The Study Of The Transmembrane Activity Of The PTD Of Tat Protein Fused At The Carboxyl-terminus Of Heterologous Protein

The protein transduction domain (PTD) of Tat (Trans-activator transcription) protein is sufficient for intracellular transduction of Tat protein and subcellular location. PTD-Tat fused at the carboxyl-terminus of heterologous protein was theoretically superior than that fused at the amino-terminus of heterologous protein in the expression of the fusion protein. So, it is necessary to study the transmembrane delivery of PTD-Tat fused at the carboxyl-terminus of heterologous protein. The expression plasmid pGEX-GFP-Tat was constructed by genetically fusing PTD-Tat to the carboxyl-terminus of green fluorescence protein (GFP),which was in the right downstream of the coding sequence of glutathione-S-transferase(GST). The fusion protein GST-GFP-Tat was expressed in E.coli BL21(DE3) and purified by Glutathione Sepharose 4B affinity chromatography. The fusion protein will be used for studying the transmembrane delivery of PTD-Tat fused at the carboxyl-terminus of heterologous protein. When HeLa cells in vitro were co-cultured with fusion protein GST-GFP-Tat,the analysis of fluorescence microscope and flow-cytometry showed the results of the experiment as follows.(A)Fusion protein GST-GFP-Tat could internalize into HeLa cells in vitro efficiently and localize to both the cytoplasm and nucleus.(B)The transmembrane of PTD-Tat into cultured HeLa cells increased in a dose/time-dependent manner. The fluorescence intensity could be detected within 15min at 37℃. (C)It seemed that the temperature had a great influence on the uptake of PTD-Tat fusion protein. A rightward shift of the signal was observed signifying a reduction of the uptake of the fusion protein GST-GFP-Tat at 4℃compared to its cellular uptake at 37℃.(D)Heparin sulfate(HS)on the cell membrane affected the internalization of GST-GFP-Tat greatly. However, metabolic inhibitors didn't induce the internalization of GST-GFP-Tat.(E)MTT colorimetric assay found no cytotoxic effect of GST-GFP-Tat to HeLa cells. Furthermore, flow-cytometry analysis found the transduction efficiency at dose 4.0 μmol/L of GST-GFP-Tat was 80.0 %. In contrast,the transduction efficiency reduced to 32.9 % at dose 4.0 μmol/L of GST-Tat-GFP of positive control, which was GST-Tat-GFP with PTD-Tat fused at the amino-terminus of GFP. The observation of fluorescence microscope was in agreement with results of flow-cytometry analysis. Finally, the animal experiments also showed the transmembrane activity of the fusion protein.(A)GST-GFP-Tat was proved to be delivered into dermis of mice by the result of the skin painting experiment.(B) The fusion protein GST-GFP-Tat could be detected in different tissues, even passed through blood-brain barrier of mice with intraperitoneal injection. And the observation of fluorescence microscope indicated the fluorescence intensity was stronger than that of GST-Tat-GFP, especially in the brain.(C)When the fusion protein was co-culture with nematode,the analysis of fluorescence microscope showed the digestive system and the coelom of nematode could be detected green fluorescence, and the fluorescence intensity was time-and protein concentration-dependent. The research findings indicated that PTD-Tat fused at the carboxyl-terminus of heterologous protein not could internalize into cells in vitro, but the transduction was successfully applied in the tissues of mice by the comparison of PTD-Tat fused at the carboxyl/ amino-terminus of GFP. Furthermore,the transmembrane activity of PTD-Tat fused at the carboxyl-terminus displayed stronger. And the fusion protein showed no cytotoxic effect to the cells. These results will provide theoretical instruction for the efficient application of PTD-Tat in the field of the delivery of protein drugs.