SEMA6C胞外域在293T细胞中的大量表达及其结构特性的研究

High-level Expression in 293T Cells and Structural Characterization of Extracellular Region of Semaphorin 6C

Sema6C是semaphorin蛋白超家族的成员之一,具有调节神经系统发生和发展的生理功能.Sema6C由胞外域、跨膜片段和胞内结构域组成.利用293T细胞表达了Sema6C的胞外域并研究了其结构特性.实验结果表明Sema6C可在293T细胞中大量表达,纯化出的该蛋白N-糖基化修饰,并具有导致背根神经节(DRG)生长冠萎缩的活性,其EC_(50)约为600 pmol/L.用分子筛层析色谱和不变性聚丙烯酰胺凝胶电泳研究该蛋白的结构特性,发现该蛋白在糖基化和去糖基化的状态下均具有稳定的结构和单一的分布形态,其相对分子量分别为190.2和168.8 kDa.此外,分析沉降平衡试验结果表明了该蛋白质形成二聚体.基于以上结果,对Sema6C胞外域的二聚体结构对其致萎缩活性的影响进行了讨论.

Semaphorin 6C （Sema6C） is a number of the semaphorin family and it can adjust nervous system form and development. Sema6C is composed of an extracellular region, a trans- membrane segment, and cytoplasmic domains. Here, the extracellular region of Sema6C has been expressed in 293T cells and its structural characterization has been investigated. The protein expressed in 293T cells is modified mainly with N-linked glycosylation, and induces growth cone collapsing of dorsal root ganglion neurons with an EC50 of 600 pmol/L. Both size exclusion chromatography and native polyacrylamide gel electrophoresis （native-PAGE） showed that the protein in the native and the deglycosylated states exists as a stable single species in solution with molecular masses of 190.2 and 168.8 kDa, respectively. Furthermore, the analytical sedimentation equilibrium experiment clearly showed that the protein forms dimer. Based on these results, the effects of the dimerization of the extracellular region of class 6 semaphorins on their collapsing activity have been discussed.