新型断裂内含肽介导的可控性C端断裂(英文)  

Non-canonical Split Intein Based Controllable C-cleavage

作　　者：齐兴梅[1] 林瑛[1] 孟清[1] 

机构地区：[1]东华大学生物科学与技术研究所,上海201620

出　　处：《中国生物化学与分子生物学报》2011年第10期956-960,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：Supported by National Natural Science Foundation of China(No.30800186 and 31070698);National High Technology Research and Development Program of China(863 Program,No.2006AA03Z451);Shanghai Key Project of Basic Program(No.10JC1400300);Ph.D. Programs Foundation of Ministry of Education of China(No.200802551026);Special Funds of Basic Scientific Research Operating Expenses from Chinese Universities(No.11D10530)~~

摘　　要：内含肽介导的蛋白质断裂被广泛地应用于蛋白质纯化、连接和环化.但目前的方法都是用传统的连续的内含肽来介导蛋白质断裂反应,因而往往存在自发性断裂、产率低等问题.本实验选择3个S1型新型断裂内含肽Ter ThyX、Ssp GryB和Rma DnaB来实现蛋白质断裂反应的可控性.在可控性C端断裂反应中,S1型断裂内含肽的C端片段(IC)与硫氧还蛋白(T)融合作为前体蛋白,加入化学合成的Ssp DnaB S1型断裂内含肽的N端小肽与二硫苏糖醇(DTT)共同诱导C端断裂反应.结果表明,该小肽可以诱导这3个不同的S1型断裂内含肽的前体蛋白发生C端断裂反应.该方法为利用内含肽C端断裂介导的蛋白质纯化提供了更多的选择,并为内含肽的结构与功能的关系研究提供了有用的线索.Intein-based protein cleavage has been used in protein purification,peptide ligation and cyclization.However,the existing methods employing conventional contiguous inteins often result in spontaneous cleavage and cause reduced yields of the desired protein products.Here we report a controllable cleavage strategy using three engineered S1 split-inteins,Ter ThyX,Ssp GryB and Rma DnaB.In this controllable C-cleavage design,the C-terminus of a S1-IC sequence was fused with thioredoxin to form a precursor protein,and a synthetic S1-IN peptide from the Ssp DnaB S1 split-intein was used together with DTT to trigger the cleavage at the C-terminus of the IC,where this peptide can trigger C-cleavage in all three inteins.This approach provided more options for protein purification using intein C-cleavage without spontaneous cleavage and might be useful to the studies of the intein structurefunction relations.

关 键 词：内含肽 断裂内含肽 蛋白质剪接 可控性断裂 

分 类 号：Q71[生物学—分子生物学]