Biotechnology and Bioprocess Engineering

, Volume 23, Issue 6, pp 670–678 | Cite as

An Efficient and Rapid Two-step Purification Method for Active Human Macrophage Colony-stimulating Factor from Escherichia coli

  • Jisu Lee
  • Jeongmin Lee
  • Eunha Hwang
  • Hye-Jung Kim
  • Sheunghun Lee
  • Hyerim LeeEmail author
  • Seong Yun Hwang
  • Sung-Jin Cho
  • Sun-Ju Yi
  • Kyunghwan Kim
Research Paper


Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that stimulates the proliferation and differentiation of mononuclear phagocytes. To be biologically active, M-CSF must form a homodimer linked by disulfide bridges. However, the refolding step of recombinant M-CSF is not only extremely laborious but also rate-limiting. Here, we describe an efficient and simplified method for refolding and purifying M-CSF from Escherichia coli. A truncated M-CSF (amino acids 36–181) was tagged with histidine, expressed, and then purified using nickel affinity columns under denaturing conditions. Our redox refolding buffer containing a mixture of reduced and oxidized glutathione and arginine correctly refolded M-CSF. Chromatography on Q-sepharose columns selectively purified the M-CSF dimer from the monomeric form. The dimeric nature of the purified M-CSF was confirmed using multi angle light scattering combined with high performance liquid chromatography. Moreover, cell proliferation and osteoclast differentiation assays using bone marrow-derived macrophages demonstrated that the recombinant M-CSF was biologically active ex vivo.


