酪氨酸激酶EphB2受体N端球状结构域的表达及纯化

根据酪氨酸激酶ＥｐｈＢ２受体的碱基序列，用ＰＣＲ方法扩增其结合配体的关键结构域Ｎ端球状区，定向克隆到融合表达质粒载体ｒＲＳＥＴ Ａ中，转化大肠杆菌ＪＭ１０９（ＤＥ３）。阳性克隆经ＩＰＴＧ诱导，由Ｔ７启动子调控表达了氨基端带６个连续组氨酸残基的融合蛋白。电泳分析表明，表达的融合蛋白主要以包含体的形式存在，约占细菌总蛋白的１４％。Ｗｅｓｔｅｒｎ印迹确证，利用Ｎｉ－ＮＴＡ金属螯合亲和色谱法在变性条件下对

Expression and purification of the N-terminal globular domain of tyrosine kinase EphB2 receptor

The N?terminal globular domain of tyrosine kinase EphB2 receptor which is important for ligand binding, was amplified by PCR method and cloned into the E.coli expression vector pRSET A, then transferred into the host strain E.coli JM109(DE3). Recombinant protein was expressed with a hexahistidine tail attacked to the N?terminal after induction with IPTG, under the control of T7 promoter, and was confirmed by Western blotting. SDS-PAGE analysis showed that the proteins were mainly aggregated into the inclusion body, and weighed 14% of the total proteins of the expression bacteria. We isolated the inclusion body from the culture under denaturing condition using metal chelate chromatography. The purity was up to 95%.