GST-TAT-GFP融合蛋白的表达、纯化及鉴定

目的 获得融合TAT短肽的融合蛋白GST—TAT-GFP，以用于阐明TAT短肽的跨膜递送机理。方法 以质粒pGEX—GFP为模板，扩增目的基因TAT—GFP，将其克隆至质粒pGEX-2T，用所构建的质粒pGEX—TAT—GFP转化大肠杆菌BL21并诱导表达，利用GS-4B亲和柱进行纯化，所得产物进行SDS-PAGE、Western blot及细胞跨膜实验鉴定。结果 大肠杆菌细胞经诱导高效表达出约54．3kD的蛋白，其相对分子质量与GST—TAT-GFP融合蛋白相符；Western blot显示该融合蛋白能够被抗GFP的抗体特异性识别；细胞实验表明融合TAT短肽的融合蛋白能跨膜进入细胞L-02、SMMC-7721、Hela和BEL-7402。结论 在大肠杆菌表达系统中高效表达了有活性的GST—TAT—GFP融合蛋白。

Expression of Recombinant GST-TAT-GFP Fusion Protein in E. Coli BL21

Objective To obtain the expression of recombinant GST-TAT-GFP fusion protein in E.coli BL21.Methods The cDNA of TAT-GFP was amplified from plasmid pGEX-GFP and cloned into pGEX-2T vector.The recombinant plasid pGEXTAT-GFP was expressed in E.coli BL21 and the products were purified by affinity resin-Glutathione Sepharose 4B(GS-4B).The specific expression was identified by SDS-PAGE,Western blot.Results The resolved GST-TAT-GFP fusion protein on 10% SDS-PAGE showed a major band at position of 54.3 kD.The fusion protein was recognized by anti-GFP antibody on PVDF membrane.The fusion protein GST-TAT-GFP can conneet into the menbranes of L-02,SMMC-7721,Hela and BEL-74021 cells efficiently.Conclusion The active recombinant GST-TAT-GFP fusion protein was expressed in E.coli cells efficiently.