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
Abstract
:1. Introduction
2. Materials and Methods
2.1. Biological Reagents
2.2. Mammalian Cell Lines and Cell Culture Conditions
2.3. SARS-CoV-2 N Plasmid Construction and Lentivirus Production
2.4. Transduction of HEK-293 Cells and Generation of SARS-CoV-2 N-Protein-Expressing Cell Pools
2.5. Assessment of SARS-CoV-2 N-Protein Expression and Degradation in the Cell Culture Supernatant of Recombinant HEK-293 Cells Cultured in Adherent Conditions
2.6. Production of the SARS-CoV-2 N and N-CD Proteins in Cell Suspension Culture
2.7. Cell Cloning of the HEK-293-N-CD-50 and HEK-293-N-CD-100 Cell Pools via Limiting Dilution
2.8. Batch Culture of the N-CD-Expressing Cell Clones Cultured in Suspension Culture and Serum-Free Medium
2.9. Simulation of Perfusion Culture of the N-CD-Expressing Cell Clones Cultured in Suspension Culture and Serum-Free Medium
2.10. Protein Purification
2.11. Protein Quantification with ELISA
2.12. SDS-PAGE and Western Blot
2.13. Statistical Analysis
3. Results
3.1. Production of the SARS-CoV-2 N Protein in Adherent HEK-293 Cells
3.2. Production of SARS-CoV-2 N and N-CD Proteins through Transient Expression in Suspension Conditions and Serum-Free Medium Using Shake Flasks
3.3. Production of the SARS-CoV-2 N and N-CD Proteins in 1 L and 3 L Stirred-Tank Bioreactors
3.4. Purification of the SARS-CoV-2 N and N-CD Proteins Produced in Suspension Culture and Serum-Free Medium
3.5. Obtaining N-CD-Expressing HEK-293 Cell Clones and Adaptation to Serum-Free Medium and Suspension Culture Conditions
3.6. Batch Culture of the N-CD-Expressing HEK-293 Cell Clone 100-2E9 and Simulation of Perfusion Conditions
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Cell Clones | Growth Phase Duration § (days) | Experiment Duration (days) | VCDmax (106 cell/mL) | IVCCmax (108 cells × h/mL) | μmax (h−1) |
---|---|---|---|---|---|
50-6D9 * | 4 | 16 | 1.7 | 314.5 | 0.0116 |
50-9B9 * | 4 | 13 | 3.1 | 632.8 | 0.0159 |
100-2E9 * | 6 | 16 | 2.5 | 592.7 | 0.0105 |
100-2E9 ** | 10 | 10 | 3.1 | 354.3 | 0.0101 |
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Lao, T.; Farnos, O.; Bueno, A.; Alvarez, A.; Rodríguez, E.; Palacios, J.; Luz, K.R.d.l.; Kamen, A.; Carpio, Y.; Estrada, M.P. 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. Biomedicines 2023, 11, 3050. https://doi.org/10.3390/biomedicines11113050
Lao T, Farnos O, Bueno A, Alvarez A, Rodríguez E, Palacios J, Luz KRdl, Kamen A, Carpio Y, Estrada MP. 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. Biomedicines. 2023; 11(11):3050. https://doi.org/10.3390/biomedicines11113050
Chicago/Turabian StyleLao, Thailin, Omar Farnos, Alexi Bueno, Anays Alvarez, Elsa Rodríguez, Julio Palacios, Kathya Rashida de la Luz, Amine Kamen, Yamila Carpio, and Mario Pablo Estrada. 2023. "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" Biomedicines 11, no. 11: 3050. https://doi.org/10.3390/biomedicines11113050
APA StyleLao, T., Farnos, O., Bueno, A., Alvarez, A., Rodríguez, E., Palacios, J., Luz, K. R. d. l., Kamen, A., Carpio, Y., & Estrada, M. P. (2023). 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. Biomedicines, 11(11), 3050. https://doi.org/10.3390/biomedicines11113050