Skip to main content
PMC home page
Cytotechnology logoLink to Cytotechnology
. 2005 Jan;47(1-3):29–36. doi: 10.1007/s10616-005-3765-4

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

Kiichiro Teruya 1,✉, Yoshihito Daimon 2, Xiao-Yan Dong 3, Yoshinori Katakura 1, Takumi Miura 2, Akira Ichikawa 4, Tsukasa Fujiki 1, Makiko Yamashita 1, Tetsuya Mori 2, Hideya Ohashi 5, Sanetaka Shirahata 1
PMCID: PMC3449826  PMID: 19003042

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.

Keywords: CHO, Cytomegalovirus promoter, ER overload response, ER signaling, GRP78/BiP, Mass production, PAP and GS hybrid system, PAP system

Full Text

The Full Text of this article is available as a PDF (260.2 KB).

Glossary

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

References

  1. Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  2. Cockett M.I., Bebbington C.R., Yarranton G.T. High level expression of tissue inhibitor of metalloproteinases in Chinese hamster ovary cells using glutamine synthetase gene amplification. Bio/Technology. 1990;8:662–667. doi: 10.1038/nbt0790-662. [DOI] [PubMed] [Google Scholar]
  3. Cudna R.E., Dickson A.J. Endoplasmic reticulum signaling as a determinant of recombinant protein expression. Biotechnol. Bioeng. 2002;81:56–65. doi: 10.1002/bit.10445. [DOI] [PubMed] [Google Scholar]
  4. Dignam J.D., Lebovitz R.M., Roeder R.G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983;11:1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dong X., Teruya K., Katakura Y., Zhang Y., Miura T., Daimon Y., Mori T., Ohashi H., Shirahata S. A hybrid system using both promoter activation and gene amplification for establishing exogenous protein hyper-producing cell lines. Cytotechnology. 2003;43:11–17. doi: 10.1023/B:CYTO.0000039901.92984.7a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Friedman J.S., Cofer C.L., Anderson C.L., Kushner J.A., Gray P.P., Chapman G.E., Stuart M.C., Lazarus L., Shine J., Kushner P. High expression in mammalian cells without amplification. Bio/Technology. 1989;7:359–362. doi: 10.1038/nbt0489-359. [DOI] [Google Scholar]
  7. Jiang H.Y., Wek S.A., McGrath B.C., Scheuner D., Kaufman R.J., Cavener D.R., Wek R.C. Phosphorylation of the α subunit of eukaryotic initiation factor 2 is required for activation of NF-κB in response to diverse cellular stresses. Mol. Cell. Biol. 2003;16:5651–5663. doi: 10.1128/MCB.23.16.5651-5663.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Katakura Y., Seto P., Miura T., Ohashi H., Teruya K., Shirahata S. Productivity enhancement of recombinant protein in CHO cells via specific promoter activation by oncogenes. Cytotechnology. 1999;31:103–109. doi: 10.1023/A:1008048928053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kemball-Cook G., Garner I., Imanaka Y., Nishimura T., O’Brien D.P., Tuddenham E.G., McVey J.H. High-level production of human blood coagulation factors VII and XI using a new mammalian expression vector. Gene. 1994;139:275–279. doi: 10.1016/0378-1119(94)90769-2. [DOI] [PubMed] [Google Scholar]
  10. Le H.V., Ramanathan L., Labdon J.E., Mays-Ichinco C.A., Syto R., Arai N., Hoy P., Takebe Y., Nagabhushan T.L., Trotta P.P. Isolation and characterization of multiple variants of recombinant human interleukin 4 expressed in mammalian cells. J. Biol. Chem. 1988;263:10817–10823. [PubMed] [Google Scholar]
  11. Miura T., Katakura Y., Seto P., Zhang Y., Teruya K., Nishimura E., Kato M., Hashizume S., Shirahata S. Availability of oncogene activated production system for mass production of light chain of human antibody in CHO cells. Cytotechnology. 2001;35:9–16. doi: 10.1023/A:1008179919857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Pahl H.L. Signal transduction from the endoplasmic reticulum to the cell nucleus. Phisiol. Rev. 1999;79:683–701. doi: 10.1152/physrev.1999.79.3.683. [DOI] [PubMed] [Google Scholar]
