Live-cell protein engineering with an ultra-short split intein
Live-cell protein engineering with an ultra-short split intein
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DOI：
10.1073/pnas.2003613117
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发表时间：
2020-06-02
影响因子：
11.1
通讯作者：
Muir, Tom W.
中科院分区：
文献类型：
--
作者：
Burton, Antony J.;Haugbro, Michael;Muir, Tom W.
断裂内含肽是蛋白质化学修饰的特殊分子支架。尽管这些多肽连接酶在体外应用中是有效的，但尚未用于活细胞中蛋白质的半合成。在这里，我们对自然分裂的内含肽VidaL进行了生物化学和结构表征。我们表明，这种分裂内含肽，其特征是已知的最短的N-末端片段，支持快速和有效的蛋白质反式剪接在一系列条件下，使半合成的修饰蛋白质在体外和哺乳动物细胞。这种蛋白质工程系统的效用说明通过无痕组装的多结构域蛋白质，其生物物理特性使它们不兼容的一个单一的表达系统，以及通过半合成的双postconstitutionally修饰的组蛋白在活细胞中。我们还利用VidaL的结构域交换功能来实现活细胞中核蛋白HP 1 α的同时修饰和易位。总的来说，我们的研究突出了VidaL系统作为一种工具，用于对细胞蛋白质进行精确的化学修饰，并进行空间和时间控制。
Split inteins are privileged molecular scaffolds for the chemical modification of proteins. Though efficient for in vitro applications, these polypeptide ligases have not been utilized for the semisynthesis of proteins in live cells. Here, we biochemically and structurally characterize the naturally split intein VidaL. We show that this split intein, which features the shortest known N-terminal fragment, supports rapid and efficient protein trans-splicing under a range of conditions, enabling semisynthesis of modified proteins both in vitro and in mammalian cells. The utility of this protein engineering system is illustrated through the traceless assembly of multidomain proteins whose biophysical properties render them incompatible with a single expression system, as well as by the semisynthesis of dual posttranslationally modified histone proteins in live cells. We also exploit the domain swapping function of VidaL to effect simultaneous modification and translocation of the nuclear protein HP1 alpha in live cells. Collectively, our studies highlight the VidaL system as a tool for the precise chemical modification of cellular proteins with spatial and temporal control.