巢蛋白样结构域蛋白的表达?纯化与鉴定

目的:重组构建原核表达载体以正确表达巢蛋白样结构域(nidogen domains,NIDO)蛋白,并进一步纯化与鉴定?方法:PCR法拼接NIDO基因,经T-A连接亚克隆至pMD19-T载体,测序鉴定?采用质粒抽提?纯化?酶切?连接?感受态细胞制备和转化等技术,构建并鉴定原核表达载体pGEX-4T-1-NIDO?药物诱导方式诱导NIDO-GST融合蛋白的表达?对表达产物进行分离,用SDS-PAGE检测上清与包涵体沉淀中目的蛋白的表达量?对包涵体进行变性和透析复性后,进行GST亲和层析纯化?采用SDS-PAGE和质谱分析,鉴定纯化的目的蛋白?结果:测序证实了NIDO基因的正确合成和pMD19-T-NIDO克隆载体的构建?原核表达系统pGEX-4T-1-NIDO构建成功,在大肠杆菌BL21中被诱导,获得高表达量的N-末端带有GST标签序列的重组融合表达蛋白(30%)?超声裂菌法分离的结果显示,目的蛋白在菌体沉淀中以包涵体形式居多?将包涵体变性?复性后用GST亲和层析纯化,纯化蛋白> 80%并经质谱鉴定证实?结论:成功构建原核表达载体pGEX-4T-1-NIDO,能正确表达NIDO-GST融合蛋白,GST亲和层析对于NIDO-GST融合蛋白有较好的纯化效果?

Cloning,expression and purification of the important NIDO Domain protein

Objective: To construct recombinant prokaryotic expression vector to express the important NIDO Domain protein and further purify and identify the NIDO protein. Methods: The NIDO gene was subcloned into pMD19-T vector for DNA sequencing analysis. Plasmid extraction,purification,enzyme,connection,competent cell preparation and transformation technology were performed to construct and identify the prokaryotic expression vector pGEX-4T-1-NIDO. The protein NIDO-GST was expressed in E.coli-BL21 as a fusion protein induced by IPTG. The expression products were isolated,and the target proteins of supernatants and inclusion precipitation were detected by 12%SDS-PAGE. After denaturation and renaturation of inclusion body,the expression products were purified by GST-affinity chromatography. The purity of NIDO-GST fusion protein was identified by 12% SDS-PAGE and the Mass-Spectrographic analysis. Results: NIDO coding region was cloned into pMD19-T vector and the sequence was confirmed. Prokaryotic expression system of pGEX-4T-1-NIDO was constructed successfully,and were induced in E.coli BL21,to obtain high expression of N-terminal with GST tag sequence of the recombinant fusion protein(NIDO-GST) expression(30%). Target protein in bacterial precipitation was in the form of inclusion body mostly after Ultrasonic crack bacteria. After denaturation and renaturation of the inclusion body and further GST affinity chromatography purification,the purity of protein NIDO-GST was more than 80% and confirmed by mass spectrum identification. Conclusion: The purified fusion protein NIDO-GST was constructed successfully by the prokaryotic expression and affinity chromatography.