Cysteine-free non-canonical C-intein for versatile protein C-terminal labeling through trans-splicing.
Cysteine-free non-canonical C-intein for versatile protein C-terminal labeling through trans-splicing.
复制标题
不含半胱氨酸的非典型 C 内含肽，用于通过反式剪接进行多功能蛋白质 C 末端标记。
DOI：
10.1007/s00253-015-6796-6
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发表时间：
2015
影响因子：
5
通讯作者：
Meng Qing
中科院分区：
文献类型：
--
作者：
Dai Xudong;Xun Qijing;Liu Xiang-Qin;Meng Qing
蛋白质位点特异性标记是蛋白质研究和工程的有力手段，然而，新的和改进的标记方法是非常需要的。断裂内含肽催化蛋白质反式剪接反应，可用于酶促和几乎无缝的蛋白质标记。非经典的S11断裂内含肽已用于蛋白质C末端标记的早期方法;然而，其与靶蛋白融合的相对较大（~150 aa）的N-内含肽通常会阻碍蛋白质的表达、折叠和溶解度。为了解决这个问题，在这里，我们已经设计并证明了一种新的蛋白质C-末端标记的方法，首先工程化的功能性非典型的S1分裂内含肽，具有一个非常小的（12 aa）N-内含肽和半胱氨酸的C-内含肽。工程改造的RmaDnaB S1分裂内含肽被修饰为具有无半胱氨酸的C-内含肽，同时仍然保留其强大的反式剪接功能，这允许C-前体中的C-内含肽具有单个半胱氨酸，以便与所需的标记基团容易和特异性地连接。所得到的新的和普遍有用的方法具有两个独特的优点：（1）极小的（12 aa）N-内含肽，其必须与靶蛋白的C末端融合，不太可能阻碍蛋白质的表达、折叠和溶解性;和（2）C-内含肽中的单个半胱氨酸可以使用市售试剂容易地连接到各种标记或修饰基团。
Site-specific protein labeling are powerful means of protein research and engineering; however, new and improved labeling methods are greatly needed. Split inteins catalyze a proteintrans-splicing reaction that can be used for enzymatic and nearly seamless protein labeling. Non-canonical S11 split intein has been used in an earlier method of protein C-terminal labeling; however, its relatively large (~150 aa) N-intein fused to the target protein often hindered protein expression, folding, and solubility. To solve this problem, here, we have designed and demonstrated a new method of protein C-terminal labeling, by first engineering a functional non-canonical S1 split intein that has an extremely small (12 aa) N-intein and a cysteine-free C-intein. An engineeredRmaDnaB S1 split intein was modified to have a cysteine-free C-intein, while still retaining its robusttrans-splicing function, which permitted the C-extein in a C-precursor to have a single cysteine for easy and specific linkage with desired labeling groups. The resulting new and generally useful method has two unique advantages: (1) The extremely small (12 aa) N-intein, which must be fused to the C terminus of the target protein, is less likely to hinder the protein expression, folding, and solubility; and (2) the single cysteine in the C-extein may be readily linked to a variety of labeling or modification groups using commercially available reagents.