Advertisement

Molecular Biotechnology

, Volume 30, Issue 2, pp 103–115 | Cite as

Combined effect of protein fusion and signal sequence greatly enhances the production of recombinant human GM-CSF in Escherichia coli

  • Palash Bhattacharya
  • Gaurav Pandey
  • Poonam Srivastava
  • Krishna Jyoti Mukherjee
Research

Abstract

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor, that has been used as a therapeutic agent in facilitating bone marrow and stem cell transplantation and in other clinical cases like neutropenia. Although biologically active recombinant GM-CSF has been successfully produced in Escherichia coli, the reported levels are extremely poor. In this study we looked into the possible reasons for poor expression and found that protein toxicity coupled with protease-based degradation was the principal reason for low productivity. To overcome this problem we attached a signal sequence, as well as an amino-terminal His-tag fusion to the GM-CSF gene. This combination had a dramatic effect on expression levels, which increased from 0.8 µg/mL in the control to 40 µg/mL. When a larger fusion partner, such as the Maltose-binding protein (MBP-tag), was used the expression levels increased further to 69.5 µg/mL, which along with the MBP-tag represented approx 12% of the total cellular protein.

Index Entries

GM-CSF E. coli signal sequence protein fusion high cell density 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Metcalf, D., Begley, C. G., Johnson, G. R., et al. (1986) Biologic properties in vitro of a recombinant human granulocyte-macrophage colony-stimulating factor. Blood 67, 37–45.PubMedGoogle Scholar
  2. 2.
    Armitage, J. (1998) Emerging applications of recombinant human granulocyte-macrophage colony-stimulating factor. Blood 92, 4491–4508.PubMedGoogle Scholar
  3. 3.
    Greenberg, P., Advani, R., Keating, A., ET AL. (1996) GM-CSF accelerates neutrophil recovery after autologous hematopoietic stem cell transplantation. Bone Marrow Transpl. 18, 1057–1064.Google Scholar
  4. 4.
    Anderson, J. E. and Appelbaum, F. R. (1994) Hematopoietic effects and clinical uses of granulocyte-macrophage colony-stimulating factor and PIXY321. Curr. Opin. Hematol. 1, 203–209.PubMedGoogle Scholar
  5. 5.
    Smith, T. and Grossberg, H. (1990) Successful use of granulocyte-macrophage colony-stimulating factor in patients with acute lymphocytic leukemia. Am. J. Med. 89, 384–386.PubMedCrossRefGoogle Scholar
  6. 6.
    Nemunaitis, J., Gordon, A., Cox, J., ET AL. (1994) Phase II pilot trial comparing neutrophil and monocyte function by microbicidal assay in oncology patients receiving rhG-CSF, rHuGM-CSF or no cytokine after cytotoxic chemotherapy. Blood 84, 135.Google Scholar
  7. 7.
    Rowe, J. M., Rubin, A., Mazza, J. J., et al. (1996) Incidence of infections in adult patients (>55 years) with acute myeloid leukemia treated with yeast-derived GM-CSF (sargramostim): results of a double-blind prospective study by the Eastern Cooperative Oncology Group, in Acute Leukemias V: Experimental Approaches and Management of Refractory Disease (Hiddemann W., ed.). Springer-Verlag, Berlin, Germany, pp. 178.Google Scholar
  8. 8.
    Bodey, G. P., Anaissie, E., Gutterman, J., and Vadhan-Raj, S. (1993) Role of granulocyte-macrophage colony-stimulating factor as adjuvant therapy of fungal infection in patients with cancer. Clin. Infect. Dis. 17, 705–707.PubMedGoogle Scholar
  9. 9.
    Dranoff, G., Jaffee, E., Lazenby, A., et al. (1993) Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc. Natl. Acad. Sci. 90, 3539–3543.PubMedCrossRefGoogle Scholar
  10. 10.
    Spitler, L. E., Grossbard, M. L., Ernstoff, M. S., et al. (1997) Adjuvant therapy of stage III and IV malignant melanoma using yeast derived, GM-CSF. Melanoma Res. 7, 160.CrossRefGoogle Scholar
  11. 11.
    Chachoua, A., Oratz, R., Liebes, L., et al. (1994) Phase IB trial of granulocyte-macrophage colony-stimulating factor combined with murine monoclonal antibody R24 in patients with metastatic melanoma. J. Immunother. Emphasis Tumor Immunol, 16, 132–141.PubMedGoogle Scholar
  12. 12.
    Yu, A. L., Batova, A., Alvarado, C., Rao, V. J., and Castleberry, R. P. (1997) Usefulness of a chimeric anti-GD2 (ch14.18) and GM-CSF for refractory neuroblastoma: a POG phase II study. Proc. Am. Soc. Clin. Oncol. 16, 513a.Google Scholar
