Skip to main content
SpringerLink
Log in

Eukaryotic Expression and Purification of Native Form of Mouse Midkine from Pichia pastoris

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

To confirm the treating effectiveness of midkine as an articular protective agent, mouse midkine (mMK) was produced for the pre-clinic long-term studies in mice. The protein was expressed under the control of the AOX1 gene promoter in Pichia pastoris, X-33 strain, and secreted into fermentation broth through high-density fermentation. Approximately 380 mg mMK, containing authentic and truncated forms, was secreted into 1 liter induction medium and 280 mg mMK was obtained after one-step purification on a 50 ml SP Sepharose Fast Flow column. The purified protein was characterized and identified to be the mature, authentic form of mMK. N-terminal five amino acid sequence was determined to be K-K-K-E-K. SDS-PAGE analysis indicated that the molecular weight of the product was about 13 KDa. The purity of the purified rmMK protein was determined to be 99 % by high performance liquid chromatography. The biological activity of final product was verified via migration assay on osteoblast-like UMR-106 cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Kadomatsu, K., Tomomura, M., & Muramatsu, T. (1988). cDNA cloning and sequencing of a new gene intensely expressed in early differentiation stages of embryonal carcinoma cells and in mid-gestation period of mouse embryogenesis. Biochemical and Biophysical Research Communications, 151, 1312–1318.

    Article  CAS  Google Scholar 

  2. Milner, P. G., Li, Y. S., Hoffman, R. M., Kodner, C. M., Siegel, N. R., & Deuel, T. F. (1989). A novel 17 kD heparin-binding growth factor (HBGF-8) in bovine uterus: purification and N-terminal amino acid sequence. Biochemical and Biophysical Research Communications, 165, 1096–1103.

    Article  CAS  Google Scholar 

  3. Fabri, L., Maruta, H., Muramatsu, H., Muramatsu, T., Simpson, R. J., Burgess, A. W., et al. (1993). Structural characterisation of native and recombinant forms of the neurotrophic cytokine MK. Journal of Chromatography, 646, 213–225.

    Article  CAS  Google Scholar 

  4. Akhter, S., Ichihara-Tanaka, K., Kojima, S., Muramatsu, H., Inui, T., Kimura, T., et al. (1998). Clusters of basic amino acids in midkine: roles in neurite-promoting activity and plasminogen activator-enhancing activity. Journal of Biochemistry, 123, 1127–1136.

    Article  CAS  Google Scholar 

  5. Kojima, S., Inui, T., Muramatsu, H., Suzuki, Y., Kadomatsu, K., Yoshizawa, M., et al. (1997). Dimerization of midkine by tissue transglutaminase and its functional implication. Journal of Biological Chemistry, 272, 9410–9416.

    Article  CAS  Google Scholar 

  6. Muramatsu, T. (2014). Structure and function of midkine as the basis of its pharmacological effects. British Journal of Pharmacology, 171, 814–826.

    Article  CAS  Google Scholar 

  7. Tsutsui, J., Kadomatsu, K., Matsubara, S., Nakagawara, A., Hamanoue, M., Takao, S., et al. (1993). A new family of heparin-binding growth/differentiation factors: increased midkine expression in Wilms’ tumor and other human carcinomas. Cancer Research, 53, 1281–1285.

    CAS  Google Scholar 

  8. Aridome, K., Tsutsui, J., Takao, S., Kadomatsu, K., Ozawa, M., Aikou, T., et al. (1995). Increased midkine gene expression in human gastrointestinal cancers. Japanese Journal of Cancer Research, 86, 655–661.

    Article  CAS  Google Scholar 

  9. Kadomatsu, K., & Muramatsu, T. (2004). Midkine and pleiotrophin in neural development and cancer. Cancer Letters, 204, 127–143.

    Article  CAS  Google Scholar 

  10. Muramatsu, T. (2010). Midkine, a heparin-binding cytokine with multiple roles in development, repair and diseases. Proceedings of the Japan Academy. Series B, Physical and Biological Sciences, 86, 410–425.

    Article  CAS  Google Scholar 

  11. Muramatsu, T. (2011). Midkine: a promising molecule for drug development to treat diseases of the central nervous system. Current Pharmaceutical Design, 17, 410–423.

    Article  CAS  Google Scholar 

  12. Zhang, Z. H., Li, H. X., Qi, Y. P., Du, L. J., Zhu, S. Y., Wu, M. Y., et al. (2010). Recombinant human midkine stimulates proliferation of articular chondrocytes. Cell Proliferation, 43, 184–194.

    Article  CAS  Google Scholar 

  13. Tsutsui, J., Uehara, K., Kadomatsu, K., Matsubara, S., & Muramatsu, T. (1991). A new family of heparin-binding factors: strong conservation of midkine (MK) sequences between the human and the mouse. Biochemical and Biophysical Research Communications, 176, 792–797.

    Article  CAS  Google Scholar 

  14. Inui, T., Bodi, J., Kubo, S., Nishio, H., Kimura, T., Kojima, S., et al. (1996). Solution synthesis of human midkine, a novel heparin-binding neurotrophic factor consisting of 121 amino acid residues with five disulphide bonds. Journal of Peptide Science, 2, 28–39.

