Molecular Biotechnology

, Volume 61, Issue 5, pp 317–324 | Cite as

HEK293 Cells Overexpressing Nuclear Factor E2-Related Factor-2 Improve Expression of Recombinant Coagulation Factor VII

  • Zahra Abbasi-Malati
  • Fatemeh Amiri
  • Mahshid Mohammadipour
  • Mehryar Habibi RoudkenarEmail author
Original paper


The mammalian expression system plays a key central role in the production of therapeutic recombinant proteins. Conspicuously, any improvements in the expression system which lead to a higher expression level would have an impact especially in bio-pharmaceutical industries. In the current study, to take steps toward the improvement of expression of recombinant protein, first, we established a stable HEK293 cell line to overexpress a well-known cytoprotective and antioxidant gene, Nrf2. Next, we transiently expressed human recombinant coagulation factor VII, as an example of human recombinant protein, in the engineered-HEK293 cell line. Our results revealed that the established cells had a higher growth rate and were able to endure to UV-induced oxidative stress. Furthermore, within our expectation, our results revealed that the expression level of recombinant FVII in Nrf2-engineered HEK293 cells (315 ng/ml) was higher than the HEK293 (198 ng/ml) cells and it was functional in a coagulation test assay. Moreover, our new cell line could be a suitable cell to express other recombinant proteins especially for large-scale production of recombinant protein under other culture condition such as lower serum and suspension culture that imposed advantages especially in terms of cost benefits in bio-pharmaceutical industries.


Mammalian expression system HEK293 cell line Nrf2 Human coagulation factor VII 



This study was supported by the Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Iran, Grant No. 1395-01-33-1933.

Author Contributions

ZAb, FA and MM collected all data, samples and also accomplished all cellular and molecular tests. MHR controlled and managed the project, wrote the manuscript and finalized it. All authors revised the article carefully and confirmed the edited version of the paper.

Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest.

Ethical Approval

There are no ethical problems for this project.


