Biochemistry (Moscow)

, Volume 80, Issue 4, pp 408–416 | Cite as

Recombinant horseradish peroxidase: Production and analytical applications

  • V. G. GrigorenkoEmail author
  • I. P. Andreeva
  • M. Yu. Rubtsova
  • A. M. Egorov


Horseradish peroxidase is a key enzyme in bio- and immunochemical analysis. New approaches in functional expression of the peroxidase gene in E. coli cells and the subsequent refolding of the resulting protein yield a recombinant enzyme that is comparable in its spectral and catalytic characteristics to the native plant peroxidase. Genetic engineering approaches allow production of recombinant peroxidase conjugates with both protein antigens and Fab antibody fragments. The present article reviews the use of recombinant horseradish peroxidase as the marker enzyme in ELISA procedures as well as in amperometric sensors based on direct electron transfer.

Key words

horseradish peroxidase recombinant conjugate amperometric biosensor 



fatty acid-binding protein


horseradish peroxidase




recombinant horseradish peroxidase


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  1. 1.
    Chance, B. (1949) The enzyme-substrate compounds of horseradish peroxidase and peroxides; kinetics of formation and decomposition of the primary and secondary complexes, Arch. Biochem., 22, 224–252.PubMedGoogle Scholar
  2. 2.
    Dunford, H. B., and Stillman, J. S. (1976) On the function and mechanism of action of peroxidases, Coord. Chem. Rev., 19, 187–251.CrossRefGoogle Scholar
  3. 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, Eur. J. Biochem., 96, 483–502.CrossRefPubMedGoogle Scholar
  4. 4.
    Ferapontova, E. E., Grigorenko, V. G., Egorov, A. M., Borchers, T., Ruzgas, T., and Gorton, L. (2001) Direct electron transfer in the system gold electrode-recombinant horseradish peroxidases, J. Electroanal. Chem., 509, 19–26.CrossRefGoogle Scholar
  5. 5.
    Ferapontova, E. E., Grigorenko, V. G., Egorov, A. M., Borchers, T., Ruzgas, T., and Gorton, L. (2001) Mediatorless biosensor for H2O2 based on recombinant forms of horseradish peroxidase directly adsorbed on polycrystalline gold, Biosens. Bioelectron., 16, 147–157.CrossRefPubMedGoogle Scholar
  6. 6.
    Presnova, G., Grigorenko, V., Egorov, A., Ruzdas, T., Lindgren, A., Gorton, L., and Borchers, T. (2000) Direct heterogeneous electron transfer of recombinant horseradish peroxidases on gold, Faraday Discuss, 116, 281–289.CrossRefPubMedGoogle Scholar
  7. 7.
    Rubtsova, M. Y., Kovba, G. V., and Egorov, A. M. (1998) Chemiluminescent biosensors based on porous supports with immobilized peroxidase, Biosens. Bioelectron., 13, 75–85.CrossRefPubMedGoogle Scholar
  8. 8.
    Rubtsova, M. Yu., Ulyashova, M. M., Edelstein, M. V., and Egorov, A. M. (2010) Oligonucleotide microarrays with horseradish peroxidase-based detection for the identification of extended-spectrum β-lactamases, Biosens. Bioelectron., 26, 1252–1260.CrossRefPubMedGoogle Scholar
  9. 9.
    Hartmann, C., and Ortiz de Montellano, P. R. (1992) Baculovirus expression and characterization of catalytically active horseradish peroxidase, Arch. Biochem. Biophys., 297, 61–72.CrossRefPubMedGoogle Scholar
  10. 10.
