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

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 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.

SpyDisplay: A Versatile Phage Display Selection System using SpyTag/SpyCatcher Technology

“…Other protein ligation technologies besides SpyTag/SpyCatcher have been established and could be used to establish analogous ‘covalent capture’ display methods. Such technologies include SnoopTag/SnoopCatcher (Veggiani et al, 2016), DogTag/DogCatcher (Keeble et al, 2022), SilkTag/SilkCatcher (Fan et al, 2022), split inteins (Vila-Perello and Muir, 2010), or enzyme-based ligation technologies such as ones based on sortase (Schmohl and Schwarzer, 2014), butelase (Nguyen et al, 2014), or peptiligase (Toplak et al, 2016).…”