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An Approach to Further Enhance the Cellular Productivity of Exogenous Protein Hyper-producing Chinese Hamster Ovary (CHO) Cells

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Abstract

The cell line D29, which was easily and rapidly established by the promoter-activated production and glutamine synthetase hybrid system, secreted recombinant human interleukin-6 (hIL-6) at a productivity rate of 39.5 μg 10−6 cells day−1, one of the highest reported levels worldwide. The productivity rate was about 130-fold higher than that of the cell line A7, which was established without both promoter activation and gene amplification. Although D29 cells had a high copy number and high mRNA level of the hIL-6 gene as well as a high secretion rate of hIL-6, large amounts of intracellular hIL-6 protein accumulated in D29 cells compared to A7 cells. Northern blotting analysis showed no change in the GRP78/BiP expression level in D29 cells. In contrast, an electrophoresis mobility shift assay revealed strong activation of NF-κB in D29 cells. These results suggest that large amounts of hIL-6 translated from large amounts of hIL-6 mRNA cause excess accumulation of intact hIL-6 in the endoplasmic reticulum (ER), and that subsequent negative feedback signals via the ER overload response inhibit hIL-6 protein secretion. To enhance the hIL-6 productivity rate of D29 cells by releasing the negative feedback signals, the effect of pyrrolidinedithiocarbamate, an inhibitor of NF-κB activation, was examined. Suppression of NF-κB activation in D29 cells produced a 25% augmentation of the hIL-6 productivity rate. Therefore, in highly productive cells like D29 cells, the release of negative feedback signals could increase the total amount of recombinant protein secretion.

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Abbreviations

CHO:

Chinese hamster ovary

dhfr:

dihydrofolate reductase

DMEM:

DulbeccoÆs modified EagleÆs medium

ECL:

enhanced chemiluminescence

ELISA:

enzyme-linked immunosorbent assay

EMSA:

electrophoretic mobility shift assay

EOR:

endoplasmic reticulum overload response

ER:

endoplasmic reticulum

FBS:

fetal bovine serum

GS:

glutamine synthetase

hIL-6:

human interleukin-6

MSX:

methionine sulphoximine

MTX:

methotrexate

PAP:

promoter-activated production

PDTC:

pyrrolidinedithiocarbamate

UPR:

unfolded protein response

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Authors and Affiliations

  1. Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581, Japan

    Kiichiro Teruya, Yoshinori Katakura, Tsukasa Fujiki, Makiko Yamashita & Sanetaka Shirahata

  2. Graduate School of Genetic Resources Technology, Kyushu University, Fukuoka, 812-8581, Japan

    Yoshihito Daimon, Takumi Miura & Tetsuya Mori

  3. Department of Chemical Engineering, Tianjin University, Tianjin, 300072, P. R. China

    Xiao-Yan Dong

  4. Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, 606-8585, Japan

    Akira Ichikawa

  5. Pharmaceutical Laboratory, Kirin Brewery Co. Ltd., Takasaki, Gunma, 370-1202, Japan

    Hideya Ohashi

Authors
  1. Kiichiro Teruya
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  2. Yoshihito Daimon
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  3. Xiao-Yan Dong
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  4. Yoshinori Katakura
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  5. Takumi Miura
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  6. Akira Ichikawa
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  7. Tsukasa Fujiki
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  8. Makiko Yamashita
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  9. Tetsuya Mori
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  10. Hideya Ohashi
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  11. Sanetaka Shirahata
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Correspondence to Kiichiro Teruya.

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Teruya, K., Daimon, Y., Dong, XY. et al. An Approach to Further Enhance the Cellular Productivity of Exogenous Protein Hyper-producing Chinese Hamster Ovary (CHO) Cells. Cytotechnology 47, 29–36 (2005). https://doi.org/10.1007/s10616-005-3765-4

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  • DOI: https://doi.org/10.1007/s10616-005-3765-4

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