Cyclic green fluorescent protein produced in vivo using an artificially split PI-PfuI intein from Pyrococcus furiosus
Cyclic green fluorescent protein produced in vivo using an artificially split PI-PfuI intein from Pyrococcus furiosus
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DOI：
10.1074/jbc.m011639200
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发表时间：
2001-05-11
影响因子：
4.8
通讯作者：
Plückthun, A
中科院分区：
文献类型：
--
作者：
Iwai, H;Lingel, A;Plückthun, A
以一种新的方法在体内使用来自Furiosus Pi-pfui的内含子产生环状蛋白，通过在内含子结构域中引入新的末端来创建前体蛋白的环状排列。用该方法对绿色荧光蛋白(GFP)进行了毫克量级的体内环化。与其他使用整合素的体外或体内环化方法不同，分离的线性或聚合物种没有副产物。环化的GFP在化学变性时以线状形式的一半速率展开，并在7M盐酸胍中需要>；2天，直到残留的快速折叠阶段(与持久的顺式脯氨酸一致)消失。环状GFP可能成为一种新的工具，用于研究末端和骨架拓扑结构在体内和体外蛋白质降解和转位等各种生物学过程中的作用。
A cyclic protein was produced in vivo using the intein from Pyrococcus furiosus PI-PfuI in a novel approach to create a circular permutation of the precursor protein by introducing new termini in the intein domain. Green fluorescent protein (GFP) was cyclized with this method in vivo on milligram scales. There was no by-product of linear or polymerized species isolated, unlike with other in vitro or in vivo cyclization methods utilizing inteins. Cyclized GFP unfolded at half the rate of the linear form upon chemical denaturation and required >2 days in 7 M guanidine hydrochloride until a residual fast folding phase (consistent with a persistent cis-proline) had disappeared. Cyclic GFP might become a novel tool for studying the role of termini and backbone topology in various biological processes such as protein degradation and translocation in vivo as well as in vitro.