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Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attr...

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Autores principales: Mehalko, Jennifer, Drew, Matthew, Snead, Kelly, Denson, John-Paul, Wall, Vanessa, Taylor, Troy, Sadtler, Kaitlyn, Messing, Simon, Gillette, William, Esposito, Dominic
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685350/
https://www.ncbi.nlm.nih.gov/pubmed/33236017
http://dx.doi.org/10.1101/2020.11.18.388868
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author Mehalko, Jennifer
Drew, Matthew
Snead, Kelly
Denson, John-Paul
Wall, Vanessa
Taylor, Troy
Sadtler, Kaitlyn
Messing, Simon
Gillette, William
Esposito, Dominic
author_facet Mehalko, Jennifer
Drew, Matthew
Snead, Kelly
Denson, John-Paul
Wall, Vanessa
Taylor, Troy
Sadtler, Kaitlyn
Messing, Simon
Gillette, William
Esposito, Dominic
author_sort Mehalko, Jennifer
collection PubMed
description The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection.
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spelling pubmed-76853502020-11-25 Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays Mehalko, Jennifer Drew, Matthew Snead, Kelly Denson, John-Paul Wall, Vanessa Taylor, Troy Sadtler, Kaitlyn Messing, Simon Gillette, William Esposito, Dominic bioRxiv Article The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection. Cold Spring Harbor Laboratory 2020-11-18 /pmc/articles/PMC7685350/ /pubmed/33236017 http://dx.doi.org/10.1101/2020.11.18.388868 Text en https://creativecommons.org/publicdomain/zero/1.0/This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license (https://creativecommons.org/publicdomain/zero/1.0/) .
spellingShingle Article
Mehalko, Jennifer
Drew, Matthew
Snead, Kelly
Denson, John-Paul
Wall, Vanessa
Taylor, Troy
Sadtler, Kaitlyn
Messing, Simon
Gillette, William
Esposito, Dominic
Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays
title Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays
title_full Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays
title_fullStr Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays
title_full_unstemmed Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays
title_short Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays
title_sort improved production of sars-cov-2 spike receptor-binding domain (rbd) for serology assays
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685350/
https://www.ncbi.nlm.nih.gov/pubmed/33236017
http://dx.doi.org/10.1101/2020.11.18.388868
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