Mechanism For Cellular Internalization And Aapplication Of The TAT Protein Transduction Domain

The protein transduction domain (PTD) of TAT protein which composed of 11 amino acid(47-57) can mediate the intracellular delivery of heterogeneous proteins. The mechanism for its cellular internalization was unclear till now. For its important application on clinic therapeusis, it's necessary to study the mechanism for cellular internalization of TAT-PTD. We constructed the recombinant protein firstly as following steps. The target gene TAT-GFP was amplified by PCR at first, then the gene identified as TAT-GFP was ligated with the plasmid pGEX-2T. When the recombinant plasmid pGEX-TAT-GFP with GST tag was successly constructed, we transformed it to E.coli BL21. The fusion protein GST-TAT-GFP with MW of 54.3kD was purified with affinity resin-Glutathione Sepharose 4B(GS-4B) after it was expressed in E.coli BL21.Secondly,the fusion protein having activity was acted on cells in vitro at differrent conditions and detected with fluorescence microscope and fluorespectrophotometer to study the internalization mechanism of TAT-PTD. We can learn from the results of the experiment as follows. (A) GST-TAT-GFP can internalize into many cells efficiently. Where it can receive includes the cytoplasm and vicinity of the nucleus. (B) The transduction into Hela was depended on concentration and time. The fusion protein GST-TAT-GFP can transduct into Hela within 15min. The amount of GST-TAT-GFP transduct into Hela would be larger along with increment of concentration. It seemed that the temperature had a great effect on the transduction of GST-TAT-GFP into Hela as the recombinant could successly transduct into cell in 37℃ and almost invalidated in 4℃. (C) The experiment about the effect of metabolic inhibitor showed that the transduction of GST-TAT-GFP didn't depond on energy. (D) The cell surface heparansulfate may contribute to the internalization of GST-TAT-GFP. Thirdly, the transduction of GST-TAT-GFP into cell was found to have no obvious cytotoxic or any destructive effect on the membrane substantially. Finally, it's the first time that the active protein was used to study the internalization of TAT-PTD in little albino rat. The active protein GST-TAT-GFP could be detected in different tissue with intraperitoneal injection and the protein was proved to can transduct into not only epidermis but also derma by the result of the skin painting experiment. In contrast, the transduction of control proein GST-GFP almost could not be detected both in the four cell lines, the tissues of the little albino rat. These results indicated that TAT-PTD was internalized into cells by combining with cell surface heparansulfate, which was one of the low affinity receptors and the transduction of TAT-PTD into cell had no effect on cell. At the same time, the transduction of TAT-PTD was successfully applied in the tissues of the animal. all the results will provided a theory basis in many fields such as cosmetics .