TAT-PTD融合蛋白可能存在的跨膜递送作用机制

为探讨TAT-PTD融合蛋白的跨膜递送作用机制,采用DNA重组技术构建pGEX-TAT-GFP-表达质粒.在E.coli- BL21表达GST-TAT-GFP,并用谷胱甘肽(glutathione) Sepharose-4B亲和柱进行纯化.GST-TAT-GFP在不同条件下与细胞的作用结果表明,GST-TAT-GFP能有效进入HeLa、SMMC-7721、L-02和BEL-7402细胞，GST-TAT-GFP在递送时对时间和浓度有依赖关系.同时,温度对GST-TAT-GFP跨膜作用具有明显的影响,GST-TAT-GFP的跨膜作用受代谢抑制剂的影响很小,肝素的存在能明显抑制GST-TAT-GFP跨膜进入细胞的能力,GST-TAT-GFP对细胞活性没有影响.这些结果说明,TAT-PTD可能是通过与细胞表面的硫酸乙酰肝素等受体相结合介导融合蛋白跨膜递送进入细胞的.

Possible Mecanism of Transmembrane of TAT-PTD Fusion Protein

To investigate the transmembrane mechanism of recombinant protein fused to protein transduction domain(PTD) of TAT protein(TAT-PTD) on cell surface,the expression plasmid pGEX-TAT-GFP was constructed by genetically fusing TAT-PTD to the amido-terminus of green fluorescence ptotein(GFP).TAT-GFP was in the downstream of the coding sequence of glutathione-S-transferase(GST). The fusion protein GST-TAT-GFP expressed in E.coli BL21 was purified by glutathione-Sepharose 4B affinity chromatography. The cells were co-cultured with GST-TAT-GFP at different conditions and the results were determined by fluorescence microscope and fluorespectrophotometer. The results revealed that the fusion protein GST-TAT-GFP could internalize into human cervical carcinoma HeLa, human liver cancer SMMC-7721, human liver L-02, and human liver cancer BEL-7402 cells efficiently. The dependence of the fusion protein concentration and time course of cellular uptake were confirmed. It seemed that the temperature had a great effect on the transduction of GST-TAT-GFP into cell. The matabolic inhibitor had little effect on the internalization of GST-TAT-GFP. The uptake of GST-TAT-GFP was significantly inhibited in the presence of heparin sulfate. In addition,the transduction of GST-TAT-GFP into cells was found to no obvious cytotoxic. These results indicate that TAT-PTD mediate fusion protein deliver into cells by combining with cell surface heparansulfate, which is one of the receptors.