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

Journal article

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.

Authors: Lim, SYT; Keeble, AH; Howarth, MR

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: American Chemical Society
• Journal: ACS Chemical Biology
• Volume: 20
• Issue: 10
• Pages: 2475-2482
• Publication date: 2025-09-15
• Acceptance date: 2025-08-25
• DOI: 10.1021/acschembio.5c00515
• EISSN: 1554-8937
• ISSN: 1554-8929
• Language: English