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Efficient Soluble Expression and Purification of Recombinant Human Acidic Fibroblast Growth Factor from Escherichia coli via Fusion with a Novel Collagen-like Protein Scl2

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Abstract

Human acidic fibroblast growth factor (haFGF) is a multifunctional protein involved in regulating a wide range of cellular processes. As a potent therapeutic agent, it is highly desirable to produce recombinant haFGF (r-haFGF) at low cost. However, the complex structure and formation of aggregation confines its high-level soluble expression and functional form. Herein, to produce r-haFGF efficiently in E. coli, we devised a novel soluble expression and cost-effective purification approach based on fusion with Scl2-M (a novel modified collagen-like protein) for the first time. By using this strategy, more than 95% of the Scl2-M-haFGF fusion protein was highly expressed in soluble form and the expression level of targeted fusion protein in shake flasks and 5-L fermenter was 0.42 g/L and 2.28 g/L, respectively. Subsequently, the recombinant Scl2-M-haFGF was readily purified through a facile process of acid precipitation and subjected to enterokinase (EK) cleavage. After Scl2-M cleavage, tag-free r-haFGF was further purified using ion-exchange chromatography. The recovery rate of the whole purification process attained 34.2%. Furthermore, the resulting high-purity (96.0%) r-haFGF was prepared by freeze-drying as a final product, and its bioactivity was confirmed to potentiate the proliferation of L929 and BALB-3T3 fibroblasts. Overall, our developed method has the potential for the massive production of the r-haFGF in the future.

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References

  1. Zhou, Y., Ren, L., Zhu, J., Yan, S., Wang, H., Song, N., Li, L., Ouyang, H., & Pang, D. (2011). Construction of a recombinant human FGF1 expression vector for mammary gland-specific expression in human breast cancer cells. Molecular and Cellular Biochemistry, 354, 39–46.

    Article  CAS  PubMed  Google Scholar 

  2. Wu, X., Kamei, K., Sato, H., Sato, S.-I., Takano, R., Ichida, M., Mori, H., & Hara, S. (2001). High-level expression of human acidic fibroblast growth factor and basic fibroblast growth factor in silkworm (Bombyx mori L) using recombinant baculovirus. Protein Expression and Purification, 21(1), 192–200.

    Article  CAS  PubMed  Google Scholar 

  3. Fantoni, A., Bill, R. M., Gustafsson, L., & Hedfalk, K. (2007). Improved yields of full-length functional human FGF1 can be achieved using the methylotrophic yeast Pichia pastoris. Protein Expression and Purification, 52(1), 31–39.

    Article  CAS  PubMed  Google Scholar 

  4. Stoykova, P., Radkova, M., Stoeva-Popova, P., Atanasov, N., Chassovnikarova, T., Wang, X., Iantcheva, A., Vlahova, M., & Atanassov, A. (2011). Expression of the human acidic fibroblast growth factor in transgenic tomato and safety assessment of transgenic lines. Biotechnology & Biotechnological Equipment, 25, 2187–2196.

    Article  CAS  Google Scholar 

  5. Sugawara, S., Ito, T., Sato, S., Sato, Y., Kasuga, K., Kojima, I., & Kobayashi, M. (2014). Identification of site-specific degradation in bacterially expressed human fibroblast growth factor 4 and generation of an amino terminally truncated, stable form. Applied Biochemistry and Biotechnology, 172(1), 206–215.

    Article  CAS  PubMed  Google Scholar 

  6. Zazo, M., Lozano, R. M., Ortega, S., Varela, J., Diaz-Orejas, R., Ramirez, J. M., & Giménez-Gallego, G. (1992). High-level synthesis in Escherichia coli of shortened and full-length human acidic fibroblast growth factor and purification in a form stable in aqueous solutions. Gene, 113(2), 231–238.

    Article  CAS  PubMed  Google Scholar 

  7. Mohammadi, M., Olsen, S. K., & Ibrahimi, O. A. (2005). Structural basis for fibroblast growth factor receptor activation. Cytokine & Growth Factor Reviews, 16(2), 107–137.

    Article  CAS  Google Scholar 

  8. Yun, Y.-R., Won, J. E., Jeon, E., Lee, S., Kang, W., Jo, H., Jang, J.-H., Shin, U. S., & Kim, H.-W. (2010). Fibroblast growth factors: biology, function, and application for tissue regeneration. Journal of Tissue Engineering, 1, 218142.

