非洲栖热腔菌(Thermosipho africanus)纤维寡糖磷酸化酶的结晶及衍射分析

Analysis of crystallization and X-ray crystallographic of the cellodextrin phosphorylase from Thermosipho africanus

为探究非洲栖热腔菌(Thermosipho africanus)纤维寡糖磷酸化酶TaCDP底物宽泛性的结构基础,通过Ni离子亲和层析-阴离子交换层析-凝胶过滤层析3步法纯化TaCDP,采用气相扩散法筛选TaCDP晶体,利用X-射线晶体学方法对TaCDP的晶体结构进行了研究。结果表明,纯化后TaCDP蛋白的纯度高,聚集状态均一,在2.2 mol·L^-1(NH 4)2HPO 4,0.1 mol·L^-1 CHES-NaOH,pH值9.2,10.0 g·L^-1 polyvinylpyrrolidone K15条件下筛选到TaCDP与纤维三糖底物的复合物晶体。该晶体经同步辐射收集衍射数据后分辨率可达0.28 nm,晶体空间群属于P4 122,晶胞参数为a=b=9.928 nm,c=56.251 nm,表明1个不对称单位有2个TaCDP分子。

To elucidate the structural basis of the broad substrate specificity of the cellodextrin phosphorylase from Thermosipho africanus(TaCDP),the TaCDP was purified with metal chelate afnity chromatography-anion exchange chromatography-size exclusion chromatography.Crystallization of TaCDP was performed by the vapour diffusion method and the crystal structures were analysed with the X-ray crystallographic method.The results showed that the purified recombinant TaCDP protein had high purity and uniform aggregation state.Composite crystals of TaCDP and fiber trisaccharide substrates were obtained under the conditions of 2.2 mol·L^-1(NH 4)2HPO 4,0.1 mol·L^-1 CHES-NaOH pH 9.2 and 10.0 g·L^-1 polyvinylpyrrolidone K15.The resolution of the crystal could reach 0.28 nm after collecting diffraction data by synchrotron radiation.The crystals belonged to space group P4 122,with unit-cell parameters a=b=9.928 nm,c=56.251 nm,suggesting that there are two TaCDP molecules in one asymmetric unit.