Expression and purification of Tat-GFP fusion protein and its cell membrane penetrating activity
10.13481/j.1671-587x.20140406
- VernacularTitle:Tat-GFP融合蛋白的表达纯化及其穿膜活性
- Author:
Xingang GUAN
;
Weiheng SU
;
Xin YU
;
Haibin TONG
;
Xin SUN
- Publication Type:Journal Article
- Keywords:
Tat-GFP;
cell-penetrating peptides;
fusion protein;
penetrating activity
- From:
Journal of Jilin University(Medicine Edition)
2014;(4):725-728
- CountryChina
- Language:Chinese
-
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 to identify 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 BL2 1 cells at low temperature;the obtained Tat-GFP protein with green fluorescence preserves the cell penetrating activity.
