GST-TAT-GFP融合蛋白的表达、纯化及鉴定

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

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54.3 kD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。

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 pGEX-TAT-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.