  1. 1.
    Berlec, A., & Štrukelj, B. (2013). Current state and recent advances in biopharmaceutical production in Escherichia coli, yeasts and mammalian cells. Journal of Industrial Microbiology & Biotechnology, 40(3–4), 257–274.CrossRefGoogle Scholar
  2. 2.
    Dalton, A. C., & Barton, W. A. (2014). Over-expression of secreted proteins from mammalian cell lines. Protein Science, 23(5), 517–525.CrossRefGoogle Scholar
  3. 3.
    Dumont, J., Euwart, D., Mei, B., Estes, S., & Kshirsagar, R. (2016). Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives. Critical Reviews in Biotechnology, 36(6), 1110–1122.CrossRefGoogle Scholar
  4. 4.
    Durocher, Y., & Butler, M. (2009). Expression systems for therapeutic glycoprotein production. Current Opinion in Biotechnology, 20(6), 700–707.CrossRefGoogle Scholar
  5. 5.
    Durocher, Y., Perret, S., & Kamen, A. (2002). High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Research, 30(2), e9–e9.CrossRefGoogle Scholar
  6. 6.
    Estes, S., & Melville, M. (2013). Mammalian cell line developments in speed and efficiency. In W. Zhou & A. Kantardjieff (Eds.), Mammalian cell cultures for biologics manufacturing (pp. 11–33). Berlin: Springer.CrossRefGoogle Scholar
  7. 7.
    Ghaderi, D., Taylor, R. E., Padler-Karavani, V., Diaz, S., & Varki, A. (2010). Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins. Nature Biotechnology, 28, 863.CrossRefGoogle Scholar
  8. 8.
    Ghaderi, D., Zhang, M., Hurtado-Ziola, N., & Varki, A. (2012). Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation. Biotechnology and Genetic Engineering Reviews, 28(1), 147–176.CrossRefGoogle Scholar
  9. 9.
    Gupta, S. K., Dangi, A. K., Smita, M., Dwivedi, S., & Shukla, P. (2019). Effectual bioprocess development for protein production. In P. Shukla (Ed.), Applied Microbiology and bioengineering (pp. 203–227). Cambridge: Academic Press.CrossRefGoogle Scholar
  10. 10.
    Gupta, S. K., Sharma, A., Kushwaha, H., & Shukla, P. (2017). Over-expression of a codon optimized yeast cytosolic pyruvate carboxylase (PYC2) in CHO cells for an augmented lactate metabolism. Frontiers in Pharmacology, 8, 463.CrossRefGoogle Scholar
  11. 11.
    Gupta, S. K., & Shukla, P. (2017). Sophisticated cloning, fermentation, and purification technologies for an enhanced therapeutic protein production: A review. Frontiers in Pharmacology, 8, 419.CrossRefGoogle Scholar
  12. 12.
    Gupta, S. K., Srivastava, S. K., Sharma, A., Nalage, V. H., Salvi, D., Kushwaha, H., Chitnis, N. B., & Shukla, P. (2017). Metabolic engineering of CHO cells for the development of a robust protein production platform. PloS ONE, 12(8), e0181455.CrossRefGoogle Scholar
  13. 13.
    Halabian, R., Roudkenar, M. H., Esmaeili, N. S., Masroori, N., Roushandeh, A., & Najafabadi, A. (2009). Establishment of a cell line expressing recombinant factor VII and its subsequent conversion to active form FVIIa through hepsin. Genetic Engineering Method.Vox Sanguinis, 96(4), 309–315.CrossRefGoogle Scholar
  14. 14.
  15. 15.
    Invitrogen Corporation, C. CA,
  16. 16.
    Gupta, K., S. and Shukla, P. (2018). Glycosylation control technologies for recombinant therapeutic proteins. Applied Microbiology and Biotechnology, 102(24), 10457–10468.CrossRefGoogle Scholar
  17. 17.
    Kayser, K., Lin, N., Allison, D., Donahue, L., & Caple, M. (2006). Cell line engineering methods for improving productivity. BioProcess International, 4(5), 6–13.Google Scholar
  18. 18.
    Kopito, R. R. (2000). Aggresomes, inclusion bodies and protein aggregation. Trends in Cell Biology, 10(12), 524–530.CrossRefGoogle Scholar
  19. 19.
    Kuriakose, A., Chirmule, N., & Nair, P. (2016). Immunogenicity of biotherapeutics: causes and association with posttranslational modifications. Journal of Immunology Research. Google Scholar
  20. 20.
    Lai, T., Yang, Y., & Ng, S. K. (2013). Advances in mammalian cell line development technologies for recombinant protein production. Pharmaceuticals, 6(5), 579–603.CrossRefGoogle Scholar
  21. 21.
    Lee, J.-M., & Johnson, J. A. (2004). An important role of Nrf2-ARE pathway in the cellular defense mechanism. BMB Reports, 37(2), 139–143.CrossRefGoogle Scholar
  22. 22.
    Liste-Calleja, L., Lecina, M., Schucht, R., Wirth, D., Hauser, H., & Cairó, J. J. (2015). “Hek293 as a recombinant protein factory: three different approaches for protein production. BMC Proceedings, 9(9): P74.CrossRefGoogle Scholar
  23. 23.
