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Refolding of lysozyme in hydrophobic interaction chromatography: Effects of hydrophobicity of adsorbent and salt concentration in mobile phase

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Abstract

The effects of salt concentration in mobile phase, elution strategy, and hydrophobicity of stationary phase on lysozyme refolding in hydrophobic interaction chromatography (HIC) were investigated. Butyl Sepharose 4 Fast Flow, the least hydrophobic HIC resin among the tested adsorbent, showed the best refolding yield. The binding efficiency of unfolded lysozyme on the adsorbent was maximized when 1 and 0.4 M of initial and final concentration of ammonium sulfate was used in mobile phase, respectively. The optimum gradient strategy for refolding and elution of lysozyme was determined as linear increase of urea concentration to 4M. The optimized condition suggests the less hydrophobic environment than conventionally used salt solutions and HIC resins. Consequently, total refolding yield was improved 1.6 times comparing with optimized dilution-based batch refolding method.

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References

  1. To, B. C. S. and A. M. Lenhoff (2007) Hydrophobic interaction chromatography of proteins 1. The effects of protein and adsorbent properties on retention and recovery. J. Chromatogr. A 1141: 191–205.

    Article  CAS  Google Scholar 

  2. Goldberg, M. E., R. Rainer, and J. Rainer (1991) A kinetic study of the competition between renaturation and aggregation during refolding of denatured-reduced egg white lysozyme. Biochemistry 30: 2790–2797.

    Article  CAS  Google Scholar 

  3. Li, J. -J., Y. Liu, F. Wang, G. Ma, and Z. Su (2004) Hydrophobic interaction chromatography correctly refolding protein assisted by glycerol and urea gradient. J. Chromatogr. A 1061: 193–199.

    Article  CAS  Google Scholar 

  4. Cho, E. K. (2007) Enhanced tolerance against freezing stress in Escherichia coli cells expressing an algal cyclophilin gene. Biotechnol. Bioprocess Eng. 12: 502–507.

    Article  CAS  Google Scholar 

  5. Kopito, R. R. (2000) Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol. 10: 524–530.

    Article  CAS  Google Scholar 

  6. Jungbauer, A. and W. Kaar (2007) Current status of technical protein refolding, J. Biotechnol. 128: 587–596.

    Article  CAS  Google Scholar 

  7. Batas, D. and J. B. Chaudhuri (1996) Protein refolding at high-concentration using size-exclusion chromatography. Biotechnol. Bioeng. 50: 16–23.

    Article  CAS  Google Scholar 

  8. Gu, Z., Z. Su, and J. C. Janson (2001) Urea gradient size-exclusion chromatography enhanced the yield of lysozyme refolding. J. Chromatogr. A 918: 311–318.

    Article  CAS  Google Scholar 

  9. Creighton, T. E. and D. L. Oxender (1986) UCLA Symposia on Molecular and Cellular Biology. pp. 249–257. A. R. Liss, NY, USA.

    Google Scholar 

  10. Glynou, K., P. C. Ioannou, and T. Christopoulos (2003) one-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography. Protein Expression Purif. 27: 384–390.

    Article  CAS  Google Scholar 

  11. Geng, X. and X. Chang (1992) High-performance hydrophobic interaction chromatography as a tool for protein refolding. J. Chromatogr. A 599: 185–194.

    Article  CAS  Google Scholar 

  12. Lienqueo, M. E., A. Mahn, J. C. Salgado, and J. A. Asenjo (2007) Current insights on protein behavior in hydrophobic interaction chromatography. J. Chromatogr. B 849: 53–68.

    Article  CAS  Google Scholar 

  13. Shugar, D. (1952) The measurement of lysozyme activity and the ultraviolet inactivation of lysozyme. Biochem. Biophys. Acta 8: 302–309.

    Article  CAS  Google Scholar 

  14. Hamada, H. and K. Shiraki (2007) L-argininamide improves the refolding more effectively than L-arginine. J. Biotechnol. 130: 153–160.

    Article  CAS  Google Scholar 

  15. Lin, F. Y., W. Y. Chen, and M. T. W. Hearn (2001) Microcalorimetric studies on the interaction mechanism between proteins and hydrophobic solid surfaces in hydrophobic interaction chromatography: effects of salts, hydrophobicity of the serbent, and structure of the protein. Anal. Chem. 73: 3875–3883.

    Article  CAS  Google Scholar 

  16. Geng, X. D. and X. Q. Chang (1992) High-performance hydrophobic interaction chromatography as a tool for protein refolding. J. Chromatogr. A. 599: 185–194.

    Article  CAS  Google Scholar 

  17. Lilli, W., C. Z. Wang, and X. D. Geng (2006) Expression, renaturation, and simultaneous purification of recombinant human stem cell factor in Escherichia coli. Biotechnol. Lett. 28: 993–997.

    Article  Google Scholar 

  18. Dan, W. U., C. Z Wang, and X. D. Geng (2007) Effect of mobile phase on the mass recovery of rhIFN-γ in protein folding liquid chromatography. Chinese J. Chromatogr. 25: 197–202.

    Article  Google Scholar 

  19. Wang, F., Y. Liu, J. Li, G. Ma, and Z. Su (2006) On-column refolding of consensus interferon at high concentration with guanidine-hydrochloride and polyethylene glycol gradients. J. Chromatogr. A 1115: 72–80.

    Article  CAS  Google Scholar 

  20. Wang, S. S.-S., Y.-T. Hung, P. Wang, and J. W. Wu (2007) The formation of amyloid fibril-like hen egg-white lysozyme species induced by temperature and urea concentration-dependent denaturation. Kor. J. Chem. Eng. 24: 787–795.

    Article  Google Scholar 

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Authors and Affiliations

  1. Department of Biological Engineering, Inha University, Incheon, 402-751, Korea

    Sung-Mi Hwang, Hye-Jin Kang, Sang-Woo Bae & Yoon-Mo Koo

  2. ERC for Advanced Bioseparation Technology, Inha University, Incheon, 402-751, Korea

    Woo-Jin Chang & Yoon-Mo Koo

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  1. Sung-Mi Hwang
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  2. Hye-Jin Kang
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  3. Sang-Woo Bae
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  4. Woo-Jin Chang
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Correspondence to Woo-Jin Chang.

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Hwang, SM., Kang, HJ., Bae, SW. et al. Refolding of lysozyme in hydrophobic interaction chromatography: Effects of hydrophobicity of adsorbent and salt concentration in mobile phase. Biotechnol Bioproc E 15, 213–219 (2010). https://doi.org/10.1007/s12257-009-0216-7

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  • DOI: https://doi.org/10.1007/s12257-009-0216-7

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