Tail-to-side-chain cyclization stabilizes proteins on thermoresponsive ELP-based nanoparticles

Thermoresponsive elastin-like polypeptide (ELP) assemblies are promising platforms for functional nanoparticles, but the elevated temperatures required for self-assembly can inactivate heat-labile proteins of interest (POIs). To address this issue, we introduced a genetically encoded SnoopTag/SnoopCatcher cyclization module into an ELP-poly(aspartic acid) (ELP-D) scaffold. Using Renilla luciferase (RLuc) as a model POI, tail-to-side-chain cyclization increased both melting temperature and half-inactivation temperature by ∼6°C, indicating enhanced thermal stability. This stabilization was preserved upon fusion to ELP-D without affecting nanoparticle size or morphology. ELP-D-cRLuc nanoparticles retained ∼70% activity after heating to 42°C, compared with ∼30% for non-cyclized constructs. These results demonstrate that genetically encoded cyclization effectively protects POIs during heat-triggered nanoparticle assembly and enables activity retention at elevated temperatures.