    Article  Google Scholar 

  9. Zakrzewska, M., Marcinkowska, E., & Wiedlocha, A. (2008). FGF-1: From biology through engineering to potential medical applications. Critical Reviews in Clinical Laboratory Sciences, 45(1), 91–135.

    Article  CAS  PubMed  Google Scholar 

  10. Wang, F., Wang, R., Wang, Y., Zhao, P., & Xia, Q. (2015). Large-scale production of bioactive recombinant human acidic fibroblast growth factor in transgenic silkworm cocoons. Scientific Reports, 5, 16323.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Nham, S.-U., Kim, H.-S., & Lee, Y.-I. (1994). Overexpression and purification of full-length acidic fibroblast growth factor in E. coli. Biotechnology Letters, 16, 661–666.

    Article  CAS  Google Scholar 

  12. Ha, J.-H., Kim, H.-N., Moon, K.-B., Jeon, J.-H., Jung, D.-H., Kim, S.-J., Mason, H. S., Shin, S.-Y., Kim, H.-S., & Park, K.-M. (2017). Recombinant human acidic fibroblast growth factor (aFGF) expressed in Nicotiana benthamiana potentially inhibits skin photoaging. Planta Medica, 83(10), 862–869.

    Article  CAS  PubMed  Google Scholar 

  13. Batra, S., Sahi, N., Mikulcik, K., Shockley, H., Turner, C., Laux, Z., Badwaik, V. D., Conte, E., & Rajalingam, D. (2011). Efficient and inexpensive method for purification of heparin binding proteins. Journal of Chromatography B, 879, 2437–2442.

    Article  CAS  Google Scholar 

  14. Zhang, D., Wei, P., Fan, L., Lian, J., Huang, L., Cai, J., & Xu, Z. (2010). High-level soluble expression of hIGF-1 fusion protein in recombinant Escherichia coli. Process Biochemistry, 45, 1401–1405.

    Article  Google Scholar 

  15. Sun, C., Li, Y., Taylor, S. E., Mao, X., Wilkinson, M. C., & Fernig, D. G. (2015). HaloTag is an effective expression and solubilisation fusion partner for a range of fibroblast growth factors. PeerJ, 3, e1060.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Huang, Y., Rao, Y., Feng, C., Li, Y., Wu, X., Su, Z., Xiao, J., Xiao, Y., Feng, W., & Li, X. (2008). High-level expression and purification of Tat-haFGF 19-154. Applied Microbiology and Biotechnology, 77(5), 1015–1022.

    Article  CAS  PubMed  Google Scholar 

  17. Wood, D. W., Derbyshire, V., Wu, W., Chartrain, M., Belfort, M., & Belfort, G. (2000). Optimized single-step affinity purification with a self-cleaving intein applied to human acidic fibroblast growth factor. Biotechnology Progress, 16(6), 1055–1063.

    Article  CAS  PubMed  Google Scholar 

  18. Yang, J., Guan, L., Guo, Y., Du, L., Wang, F., Wang, Y., Zhen, L., Wang, Q., Zou, D., & Chen, W. (2015). Expression of biologically recombinant human acidic fibroblast growth factor in Arabidopsis thaliana seeds via oleosin fusion technology. Gene, 566, 89–94.

    Article  CAS  PubMed  Google Scholar 

  19. Peng, Y. Y., Stoichevska, V., Madsen, S., Howell, L., Dumsday, G. J., Werkmeister, J. A., & Ramshaw, J. A. (2014). A simple cost-effective methodology for large-scale purification of recombinant non-animal collagens. Applied Microbiology and Biotechnology, 98(4), 1807–1815.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Peng, Y. Y., Howell, L., Stoichevska, V., Werkmeister, J. A., Dumsday, G. J., & Ramshaw, J. A. (2012). Towards scalable production of a collagen-like protein from Streptococcus pyogenes for biomedical applications. Microbial Cell Factories, 11, 146.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bellis, S. L. (2011). Advantages of RGD peptides for directing cell association with biomaterials. Biomaterials, 32(18), 4205–4210.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Zitzmann, S., Ehemann, V., & Schwab, M. (2002). Arginine-glycine-aspartic acid (RGD)-peptide binds to both tumor and tumor-endothelial cells in vivo. Cancer Research, 62, 5139–5143.

    CAS  PubMed  Google Scholar 

  23. Lian, J., Ding, S., Cai, J., Zhang, D., Xu, Z., & Wang, X. (2009). Improving aquaporin Z expression in Escherichia coli by fusion partners and subsequent condition optimization. Applied Microbiology and Biotechnology, 82(3), 463–470.

