Applying ncAA Incorporation To CRM197 Vaccine Carrier Protein Engineering

Engineering vaccine carrier proteins for conjugation has historically relied on nonspecific chemistry, introducing variability that can compromise immunogenicity and manufacturing consistency. CRM197, widely used to boost immune responses to polysaccharides and carbohydrates, presents a specific set of structural constraints: solvent accessibility, preservation of known T cell epitopes, and maintenance of the native dimer interface must all be considered when selecting conjugation sites.

Using a combination of crystal structure analysis and hydrogen-deuterium exchange mass spectrometry, researchers identified 11 candidate sites that meet these criteria simultaneously. Amber codon insertion at each position allowed site-specific incorporation of para-azido-phenylalanine, enabling precise, bioorthogonal conjugation chemistry. Expression runs at two-liter scale demonstrated the approach is compatible with process-relevant conditions.

Watch the full video below to see how site selection criteria, expression data, and conjugation outcomes translate into a practical engineering workflow.