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Studies on the Refolding Process of Recombinant Horseradish Peroxidase

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Abstract

Horseradish peroxidase (HRP) is an important heme-containing glyco-enzyme that has been used in many biotechnological fields. Valuable proteins like HRP can be obtained in sufficient amounts using Escherichia coli as an expression system. However, frequently, the expression of recombinant enzyme results in inclusion bodies, and the refolding yield is generally low for proteins such as plant peroxidases. In this study, a recombinant HRP was cloned and expressed in the form of inclusion bodies. Initially, the influence of few additives on HRP refolding was assessed by the one factor at a time method. Subsequently, factors with significant effects including glycerol, GSSG/DTT, and the enzyme concentration were selected for further optimization by means of the central composite design of response surface methodology (RSM). Under the obtained optimal condition, refolding increased about twofold. The refolding process was then monitored by the intrinsic fluorescence intensity under optimal conditions (0.35 mM GSSG, 0.044 mM DTT, 7 % glycerol, 1.7 M urea, and 2 mM CaCl2 in 20 mM Tris, pH 8.5) and the reconstitution of heme to the refolded peroxidase was detected by the Soret absorbance. Additionally, samples under unfolding and refolding conditions were analyzed by Zetasizer to determine size distribution in different media.

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References

  1. Levin, G., Mendive, F., Hector, M. T., Osvaldo, C., & Mara, V. M. (2005). Genetically engineered horseradish peroxidase for facilitated purification from baculovirus cultures by cation-exchange chromatography. Journal of Biotechnology, 118, 363–369.

    Article  CAS  Google Scholar 

  2. Gray, J. S., Yun Yang, B., & Montgomery, R. (1998). Heterogeneity of glycans at each N-glycosylation site of horseradish peroxidase. Carbohydrate Research, 311, 61–69.

    Article  CAS  Google Scholar 

  3. Welinder, K. G. (1979). Amino acid sequence studies of horseradish peroxidase: amino and carboxyl termini, cyanogen bromide and tryptic fragments, the complete sequence, and some structural characteristics of horseradish peroxidase C. European Journal of Biochemistry, 96, 483–502.

    Article  CAS  Google Scholar 

  4. Smith, A. T., Santama, N., Dacey, S., Edwards, M., Bray, R. C., Thorneley, R. N. F., et al. (1990). Expression of a synthetic gene for horseradish peroxidase C in Escherichia coli and folding and activation of the recombinant enzyme with Ca2+ and heme. Journal of Biological Chemistry, 265, 13335–13343.

    CAS  Google Scholar 

  5. Dunford, H. B. (1991). Peroxidases. In J. Everse, K. E. Everse, & M. B. Grisham (Eds.), Chemistry and biology (pp. 1–24). Boca Raton: CRC Press.

  6. Pérez-Boada, M., Doyle, W. A., Ruiz-Dueñas, F. J., Martínez, M. J., Martínez, A. T., & Smith, A. T. (2002). Expression of Pleurotus eryngii versatile peroxidase in Escherichia coli and optimisation of in vitro folding. Enzyme and Microbial Technology, 30, 518–524.

    Article  Google Scholar 

  7. Vallejo, L. F., & Rinas, U. (2004). Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins. Microbial Cell Factories, 3, 11–22.

    Article  Google Scholar 

  8. Whitwam, R., & Tien, M. (1996). Heterologous expression and reconstitution of fungal Mn peroxidase. Archives of Biochemistry and Biophysics, 333, 439–446.

    Article  CAS  Google Scholar 

  9. Doyle, W. A., & Smith, A. T. (1996). Expression of lignin peroxidase H8 in Echerichia coli: folding and activation of the recombinant enzyme with Ca 2+ and haem. Biochemical Journal, 315, 15–19.

    CAS  Google Scholar 

  10. Grigorenko, V., Chubar, T., Kapeliuch, Y., Borchers, T., Spener, F., & Egorov, A. (1999). New approaches for functional expression of recombinant horseradish peroxidase C in Escherichia coli. Biocatalysis and Biotransformation, 17, 359–379.

    Article  CAS  Google Scholar 

  11. 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 

  12. Wilson, C. M. (1983). Staining of proteins on gels: comparison of dyes and procedures. Methods in Enzymology, 91, 236–247.

