利用新型抗冻蛋白-内含肽标签高效纯化重组蛋白  

Purification of recombinant proteins with high efficiency using a new antifree protein-intein tag

作　　者：郑延蓉 王从纲 孙佳明 李宪臻[1] ZHENG Yanrong;WANG Conggang;SUN Jiaming;LI Xianzhen(School of Biological Engineering,Dalian Polytechnic University,Dalian 116034,China)

机构地区：[1]大连工业大学生物工程学院,辽宁大连116034

出　　处：《大连工业大学学报》2022年第4期251-255,共5页Journal of Dalian Polytechnic University

基　　金：辽宁省自然科学基金项目(20180550668).

摘　　要：利用抗冻蛋白AFP的冰结合特性和Mxe GyrA内含肽的可控自切割特性,构建一种以冰为纯化介质,能够可控自切割的纯化标签。以谷胱甘肽硫转移酶(GST)为对象研究其可行性,采用重叠延伸PCR技术和酶切连接技术构建表达载体pET 24a-GST-Mxe GyrA-linker-AFP,在大肠杆菌中成功可溶表达后,利用冰作为吸附介质从细胞破碎上清液中一步分离出融合蛋白GST-Mxe GyrA-linker-AFP。冰融化后,加入终浓度40 mmol/L二硫苏糖醇缓冲液,诱导内含肽N端发生断裂反应形成Mxe GyrA-linker-AFP标签和GST的混合物,再次利用冰吸附去除标签,得到目的蛋白。An ice-binding self-cleavage purification tag was constructed using an ice-binding antifreeze protein(AFP)and a self-cleavable Mxe GyrA intein.The feasibility of the scheme was studied using glutathione S-transferase(GST)as a model protein.The pET 24a-GST-Mxe GyrA-linker-AFP was constructed by using overlap extension PCR and digestion-ligation cloning.The fusion proteins were expressed as soluble in Escherichia coli.GST-Mxe GyrA-linker-AFP fusion proteins were isolated from the clarified cell lysate using ice as adsorbent.Then the ice was melted and the self-cleavage reactions of intein was induced by the addition of buffer with a final concentration of 40 mmol/L of dithiothreitol to release the Mxe GyrA-linker-AFP tag and GST.The tag was removed by ice-mediated adsorption and GST with high purity was evaluated.

关 键 词：亲和色谱 纯化标签 抗冻蛋白 内含肽 

分 类 号：Q816[生物学—生物工程]