GST-His双标签原核表达载体的构建及应用  

Construction and Application of Prokaryotic Expression Plasmid Containing GST and His Tags

作　　者：赵静[1,2] 丁丽华[1] 仇玮祎[1] 牛畅[1] 陈力权[1] 甘纯玑[2] 叶棋浓[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850 [2]福建农林大学生命科学学院,福建福州350002

出　　处：《生物技术通讯》2009年第4期520-522,共3页Letters in Biotechnology

基　　金：国家自然科学基金(30500091);国家重点基础研究发展计划(2006CB943501);全军医药卫生科研基金(06J021)

摘　　要：目的:在原有带有GST标签的pGEX-KG载体上添加His标签,构建双标签原核表达载体,以提高纯化后的融合蛋白的纯度。方法:双酶切pGEX-KG载体,将同样带有双酶切位点编码His标签的DNA序列酶切后与其连接、转化大肠杆菌DH5α、鉴定阳性克隆并测序,并将编码雌激素受体β(ERβ)的片段构建到该载体上,分别利用GST标签和His标签对ERβ蛋白进行2次纯化。结果:构建了GST-His双标签原核表达载体,将ERβ编码片段克隆入该载体中,在原核生物中得到表达;分别用GST和His抗体进行Western blot分析,均可检测到GST-His-ERβ融合蛋白的表达;利用此双标签载体纯化得到了纯度较高的ERβ蛋白。结论:GST-His双标签原核表达载体的构建对提高目的蛋白纯度具有重要意义。Objective： Adding His tag to pGEX-KG to construct prokaryotic expression plasmid containing GST and His tags, and to increase purity of purified fusion proteins. Methods： pGEX-KG was digested with two enzymes, and the digested vector was ligated with His-tag-containing DNA sequence digested with two enzymes. The ligation products were transformed into E.coli DH5α. Positive clones were selected and sequenced. Then, the estrogen receptor（ER） β coding fragment was cloned into the resulting plasmid and the ERβ fusion protein was purified sequentially with His tag and GST tag, respectively. Results： Prokaryotic expression plasmid containing GST and His tags was constructed and the ERβ fragment was cloned into the plasmid. ERβ fusion protein was expressed efficiently in prokaryotic system and successfully demonstrated by Western blot with antibodies against GST and His, respectively. We obtained much more purified ERβ fusion protein with the two-tag plasmid. Conclusion： The prokaryotic expression plasmid containing GST and His tags can be applied to increase the purity of interest protein efficiently.

关 键 词：GST-His双标签 原核表达载体 蛋白纯化 雌激素受体Β 

分 类 号：Q78[生物学—分子生物学]