Engineering Protein–Peptide Interfaces via Combinatorial Mutagenesis and Mass Photometric Screening

The SpyTag-SpyCatcher system, developed by the Howarth lab, is based on splitting the CnaB2 domain from Streptococcus pyogenes into two parts: a 13-amino-acid SpyTag and a 116-amino-acid SpyCatcher. Upon incubation, they spontaneously form a covalent isopeptide bond between Asp7 (SpyTag) and Lys31 (SpyCatcher). This study explores whether the interaction specificity can be modulated by altering hydrophobic residues within the SpyCatcher binding pocket and corresponding SpyTag positions, potentially to create orthogonal SpyTag-SpyCatcher pairs. Libraries of SpyCatcher and SpyTag were created by partial saturation mutagenesis using overlap PCR and MAX randomisation, respectively. To assess the specificity of the SpyCatcher-SpyTag interaction within the resulting protein mixtures, a novel screening strategy based on mass photometry was developed to detect isopeptide bond formation. We demonstrate tolerance to mutation in the hydrophobic binding pocket of SpyCatcher in terms of binding native SpyTag and demonstrate what to our knowledge constitutes the first example of using mass photometry to examine the interactions of small libraries of proteins with a given ligand. Mass photometry detects stable interactions whether covalent or not and so this study suggests the prospect of employing mass photometry for more general application in protein engineering.