Details
| Original language | English |
|---|---|
| Pages (from-to) | 397-402 |
| Number of pages | 6 |
| Journal | Archives of microbiology |
| Volume | 191 |
| Issue number | 5 |
| Publication status | Published - 27 Feb 2009 |
Abstract
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.
Keywords
- Basidiomycete, E. coli, Heterologous expression, In vitro refolding, Peroxidase
ASJC Scopus subject areas
- Immunology and Microbiology(all)
- Microbiology
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Biology
- Biochemistry, Genetics and Molecular Biology(all)
- Genetics
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In: Archives of microbiology, Vol. 191, No. 5, 27.02.2009, p. 397-402.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Heterologous expression of the msp2 gene from Marasmius scorodonius
AU - Zelena, Kateryna
AU - Zorn, Holger
AU - Nimtz, Manfred
AU - Berger, Ralf Günter
PY - 2009/2/27
Y1 - 2009/2/27
N2 - 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.
AB - 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.
KW - Basidiomycete
KW - E. coli
KW - Heterologous expression
KW - In vitro refolding
KW - Peroxidase
UR - http://www.scopus.com/inward/record.url?scp=67349236053&partnerID=8YFLogxK
U2 - 10.1007/s00203-009-0462-2
DO - 10.1007/s00203-009-0462-2
M3 - Article
C2 - 19247632
AN - SCOPUS:67349236053
VL - 191
SP - 397
EP - 402
JO - Archives of microbiology
JF - Archives of microbiology
SN - 0302-8933
IS - 5
ER -