Combined effect of protein fusion and signal sequence greatly enhances the production of recombinant human GM-CSF in Escherichia coli
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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 EntriesGM-CSF E. coli signal sequence protein fusion high cell density
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- 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
- 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.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
- 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
- 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
- 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
- 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.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