龋齿致病菌变形链球菌蛋白Smu.260的制备、结晶及初级晶体学分析(英文)

Protein Preparation, Crystallization and Preliminary X-ray Crystallographic Analysis ofSmu.260 From Streptococcus mutans——a Cariogenic Dental Pathogen

变形链球菌(Streptococcusmutans) 是最主要的龋齿致病菌,其基因Smu.260编码一个约23 ku (200个氨基酸) 的蛋白质. Smu.260的DNA片段被克隆到表达载体pET28a后在大肠杆菌BL21(DE3)菌株中表达得到很好的产量. 产物Smu.260蛋白通过Ni2+亲和柱和分子筛两步法纯化,并发现纯化后的蛋白以两种形式存在,二聚体(约46 ku) 和四聚体,前者呈亮黄色,后者无色。采用悬滴气象扩散法得到了二聚体形式的晶体. 晶体的X射线衍射分辨率达到2.3 魡,晶体属正交空间群P212121,晶格参数为a=89.88 魡, b=90.91 魡, c=105.17 魡. 晶胞不对称单元内估计含有一个二聚体,溶剂含量为53%.

Smu.260 encodes a putative protein of 200 residues in Streptococcus mutans, a primary pathogen for human dental caries. Smu.260 was cloned into expression vector pET28a and expressed in good amount from the E.coli strain BL21 (DE3). Smu.260 protein was purified to homogeneity in a two-step procedure of Ni(2+) chelating and size exclusion chromatography. The purified protein exists in two forms, a dimer form about 46 ku with yellow color and a tetramer form without apparent color. Crystals were obtained from the dimer protein by hanging-drop vapor-diffusion method. The crystals diffracted to about 2.3 angstrom resolution and belong to orthorhombic space group P2(1)2(1)2(1) with cell dimensions of a = 89.88 angstrom, b = 90.91 angstrom, c = 105.17 angstrom. The asymmetric unit is expected to contain two dimers with solvent content of 53%.