大肠杆菌中重组多肽Tat-PFKMVV的分离?表达和纯化

目的:构建重组表达载体pED-Tat-PFKMVV,在大肠杆菌中表达并通过纯化以获得重组多肽Tat-PFKMVV? 方法:用PCR的方法扩增出Tat-PFKMVV基因,插入含T7启动子的pED表达载体,构建重组表达载体 pED-Tat-PFKMVV,重组表达载体转化至大肠杆菌并表达,表达产物经洗涤?乙醇沉淀,酸水解和等电点沉淀纯化目的多肽?结果:克隆的Tat-PFKMVV基因与Genbank 对比,二者同源性为100%,酸水解位点正确?大肠杆菌表达的融合蛋白产物AnsB-C-Tat-PFKMVV经SDS-PAGE电泳显示相对分子量为17 000,质谱显示最终纯化产物Tat-PFKMVV蛋白相对分子量为2 659?通过GST-pull down实验证明Tat-PFKMVV蛋白能在体外解开神经元型一氧化氮合酶与肌肉型磷酸果糖激酶的偶联?结论:成功构建了表达AnsB-C-Tat-PFKMVV融合蛋白的原核表达体系,纯化得到了较高纯度的目的多肽Tat-PFKMVV?通过此过程建立了一套简单可行且高效的融合蛋白表达?水解?分离与纯化的方法?

Cloning,expression and purification of a recombinant Tat-PFKMVV peptide in Escherichia coli

Objective:We sought to construct the recombinant expression vector pED-Tat-PFKMVV to express and purify the recombinant peptide Tat-PFKMVV. Methods: The gene fragment encoding a recombinant peptide Tat-PFKMVV was constructed by PCR and ligated into expression vector for recombinant expression in a T7 RNA polymerase-based expression system. The recombinant plasmid vector was transduced into E.coli. The expressed recombinant peptide was partially purified by the wash solution of Tris-Cl/TritonX-100,alcohol precipitation,acid hydrolysis and isoelectric point precipitation. Results:The correct recombinant DNA coding fragment was validated by DNA sequencing. The 17 000 AsnB-C-Tat-PFKMVV fusion protein was highly expressed in E.coli BL21 transduced by pED-Tat-PFKMVV. The analysis of the purified Tat-PFKMVV by mass spectrometry indicated that its molecular weight was 2 659. GST-pull down experiments showed that Tat-PFKMVV in vitro disturbed the coupling of nNOS and PFK-M. Conclusion:The experiment constructed the recombinant expression vector of AnsB-C-Tat-PFKMVV,and expressed and purified the peptide Tat-PFKMVV. The novel preparation method is a potentially useful strategy for the large-scale preparations of bioactive peptides due to its high yield and simple steps.