Skip to main content

Advertisement

SpringerLink
Log in

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

  • Biotechnological products and process engineering
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Backliwal G, Hildinger M, Chenuet S, Wulhfard S, Jesus MD, Wurm FM (2008) Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions. Nucleic Acids Res 36(15):e96

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Brummelkamp TR, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296(5567):550–553

    Article  PubMed  CAS  Google Scholar 

  • Codamo J, Munro TP, Hughes BS, Song M, Gray PP (2011) Enhanced CHO cell-based transient gene expression with the epi-CHO expression system. Mol Biotechnol 48(2):109–115

    Article  PubMed  CAS  Google Scholar 

  • Daramola O, Stevenson J, Dean G, Hatton D, Pettman G, Holmes W, Field R (2014) A high-yielding CHO transient system: coexpression of genes encoding EBNA-1 and GS enhances transient protein expression. Biotechnol Prog 30(1):132–141

    Article  PubMed  CAS  Google Scholar 

  • Dumont J, Euwart D, Mei B, Estes S, Kshirsagar R (2016) Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives. Crit Rev Biotechnol 36(6):1110–1122

    Article  PubMed  CAS  Google Scholar 

  • Gaj T, Gersbach CA, Barbas CFIII (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31(7):397–405

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Geisse S (2009) Reflections on more than 10 years of TGE approaches. Protein Expr Purif 64(2):99–107

    Article  PubMed  CAS  Google Scholar 

  • Hanon GJ (2002) RNA interference. Nature 418(6894):244–251

    Article  CAS  Google Scholar 

  • Heffernan M, Dennis JW (1991) Polyoma and hamster papovavirus large T antigen-mediated replication of expression shuttle vectors in Chinese hamster ovary cells. Nucleic Acids Res 19(1):85–92

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Kang SY, Kim YG, Lee HW, Lee EG (2015) A single-plasmid vector for transgene amplification using short hairpin RNA targeting the 3′-UTR of amplifiable dhfr. Appl Microbiol Biotechnol 99(23):10117–10126

    Article  PubMed  CAS  Google Scholar 

  • Kim WH, Kim MS, Kim YG, Lee GM (2012) Development of apoptosis-resistant CHO cell line expressing PyLT for the enhancement of transient antibody production. Process Biochem 47:2557–2561

    Article  CAS  Google Scholar 

  • Kim YG, Lee GM (2009) Bcl-xL overexpression does not enhance specific erythropoietin productivity of recombinant CHO cells grown at 33 degrees C and 37 degrees C. Biotechnol Prog 25(1):252–256

    Article  PubMed  CAS  Google Scholar 

  • Kishida T, Asada H, Kubo K, Sato YT, Shin-Ya M, Imanishi J, Yoshikawa K, Mazda O (2008) Pleiotrophic functions of Epstein-Barr virus nuclear antigen-1 (EBNA-1) and oriP differentially contribute to the efficiency of transfection/expression of exogenous gene in mammalian cells. J Biotechnol 133(2):201–207

    Article  PubMed  CAS  Google Scholar 

  • Klein R, Ruttkowski B, Knapp E, Salmons B, Günzburg WH, Hohenadl C (2006) WPRE-mediated enhancement of gene expression is promoter and cell line specific. Gene 372:153–161

    Article  PubMed  CAS  Google Scholar 

  • Kunaparaju R, Liao M, Sunstrom NA (2005) Epi-CHO, an episomal expression system for recombinant protein production in CHO cells. Biotechnol Bioeng 91(6):670–677

    Article  PubMed  CAS  Google Scholar 

  • Le Hir H, Nott A, Moore MJ (2003) How introns influence and enhance eukaryotic gene expression. Trends Biochem Sci 28(4):215–220

    Article  PubMed  CAS  Google Scholar 

  • Lee JH, Lim SM, Park SH, Min JK, Lee GM, Kim YG (2017a) Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells. Process Biochem 55:182–186

    Article  CAS  Google Scholar 

  • Lee JH, Jeong YR, Kim YG, Lee GM (2017b) Understanding of decreased sialylation of Fc-fusion protein in hyperosmotic recombinant Chinese hamster ovary cell culture: N-glycosylation gene expression and N-linked glycan antennary profile. Biotechnol Bioeng 114(8):1721–1732

    Article  PubMed  CAS  Google Scholar 

  • Majors BS, Betenbaugh MJ, Pederson NE, Chiang GG (2008) Enhancement of transient gene expression and culture viability using Chinese hamster ovary cells overexpressing Bcl-x(L). Biotechnol Bioeng 101(3):567–578

    Article  PubMed  CAS  Google Scholar 

  • Makrides SC (1999) Components of vectors for gene transfer and expression in mammalian cells. Protein Expr Purif 17(2):183–202

