Tat-GFP融合蛋白的表达纯化及其穿膜活性

Expression and purification of Tat-GFP fusion protein and its cell membrane penetrating activity

目的:获得具备穿膜活性与绿色荧光蛋白(GFP)标记的Tat-GFP融合蛋白,探讨Tat-GFP在MCF-7细胞中的跨膜转运特性。方法:应用pET-24a-Tat-GFP质粒转化大肠杆菌BL21感受态细胞,检测不同异丙基硫代半乳糖苷(IPTG)浓度(0.5和1.0mmol·L-1)和不同温度(22℃和37℃)诱导融合蛋白的表达情况;利用Ni-IDA树脂亲和纯化Tat-GFP蛋白,利用GFP特异性抗体采用Western blotting法分析洗脱液中的蛋白;激光共聚焦荧光显微镜下检测Tat-GFP融合蛋白的跨膜转运活性。结果:0.5和1.0mmol·L-1 IPTG诱导出的细菌总蛋白中所含的Tat-GFP蛋白量无明显差异;低温(22℃)诱导生产的Tat-GFP蛋白量较37℃更高;Western blotting分析,GFP抗体能够特异性识别PVDF膜上的蛋白,条带灰度与Tat-GFP蛋白上样量有关联;细胞穿膜实验,绿色荧光分布于MCF-7细胞的细胞质和细胞核中。结论:低温诱导时大肠杆菌BL21菌体上清液中Tat-GFP融合蛋白量更高,所生产的Tat-GFP融合蛋白既具备穿膜活性又具有易于检测的绿色荧光。

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-7cells.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 0.5and 1.0mmol·L^-1IPTG;however,the low temperature（22℃）-induced BL21cells expressed more Tat-GFP proteins than that at37℃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-7cells.Conclusion The Tat-GFP protein highly expresses in the supenatant of Escherichia coli i BL21cells at low temperature;the obtained Tat-GFP protein with green fluorescence preserves the cell penetrating activity.