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Engineering a minimal SilkCatcher/Tag pair compatible with SpyCatcher/Tag pair for the production of native-sized spider silk

Fan, R.; Hakanpaa, J.; Elfving, K.; Taberman, H.; Linder, M. B.; Aranko, A. S.

2022-10-21 biochemistry
10.1101/2022.10.21.513168 bioRxiv
Show abstract

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 from Lactobacillus 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.

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