A Moonlighting Superpositively Charged SpyCatcher

Genetically encoded covalent peptide tagging tools such as the SpyTag/SpyCatcher reactive pair, have been demonstrated versatile and useful for protein modification. Herein, we present a superpositively charged SpyCatcher bearing a theoretical net charge of +21 capable of accomplishing multiple unrelated independent tasks to enrich this toolbox and culti-vate new functions. The SpyCatcher(+21) possessed stimuli-responsive reactivity toward SpyTag and could serve as a potent and general platform for the delivery of proteins, including RNase A into HeLa cells. Remarkably, the delivered RNase A caused substan-tial proliferation inhibition toward HeLa cells. In addi-tion, the superpositively charged SpyCatcher could form coacervate with plasmid DNA for further study of gene delivery and liquid-liquid phase separation. These findings demonstrate the robustness of the SpyTag/SpyCatcher structure against surface muta-tion and the prospect of applying supercharging tech-nology on diverse functional proteins to create moonlighting proteins.