TAT介导多肽药物过表达纯化和穿膜活性检测

Overexpression, Purification and Transmembrane Activity Assay of a TAT-Mediated Peptide NR2Pep

目的:获得NR2B羧基端多肽(NR2Pep),探讨TAT-NR2Pep在细胞中的跨膜特性。方法:利用酶切连接方法构建p Waldo-TAT-pep质粒,转化大肠杆菌BL21(DE3)感受态细胞,检测不同浓度诱导剂和不同温度对蛋白质表达的影响;利用镍柱亲和层析及分子筛方法纯化TAT-NR2Pep-GFP融合蛋白,利用GFP发荧光的特性采用In-gel检测蛋白质的表达情况;以融合蛋白C端His标签特异性抗体,通过Western印迹确定融合蛋白的表达;荧光显微镜下观察TAT-NR2Pep-GFP融合蛋白在CHO和C2C12等细胞中的跨膜活性。结果:构建了TAT-NR2Pep-GFP表达载体,重组菌经0.1 mmol/L IPTG于22℃诱导20 h能够表达较多高质量的目标蛋白;In-gel荧光检测证实融合蛋白TATNR2Pep-GFP拥有正确构象,GFP能够发出绿色荧光;Western印迹分析表明融合蛋白相对分子质量符合预期;细胞穿膜实验证实TAT能够介导多肽穿过细胞膜。结论:原核表达并纯化得到TAT-NR2Pep-GFP融合蛋白,该蛋白散发绿色荧光,能够穿过细胞膜。

Objective： To obtain peptide NR2 Pep and determine the transmembrane activity of TAT-NR2 Pep in different cell line. Methods： The p Waldo-TAT-pep expression plasmid was constructed by digestion and ligation method. The plasmid was transformed into E.coli BL21（DE3） competent cell. The concentration of IPTG and induction temprtrature for fusion protein expression were optimized. The TAT-NR2 Pep-GFP fusion protein was purified by Ni column and gel filtration method. In-gel analysis was used to determine the protein expression by detecting GFP fluorescent. Western blotting was carried out to confirm the protein expression by using anti-His-tag antibody. The CHO and C2 C12 cells were used to determine the transmembranse activity of TAT-NR2 Pep-GFP fusion protein which was analyzed under fluorescent microscope. Results： The overexpression vector of TAT-NR2 PepGFP was constructed. The expression and purification conditions of TAT-NR2 Pep-GFP were optimized. Induced with 0.1 mmol/L IPTG at 22℃ for 20 h, the protein had high expression level and quality. In-gel analysis showed that the fusion protein could fold properly with green fluorescence. Western blotting analysis confirmed the fusion protein with predicted molecular mass. Transemembrane activity analysis showed TAT-NR2 Pep-GFP could pass through the cell membrane. Conclusion： TAT-NR2 Pep-GFP was expressed as soluble protein in E.coli BL21（DE3） induced with IPTG. TAT could mediate the peptide to pass through the cell membrane.