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Glycerol-Assisted Hydrophobic Interaction Chromatography Improving Refolding of Recombinant Human Granulocyte Colony-Stimulating Factor

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Abstract

Efficient refolding of recombinant proteins in the forms of inclusion bodies at higher concentration remains challenging. Here, we report a strategy of a dual-gradient hydrophobic interaction chromatography (HIC) mode to refold recombinant human granulocyte colony-stimulating factor from its inclusion bodies at high protein concentration. The strategy was taken to meet the demand of dynamic refolding proceeding by gradually decrease the denaturant (guanidine-HCl) concentration and gradually increase the hydrophilicity of media (column of Poros PE 20) with glycerol as additive to provide a mild refolding surroundings. Compared with dilution method, this dual-gradient HIC process gave about 8.5-fold of increase in specific activity and 30% increase in soluble protein recovery. Furthermore, much higher protein concentration could be obtained at the same time.

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References

  1. Minton, A. P. (2004). Current Biology, 10, 97–99. doi:10.1016/S0960-9822(00)00301-8.

    Article  Google Scholar 

  2. Georgiou, G., & Valax, P. (1996). Current Opinion in Biotechnology, 7, 190–197. doi:10.1016/S0958-1669(96)80012-7.

    Article  CAS  Google Scholar 

  3. Guise, A. D., West, S. M., & Chaudhuri, J. B. (1996). Molecular Biotechnology, 6, 53–64. doi:10.1007/BF02762323.

    Article  CAS  Google Scholar 

  4. Middelberg, A. P. J. (2002). Trends in Biotechnology, 20, 437–443. doi:10.1016/S0167-7799(02)02047-4.

    Article  CAS  Google Scholar 

  5. Bernardez, D., & Clark, E. (2001). Current Opinion in Biotechnology, 12, 202–207. doi:10.1016/S0958-1669(00)00200-7.

    Article  Google Scholar 

  6. Hevehan, D. L., Bernardez, D., & Clark, E. (1997). Biotechnology and Bioengineering, 54, 221–230. doi:10.1002/(SICI)1097-0290(19970505)54:3<221::AID-BIT3>3.0.CO;2-H.

    Article  CAS  Google Scholar 

  7. Kiefhaber, T., Rudolph, R., Kohler, H. H., & Buchner, J. (1991). Biotechnology, 9, 825–829. doi:10.1038/nbt0991-825.

    Article  CAS  Google Scholar 

  8. Shimizu, H., Fujimoto, K., & Kawaguchi, H. (2000). Colloids and Surfaces B, 18, 137–144. doi:10.1016/S0927-7765(99)00140-X.

    Article  CAS  Google Scholar 

  9. Raman, B., Ramakrishna, T., & Rao, C. M. (1996). The Journal of Biological Chemistry, 271, 17067–17072. doi:10.1074/jbc.271.44.27595.

    Article  CAS  Google Scholar 

  10. Batas, B., & Chaudhuri, J. B. (1996). Biotechnology and Bioengineering, 50, 16–23. doi:10.1002/(SICI)1097-0290(19960405)50:1<16::AID-BIT3>3.0.CO;2-4.

    Article  CAS  Google Scholar 

  11. Gu, Z., Su, Z., & Jonson, J. C. (2001). Journal of Chromatography. A, 918, 311–318. doi:10.1016/S0021-9673(01)00766-X.

    Article  CAS  Google Scholar 

  12. Li, M., & Su, Z. (2002). Chromatographia, 56, 33–38. doi:10.1007/BF02490243.

    Article  CAS  Google Scholar 

  13. Creighton, T. E. (1985). UCLA symposia on molecular and cellular biology, vol. 39 pp. 249–257. New York: Liss.

    Google Scholar 

  14. Rogl, H., Kosemund, K., Kuhlbrandt, W., & Collinson, I. (1998). FEBS Letters, 432, 21–26. doi:10.1016/S0014-5793(98)00825-4.

