Bioprocess and Biosystems Engineering

, Volume 42, Issue 5, pp 799–806 | Cite as

Study of the mechanism for increased protein expression via transcription potency reduction of the selection marker

  • Bin Yang
  • Jiatao Zhou
  • Hui Zhao
  • Anling Wang
  • Yuanjun Lei
  • Qiuling Xie
  • Sheng XiongEmail author
Research Paper


Stable transfection of mammalian cells using various expression cassettes for exogenous gene expression has been well established. The impact of critical factors in these cassettes, such as promoter and enhancer elements, on recombinant protein production in mammalian cells has been studied extensively to optimize the expression efficiency. However, few studies on the correlation between the strength of selection marker and the expression of gene of interest (GOI) have been reported. Here we investigated the correlation between the strength of a widely used selection marker, glutamine synthetase (GS) gene, and gene of interest in which the expression of GOI is driven by mouse cytomegalovirus (mCMV) major immediate early (MIE) promoter whereas the expression of GS is controlled by SV40E (Simian vacuolating virus 40E) promoter. We used a green fluorescent protein and the adalimumab antibody (heavy and light chain) as two distinct examples for the gene of interest. We then decreased the expression of GS gene by engineering a specific region of its SV40E promoter in these expression cassettes. By comparing the expression of GS and GOI at transcription and translation level before and after the SV40E promoter was weakened, we found that lower GS expression due to weaker SV40E transcription correlated well with the higher expression of recombinant proteins, mainly by increasing the copy number of GS and GOI integration into host cell genome.


Chinese hamster ovary (CHO) cell Glutamine synthetase (GS) Simian vacuolating virus 40E (SV40E) promoter Stable transfection 



This work was supported by Guangdong Provincial Science and Tech Project (2015A020211016); the Guangzhou Industry-Academia-Research Collaborative Innovation Project (201604016009).

Compliance with ethical standards

Conflict of interest

All the authors reviewed and agreed to submit this manuscript. The authors declare that they have no conflict of interest.

Research involving human participants and/or animals

The study does not contain experiments using animals and human studies.