  13. 13.
    Costello, R. T. (1993) Therapeutic use of granulocyte-macrophage colony-stimulating factor (GM-CSF). A review of recent experience. Acta Oncol. 32, 403–408.PubMedGoogle Scholar
  14. 14.
    Berges, H., Joseph-Liauzun, E., and Fayet, O. (1996) Combined effects of the signal sequence and the major chaperone proteins on the export of human cytokines in Escherichia coli. Appl. Environ. Microbiol. 62, 55–60.PubMedGoogle Scholar
  15. 15.
    Lundell, D., Lunn, C., Greenberg, R., et al. (1990) Exploiting the cell membrane for the production of heterologous proteins in Escherichia coli. Biotechnol. Appl. Biochem. 12, 567–578.PubMedGoogle Scholar
  16. 16.
    Gronski, P., Badziong, W., Habermann, P., et al. (1988) E. coli derived human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) available for clinical trials. Behring. Inst. Mitt. 83, 246–249.PubMedGoogle Scholar
  17. 17.
    Wang, J., Zou, M., Huang, B., Zhang, M., and Mao, N. (1995) Molecular cloning and expression of human granulocyte-macrophage colony stimulating factor in Escherichia coli system. Chin. J. Biotechnol. 11, 45–81.PubMedGoogle Scholar
  18. 18.
    Ling, M., Zou, M., Xu, M., Wang, J., and Ma, X. (1995) Purification of recombinant human granulocyte-macrophage colony stimulating factor expressed in Escherichia coli. Chin. J. Biotechnol. 11, 157–162.PubMedGoogle Scholar
  19. 19.
    Hua, Z., Wang, H., Chen, D., Chen, Y., and Zhu, D. (1994) Enhancement of expression of human granulocyte-macrophage colony stimulating factor by argU gene product in Escherichia coli. Biochem Mol Biol Int. 32, 537–543.PubMedGoogle Scholar
  20. 20.
    Pluckthun, A. (1992) Mono- and bivalent antibody fragments producted in Escherichia coli: engineering, folding and antigen binding. Immunol. Rev. 130, 151–188.PubMedCrossRefGoogle Scholar
  21. 21.
    Bachmair, A., Finley, D., and Varshavsky, A. (1986) In vivo half-life of a protein is a function of its aminoterminal residue. Science 234, 179–186.PubMedCrossRefGoogle Scholar
  22. 22.
    Nygren, P. A., Stahl, S., and Uhlen, M. (1994) Engineering proteins to facilitate bioprocessing. Trends Biotechnol. 12, 184–188.PubMedCrossRefGoogle Scholar
  23. 23.
    Ujihara, M., Nomura, K., Yamada, O., Shibata, N., Kobayashi, M., and Takano, K. (2001) Granulocyte-macrophage colony-stimulating factor ensures macrophage survival and generation of the superoxide anion: a study using a monocytic-differentiated HL60 subline. Free Radic. Biol. Med. 31, 1396–1404.PubMedCrossRefGoogle Scholar
  24. 24.
    Sorensen, M. A., Kurland, C. G., and Pedersen, S. (1989) Codon usage determines translation rate in Escherichia coli. J. Mol. Biol. 207, 365–377.PubMedCrossRefGoogle Scholar
  25. 25.
    Zhang, S. P., Zubay, G., and Goldman, E. (1991) Low-usage codons in Escherichia coli, yeast, fruit fly and primates. Gene 105, 61–72.PubMedCrossRefGoogle Scholar
  26. 26.
    Petrovskaia, L. E., Kriukov, E. A., Iakimov, S. A., et al. (1995) Effect of the topography of the signal peptidase site on the effectiveness of secretion of recombinant human granulocyte-macrophage colony-stimulating factor into Escherichia coli periplasm. Bioorg. Khim. 21, 912–919.PubMedGoogle Scholar
  27. 27.
    Hua, Z., Jie, L., and Zhu, D. (1994) Expression of a biologically active human granulocyte-macrophage colony stimulating factor fusion protein in Escherichia coli. Biochem. Mol. Bio. Int. 34, 621–626.Google Scholar
  28. 28.
    Eliseev, R., Alexandrov, A., and Gunter, T. (2004) High-yield expression and purification of p18 form of Bax as an MBP fusion protein. Protein Exp. Purification 35, 206–209.CrossRefGoogle Scholar
  29. 29.
    Pryor, K. D. and Leiting, B. (1997) High-level expression of soluble protein in Escherichia coli using a His6-Tag and maltose-binding protein double-affinity fusion system. Protein Exp. Purification 10, 309–319.CrossRefGoogle Scholar
  30. 30.
    Guan, M., Su, B., Ye, C., and Yuan L. (2002) Production of extracellular domain of human tissue factor using maltose-binding protein fusion system. Protein Exp. Purification 26, 229–234.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Palash Bhattacharya
  • Gaurav Pandey
  • Poonam Srivastava
  • Krishna Jyoti Mukherjee
    • 1
  1. 1.Center for BiotechnologyJawaharlal Nehru UniversityNew Dehli, DelhiIndia

Personalised recommendations