Optimized protocol for the 2-DE of extracellular proteins from higher basidiomycetes inhabiting lignocellulose.
Identifieur interne : 000612 ( Main/Exploration ); précédent : 000611; suivant : 000613Optimized protocol for the 2-DE of extracellular proteins from higher basidiomycetes inhabiting lignocellulose.
Auteurs : Dorothea Fragner [Allemagne] ; Mojtaba Zomorrodi ; Ursula Kües ; Andrzej MajcherczykSource :
- Electrophoresis [ 1522-2683 ] ; 2009.
Descripteurs français
- KwdFr :
- Basidiomycota (composition chimique), Basidiomycota (métabolisme), Focalisation isoélectrique (MeSH), Lignine (MeSH), Mycologie (méthodes), Protéines fongiques (analyse), Protéome (analyse), Précipitation chimique (MeSH), Techniques de culture cellulaire (MeSH), Électrophorèse bidimensionnelle sur gel (méthodes), Électrophorèse sur gel de polyacrylamide (MeSH).
- MESH :
- analyse : Protéines fongiques, Protéome.
- composition chimique : Basidiomycota.
- métabolisme : Basidiomycota.
- méthodes : Mycologie, Électrophorèse bidimensionnelle sur gel.
- Focalisation isoélectrique, Lignine, Précipitation chimique, Techniques de culture cellulaire, Électrophorèse sur gel de polyacrylamide.
English descriptors
- KwdEn :
- Basidiomycota (chemistry), Basidiomycota (metabolism), Cell Culture Techniques (MeSH), Chemical Precipitation (MeSH), Electrophoresis, Gel, Two-Dimensional (methods), Electrophoresis, Polyacrylamide Gel (MeSH), Fungal Proteins (analysis), Isoelectric Focusing (MeSH), Lignin (MeSH), Mycology (methods), Proteome (analysis).
- MESH :
- chemical , analysis : Fungal Proteins, Proteome.
- chemistry : Basidiomycota.
- metabolism : Basidiomycota.
- methods : Electrophoresis, Gel, Two-Dimensional, Mycology.
- Cell Culture Techniques, Chemical Precipitation, Electrophoresis, Polyacrylamide Gel, Isoelectric Focusing, Lignin.
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.
DOI: 10.1002/elps.200800770
PubMed: 19593751
Affiliations:
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proteins from higher basidiomycetes inhabiting lignocellulose.</title><author><name sortKey="Fragner, Dorothea" sort="Fragner, Dorothea" uniqKey="Fragner D" first="Dorothea" last="Fragner">Dorothea Fragner</name><affiliation wicri:level="3"><nlm:affiliation>Büsgen Institute, Section Molecular Wood Biotechnology and Technical Mycology, Georg-August-University Göttingen, Göttingen, Germany.</nlm:affiliation><country xml:lang="fr">Allemagne</country><wicri:regionArea>Büsgen Institute, Section Molecular Wood Biotechnology and Technical Mycology, Georg-August-University Göttingen, Göttingen</wicri:regionArea><placeName><region type="land" nuts="2">Basse-Saxe</region><settlement type="city">Göttingen</settlement></placeName></affiliation></author><author><name sortKey="Zomorrodi, Mojtaba" sort="Zomorrodi, Mojtaba" uniqKey="Zomorrodi M" first="Mojtaba" last="Zomorrodi">Mojtaba Zomorrodi</name></author><author><name sortKey="Kues, Ursula" sort="Kues, Ursula" uniqKey="Kues U" first="Ursula" last="Kües">Ursula Kües</name></author><author><name sortKey="Majcherczyk, Andrzej" sort="Majcherczyk, Andrzej" uniqKey="Majcherczyk A" first="Andrzej" last="Majcherczyk">Andrzej Majcherczyk</name></author></analytic><series><title level="j">Electrophoresis</title><idno type="eISSN">1522-2683</idno><imprint><date when="2009" type="published">2009</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Basidiomycota (chemistry)</term><term>Basidiomycota (metabolism)</term><term>Cell Culture Techniques (MeSH)</term><term>Chemical Precipitation (MeSH)</term><term>Electrophoresis, Gel, Two-Dimensional (methods)</term><term>Electrophoresis, Polyacrylamide Gel (MeSH)</term><term>Fungal Proteins (analysis)</term><term>Isoelectric Focusing (MeSH)</term><term>Lignin (MeSH)</term><term>Mycology (methods)</term><term>Proteome (analysis)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Basidiomycota (composition chimique)</term><term>Basidiomycota (métabolisme)</term><term>Focalisation isoélectrique (MeSH)</term><term>Lignine (MeSH)</term><term>Mycologie (méthodes)</term><term>Protéines fongiques (analyse)</term><term>Protéome (analyse)</term><term>Précipitation chimique (MeSH)</term><term>Techniques de culture cellulaire (MeSH)</term><term>Électrophorèse bidimensionnelle sur gel (méthodes)</term><term>Électrophorèse sur gel de polyacrylamide (MeSH)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="analysis" xml:lang="en"><term>Fungal Proteins</term><term>Proteome</term></keywords><keywords scheme="MESH" qualifier="analyse" xml:lang="fr"><term>Protéines fongiques</term><term>Protéome</term></keywords><keywords scheme="MESH" qualifier="chemistry" xml:lang="en"><term>Basidiomycota</term></keywords><keywords scheme="MESH" qualifier="composition chimique" xml:lang="fr"><term>Basidiomycota</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Basidiomycota</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Electrophoresis, Gel, Two-Dimensional</term><term>Mycology</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Basidiomycota</term></keywords><keywords scheme="MESH" qualifier="méthodes" xml:lang="fr"><term>Mycologie</term><term>Électrophorèse bidimensionnelle sur gel</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Cell Culture Techniques</term><term>Chemical Precipitation</term><term>Electrophoresis, Polyacrylamide Gel</term><term>Isoelectric Focusing</term><term>Lignin</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Focalisation isoélectrique</term><term>Lignine</term><term>Précipitation chimique</term><term>Techniques de culture cellulaire</term><term>Électrophorèse sur gel de polyacrylamide</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">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.