    Vlamis-Gardikas, A., Smith, A. T., Clements, J. M., and Burke, J. F. (1992) Expression of active horseradish peroxidase in Saccharomyces cerevisiae, Biochem. Soc. Trans., 20, 111S.PubMedGoogle Scholar
  11. 11.
    Smith, A. T., Santana, N., Dacey, S., Edwards, M., Bray, R. C., Thorneley, R. N. F., and Burke, J. F. (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, J. Biol. Chem., 265, 13335–13343.PubMedGoogle Scholar
  12. 12.
    Egorov, A. M., Gazaryan, I. G., Kim, B. B., Doseeva, V. V., Kapeliuch, J. L., Veryovkin, A. N., and Fechina, V. A. (1994) Horseradish peroxidase isozyme C. A comparative study of native and recombinant enzyme produced by E. coli transformants, Ann. N. Y. Acad. Sci., 721, 73–82.CrossRefPubMedGoogle Scholar
  13. 13.
    Gajhede, M., Schuller, D. J., Henriksen, A., Smith, A. T., and Poulos, T. L. (1997) Crystal structure of horseradish peroxidase C at 2.15 Å resolution, Nature Struct. Biol., 4, 1032–1038.CrossRefPubMedGoogle Scholar
  14. 14.
    Freedman, R. B. (1995) The formation of protein disulphide bonds, Curr. Opin. Struct. Biol., 5, 85–91.CrossRefPubMedGoogle Scholar
  15. 15.
    Grigorenko, V., Chubar, T., Kapeliuch, Yu., Borchers, T., Spener, F., and Egorov, A. (1999) New approaches for functional expression of recombinant horseradish peroxidase C in Escherichia coli, Biocatal. Biotransform., 17, 359–397.CrossRefGoogle Scholar
  16. 16.
    Lindbladh, C., Mosbach, K., and Bulow, L. (1993) Use of genetically prepared enzyme conjugates in enzyme immunoassay, Trends Biochem. Sci., 18, 279–283.CrossRefPubMedGoogle Scholar
  17. 17.
    Porstman, T., and Kiessig, S. T. (1992) Enzyme immunoassay techniques. An overview, J. Immunol. Methods, 150, 5–21.CrossRefGoogle Scholar
  18. 18.
    Offensperger, W., Wahl, S., Neurath, A. R., Price, P., Strick, N., Kent, S. B., Christman, J. K., and Acs, G. (1985) Expression in Escherichia coli of a cloned DNA sequence encoding the pre-S2 region of hepatitus B virus, Proc. Natl. Acad. Sci. USA, 82, 7540–7544.CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    Peterhans, A., Mecklenburg, M., Meussdoerffer, F., and Mosbach, K. (1987) A simple competitive enzyme-linked immunosorbent assay using antigen-β-galactosidase fusions, Anal. Biochem., 163, 470–475.CrossRefPubMedGoogle Scholar
  20. 20.
    Markaryan, A. N., Mashko, S. V., Kukel, L. V., Lapidus, A. L., Bach, A. N., and Egorov, A. M. (1991) Construction of expression vectors for gene fusions on the model of beta-galactosidase-human fibroblast beta-interferon for the purpose of immunoenzyme assay, Ann. N. Y. Acad. Sci., 646, 125–135.CrossRefPubMedGoogle Scholar
  21. 21.
    Lindbladh, C., Persson, M., Bulow, L., Stahl, S., and Mosbach, K. (1987) The design of a simple competitive ELISA using human proinsulin-alkaline phosphatase conjugates prepared by gene fusion, Biochem. Biophys. Res. Commun., 149, 607–614.CrossRefPubMedGoogle Scholar
  22. 22.
    Gillet, D., Ezan, E., Ducancel, F., Gaillard, C., Ardouin, T., Istin, M., Menez, A., Boulain, J.-C., and Grogent, J.-M. (1993) Enzyme immunoassay using a rat prolactin-alkaline phosphatase recombinant tracer, Anal. Chem., 65, 1779–1784.CrossRefPubMedGoogle Scholar
