N-Glycan Insertion for Probing Protein–Protein Interactions and Epitope Mapping

Introducing an N-linked glycosylation motif into recombinant proteins at specific sites is a useful tool in probing protein–protein interactions and epitope mapping. Due to their large size, a new N-glycan can block protein–protein interactions if it is introduced by site-directed mutagenesis on the same face as a ligand or antibody binding site. Recombinant mutant proteins containing these engineered glycans can then be studied using binding or functional assays to establish if the new glycan causes steric hindrance, prevents an important protein–protein interaction, or blocks (auto)antibody binding. In this book chapter, we provide guides and protocols for inserting engineered glycans, including how to use AlphaFold models to choose amino acid residues on the surface of protein domains that are suitable for mutagenesis into N-linked glycosylation motifs as well as protocols for site-directed mutagenesis and recombinant protein expression of the N-glycan variants.