    Article  CAS  Google Scholar 

  13. 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 

  14. Ghasempur, S., Torabi, S. F., Ranaei-Siadat, S. O., Jalali-Heravi, M., Ghaemi, N., & Khajeh, K. (2007). Optimization of peroxidase-catalyzed oxidative coupling process for phenol removal from wastewater using response surface methodology. Environmental Science and Technology, 41, 7073–7079.

    Article  CAS  Google Scholar 

  15. Wagner, M., & Nicell, J. A. (2002). Detoxification of phenolic solutions with horseradish peroxidase and hydrogen peroxide. Water Reseach, 36, 4041–4052.

    Article  CAS  Google Scholar 

  16. Pappa, H. S., & Cass, A. E. G. (1993). A step towards understanding the folding mechanism of horseradish peroxidase. European Journal of Biochemistry, 212, 227–235.

    Article  CAS  Google Scholar 

  17. Hushpulian, D. M., Savitski, P. A., Rojkoval, A. M., Chubar, T. A., Fechina, V. A., Sakharov, I. Y., et al. (2003). Expression and refolding of tobacco anionic peroxidase from E. coli inclusion bodies. Biochemistry, 68, 1480–1487.

    Google Scholar 

  18. Rudolph, R., & Lilie, H. (1996). In vitro folding of inclusion body proteins. FASEB Journal, 10, 49–56.

    CAS  Google Scholar 

  19. Hevehan, D. L., & Clark, E. D. B. (1997). Oxidative renaturation of lysozyme at high concentration. Biotechnology and Bioengineering, 54, 221–230.

    Article  CAS  Google Scholar 

  20. Kiefhaber, T., Rudolph, R., Kohler, H. H., & Buchner, J. (1991). Protein aggregation in vivo: a quantitative model of the kinetic competition between folding and aggregation. Biotechnology, 9, 825–829.

    Article  CAS  Google Scholar 

  21. Dong, X. Y., Huang, Y., & Sun, Y. (2004). Refolding kinetics of denatured-reduced lysozyme in the presence of folding aids. Journal of Biotechnology, 114, 135–142.

    Article  CAS  Google Scholar 

  22. Brunet, J. E., Gonzalez, G. A., & Sotomayor, C. P. (1983). Intramolecular tryptophan heme energy transfer in horseradish peroxidase. Photochemistry and Photobiology, 38, 253–254.

    Article  CAS  Google Scholar 

  23. Das, T. K., & Mazumdar, S. (1995). pH-induced conformational perturbation in horseradish peroxidase. European Journal of Biochemistry, 227, 823–828.

    Article  CAS  Google Scholar 

  24. Shannon, L., Kay, E., & Lew, J. (1966). Peroxidase isozymes from horseradish roots, Isolation and physical properties. Journal of Biological Chemistry, 241, 2166–2172.

    CAS  Google Scholar 

  25. Thongsook, T., Whitaker, J. R., Smith, G. M., & Barret, D. M. (2007). Reactivation of Broccoli peroxidase: Structural changes of partially denatured isoenzymes. Journal of Agriculture and Food Chemistry, 55, 1009–1018.

    Article  CAS  Google Scholar 

  26. Rahimpour, F., Mamoa, G., Feyzi, F., Maghsoudi, S., & Hatti-Kaul, R. (2007). Optimizing refolding and recovery of active recombinant Bacillus halodurans xylanase in polymer–salt aqueous two-phase system using surface response analysis. Journal of Chromatography, 1141, 32–40.

    Article  CAS  Google Scholar 

  27. Ahn, J. H., Lee, Y. P., & Rhee, J. S. (1997). Investigation of refolding condition for Pseudomonas fluorescens lipase by response surface methodology. Journal of Biotechnology, 54, 151–160.

    Article  CAS  Google Scholar 

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Acknowledgments

We would like to thank the research council of Tarbiat Modares University and the University of Tehran for the financial support of this investigation.

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Authors and Affiliations

  1. Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran

    Sedigheh Asad & Nasser Ghaemi

  2. Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran

    Bahareh Dabirmanesh, Seyed Masoud Etezad & Khosro Khajeh

Authors
  1. Sedigheh Asad
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  2. Bahareh Dabirmanesh
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  3. Nasser Ghaemi
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  4. Seyed Masoud Etezad
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  5. Khosro Khajeh
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Correspondence to Khosro Khajeh.

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Asad, S., Dabirmanesh, B., Ghaemi, N. et al. Studies on the Refolding Process of Recombinant Horseradish Peroxidase. Mol Biotechnol 54, 484–492 (2013). https://doi.org/10.1007/s12033-012-9588-6

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