幽门杆菌Catalase/GST融合蛋白的表达、标签切除及鉴定

旨在利用GST融合基因表达系统表达幽门螺杆菌Catalase融合蛋白,并利用凝血酶切除GST标签.将重组质粒Catalase/pGEX-4T-1转化大肠杆菌BL21( DE3)感受态中,用IPTG进行诱导表达,菌体经反复冻融、溶菌酶裂解及超声破菌后,Catalase/GST融合蛋白以部分可溶性的形式表达在上清中.采用谷胱甘肽琼脂糖树脂Glutathione Sepharose 4B对其进行纯化,得到Catalase/GST融合蛋白,再用凝血酶进行GST标签的切除,所得产物进行Western blotting鉴定.高效表达出Catalase/GST融合蛋白的相对分子质量约85 kD,凝血酶成功地切除了GST标签,Western blotting证实Catalase蛋白能被鼠抗Catalase单克隆抗体识别.

Expression, Tag Cleavage and Identification of Catalase/GST Fusion Protein of Helicobacterpylori

It was to express Catalase-GST fusion protein by GST gene expression system and cleave GST-tag using thrombin.The recombinant Catalase/pGEX4T-1 plasmid was transformed into E.coli BL21(DE3) compenent cell and induced by IPTG.The bacterial sediment was lysed by repeating freezing and thawing,lysozyme lysis and ultrasonication.Catalase/GST fusion protein was purified by Glutathione Sepharose 4B and cleaved the GST-tag using thrombin.Then Catalase was identified by anti-Catalase monoclonal antibody using Western blotting.Results showed that the fusion Catalase/GST was partly expressed in soluble form with relative molecular mass of 8.5 kD.After cleavage of GST tag,Catalase protein was recognized by mouse anti-Catalase monoclonal antibody by Western blotting.The recombinant Catalase is successfully expressed and purified,which lay a foundation for further study on function of Catalase protein.