利用串联亲和纯化技术分离大肠杆菌GroEL蛋白复合物

Application of tandem affinity purification technique for separation of GroEL complexes in Escherichia coli

肠出血性大肠杆菌O157:H7是一种重要的致病菌,加深其致病机理的基础研究将为相关疫苗研究及疾病控制等提供新的思路和依据。串联亲和纯化(TAP)技术是最近发展的分离纯化天然状态蛋白质复合物进而研究蛋白质相互作用的新方法。用我们自己构建的原核表达串联亲和标签载体,在大肠杆菌O157:H7中表达了标签融合蛋白GroEL-TAP,建立了非变性条件下制备蛋白复合物的方法,并且对串联亲和纯化过程中的相关实验条件进行了探索和优化,最终得到了高纯度的GroEL-TAP与天然GroEL形成的嵌合型多聚体复合物。这表明我们建立的串联亲和纯化技术能高度特异地纯化靶蛋白参与形成的复合物,为后续寻找O157:H7中毒力蛋白参与形成的复合物奠定了实验基础。

Enterohemorrhagic Escherichia coli O157：H7 is a kind of important pathogenic bacterium, and deeper studies of molecular pathogenesis will contribute better to related vaccine research and disease control. Tandem affinity purification （TAP） technique is a kind of new method which was recently developed and used to separate and purify native protein complexes and so on to study interactions between proteins. In this study, a prokaryotic TAP expression vector constructed by our laboratory was successfully used to express fusion protein GroEL-TAP in Enterohemorrhagic Escherichia coli O157：H7. An efficient method for preparation of native protein complexes was developed and all experimental parameters of tandem affinity purification were optimized. The highly pure protein complexes composed of both tagged GroEL-TAP and natural GroEL were obtained with our expression and purification system. These results showed that our TAP system worked well to specifically purify protein complex participated with target protein in Enterohemorrhagic Escherichia coli O157：H7. This study provided satisfactory experimental base for follow-on works on identifying virulence protein participated complexes.