Amber Codon Mutational Scanning and Bioorthogonal PEGylation for Epitope Mapping of Antibody Binding Sites on Human Arginase-1

Epitope mapping is crucial for understanding immunological responses to protein therapeutics. Here, we combined genetic code expansion and bacterial surface display to incorporate S-allylcysteine (SAC) into human arginase-1 (hArg1) via <i>Methanococcoides burtonii</i> pyrrolysyl-tRNA synthetase. Using an amber codon deep mutational scanning and sequencing workflow, we mapped SAC incorporation efficiency across the hArg1 sequence, providing insights into structural and sequence dependencies of noncanonical amino acid incorporation. We used mutually bioorthogonal allyl/tetrazine and azide/DBCO chemistries to achieve site-specific PEGylation and fluorescent labeling of hArg1, revealing insights into SAC side chain reactivity and solvent accessibility of residues in hArg1. This system was further applied to determine the binding epitope of a monoclonal antibody on the surface of hArg1, providing high-resolution data on the impact of PEGylation residue position on antibody binding. Our method produces high dimensional data of noncanonical amino acid incorporation efficiency, site-specific functionalization enabled by mutually bioorthogonal chemistries, and epitope mapping of therapeutic proteins.