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Expression and Purification of Biologically Active Human FGF2 Containing the b′a′ Domains of Human PDI in Escherichia coli

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Abstract

Among the members of the fibroblast growth factor (FGF) family that affect the growth, differentiation, migration, and survival of many cell types, FGF2 is the most abundant in the central nervous system. Because of its wound healing effects, FGF2 has potential as a therapeutic agent. The protein is also added to the culture media to maintain stem cells. Expression and purification procedures for FGF2 that are highly efficient and low cost have been intensively investigated for the past two decades. Our current study focuses on the purification of FGF2 fused with b′a′ domains of human protein disulfide isomerase to elevate overexpression, solubility, and stability with a simplified experimental procedure using only ion exchange chromatography, as well as on the confirmation of the biological activity of FGF2 on fibroblast Balb/c 3T3 cells and hippocampal neural cells.

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References

  1. Alibolandi, M., & Mirzahoseini, H. (2011). Purification and refolding of overexpressed human basic fibroblast growth factor in Escherichia coli. Biotechnol Res Int, 2011, 973741.

    Google Scholar 

  2. Amit, M., Carpenter, M. K., Inokuma, M. S., Chiu, C. P., Harris, C. P., Waknitz, M. A., et al. (2000). Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Developmental Biology, 227, 271–278.

    Article  CAS  Google Scholar 

  3. Andrades, J. A., Wu, L. T., Hall, F. L., Nimni, M. E., & Becerra, J. (2001). Engineering, expression, and renaturation of a collagen-targeted human bFGF fusion protein. Growth Factors, 18, 261–275.

    Article  CAS  Google Scholar 

  4. Arakawa, T., Hsu, Y. R., Schiffer, S. G., Tsai, L. B., Curless, C., & Fox, G. M. (1989). Characterization of a cysteine-free analog of recombinant human basic fibroblast growth factor. Biochemical and Biophysical Research Communications, 161, 335–341.

    Article  CAS  Google Scholar 

  5. Belluardo, N., Wu, G., Mudo, G., Hansson, A. C., Pettersson, R., & Fuxe, K. (1997). Comparative localization of fibroblast growth factor receptor-1, -2, and -3 mRNAs in the rat brain: in situ hybridization analysis. The Journal of Comparative Neurology, 379, 226–246.

    Article  CAS  Google Scholar 

  6. Bikfalvi, A., Klein, S., Pintucci, G., & Rifkin, D. B. (1997). Biological roles of fibroblast growth factor-2. Endocrine Reviews, 18, 26–45.

    Article  CAS  Google Scholar 

  7. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.

    Article  CAS  Google Scholar 

  8. Busso, D., Delagoutte-Busso, B., & Moras, D. (2005). Construction of a set Gateway-based destination vectors for high-throughput cloning and expression screening in Escherichia coli. Analytical Biochemistry, 343, 313–321.

    Article  CAS  Google Scholar 

  9. Chen, X. J., Sun, F. Y., Xie, Q. L., Liao, M. D., Zhang, L., Li, Z. Y., et al. (2002). Cloning and high level nonfusion expression of recombinant human basic fibroblast growth factor in Escherichia coli. Acta Pharmacologica Sinica, 23, 782–786.

    Google Scholar 

  10. Clark, E. D. (2001). Protein refolding for industrial processes. Current Opinion in Biotechnology, 12, 202–207.

    Article  CAS  Google Scholar 

  11. Denisov, A. Y., Maattanen, P., Dabrowski, C., Kozlov, G., Thomas, D. Y., & Gehring, K. (2009). Solution structure of the bb' domains of human protein disulfide isomerase. FEBS Journal, 276, 1440–1449.

    Article  CAS  Google Scholar 

  12. Ferrer, M., Chernikova, T. N., Timmis, K. N., & Golyshin, P. N. (2004). Expression of a temperature-sensitive esterase in a novel chaperone-based Escherichia coli strain. Applied and Environmental Microbiology, 70, 4499–4504.

    Article  CAS  Google Scholar 

  13. Fox, G. M., Schiffer, S. G., Rohde, M. F., Tsai, L. B., Banks, A. R., & Arakawa, T. (1988). Production, biological activity, and structure of recombinant basic fibroblast growth factor and an analog with cysteine replaced by serine. Journal of Biological Chemistry, 263, 18452–18458.

    CAS  Google Scholar 

  14. Gage, F. H., Coates, P. W., Palmer, T. D., Kuhn, H. G., Fisher, L. J., Suhonen, J. O., et al. (1995). Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proceedings of the National Academy of Sciences of the United States of America, 92, 11879–11883.

    Article  CAS  Google Scholar 

  15. Garke, G., Deckwer, W. D., & Anspach, F. B. (2000). Preparative two-step purification of recombinant human basic fibroblast growth factor from high-cell-density cultivation of Escherichia coli. Journal of Chromatography B: Biomedical Sciences and Applications, 737, 25–38.

