Protein Scaffold-Activated Protein Trans-Splicing in Mammalian Cells

Conditional protein splicing is a
powerful biotechnological tool
that can be used to rapidly and post-translationally control the activity
of a given protein. Here we demonstrate a novel conditional splicing
system in which a genetically encoded protein scaffold induces the
splicing and activation of an enzyme in mammalian cells. In this system
the protein scaffold binds to two inactive split intein/enzyme extein
protein fragments leading to intein fragment complementation, splicing,
and activation of the firefly luciferase enzyme. We first demonstrate
the ability of antiparallel coiled-coils (CCs) to mediate splicing
between two intein fragments, effectively creating two new split inteins.
We then generate and test two versions of the scaffold-induced splicing
system using two pairs of CCs. Finally, we optimize the linker lengths
of the proteins in the system and demonstrate 13-fold activation of
luciferase by the scaffold compared to the activity of negative controls.
Our protein scaffold-triggered conditional splicing system is an effective
strategy to control enzyme activity using a protein input, enabling
enhanced genetic control over protein splicing and the potential creation
of splicing-based protein sensors and autoregulatory systems.