developed in multiple organs so that it has various clinical manifestations. Scientists have identified two tumor suppressor genes， TSC1 and TSC2， whose mutations are responsible for TSC. These two genes respectively encode two proteins forming TSC1/2 complex. This complex can integrate a variety of growth related signals upstream mTOR pathway and then negatively regulate the anabolism of cells. The 3D structure of TSC1/2 complex will contribute to revealing its regulation mechanism. We expressed TSC1 and TSC2 proteins from Schizosaccharomyces pombe (S. pombe) as well as their truncations and mutations by employing Bac-to-Bac baculovirus expression system. The purification results indicate that S. pombe TSC1 exists in the form of dimer; TSC1 binds to TSC2 and the TSC1/2 complex aggregates into a multimer whose molecular weight is over 1 000 kDa; affinity purification of Flag-fused TSC2 binding to TSC1 yields considerably pure TSC1/2 complex; TSC1 can stabilize TSC2 with the help of its coiled coil region; the co-expression of TSC1 1-718 aa or 1-799 aa with the full-length TSC2 benefits the expression of TSC2; the N-terminal and GAP domain of TSC2 can be divided， and TSC1 binds to the N-terminal of TSC2. In conclusion， our research has elucidated some structural features of S. pombe TSC1 and TSC2. Moreover，the expression and purification methods of TSC1 and TSC2 lay a solid foundation for their crystallization.

CSTR: 32200.14.cjcb.2015.11.0007