可溶性Sox2-11R重组蛋白的制备与鉴定

Preparation and function analysis of soluble Sox2-11R recombinant protein

旨在通过原核表达制备可溶性Sox2-11R重组蛋白,并验证其穿膜能力。首先,采用增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)与蛋白转导域11聚精氨酸(11 arginine,11R)的融合表达和与成纤维细胞的共孵育验证11R的穿膜能力。其次,将目的基因Sox2-11R在原核表达载体pET30a中表达,通过优化表达条件获取可溶性Sox2-11R重组蛋白。最后,利用Ni柱纯化重组蛋白,SDS-PAGE及Western blotting对其进行鉴定;并用免疫荧光染色验证重组蛋白是否具有进入细胞核的能力。结果表明,成功构建了pET30a-Sox2-11R原核表达载体,纯化的Sox2-11R重组蛋白具有穿膜能力,且在细胞培养基中具有较高溶解度,这为制备其他重编程因子的重组蛋白提供了参考,也为外源蛋白诱导iPSCs奠定了基础。

The aim of the study was to prepare soluble recombinant protein Sox2-11R with cell penetratingpeptide using prokaryotic expression system and analyze its functions. Firstly, the cell permeability of the recom-binant protein was determined by designing the 11R protein transduction domain to the C-terminal of EGFP andtreated fibroblast cells with the purified protein. Secondly, the soluble protein was obtained by expressing theSox2-11R in pET30a vector and various induction conditions were tested. Finally, the fusion protein was purifiedby Ni affinity chromatography and identified by SDS-PAGE and Western blotting. The ability of cell permeabilitywas analyzed using immunocytochemistry. The results showed that the vector pET30a-Sox2-11R was successfullyconstructed and the purified soluble recombinant protein Sox2-11R showed the biological activity, efficient cellpermeability, and relatively high solubility in the cell medium. This study laid a good foundation for preparationof other reprogramming factors and induction of iPSCs with exogenous proteins.