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Split NeissLock with Spy-Acceleration Arms Mammalian Proteins for Anhydride-Mediated Cell Ligation
Sheryl Y T Lim1, Anthony H Keeble2, Mark R Howarth1,2
1Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
ACS Chemical Biology
|September 15, 2025
Summary
Researchers developed a split NeissLock system for controlled protein modification. This system enables precise covalent labeling of proteins in mammalian cells, enhancing cellular engineering capabilities.
Area of Science:
- Biochemistry
- Molecular Biology
- Synthetic Biology
Background:
- Proteins can incorporate reactive functional groups via natural amino acids or engineered additions.
- Calcium-triggered anhydride formation in *Neisseria meningitidis* FrpC's self-processing module (SPM) was previously engineered for NeissLock ligation.
- Bacterial diversity exploration revealed a related module with exceptionally rapid anhydride formation.
Purpose of the Study:
- To dissect the swift SPM and engineer a split NeissLock system for enhanced control over protein modification.
- To enable modular ligation and site-specific covalent coupling of proteins in mammalian cells.
- To demonstrate the system's utility in labeling endogenous targets and facilitating complex cellular modifications.
Main Methods:
- Dissection of a bacterial self-processing module (SPM) to create a split NeissLock system.
- Utilizing spontaneous amidation between SpyTag003 and SpyCatcher003 to accelerate split NeissLock reconstitution.
- Applying the split NeissLock system for site-specific covalent coupling of proteins to endogenous targets in mammalian cells.
Main Results:
- A split NeissLock system was generated, offering dual control (moiety mixing and calcium addition) over anhydride generation.
- The system facilitated the expression of minimal fusion tags in mammalian cells, accommodating complex post-translational modifications and avoiding self-cleavage.
- Rapid, high-yield ligation was achieved, demonstrated by specific covalent reactions with endogenous Epidermal Growth Factor Receptor and modular ligation on living cells.
Conclusions:
- Split NeissLock provides a modular and genetically encoded platform for generating highly reactive protein functionality.
- The system offers inducibility and enhanced control for sophisticated cellular modifications.
- This technology enables precise protein labeling and conjugation in complex biological systems.
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