title: Protein scaffold-activated protein trans-splicing in mammalian cells.
abstract: 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.
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Selgrade
authors.forename: Jason J
authors.initials: JJ
authors.lastname: Lohmueller
authors.forename: Florian
authors.initials: F
authors.lastname: Lienert
authors.forename: Pamela A
authors.initials: PA
authors.lastname: Silver
mesh_terms: D002478:Cells, Cultured; D004789:Enzyme Activation; D047669:Exteins; D006801:Humans; D047668:Inteins; D008156:Luciferases / Q000737:chemistry / Q000378:metabolism; D019154:Protein Splicing; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011506:Proteins; D008156:Luciferases