Site-directed tyrosinase conjugation on engineered ferritin retains immune recognition.

Ferritin (Ft) nanoparticles are promising scaffolds for antigen display in vaccine design due to their stability, defined architecture, and biocompatibility. Enzymatic methods, such as tyrosinase catalysis, enable covalent antigen conjugation by oxidizing tyrosine residues into o-quinones that react with accessible cysteine thiols. Here, we engineered Pyrococcus furiosus ferritin (PfFt) by introducing single cysteines at defined positions (K8C, D33C, and E92C) to enable site-specific bioconjugation. All PfFt variants retained their quaternary nanoparticle structure, as confirmed by mass spectrometry, dynamic light scattering, HPLC, and mass photometry. Thiol accessibility was verified by Ellman's assay. Using tyrosinase-mediated catalysis, we conjugated two tyrosine-tagged antigens, Rift Valley fever virus Gn and SARS-CoV-2 receptor-binding domain, to the engineered cysteines. Up to 13 antigens were displayed per 24-mer nanoparticle. Conjugation was highly specific to the engineered cysteines, and the resulting antigen-PfFt conjugates bound neutralizing antibodies with nanomolar affinities (2-7 nM), comparable to their soluble antigen counterparts. This work establishes a robust and modular strategy for precise antigen display on ferritin nanocages using tyrosinase-mediated cysteine conjugation. The platform shows strong potential for next-generation protein-based vaccines and other bioconjugate therapeutics.