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Optimized protocol for the 2-DE of extracellular proteins from higher basidiomycetes inhabiting lignocellulose
- Source :
- ELECTROPHORESIS. 30:2431-2441
- Publication Year :
- 2009
- Publisher :
- Wiley, 2009.
-
Abstract
- Basidiomycetes inhabiting lignocellulose comprise an important class of filamentous fungi with ecological and biotechnological relevance. Extracellular enzymes of wood-degrading fungi such as laccases, manganese-dependent (or independent) peroxidases, cellulases and xylanases are of considerable interest for biotechnological applications. Still, proteomic studies of fungal secretomes based on 2-DE can be very challenging due to low protein concentrations and variable amounts of fungal metabolites. Comparison of different standard methods for protein precipitation has demonstrated their limited applicability to fungal secretomes. The extracellular metabolites impair standard methods for protein quantification and can result in a strong overestimation of total protein. We have developed an optimized protocol for the precipitation of extracellular proteins from liquid cultures of Coprinopsis cinerea growing in an exponential phase on a complex media. We found that a considerable amount of gelatinous material can be removed from the liquid culture supernatants by high-speed centrifugation. Fungal proteins can be effectively enriched by TCA precipitation and coprecipitated metabolites hampering 2-DE can be removed through the application of Tris/acetone. Following our protocol makes it possible to concentrate proteins from culture supernatants and to simultaneously remove most of the impeding compounds from a given sample. We have applied this procedure in the 2-DE of secretomes from the model organism C. cinerea as well as other basidiomycetes such as Pleurotus ostreatus, Phanerochaete chrysosporium, Polyporus brumalis and Schizophyllum commune.
- Subjects :
- Low protein
Proteome
Clinical Biochemistry
Cell Culture Techniques
Mycology
Cellulase
Lignin
Biochemistry
Analytical Chemistry
Microbiology
Fungal Proteins
03 medical and health sciences
Extracellular
Chemical Precipitation
Electrophoresis, Gel, Two-Dimensional
030304 developmental biology
Laccase
0303 health sciences
Fungal protein
biology
030306 microbiology
Basidiomycota
fungi
Schizophyllum commune
food and beverages
biology.organism_classification
Coprinopsis cinerea
biology.protein
Phanerochaete
Electrophoresis, Polyacrylamide Gel
Isoelectric Focusing
Subjects
Details
- ISSN :
- 01730835
- Volume :
- 30
- Database :
- OpenAIRE
- Journal :
- ELECTROPHORESIS
- Accession number :
- edsair.doi.dedup.....3f6b2440af7545d54b430e609639cb99