Engineering of Group A Streptococcus Isopeptide Bonds into Immunoglobulin-Like Protein Domains

Intramolecular isopeptide bonds, formed autocatalytically between Lys and Asn/Asp side chains, are widely present in the immunoglobulin-like domains of Gram-positive bacterial adhesins, including Group A Streptococcus, and confer considerable mechanical and chemical stability. These properties make them attractive for applications in biotechnology. Here, we detail the practical considerations that are involved in engineering isopeptide bonds into Ig-like proteins, including the choice of a site where bond-forming residues could be introduced and the appropriate methodology for mutagenesis. We specify how to determine whether an isopeptide bond has formed, what strategies can be adopted to overcome problems, and how to monitor the stability of the engineered protein.