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A Novel Bifunctional Protein TNFR2-Fc-IL-1ra (TFI): Expression, Purification and its Neutralization Activity of Inflammatory Factors

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Abstract

Tumor necrosis factor receptor (TNF) and internleukin-1 (IL-1) are the most potent proinflammatory cytokines involving in autoimmune and inflammatory human diseases. Many anti-inflammatory agents have been exploited for anti-inflammation treatments by targeting cytokines including TNF and IL-1. Theoretically, simultaneously neutralizing or blocking two important inflammatory mediators may achieve a synergistic therapeutic effect. We have developed a recombinant fusion protein, TNFR2-Fc-IL-1ra (TFI), which consists of a TNF-neutralizing domain that specifically binds to TNF-α, an IL-1 receptor antagonist domain, and a dimerization Fc portion of human IgG1, for bifunctional inflammatory inhibitor. Recombinant DNA expressing the sequence of this fusion protein was expressed in CHO-S cells. The protein product was purified using a two-step purification protocol and the identity of the protein was confirmed by western blot analysis. The purified recombinant protein had a purity of about 98 % as determined by HPLC, and a molecular mass of 164.6 kDa as determined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The results of cell binding inhibition indicate that TFI was able to strongly neutralize TNF activity and antagonize IL-1r activity, suggesting that TFI may be used as a bifunctional ligand with enhanced anti-inflammatory effect. The result obtained in this study may provide a platform for extending bifunctional anti-inflammatory drug development.

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References

  1. Ferrero-Miliani, L., Nielsen, O. H., Andersen, P. S., & Girardin, S. E. (2007). Chronic inflammation: Importance of NOD2 and NALP3 in interleukin-1beta generation. Clinical and Experimental Immunology, 147, 227–235.

    CAS  Google Scholar 

  2. Strober, W., & Fuss, I. J. (2011). Proinflammatory cytokines in the pathogenesis of inflammatory bowel diseases. Gastroenterology, 140, 1756–1767. e1.

    Article  CAS  Google Scholar 

  3. Miller, A. M., & Mcinnes, I. B. (2011). Cytokines as therapeutic targets to reduce cardiovascular risk in chronic inflammation. Current Pharmaceutical Design, 17, 1–8.

    Article  CAS  Google Scholar 

  4. Vinay, D. S., & Kwon, B. S. (2011). The tumour necrosis factor/TNF receptor superfamily: Therapeutic targets in autoimmune diseases. Clinical and Experimental Immunology, 164, 145–157.

    Article  CAS  Google Scholar 

  5. Perrier, S., Darakhshan, F., & Hajduch, E. (2006). IL-1 receptor antagonist in metabolic diseases: Dr Jekyll or Mr Hyde? FEBS Letters, 580, 6289–6294.

    Article  CAS  Google Scholar 

  6. Tracey, D., Klareskog, L., Sasso, E. H., Salfeld, J. G., & Tak, P. P. (2008). Tumor necrosis factor antagonist mechanisms of action: A comprehensive review. Pharmacology & Therapeutics, 117, 244–279.

    Article  CAS  Google Scholar 

  7. Zhang, L., Berta, T., Xu, Z. Z., Liu, T., Park, J. Y., & Ji, R. R. (2011). TNF-alpha contributes to spinal cord synaptic plasticity and inflammatory pain: Distinct role of TNF receptor subtypes 1 and 2. Pain, 152, 419–427.

    Article  CAS  Google Scholar 

  8. Mizoguchi, E., Mizoguchi, A., Takedatsu, H., Cario, E., Jong, Y. P. D., Ooi, C. J., et al. (2002). Role of tumor necrosis factor receptor 2 (TNFR2) in colonic epithelial hyperplasia and chronic intestinal inflammation in mice. Gastroenterology, 122, 134–144.

    Article  CAS  Google Scholar 

  9. Mee, J. B., Cork, M. J., Giovine, F. S., Duff, G. W., & Groves, R. W. (2006). Interleukin-1: A key inflammatory mediator in psoriasis? Cytokine, 33, 72–78.

    Article  CAS  Google Scholar 

  10. Eisenberg, S. P., Evans, R. J., Arend, W. P., Verderber, E., Brewer, M. T., Hannum, C. H., et al. (1990). Primary structure and functional expression from complementary DNA of a human interleukin-1 receptor antagonist. Nature, 343, 341–346.

    Article  CAS  Google Scholar 

  11. Dewberry, R. M., King, A. R., Crossman, D. C., & Francis, S. E. (2008). Interleukin-1 receptor antagonist (IL-1ra) modulates endothelial cell proliferation. FEBS Letters, 582, 886–890.

    Article  CAS  Google Scholar 

  12. Hook, M. A., Washburn, S. N., Moreno, G., Woller, S. A., Puga, D., Lee, K. H., et al. (2011). An IL-1 receptor antagonist blocks a morphine-induced attenuation of locomotor recovery after spinal cord injury. Brain, Behavior, and Immunity, 25, 349–359.

    Article  CAS  Google Scholar 

  13. Bresnihan, B. (2001). The prospect of treating rheumatoid arthritis with recombinant human interleukin-1 receptor antagonist. BioDrugs, 15, 87–97.

    Article  CAS  Google Scholar 

  14. Schiff, M., Bulpitt, K., Weaver, A., Kazazi, F., Joh, T., & Newmark, R. (2002). Safety of combination therapy with Kineret (anakinra) and etanercept in patients with rheumatoid arthritis. Annals of the Rheumatic Diseases, 61, 185.

