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Publicação
Tyrosinase-Mediated Conjugation for Antigen Display on Ferritin Nanoparticles
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Orientador(es)
Resumo(s)
Ferritin (Ft) nanoparticles have become versatile platforms for displaying antigens, being a promising technology for vaccine development. While genetic fusion has traditionally been the preferred method for antigen display, concerns about improper folding and steric hindrance that may compromise vaccine efficacy or stability have prompted alternative approaches. Bioconjugation offers the advantage of preserving native protein structure and function, with recent advancements improving efficiency and specificity. In this study, we used tyrosinase (TYR) to bioconjugate the receptor binding domain of the SARS-CoV-2 spike protein, tagged with a tyrosine (RBD-Y), to native cysteines on Ft, resulting in RBD-Y-Ft nanoparticles. We quantified available cysteines on ferritin using Ellman’s assay and monitored their reduction during the reactions. Denaturing analytics (via SDS-PAGE, Western blot, and LC-TOF-MS) confirmed the formation of RBD-Y-Ft monomers with an expected molecular weight of 46 kDa. Mass photometry and HPLC estimated a molecular weight of RBD-Y-Ft nanoparticles of 680 kDa, which was higher than that of nonfunctionalized ferritin (480 kDa), indicating successful binding of up to eight RBD-Y antigens per 24-mer Ft nanoparticle. This work enhances our understanding of how Ft nanoparticles can be engineered to present antigens, leveraging them as a robust scaffold for producing tailored-made candidate vaccines in a timely manner.
Descrição
Funding Information: The authors acknowledge the support from European Commission (Project PROMETEUS, grant number 823780) and Portuguese Fundac\u0327a\u0303o para a Cie\u0302ncia e a Tecnologia (FCT) through the following programs: FCT Investigator Starting Grant (IF/01704/2014), Exploratory Research and Development Project (EXPL/BBB-BIO/1541/2013), and Ph.D. fellowship (DFA/BD/8167/2020). iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020) and the Associate Laboratory LS4FUTURE (LA/P/0087/2020), programs financially supported by FCT/Ministe\u0301rio da Cie\u0302ncia, Tecnologia e Ensino Superior through national funds, are acknowledged. Publisher Copyright: © 2024 The Authors. Published by American Chemical Society.
Palavras-chave
Biotechnology Bioengineering Biomedical Engineering Pharmacology Pharmaceutical Science Organic Chemistry SDG 3 - Good Health and Well-being