    Article  PubMed  CAS  Google Scholar 

  • Mizuguchi H, Hosono T, Hayakawa T (2000) Long-term replication of Epstein-Barr virus-derived episomal vectors in the rodent cells. FEBS Lett 472(2–3):173–178

    Article  PubMed  CAS  Google Scholar 

  • Noh SM, Sathyamurthy M, Lee GM (2013) Development of recombinant Chinese hamster ovary cell lines for therapeutic protein production. Curr Opin Chem Eng 2:391–397

    Article  Google Scholar 

  • Piechaczek C, Fetzer C, Baiker A, Bode J, Lipps HJ (1999) A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells. Nucleic Acids Res 27(2):426–428

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Rajendra Y, Hougland MD, Alam R, Morehead TA, Barnard GC (2015) A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line: process development and product quality assessment. Biotechnol Bioeng 112(5):977–986

    Article  PubMed  CAS  Google Scholar 

  • Renard JM, Spagnoli R, Mazier C, Salles MF, Mandine E (1988) Evidence that monoclonal antibody production kinetics is related to the integral of the viable cells curve in batch systems. Biotechnol Lett 10:91–96

    Article  Google Scholar 

  • Silla T, Hääl I, Geimanen J, Janikson K, Abroi A, Ustav E, Ustav M (2005) Episomal maintenance of plasmids with hybrid origins in mouse cells. J Virol 79(24):15277–15288

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22(11):1393–1398

    Article  PubMed  CAS  Google Scholar 

  • Xia W, Bringmann P, McClary J, Jones PP, Manzana W, Zhu Y, Wang S, Liu Y, Harvey S, Madlansacay MR, McLean K, Rosser MP, MacRobbie J, Olsen CL, Cobb RR (2006) High levels of protein expression using different mammalian CMV promoters in several cell lines. Protein Expr Purif 45(1):115–124

    Article  PubMed  CAS  Google Scholar 

  • Yates JL, Warren N, Sugden B (1993) Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature 313(6005):812–815

    Article  Google Scholar 

  • Yin W, Xiang P, Li Q (2005) Investigations of the effect of DNA size in transient transfection assay using dual luciferase system. Anal Biochem 346(2):289–294

    Article  PubMed  CAS  Google Scholar 

  • Yoon H, Song JM, Ryu CJ, Kim YG, Lee EK, Kang S, Kim SJ (2012) An efficient strategy for cell-based antibody library selection using an integrated vector system. BMC Biotechnol 12:62

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Yu DY, Noh SM, Lee GM (2016) Limitations to the development of recombinant human embryonic kidney 293E cells using glutamine synthetase-mediated gene amplification: methionine sulfoximine resistance. J Biotechnol 231:136–140

    Article  PubMed  CAS  Google Scholar 

  • Zhu J (2012) Mammalian cell protein expression for biopharmaceutical production. Biotechnol Adv 30(5):1158–1170

    Article  PubMed  CAS  Google Scholar 

  • Zustiak MP, Jose L, Xie Y, Zhu J, Betenbaugh MJ (2014) Enhanced transient recombinant protein production in CHO cells through the co-transfection of the product gene with Bcl-xL. Biotechnol J 9(9):1164–1174

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Funding

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.

Author information

Authors and Affiliations

  1. Department of Biological Sciences, KAIST, 335 Gwahak-ro, Yuseong-gu, Daejeon, South Korea

    Joo-Hyoung Lee, Jong-Ho Park, Jee Yon Kim & Gyun Min Lee

  2. Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, South Korea

    Joo-Hyoung Lee, Jong-Ho Park, Sun-Hye Park, Sun-Hong Kim, Jeong-Ki Min & Yeon-Gu Kim

  3. Department of Bioprocess Engineering, KRIBB School of Biotechnology, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, South Korea

    Sun-Hye Park & Yeon-Gu Kim

  4. Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, UK

    Sun-Hong Kim

  5. Department of Biomolecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, South Korea

    Jeong-Ki Min

Authors
  1. Joo-Hyoung Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jong-Ho Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sun-Hye Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sun-Hong Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jee Yon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jeong-Ki Min
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Gyun Min Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yeon-Gu Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Gyun Min Lee or Yeon-Gu Kim.

Ethics declarations

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.

Electronic supplementary material

ESM 1

(PDF 259 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lee, JH., Park, JH., Park, SH. et al. Co-amplification of EBNA-1 and PyLT through dhfr-mediated gene amplification for improving foreign protein production in transient gene expression in CHO cells. Appl Microbiol Biotechnol 102, 4729–4739 (2018). https://doi.org/10.1007/s00253-018-8977-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-018-8977-6

Keywords

Search

Navigation