转录因子CSL-GST融合蛋白表达载体的构建及其表达纯化

摘要

Objective To construct the GST-CSL fusion protein prokaryotic expression vector pGEX-6p-l-CSL, and test its expression in E.coli cells. Methods CSL gene was cloned from the cDNA of pancreatic cancer cell line Hpac cells by using PCR, then it was inserted into the prokaryotic expression vector pGEX-6p-l to generate the pGEX-6p-l-CSL which could express the GST-CSL fusion protein in E.coli cells. It was then purified with Glutathione-Sepharose 4B and cut with PreScission protease, to text its biological activity of binding to its target gene by using EMSA Hes-1. Results pGEX-6p-l-CSL efficiently expressed in E.coli cells and the purified CSL protein have the basic biologic activity. Conclusion The GST-CSL fusion protein prokaryotic expression vector pGEX-6p-l-CSL has been constructed successfully.%目的 构建转录因子CSL的GST原核表达载体pGEX-6p-1-CSL,并检测其在原核生物大肠杆菌中的表达.方法 通过反转录及聚合酶链反应从人胰腺癌细胞系Hpac细胞中获得编码CSL的cDNA,定向克隆至C端带GST蛋白标签序列的原核表达载体pGEX-6p-1中,原核表达的GST-CSL蛋白经Gluta-thione-Sepharose 4B和PreScission蛋白酶纯化后用凝胶迁移实验分析其活性.结果 pGEX-6p-1-CSL经原核表达及纯化后得到了纯化的CSL蛋白.结论 成功构建了转录因子CSL的GST原核表达载体pGEX-6p-1-CSL.