金黄色葡萄球菌表面蛋白SdrE的表达、纯化及晶体生长

Expression，purification and crystallization of the surface protein SdrE from Staphylococcus aureus

摘要:

目的：对金黄色葡萄球菌表面蛋白质（serine-aspartate repeat，SdrE）进行克隆表达纯化及晶体培养，以期解析其三维结构，进而深入分析金黄色葡萄球菌感染的分子机制，为研发新的抗菌药物提供基础。方法：利用PCR技术扩增SdrE基因，构建重组质粒pW28-SdrE，并在大肠杆菌（Escherichia coli，E.coli）B834中表达，用Ni2+-NTA亲和层析柱和DEAE阴离子交换层析柱纯化SdrE蛋白质，再用Hiload Superdex 200凝胶层析柱分析其在溶液中的聚集状态，用Hampton试剂盒初筛晶体及棋盘法优化晶体。结果：（1）构建了重组质粒pW28-SdrE，并在E.coli B834中可溶性表达。（2） 获得了纯度较高（85%）的SdrE蛋白质，该蛋白质在溶液中以单体形式存在。（3）培养出晶形较好、衍射能力较强的SdrE蛋白质单晶。结论：利用经典的蛋白质纯化技术和晶体培养技术，可以获得衍射能力较强的SdrE单晶，为其三维结构的解析和基于结构的新型特异性抗菌药物的研发提供了重要的基础。

Abstract:

Objective：To determine the three dimensional structure and to analyze the molecular mechanism of staphylococcus aureus（S.aureus） infection for developing a new antibacterial drugs by cloning，expressing，purifying and crystallizing the surface protein SdrE from S.aureus. Methods：SdrE gene was amplified and cloned into the vector. Recombinant plasmid，pW28-SdrE，was trans－formed into E.coli B834 to express SdrE protein. SdrE protein was purified by Ni2+-NTA affinity chromatography column and DEAE anion-exchange chromatography column. State of aggregation in solution was analyzed by Hiload Superdex 200 gel flittration column. SdrE crystal was screened with Hampton kit and crystal was optimized with chessboard method. Results：（1）Recombinant plasmid pW28-SdrE was constructed and expressed in soluble form in E.coli B834 strain. （2）Puried SdrE exhibited as monomer in solution. （3）Single crystals of SdrE with good shape and diffraction capability were obtained. Conclusions：SdrE crystals can be grown by classic protein purification and crystal cultivation methods，which provides an important base for structural and functional study of SdrE in future and is necessary for developing new antibacterial drugs.