Dok4和Dok5 PTB结构域融合蛋白的表达和纯化  

Expression and purification of the GST fusion protein of p62Dok PTB domain

作　　者：阎志勇[1] 吴爱群[1] 张勇[2] 由振东[2] 路长林[2] 周明明[3] 何成[2] 

机构地区：[1]郑州大学医学院解剖学教研室,河南郑州450052 [2]第二军医大学神经生物学教研室,上海200433 [3]纽约大学Mount Sinai医学院生理与生物物理学系,纽约10029

出　　处：《河南医学研究》2003年第2期100-103,共4页Henan Medical Research

基　　金：国家自然科学基金 ( 3 0 0 70 167);海外青年学者合作研究基金 ( 3 0 0 2 80 0 3 );国家 973项目 (G19990 5 40 0 0 )

摘　　要：目的 :制备Dok4和Dok5PTB结构域的GST融合蛋白。方法 :用PCR方法分别扩增编码Dok4、Dok5PTB结构域的cDNA ,并将其克隆入表达载体pGEX 4T 3中 ,转化大肠杆菌BL 2 1,构建成表达GST PTB融合蛋白的菌株。经IPTG诱导表达后 ,经Glutathion Sepharose 4B亲和层析柱纯化 ,用SDS PAGE进行分析 ,该融合蛋白的表达量超过菌体总蛋白的 2 5 %。结果 :获得重组的GST 4PTB和GST 5PTB融合蛋白 ,纯度在 95 %以上 ,产物得率约 75 %。结论 :成功制备高纯度Dok4和Dok5PTB结构域的GST融合蛋白 ,为进一步研究磷酸化酪氨酸结合结构域 (PTB)Objective: To produce the GST PTB domain fusion protein. Methods: The cDNA encoding p62Dok PTB domain was amplified by using PCR method, and cloned into pGEX 4T 3 vector. The expression plasmid was introduced into E.coli strain BL 21. The GST PTB fusion protein accounted for over 25% of the total bacterial protein after IPTG induction, it was purified to homogeneity from the cell lysates via affinity chromatography; and analyzed by SDS PAGE gel.Results: The recombinant GST PTB fusion proteins were prepared with high purity, more than 95%, and high recovery, about 75%. Conclusion: The established method for preparation large quantity recombinant GST PTB fusion proteins would promote the structure function analysis of the PTB domain of p62 Dok protein.

关 键 词：Dook4 DOK5 PTB结构域 融合蛋白 酪氨酸激酶受体 大肠杆菌 纯化 

分 类 号：Q51[生物学—生物化学]