SUMO-TAT-Klf4融合蛋白的原核表达及纯化

Prokaryotic Expression and Purification of SUMO-TAT-Klf4 Fusion Protein

Klf4是体细胞诱导成多能干细胞的重要转录因子之一,参与细胞增殖分化。本研究通过将小鼠Klf4基因、穿膜肽TAT以及小分子泛素样修饰蛋白SUMO融合并转入大肠杆菌Rosseta(DE3)中进行表达,经PCR鉴定和酶切验证后,用IPTG诱导后表达出约78 k D的融合蛋白,利用Ni-NTA亲和层析胶纯化获得了高质量的SUMO-TAT-Klf4蛋白,使用Western blotting检测显示特异性良好。本研究成功构建了pET3c-SUMO-TAT-Klf4原核表达载体,有利于获得大量的Klf4蛋白,并以期提高其穿透细胞膜的能力,为后续利用重编程因子融合蛋白诱导体细胞成为iPS细胞奠定基础。

Klf4 is one of the significant transcription factors that induces somatic cells into pluripotent stem cells,which is involved in cell proliferation and differentiation.In this study,the mouse Klf4 gene,transmembrane peptides of TAT and small molecule ubiquitin-like protein of SUMO were fused and transformed into Rosseta(DE3)for expression.After identification by PCR and restriction enzyme digestion,the fusion protein about 78 kD was expressed after being inducted by IPTG.High-quality SUMO-TAT-Klf4 recombinant protein was purified by Ni-NTA affinity chromatography and showed good specificity by Western blotting.The prokaryotic expression vector pET3c-SUMO-TAT-Klf4 was successfully constructed in this research,which was conducive to obtain a large amount of Klf4 protein,and to improve its ability to penetrate the cell membrane.And this study might lay the foundation for subsequent use of reprogramming factors fusion protein to induce somatic cells to become iPS cells.