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Heterologous expression of the msp2 gene from Marasmius scorodonius

Kateryna Zelena1, Holger Zorn, Manfred Nimtz

  • 1Institut für Lebensmittelchemie, Leibniz Universität Hannover, Callinstrasse 5, Hannover, Germany.

Archives of Microbiology
|February 28, 2009
PubMed

Insights

Researchers successfully expressed the Msp2 peroxidase gene from Marasmius scorodonius in E. coli, achieving high yields of the recombinant protein. Optimized refolding conditions yielded active Msp2 peroxidase enzyme.

Area of Science:

  • Biotechnology
  • Molecular Biology
  • Enzymology

Background:

  • The msp2 gene encodes an extracellular peroxidase from the edible mushroom Marasmius scorodonius.
  • Heterologous expression is crucial for studying and potentially utilizing fungal enzymes.

Purpose of the Study:

  • To achieve heterologous expression of the Marasmius scorodonius Msp2 peroxidase in Escherichia coli.
  • To investigate methods for producing and refolding the recombinant Msp2 protein.

Main Methods:

  • Cloning the msp2 cDNA into the pBAD III expression plasmid for E. coli expression.
  • Utilizing PCR to generate constructs for native or signal peptide-containing protein expression.
  • Employing immobilized metal affinity chromatography for purification of a His-tagged fusion protein.
  • Investigating refolding parameters including glutathione-mediated oxidation, urea, Ca(2+), and hemin.

Main Results:

  • High-yield production of recombinant Msp2 protein in E. coli, primarily as insoluble inclusion bodies.
  • Successful refolding of the Msp2 protein into an active enzyme using optimized conditions.
  • Generation of a fusion protein with six His-residues for enhanced detection and purification.

Conclusions:

  • Escherichia coli is a suitable host for high-yield expression of Marasmius scorodonius Msp2 peroxidase.
  • Optimized refolding protocols are essential for obtaining active recombinant Msp2 peroxidase from inclusion bodies.

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