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Transient Expression in HEK-293 Cells in Suspension Culture as a Rapid and Powerful Tool: SARS-CoV-2 N and Chimeric SARS-CoV-2N-CD154 Proteins as a Case Study

In a previous work, we proposed a vaccine chimeric antigen based on the fusion of the SARS-CoV-2 N protein to the extracellular domain of the human CD40 ligand (CD154). This vaccine antigen was named N-CD protein and its expression was carried out in HEK-293 stably transfected cells, grown in adhere...

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Autores principales: Lao, Thailin, Farnos, Omar, Bueno, Alexi, Alvarez, Anays, Rodríguez, Elsa, Palacios, Julio, de la Luz, Kathya Rashida, Kamen, Amine, Carpio, Yamila, Estrada, Mario Pablo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10669214/
https://www.ncbi.nlm.nih.gov/pubmed/38002050
http://dx.doi.org/10.3390/biomedicines11113050
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author Lao, Thailin
Farnos, Omar
Bueno, Alexi
Alvarez, Anays
Rodríguez, Elsa
Palacios, Julio
de la Luz, Kathya Rashida
Kamen, Amine
Carpio, Yamila
Estrada, Mario Pablo
author_facet Lao, Thailin
Farnos, Omar
Bueno, Alexi
Alvarez, Anays
Rodríguez, Elsa
Palacios, Julio
de la Luz, Kathya Rashida
Kamen, Amine
Carpio, Yamila
Estrada, Mario Pablo
author_sort Lao, Thailin
collection PubMed
description In a previous work, we proposed a vaccine chimeric antigen based on the fusion of the SARS-CoV-2 N protein to the extracellular domain of the human CD40 ligand (CD154). This vaccine antigen was named N-CD protein and its expression was carried out in HEK-293 stably transfected cells, grown in adherent conditions and serum-supplemented medium. The chimeric protein obtained in these conditions presented a consistent pattern of degradation. The immunization of mice and monkeys with this chimeric protein was able to induce a high N-specific IgG response with only two doses in pre-clinical experiments. In order to explore ways to diminish protein degradation, in the present work, the N and N-CD proteins were produced in suspension cultures and serum-free media following transient transfection of the HEK-293 clone 3F6, at different scales, including stirred-tank controlled bioreactors. The results showed negligible or no degradation of the target proteins. Further, clones stably expressing N-CD were obtained and adapted to suspension culture, obtaining similar results to those observed in the transient expression experiments in HEK-293-3F6. The evidence supports transient protein expression in suspension cultures and serum-free media as a powerful tool to produce in a short period of time high levels of complex proteins susceptible to degradation, such as the SARS-CoV-2 N protein.
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spelling pubmed-106692142023-11-14 Transient Expression in HEK-293 Cells in Suspension Culture as a Rapid and Powerful Tool: SARS-CoV-2 N and Chimeric SARS-CoV-2N-CD154 Proteins as a Case Study Lao, Thailin Farnos, Omar Bueno, Alexi Alvarez, Anays Rodríguez, Elsa Palacios, Julio de la Luz, Kathya Rashida Kamen, Amine Carpio, Yamila Estrada, Mario Pablo Biomedicines Article In a previous work, we proposed a vaccine chimeric antigen based on the fusion of the SARS-CoV-2 N protein to the extracellular domain of the human CD40 ligand (CD154). This vaccine antigen was named N-CD protein and its expression was carried out in HEK-293 stably transfected cells, grown in adherent conditions and serum-supplemented medium. The chimeric protein obtained in these conditions presented a consistent pattern of degradation. The immunization of mice and monkeys with this chimeric protein was able to induce a high N-specific IgG response with only two doses in pre-clinical experiments. In order to explore ways to diminish protein degradation, in the present work, the N and N-CD proteins were produced in suspension cultures and serum-free media following transient transfection of the HEK-293 clone 3F6, at different scales, including stirred-tank controlled bioreactors. The results showed negligible or no degradation of the target proteins. Further, clones stably expressing N-CD were obtained and adapted to suspension culture, obtaining similar results to those observed in the transient expression experiments in HEK-293-3F6. The evidence supports transient protein expression in suspension cultures and serum-free media as a powerful tool to produce in a short period of time high levels of complex proteins susceptible to degradation, such as the SARS-CoV-2 N protein. MDPI 2023-11-14 /pmc/articles/PMC10669214/ /pubmed/38002050 http://dx.doi.org/10.3390/biomedicines11113050 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Lao, Thailin
Farnos, Omar
Bueno, Alexi
Alvarez, Anays
Rodríguez, Elsa
Palacios, Julio
de la Luz, Kathya Rashida
Kamen, Amine
Carpio, Yamila
Estrada, Mario Pablo
Transient Expression in HEK-293 Cells in Suspension Culture as a Rapid and Powerful Tool: SARS-CoV-2 N and Chimeric SARS-CoV-2N-CD154 Proteins as a Case Study
title Transient Expression in HEK-293 Cells in Suspension Culture as a Rapid and Powerful Tool: SARS-CoV-2 N and Chimeric SARS-CoV-2N-CD154 Proteins as a Case Study
title_full Transient Expression in HEK-293 Cells in Suspension Culture as a Rapid and Powerful Tool: SARS-CoV-2 N and Chimeric SARS-CoV-2N-CD154 Proteins as a Case Study
title_fullStr Transient Expression in HEK-293 Cells in Suspension Culture as a Rapid and Powerful Tool: SARS-CoV-2 N and Chimeric SARS-CoV-2N-CD154 Proteins as a Case Study
title_full_unstemmed Transient Expression in HEK-293 Cells in Suspension Culture as a Rapid and Powerful Tool: SARS-CoV-2 N and Chimeric SARS-CoV-2N-CD154 Proteins as a Case Study
title_short Transient Expression in HEK-293 Cells in Suspension Culture as a Rapid and Powerful Tool: SARS-CoV-2 N and Chimeric SARS-CoV-2N-CD154 Proteins as a Case Study
title_sort transient expression in hek-293 cells in suspension culture as a rapid and powerful tool: sars-cov-2 n and chimeric sars-cov-2n-cd154 proteins as a case study
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10669214/
https://www.ncbi.nlm.nih.gov/pubmed/38002050
http://dx.doi.org/10.3390/biomedicines11113050
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