Applied Microbiology and Biotechnology

, Volume 102, Issue 11, pp 4729–4739 | Cite as

Co-amplification of EBNA-1 and PyLT through dhfr-mediated gene amplification for improving foreign protein production in transient gene expression in CHO cells

  • Joo-Hyoung Lee
  • Jong-Ho Park
  • Sun-Hye Park
  • Sun-Hong Kim
  • Jee Yon Kim
  • Jeong-Ki Min
  • Gyun Min Lee
  • Yeon-Gu Kim
Biotechnological products and process engineering


Despite the relatively low transfection efficiency and low specific foreign protein productivity (qp) of Chinese hamster ovary (CHO) cell-based transient gene expression (TGE) systems, TGE-based recombinant protein production technology predominantly employs CHO cells for pre-clinical research and development purposes. To improve TGE in CHO cells, Epstein-Barr virus nuclear antigen-1 (EBNA-1)/polyoma virus large T antigen (PyLT)-co-amplified recombinant CHO (rCHO) cells stably expressing EBNA-1 and PyLT were established using dihydrofolate reductase/methotrexate-mediated gene amplification. The level of transiently expressed Fc-fusion protein was significantly higher in the EBNA-1/PyLT-co-amplified pools compared to control cultures. Increased Fc-fusion protein production by EBNA-1/PyLT-co-amplification resulted from a higher qp attributable to EBNA-1 but not PyLT expression. The qp for TGE-based production with EBNA-1/PyLT-co-amplified rCHO cells (EP-amp-20) was approximately 22.9-fold that of the control culture with CHO-DG44 cells. Rather than improved transfection efficiency, this cell line demonstrated increased levels of mRNA expression and replicated DNA, contributing to an increased qp. Furthermore, there was no significant difference in N-glycan profiles in Fc-fusion proteins produced in the TGE system. Taken together, these results showed that the use of rCHO cells with co-amplified expression of the viral elements EBNA-1 and PyLT improves TGE-based therapeutic protein production dramatically. Therefore, EBNA-1/PyLT-co-amplified rCHO cells will likely be useful as host cells in CHO cell-based TGE systems.


TGE CHO cells Gene amplification EBNA-1 PyLT Fc-fusion protein 


Funding information

This work was supported in part by a grant from the National Research Foundation of Korea (NRF) funded by the Korea government (Ministry of Science, ICT & Future Planning) (No. NRF-2017R1C1B2009642) and a grant from KRIBB Initiative Program.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_8977_MOESM1_ESM.pdf (259 kb)
ESM 1 (PDF 259 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Joo-Hyoung Lee
    • 1
    • 2
  • Jong-Ho Park
    • 1
    • 2
  • Sun-Hye Park
    • 2
    • 3
  • Sun-Hong Kim
    • 2
    • 4
  • Jee Yon Kim
    • 1
  • Jeong-Ki Min
    • 2
    • 5
  • Gyun Min Lee
    • 1
  • Yeon-Gu Kim
    • 2
    • 3
  1. 1.Department of Biological SciencesKAISTDaejeonSouth Korea
  2. 2.Biotherapeutics Translational Research CenterKorea Research Institute of Bioscience and Biotechnology (KRIBB)DaejeonSouth Korea
  3. 3.Department of Bioprocess Engineering, KRIBB School of BiotechnologyKorea University of Science and Technology (UST)DaejeonSouth Korea
  4. 4.Department of Life SciencesImperial College LondonLondon SW7 2AZUK
  5. 5.Department of Biomolecular Science, KRIBB School of BioscienceKorea University of Science and Technology (UST)DaejeonSouth Korea

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