Heterologous expression of the msp2 gene from Marasmius scorodonius.
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, Ca(2+), and hemin.
Zelena
Kateryna
K
Institut für Lebensmittelchemie, Leibniz Universität Hannover, Callinstrasse 5, Hannover, Germany.
Zorn
Holger
H
Nimtz
Manfred
M
Berger
Ralf Günter
RG
D002118:Calcium / Q000494:pharmacology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D005978:Glutathione / Q000494:pharmacology; D006427:Hemin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D055433:Marasmius / Q000235:genetics*; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D003067:Coenzymes; D018076:DNA, Complementary; D005656:Fungal Proteins; D016877:Oxidants; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D006427:Hemin; D009195:Peroxidase; D005978:Glutathione; D002118:Calcium