摘要
以Npu DnaE(Nostoc punctiforme)内含肽为研究对象,使用分子生物学方法对内含肽的N端结构(IN)和C端结构(IC)进行改造,构建了以IN为亲和配基的亲和层析介质和以IC为自断裂亲和标签的融合蛋白表达体系,形成了由断裂内含肽介导的新型蛋白纯化系统。通过构建3种IN亲和配基片段以及2种IC-GFP融合蛋白片段,研究了空间位阻对融合蛋白断裂速率的影响;通过6种组合的体外断裂及Zn^2+抑制试验,找到了最佳的断裂组合以及新的Zn^2+结合位点。利用N3亲和配基片段C末端的Cys,制备了IN亲和层析介质并对其纯化效果进行了研究,成功获得高纯度GFP蛋白。
Molecular biology techniques are used to modify the N(IN)and C(IC)fragment of Npu DnaE(Nostoc punctiforme)intein.An affinity chromatography medium with IN as the affinity ligand and a fusion protein expression system with IC as self-cleavage affinity tag were constructed.Finally,a protein purification system mediated by split intein was developed.The effect of steric hindrance on the C-terminal cleavage rate of fusion protein was studied by constructing 3 kinds of IN affinity ligands and 2 kinds of IC-GFP fusion proteins.Through the combination of in vitro cleavage and Zn^2+inhibition experiment,we found the best combination of F and the new Zn^2+binding site.The IN affinity chromatography media were successfully prepared by using cysteine at the C-terminal of N3 affinity ligands.The purification effect of it was studied,and the high purity GFP protein was obtained successfully.
作者
王玉君
王姝婧
杜夜星
冯利利
夏海锋
WANG Yujun;WANG Shujing;DU Yexing;FENG Lili;XIA Haifeng(School of Biotechnology,Jiangnan University,Wuxi 214122,China;National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214122,China)
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2019年第10期135-143,共9页
Journal of Food Science and Biotechnology
基金
江苏省自然科学基金项目(BK20151123)
关键词
断裂内含肽
自我断裂
亲和层析介质
空间位阻
克隆表达
蛋白纯化
split intein
self-cleavage
affinity chromatography medium
steric hindrance
cloning and expression
protein purification
