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

Abstract 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.