幽门螺旋杆菌肿瘤坏死因子α诱导蛋白活性分子的构建、结晶及初步晶体学研究

Crystallization and Preliminary Crystallographic Studies of Active TNF-α-Inducing Protein From Helicobacter Pylori

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°.

Tipα （TNF-α-inducing protein） from Helicobacter pflori is identified as a new carcinogenic factor. Tipα induces high expression of TNF-α through NF-KB 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 Ni^2＋-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 2.2h and 2.6h, respectively. These crystals belonged to C2 space group with similar unit-cell parameters. The native protein crystal had unit-cell parameters a=127.01h,6=47.57A,c=96.5A,α=γ=90°,β=127.5°. An attempt to solve the three-dimensional structure of this protein by MAD method is under way.