Heterologous expression of the msp2 gene from Marasmius scorodonius

  • Kateryna Zelena
  • Holger Zorn
  • Manfred Nimtz
  • Ralf Günter BergerEmail author
Original Paper


For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea, Ca2+, and hemin.


Basidiomycete E. coli Heterologous expression In vitro refolding Peroxidase 



Support of the work by the BMBF cluster Biokatalyse2021 (FKZ0315172B) is gratefully acknowledged, as are helpful discussions with S. Lunkenbein.


  1. Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421PubMedCrossRefGoogle Scholar
  2. Conesa A, van de Velde F, van Rantwijk F, Sheldon RA, van den Hondel CAMJJ, Punt PJ (2001) Expression of the Caldariomyces fumago chloroperoxidase in Aspergillus niger and characterization of the recombinant enzyme. J Biol Chem 276:17635–17640PubMedCrossRefGoogle Scholar
  3. Conesa A, Punt PJ, van den Hondel CAMJJ (2002) Fungal peroxidases: molecular aspects and applications. J Biotechnol 93:143–158PubMedCrossRefGoogle Scholar
  4. de Jong EFP, de Vries JA, Field RP, der Zwan V, de Bont JAM (1992) Isolation and screening of basidiomycetes with the peroxidase activity. Mycol Res 96:1098–1104CrossRefGoogle Scholar
  5. Gu L, Lajoie C, Kelly C (2003) Expression of a Phanerochaete chrysosporium manganese peroxidase gene in the yeast Pichia pastoris. Biotechnol Prog 19:1403–1409PubMedCrossRefGoogle Scholar
  6. Guzman LM, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130PubMedGoogle Scholar
  7. Kim SJ, Shoda M (1999) Purification and characterisation of a novel peroxidase from Geotrichum candidum Dec 1 involved in decolorization of dyes. Appl Environ Microbiol 65:1029–1035PubMedGoogle Scholar
  8. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  9. Lee N, Francklyn C, Hamilton EP (1987) Arabinose-induced binding of AraC protein to araI2 activates the araBAD operon promoter. Proc Natl Acad Sci USA 84:8814–8818PubMedCrossRefGoogle Scholar
  10. Lokman BC, Joosten V, Hovenkamp J, Gouka RJ, Verrips CT, van den Hondel CAMJJ (2003) Efficient production of Arthromyces ramosus peroxidase by Aspergillus awamori. J Biotechnol 103:183–190PubMedCrossRefGoogle Scholar
  11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  12. Lucas M, Mertens V, Corbisier AM, Vanhulle S (2008) Synthetic dyes decolourisation by white-rot fungi: development of original microtitre plate method and screening. Enzyme Microb Technol 42:97–106CrossRefGoogle Scholar
  13. Nie G, Reading NS, Aust SD (1998) Expression of the lignin peroxidase h2 gene from Phanerochaete chrysosporium in Escherichia coli. Biochem Biophys Res Commun 249:146–150PubMedCrossRefGoogle Scholar
  14. Perez-Boada M, Doyle WA, Ruiz-Dueñas FJ, Martínez MJ, Martínez AT, Smith AT (2002) Expression of Pleurotus eryngii versatile peroxidase in Escherichia coli and optimisation of in vitro folding. Enzyme Microb Technol 30:518–524CrossRefGoogle Scholar
  15. Reddy CA, D’Souza TM (1994) Physiology and molecular biology of the lignin peroxidases of Phanerochaete chrysosporium. FEMS Microbiol Rev 13:137–152PubMedCrossRefGoogle Scholar
  16. Rudolph R, Lilie H (1996) In vitro folding of inclusion body proteins. FASEB J 10:49–56PubMedGoogle Scholar
  17. Ruiz-Dueñas FJ, Aguilar A, Martínez MJ, Zorn H, Martínez AT (2007) Gene cloning, heterologous expression, in vitro reconstitution and catalytic properties of a versatile peroxidase. Biocatal Biotransform 25:276–285CrossRefGoogle Scholar
  18. Sambrook J, Russell DW (2001) Molecular cloning A laboratory manual. Cold Spring Harbor, New YorkGoogle Scholar
  19. Scheibner M (2006) Identifizierung und Charakterisierung carotinoidabbauender Enzyme aus Basidiomyceten. Dissertation, Leibniz University HannoverGoogle Scholar
  20. Scheibner M, Hülsdau B, Zelena K, Nimtz M, de Boer L, Berger RG, Zorn H (2008) Novel peroxidases of Marasmius scorodonius degrade β-carotene. Appl Microbiol Biotechnol 77:1241–1250PubMedCrossRefGoogle Scholar
  21. Stewart P, Whitwam RE, Kersten PJ, Gullen D, Tien M (1996) Efficient expression of a Phanerochaete chrysosporium manganese peroxidase gene in Aspergillus oryzae. Appl Environ Microbiol 62:860–864PubMedGoogle Scholar
  22. Sugano Y, Nakano R, Sasaki K, Shoda M (2000) Efficient heterologous expression in Aspergillus oryzae of a unique dye-decolorising peroxidase, DyP, of Geotrichum candidum Dec 1. Appl Environ Microbiol 66:1754–1758PubMedCrossRefGoogle Scholar
  23. Thomas PE, Ryan D, Levin W (1976) An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. Anal Biochem 75:168–176PubMedCrossRefGoogle Scholar
  24. Wang H, Lu F, Sun Y, Du L (2004) Heterologous expression of lignin peroxidase of Phanerochaete chrysosporium in Pichia methanolica. Biotechnol Lett 26:1569–1573PubMedCrossRefGoogle Scholar
  25. Whitwam R, Tien M (1996) Heterologous expression and reconstitution of fungal Mn peroxidase. Arch Biochem Biophys 333:439–446PubMedCrossRefGoogle Scholar
  26. Yin J, Li G, Ren X, Herrler G (2007) Select what you need: a comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes. J Biotechnol 127:335–347PubMedCrossRefGoogle Scholar
  27. Zorn H, Langhoff S, Scheibner M, Berger RG (2003) Cleavage of β,β-carotene to flavour compounds by fungi. Appl Microbiol Biotechnol 62:331–336PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Kateryna Zelena
    • 1
  • Holger Zorn
    • 2
  • Manfred Nimtz
    • 3
  • Ralf Günter Berger
    • 1
    Email author
  1. 1.Institut für LebensmittelchemieLeibniz Universität HannoverHannoverGermany
  2. 2.Institute of Food Chemistry and Food BiotechnologyJustus-Liebig-University GießenGiessenGermany
  3. 3.Helmholtz-Zentrum für Infektionsforschung, Abteilung Biophysikalische AnalytikBraunschweigGermany

Personalised recommendations