断裂内含肽介导的蛋白纯化系统的构建

Construction of A Protein Purification System Mediated by Split Intein

DOI：10.3969/j.issn.1673-1689.2019.10.019

中文关键词: 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

英文关键词: split intein,self-cleavage,affinity chromatography medium,steric hindrance,cloning and expression,protein purification

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中文摘要:

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I_N为亲和配基的亲和层析介质和以I_C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I_{2+抑制试验，找到了最佳的断裂组合以及新的Zn²⁺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。}

英文摘要:

Molecular biology techniques are used to modify the N（I_N） and C（IC） fragment of Npu DnaE（Nostoc punctiforme） intein. An affinity chromatography medium with I_Nas 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 Zn²⁺inhibition experiment，we found the best combination of F and the new Znbinding site. The I_Naffinity 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.

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