(D) Induction of splicing between EE and LZA split inteins of varying GS linker lengths by a protein scaffold. Presence of the scaffold results in the splicing and activation of firefly luciferase. (C) Recombinant inteins containing noncomplementary coiled-coil domains CC-1 and CC-2 associate only in complex with a protein scaffold containing complementary CCs, CC-1′ and CC2′.
Proteins tested are in boldface, and those connected by lines are expected to interact. (B) Schematic representation of the expected complementary CC binding for each synthetic scaffold and CC-intein/extein pair. (A) Schematic representation of the expression system used to test conditional splicing of CC-intein/extein fusions in response to CC scaffolds. Protein scaffold-activated protein trans-splicing. 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. 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. We then generate and test two versions of the scaffold-induced splicing system using two pairs of CCs. We first demonstrate the ability of antiparallel coiled-coils (CCs) to mediate splicing between two intein fragments, effectively creating two new split inteins.
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. 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. Conditional protein splicing is a powerful biotechnological tool that can be used to rapidly and post-translationally control the activity of a given protein.
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