稻瘟菌Rac1蛋白的原核表达与纯化

Prokaryotic Expression and Purification of Rac1 Protein from Magnaporthe oryzae

Ras相关的C3肉毒素底物1(Rac1)是Rho族蛋白主要成员,在稻瘟菌(Magnaporthe oryzae)的侵染致病过程中发挥重要作用。本研究目的是采用结构生物学的方法进一步探究Rac1结构与功能以及与其他蛋白互作的机制,进一步阐释其致病机制。试验以稻瘟菌的cDNA为模板,根据Ras相关的C3肉毒素底物1基因(MoRac1)序列设计特异性引物进行PCR扩增,克隆了MoRac1基因并构建原核表达载体pHAT2-Rac1,异丙基硫代半乳糖苷(IPTG)诱导表达。SDS-PAGE检测与Western blot分析表明,pHAT2-Rac1在BL21(DE3)中表达,大小为25kD。通过亲和层析、离子交换与分子筛对重组蛋白进行纯化,获得了高纯度的目的蛋白。该蛋白的表达与纯化对其结构功能的研究奠定了基础。

Ras--related C3 botulinum toxin substrate l（Racl） is a maior member of the Rho--family pro- teins, and plays important role in infection and pathogenicity in Magnaporthe oryzae. Special primers were designed based on the sequences of codon of Racl. The gene of Racl was obtained by PCR amplifica- tion with the template of M. oryzae cDNA, and cloned into the prokaryotic expression vector pHAT2-- Racl, and induced by IPTG. SDS--PAGE detection and Western blot analysis showed the vector was ex- pressed in BL21（DE3） with protein about 25 kD. The recombinant protein was purified by affinity chroma- tography, ion exchange and gel filtration, then high purity target protein was obtained. The expression and purification of the protein established an important platform and basis for further study of the protein function.