Engineering SpyCatcher Variants with Proteolytic Sites for Less-Trace Ligation

The Summary of main observation and conclusion The SpyTag/SpyCatcher reaction is a powerful tool for bioconjugation, but it leaves a complex of considerable size after ligation. To facilitate removal of the catalytic fragment, proteolytic recognition sites (such as DDDDK, AVLQ, and WELQ) were directly engineered into the first or second loop of SpyCatcher at locations after the reactive lysine to give a set of cleavable SpyCatcher variants. Among them, SpyCatcher(DDDDK) exhibits excellent reactivity with SpyTag and could still be cleaved proteolytically by enterokinase after ligation. Notably, SpyCatcher(DDDDK) is disordered in solution and forms an ordered complex upon reaction with SpyTag with a second order rate constant of 99.2 +/- 0.1 M-1 center dot s(-1), which is comparable to, if not faster than, most click reactions. The results demonstrate the high sequence plasticity of SpyCatcher and suggest that covalent bond formation may confer robustness on the folded structure against extensive mutation. These variants add to the expanding toolbox of genetically-encoded peptide-protein chemistry with diverse features.