关键词: Tat-GFP, 细胞穿膜肽, 融合蛋白, 穿膜活性

Abstract:

Abstract:Objective
To obtain the Tat-GFP fusion proteins with penetrating activity and labeled with green fluorescence protein (GFP),and to explore the cell membrane penetrating activity of Tat-GFP in MCF-7 cells.Methods The plasmid pET-24a-Tat-GFP was transformed into Escherichia coli BL21 cells.Different concentrations (0.5  and 1.0 mmol•L^-1) of  isopropyl-β-D-thiogalactopyranoside (IPTG) and cell culture temperatures (22℃ and 37℃) were used to optimize the protein expression. The Tat-GFP proteins in supernatant were purified using Ni-IDA resins. Western blotting analysis was used toidentify the Tat-GFP protein,and confocal laser scanning microscope (CLSM) was used to examine the cell penetration of Tat-GFP protein.Results There was no significant difference in the Tat-GFP protein production induced by 0.5  and 1.0 mmol•L^-1 IPTG;however,the low temperature (22℃)-induced BL21 cells expressed more Tat-GFP proteins than that at 37℃ induction.The Western blotting analysis results showed that GFP antibody could specifically recognize  the proteins in PVDF membranes in dose-dependent manner;the CLSM results indicated the distribution of green fluorescence in cytoplasm and nucleus of MCF-7 cells.Conclusion The  Tat-GFP protein highly expresses  in the supenatant of Escherichia coli i BL21 cells   at low temperature;the obtained Tat-GFP protein with green fluorescence preserves the cell penetrating activity.

Key words: Tat-GFP, cell-penetrating peptides, fusion protein, penetrating activity