【摘要】 幽门螺旋杆菌中的肿瘤坏死因子α诱导蛋白(Tipα)被鉴定为幽门螺旋杆菌致病感染中的新型致癌因子.Tipα通过NF-κB的激活诱导肿瘤坏死因子α(TNF-α)的大量表达,从而促使宿主的炎症反应以及肿瘤发生、发展的进程.Tipα的同源二聚体为其发挥生物学功能的活性形式,此二聚体以两个单体间N端半胱氨酸形成的二硫键(Cys25-Cys25与Cys27-Cys27)共价连接.Tipα(25-192)的基因克隆至载体pET22b中,并且在大肠杆菌菌株BL21(DE3)中以可溶形式高水平表达.重组蛋白经过Ni2+金属亲和层析、阳离子交换层析和凝胶阻滞层析进行分离纯化.Tipα蛋白样品分别通过悬滴和microbatch的方法进行结晶搜索和优化.母体和硒代晶体分别衍射到2.2和2.6,均属于C2空间群,并且具有相似的晶胞参数.母体晶体的晶胞参数为a=127.01,b=47.57,c=96.5,α=γ=90°,β=127.5°.更多还原

【Abstract】 Tipα(TNF-α-inducing protein) from Helicobacter pylori is identified as a new carcinogenic factor.Tipα induces high expression of TNF-α through NF-κB activation,thus promoting host inflammation and tumor progression.The homodimer of Tipα as its active form for functional performances is cross-linked by a pair of inter-molecular disulfide bridges(Cys25-Cys25 and Cys27 and Cys27).Tipα(25～192) was cloned into pET22b and expressed as soluble protein in E.coli strain BL21(DE3).Recombinant active Tipα was first purified through Ni2+-chelating chromatography,and then further purified by cation-exchange chromatography and size-exclusion chromatography to get the pure homodimer protein.Native Tipα and SeMet Tipα were crystallized and optimized using hanging drop and microbatch methods,with diffraction to 2.2 and 2.6,respectively.These crystals belonged to C2 space group with similar unit-cell parameters.The native protein crystal had unit-cell parameters a=127.01,b=47.57,c=96.5,α=γ=90°,β=127.5°.An attempt to solve the three-dimensional structure of this protein by MAD method is under way.更多还原