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

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.