水稻小GTP蛋白OsRab5A的表达、纯化和GTP结合分析

小GTP结合蛋白是一类在细胞内的运输过程中重要作用的蛋白。它们通过结合子GTP而激活 ,当GTP被水解为GDP后处于非活性状态。哺乳动物中的研究表明 ,Rab5A蛋白是细胞内的形成早期内吞体 (earlyendosome)的限速因子。证实了所克隆的水稻rab5A基因编码的蛋白具有GTP结合功能。将Osrab5A基因的编码序列按正确读码框重组到pGEX4T1载体中 ,转化大肠杆菌BL2 1(DE3) ,电泳判定插入片段大小无误后 ,进一步测序鉴定其读码也无误。将该克隆命名为pG_5AE。以IPTG诱导 pG_5AE所编码的融合蛋白GST_OsRab5A的表达。SDS_PAGE电泳与无外源质粒和表达谷胱甘肽硫转移酶 (GST)的对照相比 ,得到了预计大小的融合蛋白。以GSTrapTM亲和柱纯化融合蛋白。在不同的泳道分离纯化的融合蛋白和作为对照的含GST以及无外源蛋白的细菌总蛋白 ,电转移到硝酸纤维膜上。将该膜与α_3 2 PGTP温育 ,洗膜 ,放射自显影后 ,获得了融合蛋白结合GTP的结果 ,这表明在原核中表达出的水稻Rab5A蛋白能在体外结合GTP。

Prokaryotic Expression, Protein Purification and GTP-binding Assay of Rice rab5A Gene

Osrab5A , a rab5A gene cloned from Rice ( Oryza sativa L.), encodes a putative Rab protein with high similarity to mammalian Rab5A, which was localized on the early endosome and in the cytoplast. Rab5A was identified in mammalian cells as a rate_limiting factor for homotypic endosome fusion. The complete coding sequences of Osrab5A cDNA clone were amplified and inserted into the prokaryotic expression vector pGEX4T1. The resulting recombinant plasmid with correct reading frame, pG_5AE, was introduced into E coli. BL21 (DE3),The GST tagged fusion protein, GST_OsRab5A was induced by IPTG induction and purified on a GSTrapTM column. The GTP_binding assay of this protein showed the GST tagged OsRab5A protein has GTP_binding ability in vitro.