Functional Assembly of Split Protein Pairs via a Chemically Activated SpyLigation

Abstract

Given the critical roles proteins play in a broad variety of biological functions across all levels of life, it is in our best interest to develop innovative methods to control protein activity within biological systems. This is essential for expanding our understanding of cellular processes and facilitating novel therapeutic applications. Although genome editing technologies have advanced considerably, they still face limitations in terms of protein regulation and in providing real-time capabilities that allow us to probe biological processes across time scales. In this study, we present CASL (Chemically Activated SpyLigation) as a genetically encoded and chemically activated protein ligation method, which we utilize for triggered functional assembly of split protein pairs. By incorporating a bioorthogonal trans-cyclooctene (TCO) cage at the isopeptide-forming lysine of SpyCatcher003, we achieve on-demand, permanent conjugation of non-functional split fragments in response to tetrazine treatment. The rapid TCO click-to-uncage process facilitates swift and covalent ligation, restoring functional activity both in solution and within living cells. We demonstrated that CASL’s excellent specificity and bioorthogonality allow it to be easily adapted to cellular contexts for selective protein regulation without off-target effects or the requirement for high inducer concentrations. Looking ahead, we expect CASL to provide a powerful tool for real-time control of protein function, potentially serving as a complementary method to other inducible dimerization systems. This combined approach could lead to versatile applications for probing and directing complex cellular fates in 3D cell culture.