幽门螺杆菌黏附素HpaA重组蛋白的表达、纯化及晶体生长

Expression, purification and crystallization of recombinant HpaA

目的构建幽门螺杆菌黏附素Hpa A的原核表达载体,并用纯化的重组Hpa A蛋白进行晶体培养,为其三维结构解析、基于结构的Hpa A抗原表位分析和黏附拮抗剂研究奠定基础。方法用生物信息学方法分析Hpa A蛋白序列二级结构并预测跨膜区,利用PCR技术扩增幽门螺杆菌标准株NCTC 26695编码Hpa A蛋白的hp0797基因非跨膜区片段,构建重组质粒并p ET22b(+)-r Hpa A,在大肠杆菌BL21(DE3)中IPTG诱导表达重组蛋白,经Ni离子亲和层析、阴离子交换层析、凝胶过滤层析纯化蛋白并分析其聚集状态。用Hampton Research结晶试剂盒搜索结晶条件,并系统优化,在上海同步辐射光源进行衍射实验。结果成功构建了重组质粒p ET22b(+)-r Hpa A,并在大肠杆菌BL21(DE3)中可溶性表达;重组Hpa A蛋白在溶液中以二聚体形式存在,SDS-PAGE分析单体相对分子质量约24 000,HPLC分析纯度达95%;培养出晶形较好的重组Hpa A蛋白单晶,大小约70μm×30μm×20μm,衍射分辨率2.03魡。结论利用经典的蛋白质表达、纯化技术和晶体培养技术,获得了衍射能力较强的重组Hpa A蛋白单晶,为其三维结构解析和基于结构的幽门螺杆菌疫苗研究提供了重要基础。

The objective of this study was to produce, purify and crystallize recombinant Helicobacter pylori adhesion A（Hpa A）. The hp0797 gene segment with removal of predicted transmembrane region was cloned from H. pylori NCTC 26695 and inserted into the p ET22b（＋） vector, which was subsequently transformed into Escherichia coli strain BL21（DE3）. The recombinant Hpa A（r Hpa A） protein was purified using affinity, anion-exchange and gel filtration chromatography. Initial crystallization conditions of purified r Hpa A were screened with Hampton Research screening kits followed by optimization for certain conditions. Diffraction analysis of crystals was conducted at Shanghai Synchrotron Radiation Facility（SSRF）. Hpa A was expressed as a soluble protein with molecular mass about 24 000. The purified protein exists as dimers in solution with purity higher than 95% as revealed from an HPLC analysis. Single crystals of r Hpa A with approximate dimensions of 70 μm× 30 μm× 20 μm were obtained, the best of diffracted X-ray to 2.03 A. This work established optimal methods for expression, purification and crystallization of r Hpa A, which serves as a good starting point for structural studies and structure-based immunoreactive epitope design.