    Masroori, N., Halabian, R., Mohammadipour, M., Roushandeh, A. M., Rouhbakhsh, M., Najafabadi, A. J., Fathabad, M. E., Salimi, M., Shokrgozar, M. A., & Roudkenar, M. H. (2010). High-level expression of functional recombinant human coagulation factor VII in insect cells. Biotechnology Letters, 32(6), 803–809.CrossRefGoogle Scholar
  24. 24.
    Mohammadzadeh, M., Halabian, R., Gharehbaghian, A., Amirizadeh, N., Jahanian-Najafabadi, A., Roushandeh, A. M., & Roudkenar, M. H. (2012). “Nrf-2 overexpression in mesenchymal stem cells reduces oxidative stress-induced apoptosis and cytotoxicity. Cell Stress and Chaperones, 17(5), 553–565.CrossRefGoogle Scholar
  25. 25.
    Movahed, M., Roudkenar, M. H., Bahadori, M., Mohammadipour, M., Jalili, M. A., & Amiri, F. (2018). Establishment of Stable CHO Cell Line Expressing recombinant human haptoglobin: Toward new haptoglobin-based therapeutics. Iranian Journal of Science and Technology Transactions A: Science, 42(3), 1097–1103.CrossRefGoogle Scholar
  26. 26.
    Palomares, L. A., Estrada-Mondaca, S., & Ramirez, O. T. (2004). Production of recombinant proteins: challenges and solutions. Methods in Molecular Biology, 267, 15–52.Google Scholar
  27. 27.
    Picanço-Castro, V., Tage Biaggio, R., Tadeu Cova, D., & Swiech, K. (2013). Production of recombinant therapeutic proteins in human cells: Current achievements and future perspectives. Protein and Peptide Letters, 20(12), 1373–1381.CrossRefGoogle Scholar
  28. 28.
    Schmelzer, A. E., & Miller, W. M. (2002). “Effects of osmoprotectant compounds on NCAM polysialylation under hyperosmotic stress and elevated pCO2. Biotechnology and Bioengineering, 77(4), 359–368.CrossRefGoogle Scholar
  29. 29.
    Shukla, A. A., & Thömmes, J. (2010). Recent advances in large-scale production of monoclonal antibodies and related proteins. Trends in Biotechnology, 28(5), 253–261.CrossRefGoogle Scholar
  30. 30.
    Sun, X., Hia, H. C., Goh, P. E., & Yap, M. G. (2008). High-density transient gene expression in suspension-adapted 293 EBNA1 cells. Biotechnology and Bioengineering, 99(1), 108–116.CrossRefGoogle Scholar
  31. 31.
    Swiech, K., Kamen, A., Ansorge, S., Durocher, Y., Picanço-Castro, V., Russo-Carbolante, E. M. S., Neto, M. S. A., & Covas, D. T. (2011). Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII. BMC Biotechnology, 11, 114–114.CrossRefGoogle Scholar
  32. 32.
    Swiech, K., Picanço-Castro, V., & Covas, D. T. (2012). Human cells: new platform for recombinant therapeutic protein production. Protein Expression and Purification, 84(1), 147–153.CrossRefGoogle Scholar
  33. 33.
    Thimmulappa, R. K., Lee, H., Rangasamy, T., Reddy, S. P., Yamamoto, M., Kensler, T. W., & Biswal, S. (2016). Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis. The Journal of Clinical Investigation, 116(4), 984–995.CrossRefGoogle Scholar
  34. 34.
    Thomas, P., & Smart, T. G. (2005). HEK293 cell line: a vehicle for the expression of recombinant proteins. Journal of Pharmacological and Toxicological Methods, 51(3), 187–200.CrossRefGoogle Scholar
  35. 35.
    Walsh, G. (2014). Biopharmaceutical benchmarks 2014. Nature Biotechnology, 32, 992.CrossRefGoogle Scholar
  36. 36.
    Wurm, F. M. (2004). Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotechnology, 22(11), 1393.CrossRefGoogle Scholar
  37. 37.
    Yang, M., & Butler, M. (2000). Effect of ammonia on the glycosylation of human recombinant erythropoietin in culture. Biotechnology Progress, 16(5), 751–759.CrossRefGoogle Scholar
  38. 38.
    Zhaleh, F., Amiri, F., Mohammadzadeh-Vardin, M., Bahadori, M., Harati, M. D., Roudkenar, M. H., & Saki, S. (2016). Nuclear factor erythroid-2 related factor 2 overexpressed mesenchymal stem cells transplantation, improves renal function, decreases injuries markers and increases repair markers in glycerol-induced Acute kidney injury rats. Iranian Journal of Basic medical Sciences, 19(3), 323.Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Zahra Abbasi-Malati
    • 1
  • Fatemeh Amiri
    • 2
  • Mahshid Mohammadipour
    • 1
  • Mehryar Habibi Roudkenar
    • 3
    • 4
    • 5
    Email author
  1. 1.Blood Transfusion Research Center, High Institute for Research and Education in Transfusion MedicineTehranIran
  2. 2.Department of Medical Laboratory Sciences, School of ParamedicineHamadan University of Medical SciencesHamadanIran
  3. 3.Stem cell and Regenerative Medicine Research CenterGuilan University of Medical SciencesRashtIran
  4. 4.Neuroscience Research CenterGuilan University of Medical SciencesRashtIran
  5. 5.Medical Biotechnology Department, Paramedicine FacultyGuilan University of Medical SciencesRashtIran

Personalised recommendations