  13. Pahl H.L., Baeuerle P.A. A novel signal transduction pathway from the endoplasmic reticulum to the nucleus is mediated by transcription factor NF-κ B. EMBO J. 1995;14:2580–2588. doi: 10.1002/j.1460-2075.1995.tb07256.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Santhanam U., Ghrayeb J., Sehgal P.B., May L.T. Post-translational modifications of human interleukin-6. Arch. Biochem. Biophys. 1989;274:161–170. doi: 10.1016/0003-9861(89)90427-X. [DOI] [PubMed] [Google Scholar]
  15. Schimke R.T. Gene amplification in cultured cells. J. Biol. Chem. 1988;263:5989–5992. [PubMed] [Google Scholar]
  16. Shirahata S., Watanabe J., Teruya K., Yano T., Osada K., Ohashi H., Tachibana H., Kim E.H., Murakami H. E1A and ras oncogenes synergistically enhance recombinant protein production under control of the cytomegalovirus promoter in BHK-21 cells. Biosci. Biotechnol. Biochem. 1995;59:345–347. doi: 10.1271/bbb.59.345. [DOI] [PubMed] [Google Scholar]
  17. Tachibana H., Jiyoun K., Taniguchi K., Ushio Y., Teruya K., Osada K., Inoue Y., Shirahata S., Murakami H. Modified antigen-binding of human antibodies with glycosylation variations of the light chains produced in sugar-limited human hybridoma cultures. In Vitro Cell Dev. Biol. Anim. 1996;32:178–183. doi: 10.1007/BF02723683. [DOI] [PubMed] [Google Scholar]
  18. Tachibana H., Kim J., Shirahata S. Building high affinity human antibodies by altering the glycosylation on the light chain variable region in N-acetylglucosamine-supplemented hybridoma cultures. Cytotechnology. 1997;23:151–159. doi: 10.1023/A:1007980032042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tachibana H., Shirahata S., Murakami H. Generation of specificity-variant antibodies by alteration of carbohydrate in light chain of human monoclonal antibodies. Biochem. Biophys. Res. Commun. 1992;189:625–632. doi: 10.1016/0006-291X(92)92246-T. [DOI] [PubMed] [Google Scholar]
  20. Takebe Y., Seiki M., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. SRα promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol. Cell. Biol. 1988;8:466–472. doi: 10.1128/mcb.8.1.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Teruya K., Yano T., Shirahata S., Watanabe J., Osada K., Ohashi H., Tachibana H., Kim E.H., Murakami H. Ras amplification in BHK-21 cells produces a host cell line for further rapid establishment of recombinant protein hyper-producing cell lines. Biosci. Biotechnol. Biochem. 1995;59:341–344. doi: 10.1271/bbb.59.341. [DOI] [PubMed] [Google Scholar]
  22. Wang A., Lu S.D., Mark D.F. Site-specific mutagenesis of the human interleukin-2 gene: structure-function analysis of the cysteine residues. Science. 1984;224:1431–1433. doi: 10.1126/science.6427925. [DOI] [PubMed] [Google Scholar]
  23. Yamashita M., Ichikawa A., Katakura Y., Mochizuki Y., Teruya K., Kim E.-H., Shirahata S. Induction of basophilic and eosinophilic differentiation in the human leukemic cell line KU812. Cytotechnology. 2001;36:179–186. doi: 10.1023/A:1014001322272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Yano T., Teruya K., Shirahata S., Watanabe J., Osada K., Tachibana H., Ohashi H., Kim E.H., Murakami H. Ras oncogene enhances the production of a recombinant protein regulated by the cytomegalovirus promoter in BHK-21 cells. Cytotechnology. 1994;16:167–178. doi: 10.1007/BF00749904. [DOI] [PubMed] [Google Scholar]

Articles from Cytotechnology are provided here courtesy of Springer Science+Business Media B.V.

RESOURCES