Tyrosinase-Mediated\nConjugation for Antigen Display\non Ferritin Nanoparticles

Ferritin (Ft) nanoparticles have become versatile platforms\nfor\ndisplaying antigens, being a promising technology for vaccine development.\nWhile genetic fusion has traditionally been the preferred method for\nantigen display, concerns about improper folding and steric hindrance\nthat may compromise vaccine efficacy or stability have prompted alternative\napproaches. Bioconjugation offers the advantage of preserving native\nprotein structure and function, with recent advancements improving\nefficiency and specificity. In this study, we used tyrosinase (TYR)\nto bioconjugate the receptor binding domain of the SARS-CoV-2 spike\nprotein, tagged with a tyrosine (RBD-Y), to native cysteines on Ft,\nresulting in RBD-Y-Ft nanoparticles. We quantified available cysteines\non ferritin using Ellman’s assay and monitored their reduction\nduring the reactions. Denaturing analytics (via SDS-PAGE, Western\nblot, and LC-TOF-MS) confirmed the formation of RBD-Y-Ft monomers\nwith an expected molecular weight of 46 kDa. Mass photometry and HPLC\nestimated a molecular weight of RBD-Y-Ft nanoparticles of 680 kDa,\nwhich was higher than that of nonfunctionalized ferritin (480 kDa),\nindicating successful binding of up to eight RBD-Y antigens per 24-mer\nFt nanoparticle. This work enhances our understanding of how Ft nanoparticles\ncan be engineered to present antigens, leveraging them as a robust\nscaffold for producing tailored-made candidate vaccines in a timely\nmanner.