    Article  CAS  Google Scholar 

  15. Zahn, R., Schroetter, C. V., & Wuthrich, K. (1997). FEBS Letters, 417, 400–404. doi:10.1016/S0014-5793(97)01330-6.

    Article  CAS  Google Scholar 

  16. Geng, X. D., Bai, Q., Zhang, Y. J., Li, X., & Wu, D. J. (2004). Journal of Biotechnology, 113, 137–149. doi:10.1016/j.jbiotec.2004.06.006.

    Article  CAS  Google Scholar 

  17. Li, J. J., Liu, Y. D., Wang, F. W., Ma, G. H., & Su, Z. G. (2004). Journal of Chromatography. A, 1061, 193–199. doi:10.1016/j.chroma.2004.11.002.

    Article  CAS  Google Scholar 

  18. Wang, F. W., Liu, Y. D., Li, J. J., Ma, G. H., & Su, Z. G. (2006). Journal of Chromatography. A, 1115, 72–80. doi:10.1016/j.chroma.2006.02.075.

    Article  CAS  Google Scholar 

  19. Queiroz, J. A., Tomaz, C. T., & Cabral, J. M. S. (2001). Journal of Biotechnology, 87, 143–159. doi:10.1016/S0168-1656(01)00237-1.

    Article  CAS  Google Scholar 

  20. Geng, X., & Chang, X. (2002). Journal of Chromatography. A, 599, 185–194. doi:10.1016/0021-9673(92)85472-6.

    Article  Google Scholar 

  21. Bolen, D. W. (2001). In K. P. Murphy (Ed.), Protein structure, stability and folding p. 18. New Jersey: Human.

    Google Scholar 

  22. Russo, A. T., Rösgen, J., & Bolen, D. W. (2003). Journal of Molecular Biology, 330(4), 851–866. doi:10.1016/S0022-2836(03)00626-0.

    Article  CAS  Google Scholar 

  23. Geijn, G. J., Aarts, L. H., Erkeland, S. J., Prasher, J. M., & Touw, I. P. (2003). Reviews of Physiology, Biochemistry and Pharmacology, 149, 53–71. doi:10.1007/s10254-003-0014-0.

    Article  Google Scholar 

  24. Chu, C., Luo, T., & Tan, W. (1998). Progress in Microbiology and Immunology, 26, 1–5.

    Google Scholar 

  25. Hammerling, U., Kroon, R., & Sjodin, L. (1995). Journal of Pharmaceutical and Biomedical Analysis, 13, 9–20. doi:10.1016/0731-7085(94)00128-O.

    Article  CAS  Google Scholar 

  26. Bradford, M. M. (1976). Analytical Biochemistry, 72, 248–254. doi:10.1016/0003-2697(76)90527-3.

    Article  CAS  Google Scholar 

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Acknowledgments

The authors are thankful for the financial support from the Natural Science Foundation of China (Contract Nos. 20636010, 20576136, and 20820102036), the National 863 High-Tech Project (Contract No. 2007AA021604), and the National 973 High-Tech Project (Contract No. 2007CB714305)

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Authors and Affiliations

  1. Second Clinical College, Dalian Medical University, Dalian, 116027, China

    Fangwei Wang

  2. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, China

    Fangwei Wang, Yongdong Liu, Guanghui Ma & Zhiguo Su

Authors
  1. Fangwei Wang
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  2. Yongdong Liu
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  3. Guanghui Ma
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  4. Zhiguo Su
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Correspondence to Yongdong Liu.

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Wang, F., Liu, Y., Ma, G. et al. Glycerol-Assisted Hydrophobic Interaction Chromatography Improving Refolding of Recombinant Human Granulocyte Colony-Stimulating Factor. Appl Biochem Biotechnol 159, 634–641 (2009). https://doi.org/10.1007/s12010-008-8495-6

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  • DOI: https://doi.org/10.1007/s12010-008-8495-6

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