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Live-cell protein engineering with an ultra-short split intein

Significance Current technologies for the semisynthesis of modified proteins are optimized for in vitro reactions and are not easily transferable to more biologically relevant systems such as live cell culture. Split inteins have been a leading tool for protein chemists ever since their discovery, but as of yet have not been used broadly in live cells. Using the shortest naturally occurring split intein, VidaL, we report examples of traceless protein modification in live cells. We expect that the robust splicing activity and synthetic accessibility of VidaL will find broad utility within chemical and cellular biology. 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 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.