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Phosphate-regulated expression of the SARS-CoV-2 receptor-binding domain in the diatom Phaeodactylum tricornutum for pandemic diagnostics

The worldwide COVID-19 pandemic caused by the SARS-CoV-2 betacoronavirus has highlighted the need for a synthetic biology approach to create reliable and scalable sources of viral antigen for uses in diagnostics, therapeutics and basic biomedical research. Here, we adapt plasmid-based systems in the...

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Autores principales: Slattery, Samuel S., Giguere, Daniel J., Stuckless, Emily E., Shrestha, Arina, Briere, Lee-Ann K., Galbraith, Alexa, Reaume, Stephen, Boyko, Xenia, Say, Henry H., Browne, Tyler S., Frederick, Mallory I., Lant, Jeremy T., Heinemann, Ilka U., O’Donoghue, Patrick, Dsouza, Liann, Martin, Steven, Howard, Peter, Jedeszko, Christopher, Ali, Kinza, Styba, Garth, Flatley, Martin, Karas, Bogumil J., Gloor, Gregory B., Edgell, David R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9051505/
https://www.ncbi.nlm.nih.gov/pubmed/35487958
http://dx.doi.org/10.1038/s41598-022-11053-7
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author Slattery, Samuel S.
Giguere, Daniel J.
Stuckless, Emily E.
Shrestha, Arina
Briere, Lee-Ann K.
Galbraith, Alexa
Reaume, Stephen
Boyko, Xenia
Say, Henry H.
Browne, Tyler S.
Frederick, Mallory I.
Lant, Jeremy T.
Heinemann, Ilka U.
O’Donoghue, Patrick
Dsouza, Liann
Martin, Steven
Howard, Peter
Jedeszko, Christopher
Ali, Kinza
Styba, Garth
Flatley, Martin
Karas, Bogumil J.
Gloor, Gregory B.
Edgell, David R.
author_facet Slattery, Samuel S.
Giguere, Daniel J.
Stuckless, Emily E.
Shrestha, Arina
Briere, Lee-Ann K.
Galbraith, Alexa
Reaume, Stephen
Boyko, Xenia
Say, Henry H.
Browne, Tyler S.
Frederick, Mallory I.
Lant, Jeremy T.
Heinemann, Ilka U.
O’Donoghue, Patrick
Dsouza, Liann
Martin, Steven
Howard, Peter
Jedeszko, Christopher
Ali, Kinza
Styba, Garth
Flatley, Martin
Karas, Bogumil J.
Gloor, Gregory B.
Edgell, David R.
author_sort Slattery, Samuel S.
collection PubMed
description The worldwide COVID-19 pandemic caused by the SARS-CoV-2 betacoronavirus has highlighted the need for a synthetic biology approach to create reliable and scalable sources of viral antigen for uses in diagnostics, therapeutics and basic biomedical research. Here, we adapt plasmid-based systems in the eukaryotic microalgae Phaeodactylum tricornutum to develop an inducible overexpression system for SARS-CoV-2 proteins. Limiting phosphate and iron in growth media induced expression of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein from the P. tricornutum HASP1 promoter in the wild-type strain and in a histidine auxotrophic strain that alleviates the requirement for antibiotic selection of expression plasmids. The RBD was purified from whole cell extracts (algae-RBD) with yield compromised by the finding that 90–95% of expressed RBD lacked the genetically encoded C-terminal 6X-histidine tag. Constructs that lacked the TEV protease site between the RBD and C-terminal 6X-histidine tag retained the tag, increasing yield. Purified algae-RBD was found to be N-linked glycosylated by treatment with endoglycosidases, was cross-reactive with anti-RBD polyclonal antibodies, and inhibited binding of recombinant RBD purified from mammalian cell lines to the human ACE2 receptor. We also show that the algae-RBD can be used in a lateral flow assay device to detect SARS-CoV-2 specific IgG antibodies from donor serum at sensitivity equivalent to assays performed with RBD made in mammalian cell lines. Our study shows that P. tricornutum is a scalable system with minimal biocontainment requirements for the inducible production of SARS-CoV-2 or other coronavirus antigens for pandemic diagnostics.