    Article  Google Scholar 

  15. Genovese, M. C., Cohen, S., Moreland, L., Lium, D., Robbins, S., Newmark, R., et al. (2004). Combination therapy with etanercept and anakinra in the treatment of patients with rheumatoid arthritis who have been treated unsuccessfully with methotrexate. Arthritis and Rheumatism, 50, 1412–1419.

    Article  CAS  Google Scholar 

  16. Jia, Q., Wu, H. T., Zhou, X. J., et al. (2010). A “GC-rich” method for mammalian gene expression: A dominant role of non-coding DNA GC content in regulation of mammalian gene expression. Science China Life Sciences, 53, 94–100.

    Article  CAS  Google Scholar 

  17. Eibl, R., Kaiser, S., Lombriser, R., & Eibl, D. (2010). Disposable bioreactors: The current state-of-the-art and recommended applications in biotechnology. Applied Microbiology and Biotechnology, 86, 41–49.

    Article  CAS  Google Scholar 

  18. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.

    Article  CAS  Google Scholar 

  19. Baarsch, M. J., Wannemuehler, M. J., Molitor, T. M., & Murtaugh, M. P. (1991). Detection of tumor necrosis factor α from porcine alveolar macrophages using an L929 fibroblast bioassay. Journal of Immunological Methods, 140, 15–22.

    Article  CAS  Google Scholar 

  20. Steinkasserer, A., Solari, R., Mott, H. R., Aplin, R. T., Robinson, C. C., Willis, A. C., et al. (1992). Human interleukin-1 receptor antagonist. High yield expression in E. coli and examination of cysteine residues. FEBS Letters, 310, 63–65.

    Article  CAS  Google Scholar 

  21. Durocher, Y., & Butler, M. (2009). Expression systems for therapeutic glycoprotein production. Current Opinion in Biotechnology, 20, 700–707.

    Article  CAS  Google Scholar 

  22. Birch, J. R., & Racher, A. J. (2006). Antibody production. Advanced Drug Delivery Reviews, 58, 671–685.

    Article  CAS  Google Scholar 

  23. Jia, Q., Li, H., Hui, M., Hui, N., Joudi, A., Rishton, G., et al. (2008). A bioreactor system based on a novel oxygen transfer method. BioProcess International, 6, 66–78.

    CAS  Google Scholar 

  24. Apostolaki, M., Armaka, M., Victoratos, P., & Kollias, G. (2010). Cellular mechanisms of TNF function in models of inflammation and autoimmunity. Current Directions in Autoimmunity, 11, 1–26.

    Article  CAS  Google Scholar 

  25. Kyogoku, K., Yoshida, K., Watanabe, H., Yamashita, T., Kawabe, Y., Motono, M., et al. (2008). Production of recombinant tumor necrosis factor receptor/Fc fusion protein by genetically manipulated chickens. Journal of Bioscience and Bioengineering, 105, 454–459.

    Article  CAS  Google Scholar 

  26. Gabay, C., Lamacchia, C., & Palmer, G. (2010). IL-1 pathways in inflammation and human diseases. Nature Reviews Rheumatology, 6, 232–241.

    Article  CAS  Google Scholar 

  27. Bayry, J., Sibéril, S., Triebel, F., Tough, D. F., & Kaveri, S. V. (2007). Rescuing CD4+CD25+ regulatory T-cell functions in rheumatoid arthritis by cytokine-targeted monoclonal antibody therapy. Drug Discovery Today, 12, 548–552.

    Article  CAS  Google Scholar 

  28. Notley, C. A., & Ehrenstein, M. R. (2010). The yin and yang of regulatory T cells and inflammation in RA. Nature Reviews Rheumatology, 6, 572–577.

    Article  CAS  Google Scholar 

  29. Bayry, J. (2011). New horizons in natural TNF-α antagonist research. Trends in Molecular Medicine, 17, 538–540.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This study was supported by grants from the NKLBE-IPE-CAS Research Program (Y024021167) and Cooperation Program with BGC in Mongolia (Y180261167). Especially, the authors thank Professor Zhonglin Liu at The University of Arizona for his valuable discussions and suggestions.

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Authors and Affiliations

  1. National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, 1 Bei-er Street, Zhongguancun, Beijing, 100190, China

    Bo Xie, Shan Liu & Mizhou Hui

  2. AmProtein-China Inc, Hangzhou, 310019, China

    Shuyin Wu, Weiyuan Jin, Zhixiang Guo, Shuaidong Ye & Mizhou Hui

  3. School of Life Science & Biotechnology, Dalian University of Technology, Dalian, 116024, China

    Alan Chang

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  1. Bo Xie
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  2. Shan Liu
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  3. Shuyin Wu
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  4. Alan Chang
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  5. Weiyuan Jin
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  6. Zhixiang Guo
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  7. Shuaidong Ye
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  8. Mizhou Hui
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Correspondence to Bo Xie or Mizhou Hui.

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Xie, B., Liu, S., Wu, S. et al. A Novel Bifunctional Protein TNFR2-Fc-IL-1ra (TFI): Expression, Purification and its Neutralization Activity of Inflammatory Factors. Mol Biotechnol 54, 141–147 (2013). https://doi.org/10.1007/s12033-012-9550-7

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  • DOI: https://doi.org/10.1007/s12033-012-9550-7

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