    CAS  Google Scholar 

  15. Zhang, Z. H., Du, L. J., Xiang, D., Zhu, S. Y., Wu, M. Y., Lu, H. L., et al. (2009). Expression and purification of bioactive high-purity human midkine in Escherichia coli. Journal of Zhejiang University. Science. B, 10, 79–86.

    Article  CAS  Google Scholar 

  16. Murasugi, A., & Tohma-Aiba, Y. (2003). Production of native recombinant human midkine in the yeast, Pichia pastoris. Protein Expression and Purification, 27, 244–252.

    Article  CAS  Google Scholar 

  17. Murasugi, A., & Tohma-Aiba, Y. (2002). Characterization of partially truncated human midkine expressed in Pichia pastoris. Bioscience Biotechnology and Biochemistry, 66, 1295–1300.

    Article  CAS  Google Scholar 

  18. Qian, L., Zhu, S., Shen, J., Han, X., Gao, J., Wu, M., et al. (2012). Expression and purification of recombinant human Mig in Escherichia coli and its comparison with murine Mig. Protein Expression and Purification, 82, 205–211.

    Article  CAS  Google Scholar 

  19. Xiang, D., Zhang, J., Chen, Y., Guo, Y., Schalow, A., Zhang, Z., et al. (2010). Expressions and purification of a mature form of recombinant human Chemerin in Escherichia coli. Protein Expression and Purification, 69, 153–158.

    Article  CAS  Google Scholar 

  20. Matsuda, Y., Talukder, A. H., Ishihara, M., Hara, S., Yoshida, K., Muramatsu, T., et al. (1996). Limited proteolysis by chymotrypsin of midkine and inhibition by heparin binding. Biochemical and Biophysical Research Communications, 228, 176–181.

    Article  CAS  Google Scholar 

  21. Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., & Sternberg, M. J. (2015). The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols, 10, 845–858.

    Article  CAS  Google Scholar 

  22. Pacher, P., Beckman, J. S., & Liaudet, L. (2007). Nitric oxide and peroxynitrite in health and disease. Physiological Reviews, 87, 315–424.

    Article  CAS  Google Scholar 

  23. Maitra, U., Gan, L., Chang, S., & Li, L. (2011). Low-dose endotoxin induces inflammation by selectively removing nuclear receptors and activating CCAAT/enhancer-binding protein delta. Journal of Immunology, 186, 4467–4473.

    Article  CAS  Google Scholar 

  24. Akira, S. (2001). Toll-like receptors and innate immunity. Advances in Immunology, 78, 1–56.

    Article  CAS  Google Scholar 

  25. Malyala, P., & Singh, M. (2008). Endotoxin limits in formulations for preclinical research. Journal of Pharmaceutical Sciences, 97, 2041–2044.

    Article  CAS  Google Scholar 

  26. Qi, M., Ikematsu, S., Maeda, N., Ichihara-Tanaka, K., Sakuma, S., Noda, M., et al. (2001). Haptotactic migration induced by midkine. Involvement of protein-tyrosine phosphatase zeta. Mitogen-activated protein kinase, and phosphatidylinositol 3-kinase. Journal of Biological Chemistry, 276, 15868–15875.

    Article  CAS  Google Scholar 

  27. Narayanan, S. S., & Nampoothiri, K. M. (2012). Biochemical characterization of recombinant methionine aminopeptidases (MAPs) from Mycobacterium tuberculosis H37Rv. Molecular and Cellular Biochemistry, 365, 191–202.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Financial support for this work was provided by National Science Foundation of China (Grant No. 81173113 and Grant No. 81273573), Shanghai Science Foundation (Grant No. 11431921300) and National Post-doctor Foundation of China (Grant No. 2012M520902).

Author information

Author notes

    Authors and Affiliations

    1. School of Pharmacy, Shanghai Jiao Tong University, Room 6214, 800 Dongchuan Road, Minhang District, Shanghai, 200240, People’s Republic of China

      Jin Gao, Jingjing Li & Wei Han

    2. School of Agriculture and Biology, Shanghai Jiao Tong University, Room 3409, 800 Dongchuan Road, Minhang District, Shanghai, 200240, People’s Republic of China

      Haixia Wang & Yan Yu

    3. General regeneratives Limited, Shanghai, 200203, People’s Republic of China

      Shixiang Jia

    Authors
    1. Jin Gao
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Haixia Wang
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Jingjing Li
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Shixiang Jia
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Wei Han
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Yan Yu
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Yan Yu.

    Additional information

    Jin Gao and Haixia Wang contributed equally to this work.

    Electronic Supplementary Material

    Below is the link to the electronic supplementary material.

    Figure S1

    3D structure of rmMK. The structure was predicted with software Phyre2 and viewed with a Java viewer Jmol. (TIFF 154 kb)

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Gao, J., Wang, H., Li, J. et al. Eukaryotic Expression and Purification of Native Form of Mouse Midkine from Pichia pastoris . Appl Biochem Biotechnol 178, 490–503 (2016). https://doi.org/10.1007/s12010-015-1889-3

    Download citation

    • Received:

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s12010-015-1889-3

    Keywords

    Search

    Navigation