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Site-Specific Glycosylation of Recombinant Viral Glycoproteins Produced in Nicotiana benthamiana

There is an urgent need to establish large scale biopharmaceutical manufacturing capacity in Africa where the infrastructure for biologics production is severely limited. Molecular farming, whereby pharmaceuticals are produced in plants, offers a cheaper alternative to mainstream expression platform...

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Autores principales: Margolin, Emmanuel, Allen, Joel D., Verbeek, Matthew, van Diepen, Michiel, Ximba, Phindile, Chapman, Rosamund, Meyers, Ann, Williamson, Anna-Lise, Crispin, Max, Rybicki, Edward
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8341435/
https://www.ncbi.nlm.nih.gov/pubmed/34367227
http://dx.doi.org/10.3389/fpls.2021.709344
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author Margolin, Emmanuel
Allen, Joel D.
Verbeek, Matthew
van Diepen, Michiel
Ximba, Phindile
Chapman, Rosamund
Meyers, Ann
Williamson, Anna-Lise
Crispin, Max
Rybicki, Edward
author_facet Margolin, Emmanuel
Allen, Joel D.
Verbeek, Matthew
van Diepen, Michiel
Ximba, Phindile
Chapman, Rosamund
Meyers, Ann
Williamson, Anna-Lise
Crispin, Max
Rybicki, Edward
author_sort Margolin, Emmanuel
collection PubMed
description There is an urgent need to establish large scale biopharmaceutical manufacturing capacity in Africa where the infrastructure for biologics production is severely limited. Molecular farming, whereby pharmaceuticals are produced in plants, offers a cheaper alternative to mainstream expression platforms, and is amenable to rapid large-scale production. However, there are several differences along the plant protein secretory pathway compared to mammalian systems, which constrain the production of complex pharmaceuticals. Viral envelope glycoproteins are important targets for immunization, yet in some cases they accumulate poorly in plants and may not be properly processed. Whilst the co-expression of human chaperones and furin proteases has shown promise, it is presently unclear how plant-specific differences in glycosylation impact the production of these proteins. In many cases it may be necessary to reproduce features of their native glycosylation to produce immunologically relevant vaccines, given that glycosylation is central to the folding and immunogenicity of these antigens. Building on previous work, we transiently expressed model glycoproteins from HIV and Marburg virus in Nicotiana benthamiana and mammalian cells. The proteins were purified and their site-specific glycosylation was determined by mass-spectrometry. Both glycoproteins yielded increased amounts of protein aggregates when produced in plants compared to the equivalent mammalian cell-derived proteins. The glycosylation profiles of the plant-produced glycoproteins were distinct from the mammalian cell produced proteins: they displayed lower levels of glycan occupancy, reduced complex glycans and large amounts of paucimannosidic structures. The elucidation of the site-specific glycosylation of viral glycoproteins produced in N. benthamiana is an important step toward producing heterologous viral glycoproteins in plants with authentic human-like glycosylation.
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spelling pubmed-83414352021-08-06 Site-Specific Glycosylation of Recombinant Viral Glycoproteins Produced in Nicotiana benthamiana Margolin, Emmanuel Allen, Joel D. Verbeek, Matthew van Diepen, Michiel Ximba, Phindile Chapman, Rosamund Meyers, Ann Williamson, Anna-Lise Crispin, Max Rybicki, Edward Front Plant Sci Plant Science There is an urgent need to establish large scale biopharmaceutical manufacturing capacity in Africa where the infrastructure for biologics production is severely limited. Molecular farming, whereby pharmaceuticals are produced in plants, offers a cheaper alternative to mainstream expression platforms, and is amenable to rapid large-scale production. However, there are several differences along the plant protein secretory pathway compared to mammalian systems, which constrain the production of complex pharmaceuticals. Viral envelope glycoproteins are important targets for immunization, yet in some cases they accumulate poorly in plants and may not be properly processed. Whilst the co-expression of human chaperones and furin proteases has shown promise, it is presently unclear how plant-specific differences in glycosylation impact the production of these proteins. In many cases it may be necessary to reproduce features of their native glycosylation to produce immunologically relevant vaccines, given that glycosylation is central to the folding and immunogenicity of these antigens. Building on previous work, we transiently expressed model glycoproteins from HIV and Marburg virus in Nicotiana benthamiana and mammalian cells. The proteins were purified and their site-specific glycosylation was determined by mass-spectrometry. Both glycoproteins yielded increased amounts of protein aggregates when produced in plants compared to the equivalent mammalian cell-derived proteins. The glycosylation profiles of the plant-produced glycoproteins were distinct from the mammalian cell produced proteins: they displayed lower levels of glycan occupancy, reduced complex glycans and large amounts of paucimannosidic structures. The elucidation of the site-specific glycosylation of viral glycoproteins produced in N. benthamiana is an important step toward producing heterologous viral glycoproteins in plants with authentic human-like glycosylation. Frontiers Media S.A. 2021-07-22 /pmc/articles/PMC8341435/ /pubmed/34367227 http://dx.doi.org/10.3389/fpls.2021.709344 Text en Copyright © 2021 Margolin, Allen, Verbeek, van Diepen, Ximba, Chapman, Meyers, Williamson, Crispin and Rybicki. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Plant Science
Margolin, Emmanuel
Allen, Joel D.
Verbeek, Matthew
van Diepen, Michiel
Ximba, Phindile
Chapman, Rosamund
Meyers, Ann
Williamson, Anna-Lise
Crispin, Max
Rybicki, Edward
Site-Specific Glycosylation of Recombinant Viral Glycoproteins Produced in Nicotiana benthamiana
title Site-Specific Glycosylation of Recombinant Viral Glycoproteins Produced in Nicotiana benthamiana
title_full Site-Specific Glycosylation of Recombinant Viral Glycoproteins Produced in Nicotiana benthamiana
title_fullStr Site-Specific Glycosylation of Recombinant Viral Glycoproteins Produced in Nicotiana benthamiana
title_full_unstemmed Site-Specific Glycosylation of Recombinant Viral Glycoproteins Produced in Nicotiana benthamiana
title_short Site-Specific Glycosylation of Recombinant Viral Glycoproteins Produced in Nicotiana benthamiana
title_sort site-specific glycosylation of recombinant viral glycoproteins produced in nicotiana benthamiana
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8341435/
https://www.ncbi.nlm.nih.gov/pubmed/34367227
http://dx.doi.org/10.3389/fpls.2021.709344
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