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">19593751</PMID><DateCompleted><Year>2009</Year><Month>10</Month><Day>27</Day></DateCompleted><DateRevised><Year>2009</Year><Month>11</Month><Day>19</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Electronic">1522-2683</ISSN><JournalIssue CitedMedium="Internet"><Volume>30</Volume><Issue>14</Issue><PubDate><Year>2009</Year><Month>Jul</Month></PubDate></JournalIssue><Title>Electrophoresis</Title><ISOAbbreviation>Electrophoresis</ISOAbbreviation></Journal><ArticleTitle>Optimized protocol for the 2-DE of extracellular proteins from higher basidiomycetes inhabiting lignocellulose.</ArticleTitle><Pagination><MedlinePgn>2431-41</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1002/elps.200800770</ELocationID><Abstract><AbstractText>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.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Fragner</LastName><ForeName>Dorothea</ForeName><Initials>D</Initials><AffiliationInfo><Affiliation>Büsgen Institute, Section Molecular Wood Biotechnology and Technical Mycology, Georg-August-University Göttingen, Göttingen, Germany.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Zomorrodi</LastName><ForeName>Mojtaba</ForeName><Initials>M</Initials></Author><Author ValidYN="Y"><LastName>Kües</LastName><ForeName>Ursula</ForeName><Initials>U</Initials></Author><Author ValidYN="Y"><LastName>Majcherczyk</LastName><ForeName>Andrzej</ForeName><Initials>A</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>Germany</Country><MedlineTA>Electrophoresis</MedlineTA><NlmUniqueID>8204476</NlmUniqueID><ISSNLinking>0173-0835</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D005656">Fungal Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D020543">Proteome</NameOfSubstance></Chemical><Chemical><RegistryNumber>11132-73-3</RegistryNumber><NameOfSubstance UI="C036909">lignocellulose</NameOfSubstance></Chemical><Chemical><RegistryNumber>9005-53-2</RegistryNumber><NameOfSubstance UI="D008031">Lignin</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D001487" MajorTopicYN="N">Basidiomycota</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="Y">chemistry</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D018929" MajorTopicYN="N">Cell Culture Techniques</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D011232" MajorTopicYN="N">Chemical Precipitation</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D015180" MajorTopicYN="N">Electrophoresis, Gel, Two-Dimensional</DescriptorName><QualifierName UI="Q000379" MajorTopicYN="Y">methods</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004591" MajorTopicYN="N">Electrophoresis, Polyacrylamide Gel</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D005656" MajorTopicYN="N">Fungal Proteins</DescriptorName><QualifierName UI="Q000032" MajorTopicYN="Y">analysis</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D007525" MajorTopicYN="N">Isoelectric Focusing</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D008031" MajorTopicYN="Y">Lignin</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D009172" MajorTopicYN="N">Mycology</DescriptorName><QualifierName UI="Q000379" MajorTopicYN="Y">methods</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D020543" MajorTopicYN="N">Proteome</DescriptorName><QualifierName UI="Q000032" MajorTopicYN="Y">analysis</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="entrez"><Year>2009</Year><Month>7</Month><Day>14</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2009</Year><Month>7</Month><Day>14</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2009</Year><Month>10</Month><Day>29</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">19593751</ArticleId><ArticleId IdType="doi">10.1002/elps.200800770</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>Allemagne</li></country><region><li>Basse-Saxe</li></region><settlement><li>Göttingen</li></settlement></list><tree><noCountry><name sortKey="Kues, Ursula" sort="Kues, Ursula" uniqKey="Kues U" first="Ursula" last="Kües">Ursula Kües</name><name sortKey="Majcherczyk, Andrzej" sort="Majcherczyk, Andrzej" uniqKey="Majcherczyk A" first="Andrzej" last="Majcherczyk">Andrzej Majcherczyk</name><name sortKey="Zomorrodi, Mojtaba" sort="Zomorrodi, Mojtaba" uniqKey="Zomorrodi M" first="Mojtaba" last="Zomorrodi">Mojtaba Zomorrodi</name></noCountry><country name="Allemagne"><region name="Basse-Saxe"><name sortKey="Fragner, Dorothea" sort="Fragner, Dorothea" uniqKey="Fragner D" first="Dorothea" last="Fragner">Dorothea Fragner</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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