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 SpyCatche...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 cultivate 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 RNaseAinto HeLa cells.Remarkably,the delivered RNase A caused substantial proliferation inhibition toward HeLa cells.In addition,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 mutation and the prospect of applying supercharging technology on diverse functional proteins to create moonlighting proteins.展开更多
基金support fromthe National Key R&D Program of China(grant nos.2020YFA0908100,2020AAA0105200)the National Natural Science Foundation of China(NSFC,grant nos.21991132,21925102,92056118,8200907120,8200907121)Beijing National Laboratory for Molecular Sciences(BNLMS,grant no.BNLMS-CXXM-202006).
文摘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 cultivate 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 RNaseAinto HeLa cells.Remarkably,the delivered RNase A caused substantial proliferation inhibition toward HeLa cells.In addition,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 mutation and the prospect of applying supercharging technology on diverse functional proteins to create moonlighting proteins.
