Homologous expression of recombinant cellobiose dehydrogenase in Phanerochaete chrysosporium.
Identifieur interne : 000A80 ( Main/Exploration ); précédent : 000A79; suivant : 000A81Homologous expression of recombinant cellobiose dehydrogenase in Phanerochaete chrysosporium.
Auteurs : B. Li [États-Unis] ; F A Rotsaert ; M H Gold ; V. RenganathanSource :
- Biochemical and biophysical research communications [ 0006-291X ] ; 2000.
Descripteurs français
- KwdFr :
- Carbohydrate dehydrogenases (biosynthèse), Carbohydrate dehydrogenases (génétique), Carbohydrate dehydrogenases (isolement et purification), Cellobiose (métabolisme), Cytochromes de type c (métabolisme), Dichloro-2,6 indophénol (métabolisme), Phanerochaete (enzymologie), Phanerochaete (génétique), Protéines recombinantes (biosynthèse), Transformation génétique (MeSH), Vecteurs génétiques (MeSH).
- MESH :
- biosynthèse : Carbohydrate dehydrogenases, Protéines recombinantes.
- enzymologie : Phanerochaete.
- génétique : Carbohydrate dehydrogenases, Phanerochaete.
- isolement et purification : Carbohydrate dehydrogenases.
- métabolisme : Cellobiose, Cytochromes de type c, Dichloro-2,6 indophénol.
- Transformation génétique, Vecteurs génétiques.
English descriptors
- KwdEn :
- 2,6-Dichloroindophenol (metabolism), Carbohydrate Dehydrogenases (biosynthesis), Carbohydrate Dehydrogenases (genetics), Carbohydrate Dehydrogenases (isolation & purification), Cellobiose (metabolism), Cytochrome c Group (metabolism), Genetic Vectors (MeSH), Phanerochaete (enzymology), Phanerochaete (genetics), Recombinant Proteins (biosynthesis), Transformation, Genetic (MeSH).
- MESH :
- chemical , biosynthesis : Carbohydrate Dehydrogenases, Recombinant Proteins.
- chemical , genetics : Carbohydrate Dehydrogenases.
- chemical , isolation & purification : Carbohydrate Dehydrogenases.
- chemical , metabolism : 2,6-Dichloroindophenol, Cellobiose, Cytochrome c Group.
- enzymology : Phanerochaete.
- genetics : Phanerochaete.
- Genetic Vectors, Transformation, Genetic.
Abstract
Cellobiose dehydrogenase (CDH) is a novel extracellular hemoflavoenzyme from Phanerochaete chrysosporium and is produced only in cultures supplemented with cellulose. In this report, CDH from P. chrysosporium has been homologously expressed in cultures supplemented with glucose as the sole carbon source when no endogenous CDH is expressed. This was achieved by placing the cdh-1 gene under the control of the D-glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter (1.1 kb) fused upstream of the ATG start codon of cdh-1. The gpd promoter-chd-1 construct was inserted into the multiple cloning site of the expression vector pOGI18, which contained the Schizophyllum commune ade5 as a selectable marker. The P. chrysosporium ade1 auxotrophic strain OGC107-1 was transformed with the pAGC1 construct, and the prototrophic transformants were assayed for CDH activity. Approximately 50% of the Ade(+) transformants exhibited CDH activity in the extracellular medium of stationary cultures. At least one of the transformants produced high levels (500-600 U/liter) of recombinant CDH (rCDH). Purification by ammonium sulfate precipitation, Sephacryl S-200 chromatography, and FPLC using a Mono-Q 5/5 column yielded homogeneous rCDH. Physical, spectral, and kinetic characteristics of purified homologously expressed rCDH were similar to those of wild-type CDH. This expression system will enable site-directed mutagenesis studies to be carried out on CDH.
DOI: 10.1006/bbrc.2000.2381
PubMed: 10733918
Affiliations:
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Walker Road, Beaverton, Oregon 97006-8921</wicri:regionArea><wicri:noRegion>Oregon 97006-8921</wicri:noRegion></affiliation></author><author><name sortKey="Rotsaert, F A" sort="Rotsaert, F A" uniqKey="Rotsaert F" first="F A" last="Rotsaert">F A Rotsaert</name></author><author><name sortKey="Gold, M H" sort="Gold, M H" uniqKey="Gold M" first="M H" last="Gold">M H Gold</name></author><author><name sortKey="Renganathan, V" sort="Renganathan, V" uniqKey="Renganathan V" first="V" last="Renganathan">V. 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Li</name><affiliation wicri:level="1"><nlm:affiliation>Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, 20000 N.W. Walker Road, Beaverton, Oregon 97006-8921, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, 20000 N.W. Walker Road, Beaverton, Oregon 97006-8921</wicri:regionArea><wicri:noRegion>Oregon 97006-8921</wicri:noRegion></affiliation></author><author><name sortKey="Rotsaert, F A" sort="Rotsaert, F A" uniqKey="Rotsaert F" first="F A" last="Rotsaert">F A Rotsaert</name></author><author><name sortKey="Gold, M H" sort="Gold, M H" uniqKey="Gold M" first="M H" last="Gold">M H Gold</name></author><author><name sortKey="Renganathan, V" sort="Renganathan, V" uniqKey="Renganathan V" first="V" last="Renganathan">V. Renganathan</name></author></analytic><series><title level="j">Biochemical and biophysical research communications</title><idno type="ISSN">0006-291X</idno><imprint><date when="2000" type="published">2000</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>2,6-Dichloroindophenol (metabolism)</term><term>Carbohydrate Dehydrogenases (biosynthesis)</term><term>Carbohydrate Dehydrogenases (genetics)</term><term>Carbohydrate Dehydrogenases (isolation & purification)</term><term>Cellobiose (metabolism)</term><term>Cytochrome c Group (metabolism)</term><term>Genetic Vectors (MeSH)</term><term>Phanerochaete (enzymology)</term><term>Phanerochaete (genetics)</term><term>Recombinant Proteins (biosynthesis)</term><term>Transformation, Genetic (MeSH)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Carbohydrate dehydrogenases (biosynthèse)</term><term>Carbohydrate dehydrogenases (génétique)</term><term>Carbohydrate dehydrogenases (isolement et purification)</term><term>Cellobiose (métabolisme)</term><term>Cytochromes de type c (métabolisme)</term><term>Dichloro-2,6 