幽门螺杆菌Catalas/GST融合蛋白的可溶性表达及凝血酶柱上切割GST标签  

Soluble expression of Catalase /GST fusion protein of helicobacter pylori and thrombin cleavage of GST tag on column

作　　者：李妍[1] 姜茵[1] 奚月[1] 何殿殿[1] 宁云山[1] 

机构地区：[1]南方医科大学生物技术学院生物治疗研究所,广州510515

出　　处：《现代预防医学》2012年第21期5614-5616,5619,共4页Modern Preventive Medicine

基　　金：国家自然科学基金面上项目(31070119);广东省自然科学基金自由申请项目(S2011010006070)

摘　　要：目的利用GST融合基因表达系统表达幽门螺杆菌Catalase/GST融合蛋白,并利用凝血酶柱上切割GST标签。方法将重组表达质粒Catalase/pGEX-4T-1转化大肠杆菌BL21(DE3)感受态中并用IPTG进行诱导表达,菌体经反复冻融、溶菌酶裂解及超声破菌进行裂解。采用谷胱甘肽琼脂糖树脂Glutathione Sepharose4B对Catalase/GST融合蛋白可溶性表达上清进行纯化,并同时利用凝血酶柱上切割GST标签,用鼠抗Catalase抗体对纯化产物进行Western blot鉴定。结果高效表达出相对分子质量约85kDa的Catalase/GST融合蛋白,以部分可溶性的形式表达,凝血酶柱上成功地切割了GST标签,Catalase蛋白能被鼠抗Catalase单克隆抗体识别。结论成功表达了Catalase/GST融合蛋白并柱上切割了GST标签,为深入研究Catalase的功能奠定了基础。OBJECTIVE To express Catalase /GST fusion protein by GST gene expression system and cleave GST-tag on column using thrombin.METHODS The recombinant expression plasmid Catalase /pGEX4T-1 was transformed into E.coli BL21（DE3） component 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 on column using thrombin.Purified Catalase was identified by anti-Catalase monoclonal antibody（mAb） using Western blot.RESULTS The fusion Catalase /GST was partly expressed in soluble form with relative molecular mass of 85kDa.Thrombin cleaved the GST tag on column and Catalase protein was recognized by mouse anti-Catalase mAb.CONCLUSION The recombinant Catalase /GST is successfully expressed and GST-tag is cleaved on column,which lays a foundation for further study on function of Catalase protein.

关 键 词：幽门螺杆菌 CATALASE 融合表达 GST标签 切割 

分 类 号：R34[医药卫生—基础医学]