  1. 1.
    Matsuyama R, Yamano N, Kawamura N, Omasa T (2017) Lengthening of high-yield production levels of monoclonal antibody-producing Chinese hamster ovary cells by downregulation of breast cancer 1. J Biosci Bioeng 123(3):382–389. Google Scholar
  2. 2.
    Deschenes I, Finkle CD, Winocour PD (1998) Effective use of BCH-2763, a new potent injectable direct thrombin inhibitor, in combination with tissue plasminogen activator (tPA) in a rat arterial thrombolysis model. Thromb Haemost 80(1):186–191Google Scholar
  3. 3.
    Walsh G (2010) Biopharmaceutical benchmarks 2010. Nat Biotechnol 28(9):917–924. Google Scholar
  4. 4.
    Chu L, Robinson DK (2001) Industrial choices for protein production by large-scale cell culture. Curr Opin Biotechnol 12(2):180–187Google Scholar
  5. 5.
    Zhu J (2012) Mammalian cell protein expression for biopharmaceutical production. Biotechnol Adv 30(5):1158–1170. Google Scholar
  6. 6.
    Jayapal KP, Lian W, Glod F, Sherman DH, Hu WS (2007) Comparative genomic hybridizations reveal absence of large Streptomyces coelicolor genomic islands in Streptomyces lividans. BMC Genomics 8:229. Google Scholar
  7. 7.
    Kim JY, Kim YG, Lee GM (2012) CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Appl Microbiol Biotechnol 93(3):917–930. Google Scholar
  8. 8.
    Miki H, Takagi M (2015) Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media. Cytotechnology 67(4):689–697. Google Scholar
  9. 9.
    Hacker DL, De Jesus M, Wurm FM (2009) 25 years of recombinant proteins from reactor-grown cells - where do we go from here? Biotechnol Adv 27(6):1023–1027. Google Scholar
  10. 10.
    Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22(11):1393–1398. Google Scholar
  11. 11.
    McKnight S, Tjian R (1986) Transcriptional selectivity of viral genes in mammalian cells. Cell 46(6):795–805Google Scholar
  12. 12.
    Yew NS, Wysokenski DM, Wang KX, Ziegler RJ, Marshall J, McNeilly D, Cherry M, Osburn W, Cheng SH (1997) Optimization of plasmid vectors for high-level expression in lung epithelial cells. Human Gene Ther 8(5):575–584. Google Scholar
  13. 13.
    HHa WagnerR (1997) Mammalian cell biotechnology in protein production. Walter de GruyterGoogle Scholar
  14. 14.
    Tian ZW, Xu DH, Wang TY, Wang XY, Xu HY, Zhao CP, Xu GH (2018) Identification of a potent MAR element from the human genome and assessment of its activity in stably transfected CHO cells. J Cell Mol Med 22(2):1095–1102. Google Scholar
  15. 15.
    Girod PA, Nguyen DQ, Calabrese D, Puttini S, Grandjean M, Martinet D, Regamey A, Saugy D, Beckmann JS, Bucher P, Mermod N (2007) Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells. Nat Methods 4(9):747–753. Google Scholar
  16. 16.
    Kim JD, Yoon Y, Hwang HY, Park JS, Yu S, Lee J, Baek K, Yoon J (2005) Efficient selection of stable chinese hamster ovary (CHO) cell lines for expression of recombinant proteins by using human interferon beta SAR element. Biotechnol Prog 21(3):933–937. Google Scholar
  17. 17.
    Zhang F, Thornhill SI, Howe SJ, Ulaganathan M, Schambach A, Sinclair J, Kinnon C, Gaspar HB, Antoniou M, Thrasher AJ (2007) Lentiviral vectors containing an enhancer-less ubiquitously acting chromatin opening element (UCOE) provide highly reproducible and stable transgene expression in hematopoietic cells. Blood 110(5):1448–1457. Google Scholar
  18. 18.
    Bebbington CR, Renner G, Thomson S, King D, Abrams D, Yarranton GT (1992) High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Bio/technology 10(2):169–175Google Scholar
  19. 19.
    Cockett MI, Bebbington CR, Yarranton GT (1990) High level expression of tissue inhibitor of metalloproteinases in Chinese hamster ovary cells using glutamine synthetase gene amplification. Bio/technology 8(7):662–667Google Scholar
  20. 20.
    Fan L, Kadura I, Krebs LE, Larson JL, Bowden DM, Frye CC (2013) Development of a highly-efficient CHO cell line generation system with engineered SV40E promoter. J Biotechnol 168(4):652–658. Google Scholar
  21. 21.
    Fan L, Kadura I, Krebs LE, Hatfield CC, Shaw MM, Frye CC (2012) Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells. Biotechnol Bioeng 109(4):1007–1015. Google Scholar
  22. 22.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408. Google Scholar
  23. 23.
    Byrne BJ, Davis MS, Yamaguchi J, Bergsma DJ, Subramanian KN (1983) Definition of the simian virus 40 early promoter region and demonstration of a host range bias in the enhancement effect of the simian virus 40 72-base-pair repeat. Proc Natl Acad Sci USA 80(3):721–725Google Scholar
  24. 24.
    Dorai H, Csirke B, Scallon B, Ganguly S (2006) Correlation of heavy and light chain mRNA copy numbers to antibody productivity in mouse myeloma production cell lines. Hybridoma (Larchmt) 25(1):1–9. Google Scholar
  25. 25.
    Matasci M, Hacker DL, Baldi L, Wurm FM (2008) Recombinant therapeutic protein production in cultivated mammalian cells: current status and future prospects. Drug Discov Today Technol 5(2–3):e37–e42. Google Scholar
  26. 26.
    Butler M (2005) Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals. Appl Microbiol Biotechnol 68(3):283–291. Google Scholar
  27. 27.
    Brown AJ, Sweeney B, Mainwaring DO, James DC (2015) NF-kappaB, CRE and YY1 elements are key functional regulators of CMV promoter-driven transient gene expression in CHO cells. Biotechnol J 10(7):1019–1028. Google Scholar
  28. 28.
    Wang F, Wang TY, Tang YY, Zhang JH, Yang XJ (2012) Different matrix attachment regions flanking a transgene effectively enhance gene expression in stably transfected Chinese hamster ovary cells. Gene 500(1):59–62. Google Scholar
  29. 29.
    Roman R, Miret J, Scalia F, Casablancas A, Lecina M, Cairo JJ (2016) Enhancing heterologous protein expression and secretion in HEK293 cells by means of combination of CMV promoter and IFNalpha2 signal peptide. J Biotechnol 239:57–60. Google Scholar
  30. 30.
    Zahn-Zabal M, Kobr M, Girod PA, Imhof M, Chatellard P, de Jesus M, Wurm F, Mermod N (2001) Development of stable cell lines for production or regulated expression using matrix attachment regions. J Biotechnol 87(1):29–42Google Scholar
  31. 31.
    Noh SM, Shin S, Lee GM (2018) Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies. Sci Rep 8(1):5361. Google Scholar
  32. 32.
    Tyo KE, Ajikumar PK, Stephanopoulos G (2009) Stabilized gene duplication enables long-term selection-free heterologous pathway expression. Nat Biotechnol 27(8):760–765. Google Scholar
  33. 33.
    Schimke RT (1984) Gene amplification in cultured animal cells. Cell 37(3):705–713Google Scholar
  34. 34.
    Jun SC, Kim MS, Hong HJ, Lee GM (2006) Limitations to the development of humanized antibody producing Chinese hamster ovary cells using glutamine synthetase-mediated gene amplification. Biotechnol Prog 22(3):770–780. Google Scholar
  35. 35.
    Lai T, Yang Y, Ng SK (2013) Advances in Mammalian cell line development technologies for recombinant protein production. Pharmaceuticals 6(5):579–603. Google Scholar
  36. 36.
    Huang Y, Li Y, Wang YG, Gu X, Wang Y, Shen BF (2007) An efficient and targeted gene integration system for high-level antibody expression. J Immunol Methods 322(1–2):28–39. Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Bin Yang
    • 1
  • Jiatao Zhou
    • 2
  • Hui Zhao
    • 1
  • Anling Wang
    • 1
  • Yuanjun Lei
    • 1
  • Qiuling Xie
    • 1
  • Sheng Xiong
    • 1
    Email author
  1. 1.Department of Cell Biol, Institute of Biomedicine & National Engineering Research Center of Genetic Medicine, College of Life Science and TechnologyJinan UniversityGuangzhouPeople’s Republic of China
  2. 2.Sunshine Lake Pharma Co., Ltd.DongguanPeople’s Republic of China

Personalised recommendations