v-Src蛋白在大肠杆菌中的高效表达、纯化及其活性分析

 目的表达高纯度具有激酶活性的Src蛋白,为抗肿瘤药物研究提供蛋白质靶标。方法利用高效表达质粒pGEX-KT克隆v-src全基因,诱导表达得到的GST-Src融合蛋白经GlutationeSepharose4B亲和层析柱纯化后,采用Westernblotting和以polyGlu:Try(4:1)为底物的ELISA法鉴定其免疫原性和酪氨酸激酶活性。结果GST-Src蛋白表达量约占菌体总蛋白的30%,其中包涵体与可溶性蛋白的表达量分别约13.5和2mg·L^-1,纯度分别约98%和85%,比活约1.13和0.62nmol·min^-1·mg^-1。结论本法表达的GST-Src融合蛋白表达量高、纯化效果好,且具有较高的激酶比活。

High efficient expression, purification and activity analysis of v-Src from Escherichia coli expression system

OBJECTIVE To clone and express the active v-Src protein, provide a PTK source for screening anti-tumor agents via Src inhibitors. METHODS The pGEX-KT vector was employed in the cloning of v-src gene. The expressed protein was purified, using GSH Sepharose 4B, and its characteristics were identified by Western blot analysis and ELISA technique. RESULTS The amount of GST-Src protein expressed from BL21 (DE₃)pLysS culture exceeded 30% in relation to the coproteins. In 1 L medium culture, approximately 2 mg GST-Src fusion proteins with 85% purity and 13.5 mg GST-Src inclusion bodies with 98% purity were obtained. More-over, their tyrosine kinase ac-tivies reached about 0.62 nmol·min^-1·mg^-1 and 1.13 nmol·min^-1·mg^-1, respectively. CONCLUSION The GST-Src fusion protein expressed in this study was of high product, easy to be purified and prefer-able in the kinase activity.