GST-β-catenin-His双标签融合蛋白的原核表达、纯化及鉴定

doi:10.7523/j.issn.2095-6134.2012.6.019

中国科学院大学学报 ›› 2012, Vol. ›› Issue (6): 847-852.DOI: 10.7523/j.issn.2095-6134.2012.6.019

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GST-β-catenin-His双标签融合蛋白的原核表达、纯化及鉴定

尹会龙, 袁莉

中国科学院研究生院生命科学学院, 北京 100049

收稿日期:2011-11-10 修回日期:2012-01-13 发布日期:2012-11-15
通讯作者: 袁莉
基金资助:
国家自然科学基金(Y11101L1A1)和中国科学院研究生院院长基金(095101GN00)资助

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

YIN Hui-Long, YUAN Li

College of Life Sciences, Graduate University, Chinese Academy of Sciences, Beijing 100049, China

Received:2011-11-10 Revised:2012-01-13 Published:2012-11-15

摘要/Abstract

摘要： 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.

关键词: β-连环蛋白, 纯化, GST-His双标签, Western Blotting

Abstract: 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 BL21pLysS 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.

Key words: β-catenin, purification, GST and His tags, Western Blotting

中图分类号:

R346

尹会龙, 袁莉. GST-β-catenin-His双标签融合蛋白的原核表达、纯化及鉴定[J]. 中国科学院大学学报, 2012, (6): 847-852.

YIN Hui-Long, YUAN Li. Prokaryotic expression, purification, and identification of GST-β-catenin-His double labeled fusion protein[J]. , 2012, (6): 847-852.

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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

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