    Article  CAS  Google Scholar 

  16. Gasparian, M. E., Elistratov, P. A., Drize, N. I., Nifontova, I. N., Dolgikh, D. A., & Kirpichnikov, M. P. (2009). Overexpression in Escherichia coli and purification of human fibroblast growth factor (FGF-2). Biochemistry (Mosc), 74, 221–225.

    Article  CAS  Google Scholar 

  17. Gospodarowicz, D., Ferrara, N., Schweigerer, L., & Neufeld, G. (1987). Structural characterization and biological functions of fibroblast growth factor. Endocrine Reviews, 8, 95–114.

    Article  CAS  Google Scholar 

  18. Gritti, A., Parati, E. A., Cova, L., Frolichsthal, P., Galli, R., Wanke, E., et al. (1996). Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor. Journal of Neuroscience, 16, 1091–1100.

    CAS  Google Scholar 

  19. Grothe, C., & Wewetzer, K. (1996). Fibroblast growth factor and its implications for developing and regenerating neurons. International Journal of Developmental Biology, 40, 403–410.

    CAS  Google Scholar 

  20. Hartley, J. L., Temple, G. F., & Brasch, M. A. (2000). DNA cloning using in vitro site-specific recombination. Genome Research, 10, 1788–1795.

    Article  CAS  Google Scholar 

  21. Ke, Y., Wilkinson, M. C., Fernig, D. G., Smith, J. A., Rudland, P. S., & Barraclough, R. (1992). A rapid procedure for production of human basic fibroblast growth factor in Escherichia coli cells. Biochimica et Biophysica Acta, 1131, 307–310.

    Article  CAS  Google Scholar 

  22. Klappa, P., Ruddock, L. W., Darby, N. J., & Freedman, R. B. (1998). The b' domain provides the principal peptide-binding site of protein disulfide isomerase but all domains contribute to binding of misfolded proteins. EMBO Journal, 17, 927–935.

    Article  CAS  Google Scholar 

  23. Komi-Kuramochi, A., Kawano, M., Oda, Y., Asada, M., Suzuki, M., Oki, J., et al. (2005). Expression of fibroblast growth factors and their receptors during full-thickness skin wound healing in young and aged mice. Journal of Endocrinology, 186, 273–289.

    Article  CAS  Google Scholar 

  24. Kroiher, M., Raffioni, S., & Steele, R. E. (1995). Single step purification of biologically active recombinant rat basic fibroblast growth factor by immobilized metal affinity chromatography. Biochimica et Biophysica Acta, 1250, 29–34.

    Article  Google Scholar 

  25. 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 

  26. LaVallie, E. R., DiBlasio, E. A., Kovacic, S., Grant, K. L., Schendel, P. F., & McCoy, J. M. (1993). A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology (N. Y), 11, 187–193.

    Article  CAS  Google Scholar 

  27. Lee, J. W., & Helmann, J. D. (2006). The PerR transcription factor senses H2O2 by metal-catalysed histidine oxidation. Nature, 440, 363–367.

    Article  CAS  Google Scholar 

  28. Lemaitre, G., Laaroubi, K., Soulet, L., Barritault, D., & Miskulin, M. (1995). Production and purification of active FGF2 via recombinant fusion protein. Biochimie, 77, 162–6.

    Article  CAS  Google Scholar 

  29. Liao, S., Bodmer, J., Pietras, D., Azhar, M., Doetschman, T., & Schultz Jel, J. (2009). Biological functions of the low and high molecular weight protein isoforms of fibroblast growth factor-2 in cardiovascular development and disease. Developmental Dynamics, 238, 249–264.

    Article  CAS  Google Scholar 

  30. Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65, 55–63.

    Article  CAS  Google Scholar 

  31. Murakami, S. (2011). Periodontal tissue regeneration by signaling molecule(s): what role does basic fibroblast growth factor (FGF-2) have in periodontal therapy? Periodontology 2000, 2000(56), 188–208.

    Article  Google Scholar 

  32. Nallamsetty, S., & Waugh, D. S. (2006). Solubility-enhancing proteins MBP and NusA play a passive role in the folding of their fusion partners. Protein Expression and Purification, 45, 175–182.

    Article  CAS  Google Scholar 

  33. Nygren, P. A., Stahl, S., & Uhlen, M. (1994). Engineering proteins to facilitate bioprocessing. Trends in Biotechnology, 12, 184–188.

    Article  CAS  Google Scholar 

  34. Palmer, T. D., Takahashi, J., & Gage, F. H. (1997). The adult rat hippocampus contains primordial neural stem cells. Molecular and Cellular Neuroscience, 8, 389–404.

    Article  CAS  Google Scholar 

  35. Pryor, K. D., & 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 Expression and Purification, 10, 309–319.

    Article  CAS  Google Scholar 

  36. Schultz, G. S., & Wysocki, A. (2009). Interactions between extracellular matrix and growth factors in wound healing. Wound Repair and Regeneration, 17, 153–162.