  23. 23.
    Ezan, E., Ducancel, F., Gillet, D., Drevet, P., Menez, A., Grognet, J. M., and Boulain, J. C. (1994) Recombinant technology in the preparation of immunogen and enzymatic tracer. Application to the development of an enzyme immunoassay for rat prolactin, J. Immunol. Methods, 169, 205–211.CrossRefPubMedGoogle Scholar
  24. 24.
    Ramanathan, S., Lewis, J. C., Kindy, M. S., and Daunert, S. (1998) Heterogeneous bioluminescence binding assay for an octapeptide using recombinant aequorin, Anal. Chim. Acta, 369, 181–188.CrossRefGoogle Scholar
  25. 25.
    Lewis, J. C., and Daunert, S. (1999) Dual detection of peptides in a fluorescence binding assay by employing genetically fused GFP and BFP mutants, Anal. Chem., 71, 4321–4327.CrossRefPubMedGoogle Scholar
  26. 26.
    Baneyx, F., and Georgiu, G. (1989) Expression, purification and enzymatic characterization of a protein A-β-lactamase hybrid protein, Enzyme Microbial Technol., 11, 559–567.CrossRefGoogle Scholar
  27. 27.
    Lindbladh, C., Mosbach, K., and Bulow, L. (1991) Preparation of a genetically fused protein A/luciferase conjugate for use in bioluminescent immunoassays, J. Immunol. Methods, 137, 199–207.CrossRefPubMedGoogle Scholar
  28. 28.
    Wittkowski, A., Daunert, S., Kindy, M. S., and Bachas, L. G. (1993) Enzyme-linked immunosorbent assay for an octapeptide based on a genetically engineered fusion protein, Anal. Chem., 65, 1147–1151.CrossRefGoogle Scholar
  29. 29.
    Schreiber, A., Specht, B., Pelsers, M. M. A. L., Glatz, J. F. C., Borchers, T., and Spener, F. (1998) Recombinant human heart-type fatty acid-binding protein as standard in immunochemical assays, Clin. Chem. Lab. Med., 36, 283–288.CrossRefPubMedGoogle Scholar
  30. 30.
    Rau, D., Kramer, K., and Hock, B. (2002) Single-chain Fv antibody-alkaline phosphatase fusion proteins produced by one-step cloning as rapid detection tools for ELISA, J. Immunoassay Immunochem., 23, 129–143.CrossRefPubMedGoogle Scholar
  31. 31.
    Tachibana, H., Takekoshi, M., Cheng, X. J., Nakata, Y., Takeuchi, T., and Ihara, S. (2004) Bacterial expression of a human monoclonal antibody-alkaline phosphatase conjugate specific for Entamoeba histolytica, Clin. Diagn. Lab. Immunol., 11, 216–218.PubMedCentralPubMedGoogle Scholar
  32. 32.
    Mousli, M., Turki, I., Kharmachi, H., Saadi, M., and Dellagi, K. (2007) Recombinant single-chain Fv antibody fragment-alkaline phosphatase conjugate: a novel in vitro tool to estimate rabies viral glycoprotein antigen in vaccine manufacture, J. Virol. Methods, 146, 246–256.CrossRefPubMedGoogle Scholar
  33. 33.
    Dong, J. X., Li, Z. F., Lei, H. T., Sun, Y. M., Ducancel, F., Xu, Z. L., Boulain, J. C., Yang, J. Y., Shen, Y. D., and Wang, H. (2012) Development of a single-chain variable fragment-alkaline phosphatase fusion protein and a sensitive direct competitive chemiluminescent enzyme immunoassay for detection of ractopamine in pork, Anal. Chim. Acta, 736, 85–91.CrossRefPubMedGoogle Scholar
  34. 34.
