Department of Chemistry, Frick Chemistry Laboratory, Princeton University, Princeton, NJ 08544.
Department of Chemistry, Frick Chemistry Laboratory, Princeton University, Princeton, NJ 08544
Proc Natl Acad Sci U S A. 2020 Jun 2;117(22):12041-12049. doi: 10.1073/pnas.2003613117. Epub 2020 May 18.
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 -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 HP1α 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.
分裂内含肽是蛋白质化学修饰的专用分子支架。尽管这些多肽连接酶在体外应用中非常有效，但它们尚未用于活细胞中蛋白质的半合成。在这里，我们对天然分裂内含肽 VidaL 进行了生物化学和结构表征。我们表明，这种分裂内含肽具有已知最短的 N 端片段，可在多种条件下支持快速有效的蛋白质剪接，从而能够在体外和哺乳动物细胞中进行修饰蛋白的半合成。该蛋白质工程系统的实用性通过无痕组装多结构域蛋白得到了说明，这些蛋白的生物物理特性使得它们无法在单个表达系统中表达，并且可以在活细胞中半合成双重翻译后修饰的组蛋白蛋白。我们还利用 VidaL 的结构域交换功能在活细胞中实现核蛋白 HP1α 的同时修饰和转位。总的来说，我们的研究强调了 VidaL 系统作为一种具有时空控制的精确化学修饰细胞蛋白的工具。
