Biotechnology and Bioprocess Engineering

, Volume 24, Issue 4, pp 658–665 | Cite as

Refolding with Simultaneous Purification of Recombinant Core Streptavidin Using Single-step High-performance Hydrophobic Interaction Chromatography

  • Siyao Wang
  • Yuejuan Zhang
  • Dong Gao
  • Jing Zi
  • Wenpeng Wang
  • Nianzhe Zhang
  • Yi WanEmail author
  • Lili WangEmail author
Research Paper Bioprocess Engineering


Streptavidin has applied to many areas including detection, purification, labeling, crosslinking and immobilization resulting in a high demand on its production. In this study, we report a method for preparation of recombinant core streptavidin (cSAV) protein using highperformance hydrophobic interaction chromatography (HPHIC). Firstly the cSAV was successfully cloned and expressed in Escherichia coli as inclusion bodies. A bifunctional stationary phase mainly working as HIC mode accompanied by weak anion exchange chromatography (WAX) was prepared using β-phenylethylamine (PEA) as a ligand. The denatured cSAV was then refolded and simultaneously purified by PEA hydrophobic interaction chromatography (PEA-HIC). The mass recovery and purity of cSAV by single-step were 30.2% and 98%, respectively. The bioactivity was determined to be 13.2 U/mg by biotin binding capacity assay. This method provides a new possibility for fast separation with simultaneous renaturation of cSAV.


core streptavidin high-performance hydrophobic interaction chromatography β-phenylethylamine inclusion body 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.



This work was supported by the Science Foundation of Shaanxi Academy of Sciences (No. 2014K-16), and the Science and Technology Research Project of Shaanxi Province Academy of Sciences (No. 2018nk-01), and the Foundation of Key Laboratory of Modern Separation Science in Shaanxi Province (2010JS104; 16JS115), and the Foundation of Science and Technology in Shaanxi Province (No. 2013SZS18-Z01).


