人红细胞生成素受体膜外结构域在大肠杆菌中的融合表达及其纯化

FUSION EXPRESSION AND PURIFICATION OF THE EXTRAMEMBRANE DOMAIN OF HUMAN ERYTHROPOIETIN RECEPTOR IN E.COLI

将插入ｐＢｌｕｅｓｃｒｉｂｅ的人红细胞生成素受体ｃＤＮＡ，用ＥｃｏＲＩ和ＢａｍＨＩ酶解，分离得到的基因片段，插入到经ＥｃｏＲＩ和ＲａｍＨＩ消解的表达载体ｐＧＥＸ－３Ｘ中，得到重组表达质粒ｐＧＥＸ－３Ｘ／ｈＥＰＯＲ。重组质粒ｐＧＥＸ－３Ｘ／ｈＥＰＯＲ含有ｔａｃ启动子，ＥＰＯＲ膜外结构域基因５端与谷胱甘肽转移酶（ＧＳＴ）编码基因融合，阳性重组子在大肠杆菌中经ＩＰＴＧ诱导表达ＧＳＴ－ｈＥＰＯＲ，重组表达菌裂解上清液经ＧＳＨ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和柱一步纯化，能除去绝大部分杂蛋白，基本达到纯化的效果。ＳＤＳ－ＰＡＧＥ和免疫杂交分析显示。

The DNA fragment,which encodes the extramembrane domain of human erythropoietin receptor(hEPORx),was isolated from the full length human erythropoietin receptor cDNA by EcoRI and BamHI digestion.The obtained fragment was then cloned into the expression vector pGEX3X.In the resulting plasmid pGEX3X/HEPOR,the hEPORx encoding gene was fused to the gene encoding glutathione Stransferase and the fusion protein was under the control of tac promoter.Upon IPTG induction,SThEPORx was expressed in E.coli JM103.The expressin product was primarily purified with GlutathioneSepharose 4B affinity chromatography from cellular lysate.SDSPAGE and immunodotblot analysis showed the expression product to be human erythropoietin receptor.