    Xu, Z. L., Dong, J. X., Wang, H., Li, Z. F., Beier, R. C., Jiang, Y. M., Lei, H. T., Shen, Y. D., Yang, J. Y., and Sun, Y. M. (2012) Production and characterization of a single-chain variable fragment linked alkaline phosphatase fusion protein for detection of O,O-diethyl organophosphorus pesticides in a one-step enzyme-linked immunosorbent assay, J. Agric. Food Chem., 60, 5076–5083.CrossRefPubMedGoogle Scholar
  35. 35.
    Patel, K. G., Ng, P. P., Kuo, C. C., Levy, S., Levy, R., and Swartz, J. R. (2009) Cell-free production of Gaussian princeps luciferase-antibody fragment bioconjugates for ex vivo detection of tumor cells, Biochem. Biophys. Res. Commun., 390, 971–976.CrossRefPubMedGoogle Scholar
  36. 36.
    Joosten, V., Roelofs, M. S., van den Dries, N., Goosen, T., Verrips, C. T., van den Hondel, C. A., and Lokman, B. C. (2005) Production of bifunctional proteins by Aspergillus awamori: llama variable heavy chain antibody fragment (V(HH)) R9 coupled to Arthromyces ramosus peroxidase (ARP), J. Biotechnol., 120, 347–359.CrossRefPubMedGoogle Scholar
  37. 37.
    Gillet, D., Ducancel, F., Pradel, E., Leonetti, M., Menez, A., and Boulain, J. C. (1992) Insertion of a disulfide-containing neurotoxin into E. coli alkaline phosphatase: the hybrid retains both biological activities, Protein Eng., 5, 273–278.CrossRefPubMedGoogle Scholar
  38. 38.
    Chanussot, C., Bellanger, L., Ligny-Lemaire, C., Seguin, P., Menez, A., and Boulain, J. C. (1996) Engineering of a recombinant colorimetric fusion protein for immunodiagnosis of insulin, J. Immunol. Methods, 197, 39–49.CrossRefPubMedGoogle Scholar
  39. 39.
    Kerschbaumer, R. J., Hirschl, S., Schwager, C., Ibl, M., and Himmler, G. (1996) pDAP2: a vector for construction of alkaline phosphatase fusion-proteins, Immunotechnology, 2, 145–150.CrossRefPubMedGoogle Scholar
  40. 40.
    Grigorenko, V., Andreeva, I., Borchers, T., Spener, F., and Egorov, A. (2001) A genetically engineered fusion protein with horseradish peroxidase as a marker enzyme for use in competitive immunoassays, Anal. Chem., 73, 1134–1139.CrossRefPubMedGoogle Scholar
  41. 41.
    Wodzig, K. W. H., Pelsers, M. M. A. L., van der Vusse, G. J., Roos, W., and Glatz, J. F. C. (1997) One-step enzyme-linked immunosorbent assay (ELISA) for plasma fatty acid-binding protein, Ann. Clin. Biochem., 34, 263–268.CrossRefPubMedGoogle Scholar
  42. 42.
    Nakane, P. K., and Kawaoi, A. (1974) Peroxidase-labeled antibody. A new method of conjugation, J. Histochem. Cytochem., 22, 1084–1091.CrossRefPubMedGoogle Scholar
  43. 43.
    Robin, S., Petrov, K., Dintinger, T., Kujumdzieva, A., Tellier, C., and Dion, M. (2003) Comparison of three microbial hosts for the expression of an active catalytic scFv, Mol. Immunol., 39, 729–738.CrossRefPubMedGoogle Scholar
  44. 44.
    Cupit, P. M., Whyte, J. A., Porter, A. J., Browne, M. J., Holmes, S. D., Harris, W. J., and Cunningham, C. (1999) Cloning and expression of single chain antibody fragments in Escherichia coli and Pichia pastoris, Lett. Appl. Microbiol., 29, 273–277.CrossRefPubMedGoogle Scholar
  45. 45.
    Morawski, B., Lin, Z., Cirino, P., Joo, H., Bandara G., and Arnold, F. H. (2000) Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris, Protein Eng., 13, 377–384.CrossRefPubMedGoogle Scholar
  46. 46.