    Article  Google Scholar 

  37. Schwartz, S. M., & Liaw, L. (1993). Growth control and morphogenesis in the development and pathology of arteries. Journal of Cardiovascular Pharmacology, 21(Suppl 1), S31–49.

    Article  CAS  Google Scholar 

  38. Seeger, A., & Rinas, U. (1996). Two-step chromatographic procedure for purification of basic fibroblast growth factor from recombinant Escherichia coli and characterization of the equilibrium parameters of adsorption. Journal of Chromatography. A, 746, 17–24.

    Article  CAS  Google Scholar 

  39. Seno, M., Sasada, R., Iwane, M., Sudo, K., Kurokawa, T., Ito, K., et al. (1988). Stabilizing basic fibroblast growth factor using protein engineering. Biochemical and Biophysical Research Communications, 151, 701–708.

    Article  CAS  Google Scholar 

  40. Sheng, Z., Chang, S. B., & Chirico, W. J. (2003). Expression and purification of a biologically active basic fibroblast growth factor fusion protein. Protein Expression and Purification, 27, 267–271.

    Article  CAS  Google Scholar 

  41. Squires, C. H., Childs, J., Eisenberg, S. P., Polverini, P. J., & Sommer, A. (1988). Production and characterization of human basic fibroblast growth factor from Escherichia coli. Journal of Biological Chemistry, 263, 16297–16302.

    CAS  Google Scholar 

  42. Thompson, S. A. (1992). The disulfide structure of bovine pituitary basic fibroblast growth factor. Journal of Biological Chemistry, 267, 2269–2273.

    CAS  Google Scholar 

  43. Thompson, S. A., Protter, A. A., Bitting, L., Fiddes, J. C., & Abraham, J. A. (1991). Cloning, recombinant expression, and characterization of basic fibroblast growth factor. Methods in Enzymology, 198, 96–116.

    Article  CAS  Google Scholar 

  44. Vera, A., Gonzalez-Montalban, N., Aris, A., & Villaverde, A. (2007). The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures. Biotechnology and Bioengineering, 96, 1101–1106.

    Article  CAS  Google Scholar 

  45. Wu, X., Liu, X., Xiao, Y., Huang, Z., Xiao, J., Lin, S., et al. (2007). Purification and modification by polyethylene glycol of a new human basic fibroblast growth factor mutant-hbFGF(Ser25,87,92). Journal of Chromatography. A, 1161, 51–55.

    Article  CAS  Google Scholar 

  46. Yu, S. W., Baek, S. H., Brennan, R. T., Bradley, C. J., Park, S. K., Lee, Y. S., et al. (2008). Autophagic death of adult hippocampal neural stem cells following insulin withdrawal. Stem Cells, 26, 2602–2610.

    Article  CAS  Google Scholar 

  47. Zechel, S., Werner, S., Unsicker, K., Von Bohlen, & Halbach, O. (2010). Expression and functions of fibroblast growth factor 2 (FGF-2) in hippocampal formation. The Neuroscientist, 16, 357–373.

    Article  CAS  Google Scholar 

  48. Zhang, Y., Olsen, D. R., Nguyen, K. B., Olson, P. S., Rhodes, E. T., & Mascarenhas, D. (1998). Expression of eukaryotic proteins in soluble form in Escherichia coli. Protein Expression and Purification, 12, 159–165.

    Article  CAS  Google Scholar 

  49. Zhu, X., Komiya, H., Chirino, A., Faham, S., Fox, G. M., Arakawa, T., et al. (1991). Three-dimensional structures of acidic and basic fibroblast growth factors. Science, 251, 90–93.

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by the Korea Research Foundation (2010-0029522 and 2010-0029521), the Brain Research Center of the 21st Century Frontier Research Program (2012K001122) funded by the Ministry of Education, Science and Technology, the Republic of Korea.

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Authors and Affiliations

  1. Department of Physiology and Bio-medical Institute of Technology, University of Ulsan College of Medicine, Seoul, 138-736, South Korea

    Jung-A Song, Bon-Kyung Koo, Seon Ha Chong, Han-Bong Ryu, Minh Tan Nguyen, Thu Trang Thi Vu, Boram Jeong & Han Choe

  2. Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul, 138-736, South Korea

    Jihye Kwak & Seong Who Kim

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  1. Jung-A Song
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  2. Bon-Kyung Koo
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Correspondence to Han Choe.

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J.-A. Song and B.-K. Koo contributed equally to this work.

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Song, JA., Koo, BK., Chong, S.H. et al. Expression and Purification of Biologically Active Human FGF2 Containing the b′a′ Domains of Human PDI in Escherichia coli . Appl Biochem Biotechnol 170, 67–80 (2013). https://doi.org/10.1007/s12010-013-0140-3

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