  1. 1.
    Bayer, E. A. and M. Wilchek (1990) Application of avidin-biotin technology to affinity-based separations. J. Chromatogr. 510: 3–11.CrossRefGoogle Scholar
  2. 2.
    Green, N. M. (1990) Avidin and streptavidin. Methods Enzymol. 184: 51–67.CrossRefGoogle Scholar
  3. 3.
    Sano, T., M. W. Pandori, X. Chen, C. L. Smith, and C. R. Cantor (1995) Recombinant core streptavidins. A minimum-sized core streptavidin has enhanced structural stability and higher accessibility to biotinylated macromolecules. J. Biol. Chem. 270: 28204–28209.CrossRefGoogle Scholar
  4. 4.
    Gonzalez, M., C. E. Argarana, and G. D. Fidelio (1999) Extremely high thermal stability of streptavidin and avidin upon biotin binding. Biomol. Eng. 16: 67–72.CrossRefGoogle Scholar
  5. 5.
    Dundas, C. M., D. Demonte, and S. Park (2013) Streptavidin-biotin technology: improvements and innovations in chemical and biological applications. Appl. Microbiol. Biotechnol. 97: 9343–9353.CrossRefGoogle Scholar
  6. 6.
    Laitinen, O. H., V. P. Hytönen, H. R. Nordlund, and M. S. Kulomaa (2006) Genetically engineered avidins and streptavidins. Cell Mol. Life Sci. 63: 2992–3017.CrossRefGoogle Scholar
  7. 7.
    Demonte, D., C. M. Dundas, and S. Park (2014) Expression and purification of soluble monomeric streptavidin in Escherichia coli. Appl. Microbiol. Biotechnol. 98: 6285–6295.CrossRefGoogle Scholar
  8. 8.
    Bayer, E. A., H. Ben-Hur, G. Gitlin, and M. Wilchek (1986) An improved method for the single-step purification of streptavidin. J. Biochem. Biophys. Methods. 13: 103–112.CrossRefGoogle Scholar
  9. 9.
    Pahler, A., W. A. Hendrickson, M. A. Kolks, C. E. Argarana, and C. R. Cantor (1987) Characterization and crystallization of core streptavidin. J. Biol. Chem. 262: 13933–13937.PubMedGoogle Scholar
  10. 10.
    Thompson, L. D. and P. C. Weber (1993) Construction and expression of a synthetic streptavidin-encoding gene in Escherichia coli. Gene. 136: 243–246.CrossRefGoogle Scholar
  11. 11.
    Karp, M., C. Lindqvist, R. Nissinen, S. Wahlbeck, K. Akerman, and C. Oker-Blom (1996) Identification of biotinylated molecules using a baculovirus-expressed luciferase-streptavidin fusion protein. Biotechniques. 20: 452–456, 458–459.CrossRefGoogle Scholar
  12. 12.
    Gallizia, A., C. de Lalla, E. Nardone, P. Santambrogio, A. Brandazza, A. Sidoli, and P. Arosio (1998) Production of a soluble and functional recombinant streptavidin in Escherichia coli. Protein Exp. Purif. 14: 192–196.CrossRefGoogle Scholar
  13. 13.
    Casteluber, M. C., L. M. Damasceno, W. B. da Silveira, R. H. Diniz, F. J. Passos, and F. M. Passos (2012) Cloning and expression of a functional core streptavidin in Pichia pastoris: strategies to increase yield. Biotechnol. Prog. 28: 1419–1425.CrossRefGoogle Scholar
  14. 14.
    Wetzel, D., J. M. Muller, E. Flaschel, K. Friehs, and J. M. Risse (2016) Fed-batch production and secretion of streptavidin by Hansenula polymorpha: Evaluation of genetic factors and bioprocess development. J. Biotechnol. 225: 3–9.CrossRefGoogle Scholar
  15. 15.
    Choi, J. H., K. C. Keum, and S. Y. Lee (2006) Production of recombinant proteins by high cell density culture of Escherichia coli. Chem. Eng. Sci. 61: 876–885.CrossRefGoogle Scholar
  16. 16.
    Chua, L. H., S. C. Tan, and M. W. O. Liew (2018) Process intensification of core streptavidin production through high-cell-density cultivation of recombinant E. coli and a temperature-based refolding method. J. Biotechnol. 276–277: 34–41.CrossRefGoogle Scholar
  17. 17.
    Chaiet, L. and F. J. Wolf (1964) The properties of streptavidin, a biotin-binding protein produced by streptomycetes. Arch. Biochem. Biophys. 106: 1–5.CrossRefGoogle Scholar
  18. 18.
    Rosano, G. L. and E. A. Ceccarelli (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol. 5: 172-.PubMedPubMedCentralGoogle Scholar
  19. 19.
    Yuan, J., H. Zhou, Y. Yang, W. Li, Y. Wan, and L. Wang (2015) Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein-folding liquidchromatography columns. Biomed. Chromatogr. 29: 777–782.CrossRefGoogle Scholar
  20. 20.
    Sano, T. and C. R. Cantor (1990) Expression of a cloned streptavidin gene in Escherichia coli. Proc. Natl. Acad. Sci. USA. 87: 142–146.CrossRefGoogle Scholar
  21. 21.
