Tat-GFP融合蛋白的表达纯化及其穿膜活性

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1.0 mmol•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1.0 mmol•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。

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

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.