Expression of fungal MN peroxidase in E. coli, and refolding to yield active enzyme

Rose E. Whitwam; Irina G. Gazarian; Ming Tien

doi:10.1006/bbrc.1995.2721

Expression of fungal MN peroxidase in E. coli, and refolding to yield active enzyme

Rose E. Whitwam, Irina G. Gazarian, Ming Tien

Biochemistry & Molecular Biology

Research output: Contribution to journal › Article › peer-review

52 Scopus citations

Abstract

The cDNA encoding Mn peroxidase isozyme H4 from Phanerochoete 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 H₂0₂.

Original language	English (US)
Pages (from-to)	1013-1017
Number of pages	5
Journal	Biochemical and Biophysical Research Communications
Volume	216
Issue number	3
DOIs	https://doi.org/10.1006/bbrc.1995.2721
State	Published - Nov 22 1995

All Science Journal Classification (ASJC) codes

Biophysics
Biochemistry
Molecular Biology
Cell Biology

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10.1006/bbrc.1995.2721

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abstract = "The cDNA encoding Mn peroxidase isozyme H4 from Phanerochoete 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 H202.",

author = "Whitwam, {Rose E.} and Gazarian, {Irina G.} and Ming Tien",

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AU - Whitwam, Rose E.

AU - Gazarian, Irina G.

AU - Tien, Ming

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Y1 - 1995/11/22

N2 - The cDNA encoding Mn peroxidase isozyme H4 from Phanerochoete 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 H202.

AB - The cDNA encoding Mn peroxidase isozyme H4 from Phanerochoete 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 H202.

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ER -

Expression of fungal MN peroxidase in E. coli, and refolding to yield active enzyme

Abstract

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