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Bioconjug Chem. 2016 Sep 21. [Epub ahead of print]
Bioorthogonal Strategy for Bioprocessing of Specific-site-functionalized Enveloped Influenza-virus-like Particles.
Carvalho SB, Freire JM, Moleirinho MG, Monteiro F, Gaspar DM, Castanho MA, Carrondo MJ, Alves PM, Bernardes GJ, Peixoto C.
Abstract
Virus-like particles (VLPs) constitute a promising platform in vaccine development and targeted drug delivery. To date, most applications use simple non-enveloped VLPs as human papillomavirus or hepatitis B vaccines even though the envelope is known to be critical to retain the native protein folding and biological function. Here we present tagged enveloped VLPs (TagE-VLPs) as a valuable strategy for the downstream processing and monitoring of the in vivo production of specific-site-functionalized enveloped Influenza VLPs. This two-step procedure allows bioorthogonal functionalization of azide-tagged nascent influenza type A hemagglutinin proteins in the envelope of VLPs through strain-promoted [3+2] alkyne-azide cycloadditioneaction. Importantly, labelling does not influence VLP production and allows for construction of functionalized VLPs without deleterious effects on their biological function. Refined discrimination and separation between VLP and baculovirus - the major impurity of the process - is achieved when this technique is combined with flow cytometry analysis as demonstrated by atomic force microscopy. TagE-VLPs is a versatile tool broadly applicable to the production, monitoring and purification of functionalized enveloped VLPs for vaccine design trial runs, targeted drug-delivery, and molecular imaging.
PMID: 27652605 DOI: 10.1021/acs.bioconjchem.6b00372
[PubMed - as supplied by publisher]
Bioorthogonal Strategy for Bioprocessing of Specific-site-functionalized Enveloped Influenza-virus-like Particles.
Carvalho SB, Freire JM, Moleirinho MG, Monteiro F, Gaspar DM, Castanho MA, Carrondo MJ, Alves PM, Bernardes GJ, Peixoto C.
Abstract
Virus-like particles (VLPs) constitute a promising platform in vaccine development and targeted drug delivery. To date, most applications use simple non-enveloped VLPs as human papillomavirus or hepatitis B vaccines even though the envelope is known to be critical to retain the native protein folding and biological function. Here we present tagged enveloped VLPs (TagE-VLPs) as a valuable strategy for the downstream processing and monitoring of the in vivo production of specific-site-functionalized enveloped Influenza VLPs. This two-step procedure allows bioorthogonal functionalization of azide-tagged nascent influenza type A hemagglutinin proteins in the envelope of VLPs through strain-promoted [3+2] alkyne-azide cycloadditioneaction. Importantly, labelling does not influence VLP production and allows for construction of functionalized VLPs without deleterious effects on their biological function. Refined discrimination and separation between VLP and baculovirus - the major impurity of the process - is achieved when this technique is combined with flow cytometry analysis as demonstrated by atomic force microscopy. TagE-VLPs is a versatile tool broadly applicable to the production, monitoring and purification of functionalized enveloped VLPs for vaccine design trial runs, targeted drug-delivery, and molecular imaging.
PMID: 27652605 DOI: 10.1021/acs.bioconjchem.6b00372
[PubMed - as supplied by publisher]