Skip to main content
SpringerLink
Log in

Improved recombinant protein production by regulation of transcription and protein transport in Chinese hamster ovary cells

  • Original Research Paper
  • Published:
Biotechnology Letters Aims and scope Submit manuscript

Abstract

Objectives

To identify genes that affected protein expression in Chinese hamster ovary (CHO) cells was significant, and we identified the changes in the transcriptome and the functional gene sets that would contribute to increase expression of recombinant protein.

Results

Here two sub-clones from a methotrexate-treated parental recombinant CHO cell line were selected. The two sub-clones, with different expression levels (qp were 42.8 pg/cell/day and 14.0 pg/cell/day), were analyzed through RNA-seq. More than 600 genes were identified as differently expressed, and we found that the differentially expressed genes were involved in processes such as RNA processing, transcription, protein catabolism, and protein transport. Among these, we cloned genes encoding proteins that were involved in transcription and protein transport to investigate their effect on protein production.

Conclusions

We found that some genes involved in transcription and protein transport would improve recombinant protein production in CHO cells.

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

Abbreviations

CHO cells:

Chinese hamster ovary cells

MTX:

Methotrexate

TACI:

The transmembrane activator and CAML interactor

Fc:

The fragment crystallizable

ELISA:

Enzyme linked immunosorbent assay

IMDM:

Iscove’s modified Dulbecco’s medium

FBS:

Fetal bovine serum

qPCR:

Quantitative polymerase chain reaction

References

  • Abramian DS, Glebov OK (1981) Intraclonal heterogeneity and instability of CHO-K1 Chinese hamster cells in sensitivity to ultraviolet light and resistance to 8-azaguanine. Tsitologiia 23(9):1031–1040

    CAS  PubMed  Google Scholar 

  • Allegue C, Coll M, Mates J, Campuzano O, Iglesias A, Sobrino B, Brion M, Amigo J, Carracedo A, Brugada P, Brugada J, Brugada R (2015) Genetic analysis of arrhythmogenic diseases in the era of NGS: the complexity of clinical decision-making in brugada syndrome. PLoS ONE 10(7):e0133037

    Article  PubMed  PubMed Central  Google Scholar 

  • Allen GC, Spiker S, Thompson WF (2000) Use of matrix attachment regions (MARs) to minimize transgene silencing. Plant Mol Biol 43:361–376

    Article  CAS  PubMed  Google Scholar 

  • Beckmann TF, Kramer O, Klausing S, Heinrich C, Thute T, Buntemeyer H, Hoffrogge R, Noll T (2012) Effects of high passage cultivation on CHO cells: a global analysis. Appl Microbiol Biotechnol 94(3):659–671

    Article  CAS  PubMed  Google Scholar 

  • Benton T, Chen T, McEntee M, Fox B, King D, Crombie R, Thomas TC, Bebbington C (2002) The use of UCOE vectors in combination with a preadapted serum free, suspension cell line allows for rapid production of large quantities of protein. Cytotechnology 38:43–46

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Butler M, Meneses-Acosta A (2012) Recent advances in technology supporting biopharmaceutical production from mammalian cells. Appl Microbiol Biotechnol 96(4):885–894

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen K, Li D, Li H, Li B, Li J (2017) Genetic analysis of heterogeneous sub-clones in recombinant Chinese hamster ovary cells. Appl Microbiol Biotechnol 101(14):5785–5797

    Article  CAS  PubMed  Google Scholar 

  • Davies SL, Lovelady CS, Grainger RK, Racher AJ, Young RJ, James DC (2013) Functional heterogeneity and heritability in CHO cell populations. Biotechnol Bioeng 110(1):260–274

    Article  CAS  PubMed  Google Scholar 

  • Feichtinger J, Hernandez I, Fischer C, Hanscho M, Auer N, Hackl M, Jadhav V, Baumann M, Krempl PM, Schmidl C, Farlik M, Schuster M, Merkel A, Sommer A, Heath S, Rico D, Bock C, Thallinger GG, Borth N (2016) Comprehensive genome and epigenome characterization of CHO cells in response to evolutionary pressures and over time. Biotechnol Bioeng 113(10):2241–2253

