| Téléchargement | - Voir les données supplémentaires partie 1 : Rapid, high-yield production of full-length SARS-CoV-2 spike ectodomain by transient gene expression in CHO cells (PDF, 236 Kio)
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- Voir les données supplémentaires partie 3 : Rapid, high-yield production of full-length SARS-CoV-2 spike ectodomain by transient gene expression in CHO cells (PDF, 417 Kio)
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| DOI | Trouver le DOI : https://doi.org/10.1016/j.jbiotec.2020.12.005 |
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| Auteur | Rechercher : Stuible, Matthew1; Rechercher : Gervais, Christian1; Rechercher : Lord-Dufour, Simon1; Rechercher : Perret, Sylvie1; Rechercher : L'Abbé, Denis1; Rechercher : Schrag, Joseph1; Rechercher : St-Laurent, Gilles1; Rechercher : Durocher, Yves1Identifiant ORCID : https://orcid.org/0000-0002-2268-4111 |
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| Affiliation | - Conseil national de recherches du Canada. Thérapeutique en santé humaine
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| Format | Texte, Article |
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| Sujet | SARS-CoV-2; trimeric spike; HEK293; CHO; transient gene expression; polyethylenimine |
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| Résumé | Recombinant forms of the spike protein of SARS-CoV-2 and related viruses have proven difficult to produce with good yields in mammalian cells. Given the panoply of potential COVID-19 diagnostic tools and therapeutic candidates that require purified spike protein and its importance for ongoing SARS-CoV-2 research, we have explored new approaches for spike production and purification. Three transient gene expression methods based on PEI-mediated transfection of CHO or HEK293 cells in suspension culture in chemically-defined media were compared for rapid production of full-length SARS-CoV-2 spike ectodomain. A high-cell-density protocol using DXB11-derived CHO BRI/55E1 cells gave substantially better yields than the other methods. Different forms of the spike ectodomain were expressed, including the wild-type SARS-CoV-2 sequence and a mutated form (to favor expression of the full-length spike ectodomain stabilized in pre-fusion conformation), with and without fusion to putative trimerization domains. An efficient two-step affinity purification method was also developed. Ultimately, we have been able to produce highly homogenous preparations of full-length spike, both monomeric and trimeric, with yields of 100−150 mg/L in the harvested medium. The speed and productivity of this method support further development of CHO-based approaches for recombinant spike protein manufacturing. |
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| Date de publication | 2020-12-07 |
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| Maison d’édition | Elsevier |
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| Dans | |
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| Langue | anglais |
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| Publications évaluées par des pairs | Oui |
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| Numéro du CNRC | NRC-HHT_53506FT |
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| Exporter la notice | Exporter en format RIS |
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| Signaler une correction | Signaler une correction (s'ouvre dans un nouvel onglet) |
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| Identificateur de l’enregistrement | e1925bd3-b7b4-41cd-9e1b-c79e9000c46a |
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| Enregistrement créé | 2021-01-06 |
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| Enregistrement modifié | 2021-01-06 |
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