M-CSF refolding osteoclastogenesis purification 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Stanley, E. R. and P. M. Heard (1977) Factors regulating macrophage production and growth. Purification and some properties of the colony stimulating factor from medium conditioned by mouse L cells. J. Biol. Chem. 252: 4305–4312.Google Scholar
  2. 2.
    Stanley, E. R., D. M. Chen, and H. S. Lin (1978) Induction of macrophage production and proliferation by a purified colony stimulating factor. Nature 274: 168–170.CrossRefGoogle Scholar
  3. 3.
    Stanley, E. R., K. L. Berg, D. B. Einstein, P. S. Lee, F. J. Pixley, Y. Wang, and Y. G. Yeung (1997) Biology and action of colony––stimulating factor–1. Mol. Reprod. Dev. 46: 4–10.CrossRefGoogle Scholar
  4. 4.
    Sapi, E. (2004) The role of CSF–1 in normal physiology of mammary gland and breast cancer: an update. Exp. Biol. Med. 229: 1–11.CrossRefGoogle Scholar
  5. 5.
    Ushach, I. and A. Zlotnik (2016) Biological role of granulocyte macrophage colony–stimulating factor (GM–CSF) and macrophage colony–stimulating factor (M–CSF) on cells of the myeloid lineage. J. Leukocyte Biol. 100: 481–489.CrossRefGoogle Scholar
  6. 6.
    Toh, M. L., J. Y. Bonnefoy, N. Accart, S. Cochin, S. Pohle, H. Haegel, M. De Meyer, C. Zemmour, X. Preville, C. Guillen, C. Thioudellet, P. Ancian, A. Lux, B. Sehnert, F. Nimmerjahn, R. E. Voll, and G. Schett (2014) Bone–and cartilage–protective effects of a monoclonal antibody against colony–stimulating factor 1 receptor in experimental arthritis. Arthritis Rheumatol. 66: 2989–3000.CrossRefGoogle Scholar
  7. 7.
    Klebl, F. H., J. E. Olsen, S. Jain, and W. F. Doe (2001) Expression of macrophage–colony stimulating factor in normal and inflammatory bowel disease intestine. J. Pathol. 195: 609–615.CrossRefGoogle Scholar
  8. 8.
    Menke, J., Y. Iwata, W. A. Rabacal, R. Basu, E. R. Stanley, and V. R. Kelley (2011) Distinct roles of CSF–1 isoforms in lupus nephritis. J. Am. Soc. Nephrol: JASN 22: 1821–1833.CrossRefGoogle Scholar
  9. 9.
    Ide, H., K. Hatake, Y. Terado, H. Tsukino, T. Okegawa, K. Nutahara, E. Higashihara, and S. Horie (2008) Serum level of macrophage colony–stimulating factor is increased in prostate cancer patients with bone metastasis. Human Cell 21: 1–6.CrossRefGoogle Scholar
  10. 10.
    Ohno, H., Y. Uemura, H. Murooka, H. Takanashi, T. Tokieda, Y. Ohzeki, K. Kubo, and I. Serizawa (2008) The orally–active and selective c–Fms tyrosine kinase inhibitor Ki20227 inhibits disease progression in a collagen–induced arthritis mouse model. Eur. J. Immunol. 38: 283–291.CrossRefGoogle Scholar
  11. 11.
    Hume, D. A. and K. P. MacDonald (2012) Therapeutic applications of macrophage colony–stimulating factor–1 (CSF–1) and antagonists of CSF–1 receptor (CSF–1R) signaling. Blood 119: 1810–1820.CrossRefGoogle Scholar
  12. 12.
    Hamilton, J. A. (2008) Colony–stimulating factors in inflammation and autoimmunity. Nat. Rev. Immunol. 8: 533–544.CrossRefGoogle Scholar
  13. 13.
    Polito, A., A. Fabbri, A. Ferro–Luzzi, M. Cuzzolaro, L. Censi, D. Ciarapica, E. Fabbrini, and D. Giannini (2000) Basal metabolic rate in anorexia nervosa: relation to body composition and leptin concentrations. Am. J. Clin. Nutrit. 71: 1495–1502.CrossRefGoogle Scholar
  14. 14.
    Yamanishi, K., M. Takahashi, T. Nishida, Y. Ohmoto, M. Takano, S. Nakai, and Y. Hirai (1991) Renaturation, purification, and characterization of human truncated macrophage colony–stimulating factor expressed in Escherichia coli. J. Biochem. 109: 404–409.CrossRefGoogle Scholar
  15. 15.
    Pandit, J., A. Bohm, J. Jancarik, R. Halenbeck, K. Koths, and S. H. Kim (1992) Three–dimensional structure of dimeric human recombinant macrophage colony–stimulating factor. Science 258: 1358–1362.CrossRefGoogle Scholar
  16. 16.
    Kim, K., V. Punj, J. M. Kim, S. Lee, T. S. Ulmer, W. Lu, J. C. Rice, and W. An (2016) MMP–9 facilitates selective proteolysis of the histone H3 tail at genes necessary for proficient osteoclastogenesis. Genes Dev. 30: 208–219.Google Scholar
  17. 17.
    An, D., K. Kim, and W. Lu (2014) Defective entry into mitosis 1 (Dim1) negatively regulates osteoclastogenesis by inhibiting the expression of nuclear factor of activated T–cells, cytoplasmic, calcineurin–dependent 1 (NFATc1). J. Biol. Chem. 289: 24366–24373.CrossRefGoogle Scholar
  18. 18.
    Ladner, M. B., G. A. Martin, J. A. Noble, D. M. Nikoloff, R. Tal, E. S. Kawasaki, and T. J. White (1987) Human CSF–1: gene structure and alternative splicing of mRNA precursors. EMBO J. 6: 2693–2698.CrossRefGoogle Scholar
  19. 19.
    Halenbeck, H., Robert, E. Kawasaki, J. Wrin, and K. Koths (1989) Renaturation and purification of biologically active recombinant human macrophage colony–stimulating factor expressed in E. coli. Bio/Technology 7: 710.Google Scholar
  20. 20.
    Glocker, M. O., B. Arbogast, J. Schreurs, and M. L. Deinzer (1993) Assignment of the inter–and intramolecular disulfide linkages in recombinant human macrophage colony stimulating factor using fast atom bombardment mass spectrometry. Biochemistry 32: 482–488.CrossRefGoogle Scholar
  21. 21.
    Baynes, B. M., D. I. Wang, and B. L. Trout (2005) Role of arginine in the stabilization of proteins against aggregation. Biochemistry 44: 4919–4925.CrossRefGoogle Scholar
  22. 22.
    Lee, J. C. and S. N. Timasheff (1981) The stabilization of proteins by sucrose. J. Biol. Chem. 256: 7193–7201.Google Scholar
  23. 23.
    Cleland, J. L., S. E. Builder, J. R. Swartz, M. Winkler, J. Y. Chang, and D. I. Wang (1992) Polyethylene glycol enhanced protein refolding. Bio/Technology 10: 1013–1019.Google Scholar
  24. 24.
    Stanley, E. R., K. L. Berg, D. B. Einstein, P. S. Lee, and Y. G. Yeung (1994) The biology and action of colony stimulating factor–1. Stem Cells 12 Suppl 1: 15–24; discussion 25.Google Scholar
  25. 25.
    Halenbeck, R., E. Kawasaki, J. Wrin, and K. Koths (1989) Renaturation and purification of biologically active recombinant human macrophage colony–stimulating factor expressed in E. coli. Nat. Biotechnol. 7: 710–715.CrossRefGoogle Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Jisu Lee
    • 1
  • Jeongmin Lee
    • 1
  • Eunha Hwang
    • 2
  • Hye-Jung Kim
    • 3
  • Sheunghun Lee
    • 1
  • Hyerim Lee
    • 1
    Email author
  • Seong Yun Hwang
    • 1
  • Sung-Jin Cho
    • 1
  • Sun-Ju Yi
    • 1
  • Kyunghwan Kim
    • 1
  1. 1.School of Biological Sciences, College of Natural SciencesChungbuk National UniversityCheongjuKorea
  2. 2.Korea Basic Science InstituteCheongjuKorea
  3. 3.New Drug Development CenterKBIO Osong Medical Innovation FoundationCheongjuKorea

Personalised recommendations