机构：
江南大学生物工程学院工业生物技术教育部重点实验室;江南大学粮食发酵工艺与技术国家工程实验室;
中文关键词：
Cfa断裂内含肽;;蛋白纯化;;亲和层析;;剪切;;无标签纯化
中文摘要：
应用Cfa断裂内含肽构建了一种新型的亲和纯化系统,并完成对目的蛋白无标签纯化。首先对Cfa断裂内含肽的IN片段与IC片段进行分子改造,通过研究在自由混合条件下的断裂情况,测定了其断裂速率。其次以改造后的IN片段作为配体与环氧活化的琼脂糖微球载体偶联,制备了IN亲和层析介质;IC片段作为亲和标签与目的蛋白GFP融合,并利用IN亲和层析介质实现对目的蛋白进行无标签纯化。结果表明,将IN与IC-GFP片段混合后,30s内即可断裂超过50%,4 h内即可完全断裂;IN亲和层析介质的静态载量为66.78 mg×m L~(-1),动态吸附载量约为30 mg×m L~(-1);纯化后的绿色荧光蛋白(GFP)纯度达到了96.7%,回收率为29.3%。Cfa断裂内含肽的应用为开发新型亲和纯化技术提供了一种潜在的方法。
英文篇名：
Application of chromatographic resins and affinity tag mediated by split intein
英文作者：
DU Ye-xing;ZHOU Ting-jun;CHEN Hao-ran;XIA Hai-feng;The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology,Jiangnan University;National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University;
英文摘要：
A new affinity purification system mediated by Cfa split intein was constructed, and the target protein which was tag-removed was purified in this work. IN and IC was first modified and the cleavage efficiency was measured. The IN segment of Cfa DnaE intein as an affinity ligand was then introduced to the epoxy-activated medium, and the IC segment as the cleavable tag was introduced to the target protein GFP. The results show that more than 50% of IC-GFP can be cleaved in free state after 30 seconds of incubation, and complete cleavage can be achieved after 4 hours. The static adsorption capacity was 66.78 mg×mL~(-1) and the dynamic adsorption capacity was 30 mg×m L~(-1). The purity of GFP reached 96.7% and the recovery was 29.3%. This study provides a potential method for the development of novel affinity technology.
英文关键词：
Cfa Dna E split intein;;protein purification;;affinity chromatography;;cleavage;;none-tagged
