Expression of fungal Mn peroxidase in E. coli and refolding to yield active enzyme.
The cDNA encoding Mn peroxidase isozyme H4 from Phanerochaete chrysosporium was expressed in Escherichia coli. The portion of the cDNA encoding the enzyme's signal peptide, not found in the processed holoenzyme, was deleted from the cDNA. The polypeptide was produced as inactive inclusion bodies that could be solubilized in 8 M urea and the reducing agent dithiothreitol. Reconstitution of activity was accomplished by diluting the urea concentration to 2M in the presence of hemin, calcium, and oxidized glutathione. All of the additives were required for recovery of activity. The activity of the recombinant enzyme was dependent on both Mn2+ and H2O2.
D001483:Base Sequence; D001487:Basidiomycota / Q000201:enzymology* / Q000235:genetics; D002118:Calcium / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D004229:Dithiothreitol; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005978:Glutathione / Q000494:pharmacology; D006427:Hemin / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D014508:Urea
D018076:DNA, Complementary; D011994:Recombinant Proteins; D008345:Manganese; D006427:Hemin; D014508:Urea; D006861:Hydrogen Peroxide; D010544:Peroxidases; C051129:manganese peroxidase; D005978:Glutathione; D002118:Calcium; D004229:Dithiothreitol