    Article  CAS  PubMed  Google Scholar 

  24. Peng, L., Xu, Z., Fang, X., Wang, F., & Cen, P. (2004). High-level expression of soluble human β-defensin-2 in Escherichia coli. Process Biochemistry, 39, 2199–2205.

    Article  CAS  Google Scholar 

  25. Tang, Y. (2016). Efficient production of hydroxylated human-like collagen via the co-expression of three key genes in Escherichia coli Origami (DE3). Applied Biochemistry and Biotechnology, 178(7), 1458–1470.

    Article  CAS  PubMed  Google Scholar 

  26. 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(1), 175–182.

    Article  CAS  PubMed  Google Scholar 

  27. Wu, X., Su, Z., Li, X., Zheng, Q., Huang, Y., & Yuan, H. (2005). High-level expression and purification of a nonmitogenic form of human acidic fibroblast growth factor in Escherichia coli. Protein Expression and Purification, 42(1), 7–11.

    Article  CAS  PubMed  Google Scholar 

  28. Tan, Y., Wang, K. Y., Wang, N., Li, G., & Liu, D. (2014). Ectopic expression of human acidic fibroblast growth factor 1 in the medicinal plant, Salvia miltiorrhiza, accelerates the healing of burn wounds. BMC Biotechnology, 14, 74.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Cao, Y., & Pettersson, R. F. (1990). Human acidic fibroblast growth factor overexpressed in insect cells is not secreted into the medium. Growth Factors, 3(1), 1–13.

    Article  CAS  PubMed  Google Scholar 

  30. Huang, L., Xu, Z., Zhong, Z., Peng, L., Chen, H., & Cen, P. (2007). Enhanced expression and primary purification of soluble HBD3 fusion protein in Escherichia coli. Applied Biochemistry and Biotechnology, 142(2), 139–147.

    Article  CAS  PubMed  Google Scholar 

  31. Malik, A. (2016). Protein fusion tags for efficient expression and purification of recombinant proteins in the periplasmic space of E. coli. 3. Biotech, 6, 44.

    Google Scholar 

  32. Kowalczyk, T., Hnatuszko-Konka, K., Gerszberg, A., & Kononowicz, A. K. (2014). Elastin-like polypeptides as a promising family of genetically-engineered protein based polymers. World Journal of Microbiology and Biotechnology, 30(8), 2141–2152.

    Article  CAS  PubMed  Google Scholar 

  33. Li, X., Tu, C., Peng, H., Yuan, S., & Wu, F. (1999). High-level expression of human acidic fibroblast growth factor in E. coli and its purification. Journal of West China University of Medical Sciences, 30(3), 249–252.

    CAS  PubMed  Google Scholar 

  34. Gambarini, A. G., Miyamoto, C. A., Lima, G. A., Nader, H. B., & Dietrich, C. P. (1993). Mitogenic activity of acidic fibroblast growth factor is enhanced by highly sulfated oligosaccharides derived from heparin and heparan sulfate. Molecular and Cellular Biochemistry, 124(2), 121–129.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (No. 21576232, 21606205 & 21808199), the National Key Research and Development Program of China (No. 2018YFC1603900), and the Natural Science Foundation of Zhejiang Province (No. LY18B060002).

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Authors and Affiliations

  1. College of Biosystems Engineering and Food Science, Zhejiang University, Fuli Institute of Food Science, Hangzhou, 310058, China

    Inam ur Rahman & Xiaodong Zheng

  2. College of Chemical and Biological Engineering, Zhejiang University, Institute of Biological Engineering, Hangzhou, 310027, China

    Inam ur Rahman, Wei Liu, Lei Huang & Zhinan Xu

  3. Center for Synthetic Biology, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China

    Wei Liu & Zhinan Xu

  4. Hangzhou Zhongmei Huadong Pharmaceutical Co., Ltd., Hangzhou, China

    Zhang Wei & Lina Fang

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  1. Inam ur Rahman
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Correspondence to Lei Huang or Zhinan Xu.

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Rahman, I.u., Liu, W., Wei, Z. et al. Efficient Soluble Expression and Purification of Recombinant Human Acidic Fibroblast Growth Factor from Escherichia coli via Fusion with a Novel Collagen-like Protein Scl2. Appl Biochem Biotechnol 191, 1562–1579 (2020). https://doi.org/10.1007/s12010-020-03269-y

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