| Summary: | 碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 93 === Delivery of macromolecules mediated by cell penetrating peptides (CPPs) or protein transduction domains (PTDs) attracts a lot of interest due to its therapeutic and biotechnological potential. Despite significant progress in the cytoplasmic and nuclear delivery of various cargo molecules using PTDs, the underlying mechanisms remain under active debate. Because of CPPs are the high polar molecules, its can fast and efficiency direct translocation the lipid bilayer in the cell surface.
Internalization of proteins into mammalian cells is a useful method for analyzing and regulating cellular function. In this study, we developed a novel method for the delivery of cargos into cells using the TAT-fused protein. This fusion protein consists of three functional domains, the protein transduction domain of HIV-1 TAT；the B、C domain of staphylococcal protein A，which has an ability to bind to the IgG；and the core region of the streptavidin，which has an ability to bind to the biotinylation molecules. The TAT–PAST fusion protein was mixed with fluorescence-labeled rabbit IgG and added to cells. The internalization of antibody was analyzed using confocal microscopy and flow cytometry in living cells. As a result, fluorescence labeled IgG with the TAT–PAST fusion protein was observed intracellularly. Flow cytometry results demonstrated time course and dose dependence relationships of antibody internalization. we demonstrate that the entry of Tat-PAST peptide fusion protein into HeLa cells is serum-dependent、ATP-independent and temperature dependent, indicating the involvement of endocytosis. Specific inhibitors of lipid- raft-dependent endocytosis partially inhibit Tat-PAST fusion protein uptake, implicating this pathway in TAT peptide entry. In contrast, the caveolin-dependent pathway and macropinocytosis pathway are not essential for the uptake of Tat-PAST fusion protein. It proves to be a useful technique for screening functional proteins and peptides from cell or cell-free lysate without purification. Based on this possibility, we have developed a novel method for the internalization of antibodies using the TAT-fused protein.
|
|---|