    Pennell, C. A., and Eldin, P. (1998) In vitro production of recombinant antibody fragments in Pichia pastoris, Res. Immunol., 149, 599–603.CrossRefPubMedGoogle Scholar
  47. 47.
    Fischer, R., Drossard, J., Emans, N., Commandeur, U., and Hellwig, S. (1999) Towards molecular farming in the future: Pichia pastoris-based production of single-chain antibody fragments, Biotechnol. Appl. Biochem., 30, 112–117.Google Scholar
  48. 48.
    Freyre, F. M., Vazquez, J. E., Ayala, M., Canaan-Haden, L., Bell, H., Rodriguez, I., Gonzalez, A., Cintado, A., and Gavilondo, J. V. (2000) Very high expression of an anti-carcinoembryonic antigen single chain Fv antibody fragment in the yeast Pichia pastoris, J. Biotechnol., 76, 157–163.CrossRefPubMedGoogle Scholar
  49. 49.
    Takahashi, K., Yuuki, T., Takai, T., Ra, C., Okumura, K., Yokota, T., and Okumura, Y. (2000) Production of humanized Fab fragment against human high affinity IgE receptor in Pichia pastoris, Biosci. Biotechnol. Biochem., 64, 2138–2144.CrossRefPubMedGoogle Scholar
  50. 50.
    Andrade, E. V., Albuquerque, F. C., Moraes, L. M., Brigido, M. M., and Santos-Silva, M. A. (2000) Singlechain Fv with Fc fragment of the human IgG1 tag: construction, Pichia pastoris expression and antigen binding characterization, J. Biochem. (Tokyo), 128, 891–895.CrossRefGoogle Scholar
  51. 51.
    Luo, D., Geng, M., Schultes, B., Ma, J., Xu, D. Z., Hamza, N., Qi, W., Noujaim, A. A., and Madiyalakan, R. (1998) Expression of a fusion protein of scFv-biotin mimetic peptide for immunoassay, J. Biotechnol., 65, 225–228.CrossRefPubMedGoogle Scholar
  52. 52.
    Powers, D. B., Amersdorfer, P., Poul, M., Nielsen, U. B., Shalaby, M. R., Adams, G. P., Weiner, L. M., and Marks, J. D. (2001) Expression of single-chain Fv-Fc fusions in Pichia pastoris, J. Immunol. Methods, 251, 123–135.CrossRefPubMedGoogle Scholar
  53. 53.
    Hellwig, S., Emde, F., Raven, N. P., Henke, M., van der Logt, P., and Fischer, R. (2001) Analysis of single-chain antibody production in Pichia pastoris using on-line methanol control in fed-batch and mixed-feed fermentations, Biotechnol. Bioeng., 74, 344–352.CrossRefPubMedGoogle Scholar
  54. 54.
    Lange, S., Schmitt, J., and Schmid, R. D. (2001) High-yield expression of the recombinant, atrazine-specific Fab fragment K411B by the methylotrophic yeast Pichia pastoris, J. Immunol. Methods, 255, 103–114.CrossRefPubMedGoogle Scholar
  55. 55.
    Koliasnikov, O. V., Grigorenko, V. G., Egorov, A. M., Lange, S., and Schmid, R. D. (2011) Recombinant production of horseradish peroxidase conjugates with Fab anti-bodies in Pichia pastoris for analytical applications, Acta Naturae, 3, 85–92.PubMedCentralPubMedGoogle Scholar
  56. 56.
    Kramer, K., and Hock, B. (1996) Recombinant single-chain antibodies against s-triazines, Food Agric. Immunol., 8, 97–109.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2015

Authors and Affiliations

  • V. G. Grigorenko
    • 1
    Email author
  • I. P. Andreeva
    • 1
  • M. Yu. Rubtsova
    • 1
  • A. M. Egorov
    • 1
    • 2
  1. 1.Chemical FacultyLomonosov Moscow State UniversityMoscowRussia
  2. 2.Microbiology DepartmentRussian Medical Academy of Post-Graduate EducationMoscowRussia

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