A type of protein/peptide pair known as Catcher/Tag pair spontaneously forms an intermolecular isopeptide bond which can be applied for biomolecular click reactions. Covalent protein conjugation using Catcher/Tag pairs has turned out to be a valuable tool in biotechnology and biomedicines, but it is essential to increase the current toolbox of orthogonal Catcher/Tag pairs to expand the range of applications further, for example, for controlled multiple‐fragment ligation. We report here the engineering of novel Catcher/Tag pairs for protein ligation, aided by a crystal structure of a minimal CnaB domain from Lactobacillus plantarum. We show that a newly engineered pair, called SilkCatcher/Tag enables efficient pH‐inducible protein ligation in addition to being compatible with the widely used SpyCatcher/Tag pair. Finally, we demonstrate the use of the SilkCatcher/Tag pair in the production of native‐sized highly repetitive spider‐silk‐like proteins with >90 % purity, which is not possible by traditional recombinant production methods.

Protein/peptide pairs, called Catcher/Tag pairs, are applied for biological isopeptide-bond mediated Click-reactions. Covalent protein ligation using Catcher/Tag pairs has turned out to be a valuable tool in biotechnology and biomedicines. It is essential to increase the current toolbox of Catcher/Tag pairs to expand the range of applications further, e.g., for multiple-fragment ligation that requires several orthogonal ligases. We report here engineering of novel Catcher/Tag pairs for protein ligation, aided by a new crystal structure of a minimal CnaB domain fromLactobacillus plantarum. We engineer several split variants, characterize in detail one of them, named SilkCatcher/Tag pair, and show that the newly engineered SilkCatcher/Tag pair is orthogonal and compatible with the widely used SpyCatcher/Tag pair. Finally, we demonstrate the use of the new SilkCatcher/Tag pair in the production of native-sized highly repetitive spider-silk-like proteins with >90% purity, which is not possible with the traditional recombinant production.

A type of protein/peptide pair known as Catcher/Tag pair spontaneously forms an intermolecular isopeptide bond which can be applied for biomolecular click reactions. Covalent protein conjugation using Catcher/Tag pairs has turned out to be a valuable tool in biotechnology and biomedicines, but it is essential to increase the current toolbox of orthogonal Catcher/Tag pairs to expand the range of applications further, for example, for controlled multiple‐fragment ligation. We report here the engineering of novel Catcher/Tag pairs for protein ligation, aided by a crystal structure of a minimal CnaB domain from Lactobacillus plantarum. We show that a newly engineered pair, called SilkCatcher/Tag enables efficient pH‐inducible protein ligation in addition to being compatible with the widely used SpyCatcher/Tag pair. Finally, we demonstrate the use of the SilkCatcher/Tag pair in the production of native‐sized highly repetitive spider‐silk‐like proteins with >90 % purity, which is not possible by traditional recombinant production methods.