Expression and purification of the receptor-binding domain of SARS-CoV-2 spike protein in mammalian cells for immunological assays
© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies..
The receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 virus mediates the interaction with the host cell and is required for virus internalization. It is, therefore, the primary target of neutralizing antibodies. The receptor-binding domain soon became the major target for COVID-19 research and the development of diagnostic tools and new-generation vaccines. Here, we provide a detailed protocol for high-yield expression and one-step affinity purification of recombinant RBD from transiently transfected Expi293F cells. Expi293F mammalian cells can be grown to extremely high densities in a specially formulated serum-free medium in suspension cultures, which makes them an excellent tool for secreted protein production. The highly purified RBD is glycosylated, structurally intact, and forms homomeric complexes. With this quick and easy method, we are able to produce large quantities of RBD (80 mg·L-1 culture) that we have successfully used in immunological assays to examine antibody titers and seroconversion after mRNA-based vaccination of mice.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:14 |
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| Enthalten in: |
FEBS open bio - 14(2024), 3 vom: 24. März, Seite 380-389 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Ábrahám, Edit [VerfasserIn] |
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| Links: |
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| Themen: |
Antibodies, Viral |
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| Anmerkungen: |
Date Completed 05.03.2024 Date Revised 06.03.2024 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1002/2211-5463.13754 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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NLM366198378 |
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| 500 | |a Citation Status MEDLINE | ||
| 520 | |a © 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. | ||
| 520 | |a The receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 virus mediates the interaction with the host cell and is required for virus internalization. It is, therefore, the primary target of neutralizing antibodies. The receptor-binding domain soon became the major target for COVID-19 research and the development of diagnostic tools and new-generation vaccines. Here, we provide a detailed protocol for high-yield expression and one-step affinity purification of recombinant RBD from transiently transfected Expi293F cells. Expi293F mammalian cells can be grown to extremely high densities in a specially formulated serum-free medium in suspension cultures, which makes them an excellent tool for secreted protein production. The highly purified RBD is glycosylated, structurally intact, and forms homomeric complexes. With this quick and easy method, we are able to produce large quantities of RBD (80 mg·L-1 culture) that we have successfully used in immunological assays to examine antibody titers and seroconversion after mRNA-based vaccination of mice | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a ELISA | |
| 650 | 4 | |a Expi293F mammalian cells | |
| 650 | 4 | |a SARS-CoV-2 spike | |
| 650 | 4 | |a mRNA-LNP vaccination | |
| 650 | 4 | |a protein purification | |
| 650 | 4 | |a recombinant RBD | |
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| 650 | 7 | |a Spike Glycoprotein, Coronavirus |2 NLM | |
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| 700 | 1 | |a Marton, Annamária |e verfasserin |4 aut | |
| 700 | 1 | |a Borics, Attila |e verfasserin |4 aut | |
| 700 | 1 | |a Mdluli, Thandiswa |e verfasserin |4 aut | |
| 700 | 1 | |a Pardi, Norbert |e verfasserin |4 aut | |
| 700 | 1 | |a Lipinszki, Zoltán |e verfasserin |4 aut | |
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