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fomina-Yadlin D, Mujacic M, Maggiora K, Quesnell G, Saleem R, McGrew JT (2015) Transcriptome analysis of a CHO cell line expressing a recombinant therapeutic protein treated with inducers of protein expression. J Biotechnol 212:106–115

    Article  CAS  PubMed  Google Scholar 

  • Gan W, Zhang C, Siu KY, Satoh A, Tanner JA, Yu S (2017) ULK1 phosphorylates Sec23A and mediates autophagy-induced inhibition of ER-to-Golgi traffic. BMC Cell Biol 18(1):22

    Article  PubMed  PubMed Central  Google Scholar 

  • Hammond S, Lee KH (2012) RNA interference of cofilin in Chinese hamster ovary cells improves recombinant protein productivity. Biotechnol Bioeng 109(2):528–535

    Article  CAS  PubMed  Google Scholar 

  • Huang DW, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4(1):44–57

    Article  CAS  Google Scholar 

  • Kaas CS, Kristensen C, Betenbaugh MJ, Andersen MR (2015) Sequencing the CHO DXB11 genome reveals regional variations in genomic stability and haploidy. BMC Genomics 16:160

    Article  PubMed  PubMed Central  Google Scholar 

  • Kantardjieff A, Jacob NM, Yee JC, Epstein E, Kok YJ, Philp R, Betenbaugh M, Hu WS (2010) Transcriptome and proteome analysis of Chinese hamster ovary cells under low temperature and butyrate treatment. J Biotechnol 145(2):143–159

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Lee KH, Tsutsui T, Honda K, Asano R, Kumagai I, Ohtake H, Omasa T (2013) Generation of high- producing cell lines by overexpression of cell division cycle 25 homolog A in Chinese hamster ovary cells. J Biosci Bioeng 116(6):754–760

    Article  CAS  PubMed  Google Scholar 

  • Li D, Xie K, Ding G, Li J, Chen K, Li H, Qian J, Jiang C, Fang J (2014) Tumor resistance to anti-VEGF therapy through up-regulation of VEGF-C expression. Cancer Lett 346(1):45–52

    Article  CAS  PubMed  Google Scholar 

  • Li H, Chen K, Wang Z, Li D, Lin J, Yu C, Yu F, Wang X, Huang L, Jiang C, Gu H, Fang J (2016) Genetic analysis of the clonal stability of Chinese hamster ovary cells for recombinant protein production. Mol BioSyst 12(1):102–109

    Article  CAS  PubMed  Google Scholar 

  • Nagalakshmi U, Wang Z, Waern K, Shou C, Raha D, Gerstein M, Snyder M (2008) The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 320(5881):1344–1349

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nishimiya D, Ogura Y, Sakurai H, Takahashi T (2012) Identification of antibody-interacting proteins that contribute to the production of recombinant antibody in mammalian cells. Appl Microbiol Biotechnol 96(4):971–979

    Article  CAS  PubMed  Google Scholar 

  • Nishimiya D, Mano T, Miyadai K, Yoshida H, Takahashi T (2013) Overexpression of CHOP alone and in combination with chaperones is effective in improving antibody production in mammalian cells. Appl Microbiol Biotechnol 97(6):2531–2539

    Article  CAS  PubMed  Google Scholar 

  • Omasa T, Takami T, Ohya T, Kiyama E, Hayashi T, Nishii H, Miki H, Kobayashi K, Honda K, Ohtake H (2008) Overexpression of GADD34 enhances production of recombinant human antithrombin III in Chinese hamster ovary cells. J Biosci Bioeng 106(6):568–573

    Article  CAS  PubMed  Google Scholar 

  • Park JH, Wang Z, Jeong HJ, Park HH, Kim BG, Tan WS, Choi SS, Park TH (2012) Enhancement of recombinant human EPO production and glycosylation in serum-free suspension culture of CHO cells through expression and supplementation of 30Kc19. Appl Microbiol Biotechnol 96(3):671–683

