Split NeissLock with Spy-Acceleration Arms Mammalian Proteins for Anhydride-Mediated Cell Ligation

Reactive functional groups may be incorporated into proteins or may emerge from natural amino acids in exceptional architectures. Anhydride formation is triggered by calcium in the self-processing module (SPM) of Neisseria meningitidis FrpC, which we previously engineered for "NeissLock" ligation to an unmodified target protein. Here, we explored bacterial diversity, discovering a related module with ultrafast anhydride formation. We dissected this swift SPM to generate a split NeissLock system, providing a second layer of control of anhydride generation: first mixing N- and C-terminal NeissLock moieties and second adding millimolar amounts of calcium. Split NeissLock generated a minimal fusion tag, permitting binder expression in mammalian cells with complex post-translational modifications and avoiding self-cleavage while transiting the calcium-rich secretory pathway. Employing spontaneous amidation between SpyTag003 and SpyCatcher003, we dramatically accelerated split NeissLock reconstitution, allowing a rapid high-yield reaction to naturally occurring targets. We established a specific covalent reaction to endogenous Epidermal Growth Factor Receptor using split NeissLock via Transforming Growth Factor-α secreted from mammalian cells. Modular ligation was demonstrated on living cells through site-specific coupling of the clot-busting enzyme tissue plasminogen activator or a computationally designed cytokine. Split NeissLock provides a modular architecture to generate highly reactive functionality, with inducibility and simple genetic encoding for enhanced cellular modification.