GST-β-catenin-His双标签融合蛋白的原核表达、纯化及鉴定

Prokaryotic expression,purification,and identification of GST-β-catenin-His double labeled fusion protein

应用RT-PCR技术扩增β-catenin的cDNA序列,并且在基因下游引入6×His标签序列,克隆至表达载体pGEX-4T-1.经测序鉴定后,将重组质粒转入BL21pLysS感受态细菌,经IPTG诱导表达,采用Glutathione Sepharose 4B和Ni柱分步纯化GST-β-catenin-His融合蛋白.所得产物经SDS-PAGE检测,在114 kDa处显示特异条带,与GST-β-catenin-His融合蛋白预期分子量相符.经Western Blotting鉴定,所纯化蛋白能被β-catenin抗体、GST抗体和His抗体识别,为进一步研究β-catenin的功能提供了基础和前提.

The cDNA sequence of β-catenin was amplified through RT-PCR with 6xHis tag added in the gene downstream,cloned into the vector pGEX-4T-1.The plasmid transformed into BL21 pLysS was induced by IPTG.The induced fusion protein was purified stepwise using Glutathione Sepharose 4B and Ni columns.The final protein ran on SDS-PAGE with a specific band at the expected size of 114 kDa.Western Blotting showed that the purified protein can be recognized by the anti-β-catenin,anti-GST,and anti-His antibodies,respectively.All these results provide a basis for further study of β-catenin function.