α-OGG1与ACO2蛋白的表达纯化及其相互作用鉴定

Expression and purification of α-OGG1 and ACO2 in vitro and interaction identification of both proteins

[目的]原核表达纯化获得可溶性α-OGG1,ACO2蛋白并初步鉴定其相互作用.[方法]将α-OGG1,ACO2基因构建到pGEX-4T-2-TEV和pET-28a-TEV表达载体上,转化至BL21(DE3)大肠杆菌.IPTG诱导表达融合蛋白,通过Ni柱和GST柱亲和层析法纯化目标融合蛋白;采用SDS-PAGE检测表达产物;采用GST-Pulldown方法鉴定蛋白与蛋白的相互作用;采用双质粒共转化,表达纯化α-OGG1,ACO2蛋白复合体.[结果]成功克隆并构建了α-OGG1及其N-半段、C-半段基因和ACO2基因的原核表达载体,并转化至大肠杆菌表达.通过Ni柱和GST柱亲和层析法得到可溶性表达的6×His_α-OGG1(1-326)和GST_ACO2(29-780)蛋白,SDS-PAGE检测结果表明2个蛋白条带均与理论大小相符.GSTPulldown实验结果证实α-OGG1与ACO2直接相互作用.通过共转化、表达纯化得到了α-OGG1与ACO2的复合物.[结论]采用原核表达和亲和层析纯化得到了可溶性6×His_α-OGG1,GST_ACO2融合蛋白及两者的复合体,并初步证实α-OGG1与ACO2存在直接相互作用.

OBJECTIVETo gain the solubleα-OGG1 and ACO2 in vitro through prokaryotic expression followed by purification and identify the interaction of both proteins.METHODS Bothα-OGG1 and ACO2 genes were constructed into the prokaryotic expression vectors pGEX-4T-2-TEV and pET-28a-TEV.Then each recombinant vector was transformed into E.coli BL21（DE3）,and the target recombinant proteins were induced by the addition of IPTG.The target proteins were obtained by the affinity chromatography system including Ni or GST columns.The SDS-PAGE was used to detect the expressed products.The GST-Pulldown assay was utilized to identify the protein-protein interaction.Finally the complex ofα-OGG1 and ACO2 were co-expressed and purified by the transformation of two recombinant plasmids into E.coli BL21（DE3）.RESULTS The recombinant prokaryotic expression vectors ofα-OGG1,its half in N terminal,its half in C terminal and ACO2 were constructed and transformed into E.coli BL21（DE3）successfully.The soluble 6× His_α-OGG1（1-326）and GST_ACO2（29-780）were gained by Ni and GST column affinity chromatography,respectively.The results of SDS-PAGE showed that each protein band was in accordance with its theoretical size.The GST-Pulldown assays testified that a direct interaction existed between α-OGG1 and ACO2.Finally,the complex ofα-OGG1 and ACO2 was obtained by transformation of the two recombinant plasmids into E.coli BL21（DE3）followed by co-expression and purification.CONCLUSION The soluble 6× His_α-OGG1,GST_ACO2 and the complex of both fusion proteins are expressed in prokaryotic expression and purified by the affinity chromatography.The direct interaction betweenα-OGG1 and ACO2 is preliminarily demonstrated.