Work

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

Year: 2025

Type: article

Abstract: 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 Methanococcoides burtonii 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 non-canonical 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 non-canonical amino acid incorporation efficiency, site-specific functionalization enabled by mutually bioorthogonal chemistries, and epitope mapping of therapeutic proteins.

Source: ACS Chemical Biology

DOI: 10.1021/acschembio.4c00692

Authors: Jaime Fernández de Santaella, Nikolaj G. Koch, Lorenz Widmer, Michael A. Nash

Institutions: University of Basel, ETH Zurich, Institute of Physical Chemistry, Institute of Physical Chemistry

Language: English

Topic: RNA and protein synthesis mechanisms

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: green

Funders: Universität Basel, Eidgenössische Technische Hochschule Zürich, Staatssekretariat für Bildung, Forschung und Innovation

Award: G697443731