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spelling pubmed-90515052022-04-29 Phosphate-regulated expression of the SARS-CoV-2 receptor-binding domain in the diatom Phaeodactylum tricornutum for pandemic diagnostics Slattery, Samuel S. Giguere, Daniel J. Stuckless, Emily E. Shrestha, Arina Briere, Lee-Ann K. Galbraith, Alexa Reaume, Stephen Boyko, Xenia Say, Henry H. Browne, Tyler S. Frederick, Mallory I. Lant, Jeremy T. Heinemann, Ilka U. O’Donoghue, Patrick Dsouza, Liann Martin, Steven Howard, Peter Jedeszko, Christopher Ali, Kinza Styba, Garth Flatley, Martin Karas, Bogumil J. Gloor, Gregory B. Edgell, David R. Sci Rep Article The worldwide COVID-19 pandemic caused by the SARS-CoV-2 betacoronavirus has highlighted the need for a synthetic biology approach to create reliable and scalable sources of viral antigen for uses in diagnostics, therapeutics and basic biomedical research. Here, we adapt plasmid-based systems in the eukaryotic microalgae Phaeodactylum tricornutum to develop an inducible overexpression system for SARS-CoV-2 proteins. Limiting phosphate and iron in growth media induced expression of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein from the P. tricornutum HASP1 promoter in the wild-type strain and in a histidine auxotrophic strain that alleviates the requirement for antibiotic selection of expression plasmids. The RBD was purified from whole cell extracts (algae-RBD) with yield compromised by the finding that 90–95% of expressed RBD lacked the genetically encoded C-terminal 6X-histidine tag. Constructs that lacked the TEV protease site between the RBD and C-terminal 6X-histidine tag retained the tag, increasing yield. Purified algae-RBD was found to be N-linked glycosylated by treatment with endoglycosidases, was cross-reactive with anti-RBD polyclonal antibodies, and inhibited binding of recombinant RBD purified from mammalian cell lines to the human ACE2 receptor. We also show that the algae-RBD can be used in a lateral flow assay device to detect SARS-CoV-2 specific IgG antibodies from donor serum at sensitivity equivalent to assays performed with RBD made in mammalian cell lines. Our study shows that P. tricornutum is a scalable system with minimal biocontainment requirements for the inducible production of SARS-CoV-2 or other coronavirus antigens for pandemic diagnostics. Nature Publishing Group UK 2022-04-29 /pmc/articles/PMC9051505/ /pubmed/35487958 http://dx.doi.org/10.1038/s41598-022-11053-7 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Slattery, Samuel S.
Giguere, Daniel J.
Stuckless, Emily E.
Shrestha, Arina
Briere, Lee-Ann K.
Galbraith, Alexa
Reaume, Stephen
Boyko, Xenia
Say, Henry H.
Browne, Tyler S.
Frederick, Mallory I.
Lant, Jeremy T.
Heinemann, Ilka U.
O’Donoghue, Patrick
Dsouza, Liann
Martin, Steven
Howard, Peter
Jedeszko, Christopher
Ali, Kinza
Styba, Garth
Flatley, Martin
Karas, Bogumil J.
Gloor, Gregory B.
Edgell, David R.
Phosphate-regulated expression of the SARS-CoV-2 receptor-binding domain in the diatom Phaeodactylum tricornutum for pandemic diagnostics
title Phosphate-regulated expression of the SARS-CoV-2 receptor-binding domain in the diatom Phaeodactylum tricornutum for pandemic diagnostics
title_full Phosphate-regulated expression of the SARS-CoV-2 receptor-binding domain in the diatom Phaeodactylum tricornutum for pandemic diagnostics
title_fullStr Phosphate-regulated expression of the SARS-CoV-2 receptor-binding domain in the diatom Phaeodactylum tricornutum for pandemic diagnostics
title_full_unstemmed Phosphate-regulated expression of the SARS-CoV-2 receptor-binding domain in the diatom Phaeodactylum tricornutum for pandemic diagnostics
title_short Phosphate-regulated expression of the SARS-CoV-2 receptor-binding domain in the diatom Phaeodactylum tricornutum for pandemic diagnostics
title_sort phosphate-regulated expression of the sars-cov-2 receptor-binding domain in the diatom phaeodactylum tricornutum for pandemic diagnostics
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9051505/
https://www.ncbi.nlm.nih.gov/pubmed/35487958
http://dx.doi.org/10.1038/s41598-022-11053-7
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