Engineering a Thermally Activatable SpyCatcher/SpyTag Protein Ligation for Injectable and In Situ‐Forming Hydrogels

Injectable and in situ-forming hydrogels are attractive for tissue engineering and drug delivery, yet challenges remain in balancing gelation speed, mechanical strength, and cytocompatibility. Here, we report a thermally activatable SpyCatcher/SpyTag (TASpy) system that undergoes rapid chemical crosslinking at physiological temperature. Inspired by the CnaB2 domain, thermally activatable SpyCatcher (TASpyC) fuses SpyCatcher with a non-reactive SpyTag mutant, creating a single polypeptide that remains folded and minimally reactive at lower temperatures yet partially unfolds at 37 °C to expose a covalent binding site. This design enables controlled isopeptide-bond formation, facilitating the formation of robust injectable protein hydrogels. We demonstrate improved mechanical properties, enhanced cell encapsulation, and accelerated gelation upon heating-supporting high cell viability and in vivo retention. Notably, subcutaneous injections of TASpy-based precursors solidify within minutes, degrade over five weeks, and exhibit excellent biocompatibility, underscoring the potential of TASpy hydrogels for advanced biomedical applications.