indophénol (métabolisme)</term><term>Phanerochaete (enzymologie)</term><term>Phanerochaete (génétique)</term><term>Protéines recombinantes (biosynthèse)</term><term>Transformation génétique (MeSH)</term><term>Vecteurs génétiques (MeSH)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>Carbohydrate Dehydrogenases</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Carbohydrate Dehydrogenases</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Carbohydrate Dehydrogenases</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>2,6-Dichloroindophenol</term><term>Cellobiose</term><term>Cytochrome c Group</term></keywords><keywords scheme="MESH" qualifier="biosynthèse" xml:lang="fr"><term>Carbohydrate dehydrogenases</term><term>Protéines recombinantes</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Phanerochaete</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Phanerochaete</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Phanerochaete</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Carbohydrate dehydrogenases</term><term>Phanerochaete</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Carbohydrate dehydrogenases</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Cellobiose</term><term>Cytochromes de type c</term><term>Dichloro-2,6 indophénol</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Genetic Vectors</term><term>Transformation, Genetic</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Transformation génétique</term><term>Vecteurs génétiques</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Cellobiose dehydrogenase (CDH) is a novel extracellular hemoflavoenzyme from Phanerochaete chrysosporium and is produced only in cultures supplemented with cellulose. In this report, CDH from P. chrysosporium has been homologously expressed in cultures supplemented with glucose as the sole carbon source when no endogenous CDH is expressed. This was achieved by placing the cdh-1 gene under the control of the D-glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter (1.1 kb) fused upstream of the ATG start codon of cdh-1. The gpd promoter-chd-1 construct was inserted into the multiple cloning site of the expression vector pOGI18, which contained the Schizophyllum commune ade5 as a selectable marker. The P. chrysosporium ade1 auxotrophic strain OGC107-1 was transformed with the pAGC1 construct, and the prototrophic transformants were assayed for CDH activity. Approximately 50% of the Ade(+) transformants exhibited CDH activity in the extracellular medium of stationary cultures. At least one of the transformants produced high levels (500-600 U/liter) of recombinant CDH (rCDH). Purification by ammonium sulfate precipitation, Sephacryl S-200 chromatography, and FPLC using a Mono-Q 5/5 column yielded homogeneous rCDH. Physical, spectral, and kinetic characteristics of purified homologously expressed rCDH were similar to those of wild-type CDH. This expression system will enable site-directed mutagenesis studies to be carried out on CDH.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">10733918</PMID><DateCompleted><Year>2000</Year><Month>05</Month><Day>04</Day></DateCompleted><DateRevised><Year>2013</Year><Month>11</Month><Day>21</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0006-291X</ISSN><JournalIssue CitedMedium="Print"><Volume>270</Volume><Issue>1</Issue><PubDate><Year>2000</Year><Month>Apr</Month><Day>02</Day></PubDate></JournalIssue><Title>Biochemical and biophysical research communications</Title><ISOAbbreviation>Biochem Biophys Res Commun</ISOAbbreviation></Journal><ArticleTitle>Homologous expression of recombinant cellobiose dehydrogenase in Phanerochaete chrysosporium.</ArticleTitle><Pagination><MedlinePgn>141-6</MedlinePgn></Pagination><Abstract><AbstractText>Cellobiose dehydrogenase (CDH) is a novel extracellular hemoflavoenzyme from Phanerochaete chrysosporium and is produced only in cultures supplemented with cellulose. In this report, CDH from P. chrysosporium has been homologously expressed in cultures supplemented with glucose as the sole carbon source when no endogenous CDH is expressed. This was achieved by placing the cdh-1 gene under the control of the D-glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter (1.1 kb) fused upstream of the ATG start codon of cdh-1. The gpd promoter-chd-1 construct was inserted into the multiple cloning site of the expression vector pOGI18, which contained the Schizophyllum commune ade5 as a selectable marker. The P. chrysosporium ade1 auxotrophic strain OGC107-1 was transformed with the pAGC1 construct, and the prototrophic transformants were assayed for CDH activity. Approximately 50% of the Ade(+) transformants exhibited CDH activity in the extracellular medium of stationary cultures. At least one of the transformants produced high levels (500-600 U/liter) of recombinant CDH (rCDH). Purification by ammonium sulfate precipitation, Sephacryl S-200 chromatography, and FPLC using a Mono-Q 5/5 column yielded homogeneous rCDH. Physical, spectral, and kinetic characteristics of purified homologously expressed rCDH were similar to those of wild-type CDH. This expression system will enable site-directed mutagenesis studies to be carried out on CDH.</AbstractText><CopyrightInformation>Copyright 2000 Academic Press.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Li</LastName><ForeName>B</ForeName><Initials>B</Initials><AffiliationInfo><Affiliation>Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, 20000 N.W. 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Renganathan</name><name sortKey="Rotsaert, F A" sort="Rotsaert, F A" uniqKey="Rotsaert F" first="F A" last="Rotsaert">F A Rotsaert</name></noCountry><country name="États-Unis"><noRegion><name sortKey="Li, B" sort="Li, B" uniqKey="Li B" first="B" last="Li">B. Li</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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