    Article  CAS  PubMed  Google Scholar 

  • Peng RW, Abellan E, Fussenegger M (2011) Differential effect of exocytic SNAREs on the production of recombinant proteins in mammalian cells. Biotechnol Bioeng 108(3):611–620

    Article  CAS  PubMed  Google Scholar 

  • Pilbrough W, Munro TP, Gray P (2009) Intraclonal protein expression heterogeneity in recombinant CHO cells. PLoS ONE 4(12):e8432

    Article  PubMed  PubMed Central  Google Scholar 

  • Takasea K, Ohtsukia T, Migitaa O, Torub M, Inadac T, Yamakawa-Kobayashia K, Arinamia T (2001) Association of ZNF74 gene genotypes with age-at-onset of schizophrenia. Schizophr Res 52:161–165

    Article  Google Scholar 

  • Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28(5):511–515

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7(3):562–578

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Vishwanathan N, Le H, Jacob NM, Tsao Y-S, Ng S-W, Loo B, Liu Z, Kantardjieff A, Hu W-S (2014) Transcriptome dynamics of transgene amplification in Chinese Hamster Ovary Cells. Biotechnol Bioeng 111(3):518–528

    Article  CAS  PubMed  Google Scholar 

  • Xu X, Nagarajan H, Lewis NE, Pan S, Cai Z, Liu X, Chen W, Xie M, Wang W, Hammond S, Andersen MR, Neff N, Passarelli B, Koh W, Fan HC, Wang J, Gui Y, Lee KH, Betenbaugh MJ, Quake SR, Famili I, Palsson BO, Wang J (2011) The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line. Nat Biotechnol 29(8):735–741

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This study was supported by grants from the National Basic Research Program of China (973 Program) 2015CB553706, the National Natural Science Foundation of China (NSFC31270987 and NSFC31470896), Suzhou Applicational Basic Project, China (SYG201509), the Natural Science Foundation of Jiangsu Province(BK201150278), and the Fundamental Research Funds for the Central Universities 22120170036. We would like to thank Editage [www.editage.cn] for English language editing.

Supporting information

Supplementary Table 1—Primers used for amplifying each gene for construction of the expression plasmids.

Supplementary Table 2—Overall transcriptome and gene ontology (GO) analysis of the genes differentially expressed by the CHO-L and CHO-H cell lines.

Supplementary Table 3—All genes differentially expressed in the CHO-L and CHO-H cell lines.

Supplementary Fig. 1—Effect of selected genes on the expression of the Fc fragment in CHO-K1 cells

Supplementary Fig. 2—Effect of selected genes on the expression of the Herceptin in CHO-K1 cells.

Author information

Authors and Affiliations

  1. School of Life Sciences and Technology, Tongji University, Shanghai, China

    Kaiming Chen, Dong Li, Hongwen Li, Bing Li, Ruiqin Wang, Lingxiao Jiang, Lei Huang, Xiaoqing Xu, Jie Li, Cizhong Jiang, Hua Gu & Jianmin Fang

  2. Shanghai Tongji Hospital, Tongji University, Shanghai, China

    Fei Teng & Jianmin Fang

  3. Tongji University Suzhou Institute, Suzhou, Jiangsu, China

    Jianmin Fang

  4. Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, China

    Jianmin Fang

Authors
  1. Kaiming Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hongwen Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bing Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ruiqin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lingxiao Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lei Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Xiaoqing Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jie Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Fei Teng
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Cizhong Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Hua Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Jianmin Fang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jianmin Fang.

Ethics declarations

Conflicts of interest

The authors declare that they have no conflicts of interest with the contents of this article. This article does not contain any studies with human participants performed by any of the authors.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 2416 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, K., Li, D., Li, H. et al. Improved recombinant protein production by regulation of transcription and protein transport in Chinese hamster ovary cells. Biotechnol Lett 41, 719–732 (2019). https://doi.org/10.1007/s10529-019-02681-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-019-02681-7

Keywords

Search

Navigation