    Clark, E. D. (2001) Protein refolding for industrial processes. Curr. Opin. Biotechnol. 12: 202–207.CrossRefGoogle Scholar
  22. 22.
    Chen, Y. and S. S. Leong (2009) Adsorptive refolding of a highly disulfide-bonded inclusion body protein using anion-exchange chromatography. J. Chromatogr. A. 1216: 4877–4886.CrossRefGoogle Scholar
  23. 23.
    Wang, L. and X. Geng (2014) Protein renaturation with simultaneous purification by protein folding liquid chromatography: recent developments. Amino Acids. 46: 153–165.CrossRefGoogle Scholar
  24. 24.
    Geng, X. and X. Chang (1992) High-performance hydrophobic interaction chromatography as a tool for protein refolding. J. Chromatogr. A. 599: 185–194.CrossRefGoogle Scholar
  25. 25.
    Chong, F. C., W. S. Tan, D. R. A. Biak, T. C. Ling, and B. T. Tey (2010) A preparative hydrophobic interaction chromatography for purification of recombinant nucleocapsid protein of Nipah virus from clarified Escherichia coli homogenate. Sep. Purif. Technol. 71: 97–101.CrossRefGoogle Scholar
  26. 26.
    Mahn, A., M. E. Lienqueo, and J. A. Asenjo (2004) Effect of surface hydrophobicity distribution on retention of ribonucleases in hydrophobic interaction chromatography. J. Chromatogr. A. 1043: 47–55.CrossRefGoogle Scholar
  27. 27.
    Ueberbacher, R., E. Haimer, R. Hahn, and A. Jungbauer (2008) Hydrophobic interaction chromatography of proteins V. Quantitative assessment of conformational changes. J. Chromatogr. A. 1198–1199: 154–163.CrossRefGoogle Scholar
  28. 28.
    Zhao, K., F. Yang, H. Xia, F. Wang, Q. Song, and Q. Bai (2015) Preparation of a weak anion exchange/hydrophobic interaction dual-function mixed-mode chromatography stationary phase for protein separation using click chemistry. J. Sep. Sci. 38: 703–710.CrossRefGoogle Scholar
  29. 29.
    Yang, Y., Q. Qu, W. Li, J. Yuan, Y. Ren, and L. Wang (2016) Preparation of a silica-based high-performance hydrophobic interaction chromatography stationary phase for protein separation and renaturation. J. Sep. Sci. 39: 2481–2490.CrossRefGoogle Scholar
  30. 30.
    Wang, C., X. Geng, D. Wang, and B. Tian (2004) Purification of recombinant bovine normal prion protein PrP(104-242) by HPHIC. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 806: 185–190.CrossRefGoogle Scholar
  31. 31.
    Gao, D., F. C. Tan, W. P. Wang, and L. L. Wang (2013) Resolution enhancement in hydrophobic interaction chromatography via electrostatic interactions. Chin. Chem. Lett. 24: 419–421.CrossRefGoogle Scholar
  32. 32.
    Regnier, F. E. and R. Noel (1976) Glycerolpropylsilane bonded phases in the steric exclusion chromatography of biological macromolecules. J. Chromatogr. Sci. 14: 316–320.CrossRefGoogle Scholar
  33. 33.
    Green, N. M. (1970) Spectrophotometric determination of avidin and biotin. Meth. Enzymol. 18: 418–424.CrossRefGoogle Scholar
  34. 34.
    Hamada, H., T. Arakawa, and K. Shiraki (2009) Effect of additives on protein aggregation. Curr. Pharm. Biotechnol. 10: 400–407.CrossRefGoogle Scholar
  35. 35.
    Trivedi, V. D., B. Raman, C. M. Rao, and T. Ramakrishna (1997) Co-refolding denatured-reduced hen egg white lysozyme with acidic and basic proteins. FEBS Lett. 418: 363–366.CrossRefGoogle Scholar
  36. 36.
    Wang, G. Z., X. Y. Dong, and Y. Sun (2011) Ion-exchange resins greatly facilitate refolding of like-charged proteins at high concentrations. Biotechnol. Bioeng. 108: 1068–1077.CrossRefGoogle Scholar
  37. 37.
    Liu, H., X. Dong, and Y. Sun (2016) Grafting iminodiacetic acid on silica nanoparticles for facilitated refolding of like-charged protein and its metal-chelate affinity purification. J. Chromatogr. A. 1429: 277–283.CrossRefGoogle Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer 2019

Authors and Affiliations

  • Siyao Wang
    • 2
  • Yuejuan Zhang
    • 1
    • 3
  • Dong Gao
    • 2
  • Jing Zi
    • 1
    • 3
  • Wenpeng Wang
    • 2
  • Nianzhe Zhang
    • 2
  • Yi Wan
    • 1
    • 3
    Email author
  • Lili Wang
    • 2
    Email author
  1. 1.Microbiology Institute of ShaanxiXi’anChina
  2. 2.Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Shaanxi Key Laboratory of Modern Separation Science, Institute of Modern Separation ScienceNorthwest UniversityXi’anChina
  3. 3.Engineering Center of Qinling Mountains Natural ProductsShaanxi Academy of SciencesXi’anChina

Personalised recommendations