Creation of novel proteins for peptide recognition and interaction

Abstract

The SpyTag-SpyCatcher system, developed by the Howarth lab at the University of Oxford, is based on splitting the immunoglobulin-like domain CnaB2 from the fibronectin-binding protein FbaB of Streptococcus pyogenes into two functional components: the 13-amino-acid SpyTag peptide and the 116-amino-acid SpyCatcher protein. Upon incubation, SpyTag and SpyCatcher spontaneously form a covalent isopeptide bond between Asp7 of SpyTag and Lys31 of SpyCatcher. This study investigates whether the specificity of the SpyTag-SpyCatcher interaction can be modulated through targeted substitutions within the hydrophobic binding pocket of SpyCatcher and corresponding SpyTag residues, by exploring alternative hydrophobic residues, with the aim to develop orthogonal SpyTag-SpyCatcher pairs. Molecular modelling guided the design of positionally-fixed SpyCatcher and SpyTag libraries, constructed using overlap PCR and MAX randomisation. To assess the specificity, a novel screening strategy was developed, combining SDS-PAGE for semi-quantitative binding analysis with mass photometry for precise interaction detection within complex mixtures. Screening the SpyCatcher variants against SpyTag libraries demonstrated that substitution of larger, hydrophobic, aliphatic residues is well-tolerated, while aromatic bulky residues can eliminate the interaction. Comparative analyses between SDS-PAGE and mass photometry demonstrated strong consistency, highlighting the reliability of both methods in assessing binding specificity. The findings underscore the structural importance of specific residues in the SpyTag-SpyCatcher interaction and validate mass photometry as a transformative tool for screening combinatorial protein libraries. This represents the first application of mass photometry for screening protein libraries, showcasing its potential to evaluate protein-ligand specificity with minimal sample requirements. This study opens new avenues for the application of mass photometry by validating both the quality and specificity of the constructed libraries and opening of new avenues for its use in protein engineering.Key words: SpyTag, SpyCatcher, combinatorial libraries, MAX randomisation, overlap PCR, orthogonal pair, mass photometry.