Enhancement of poplar glutaredoxin expression by optimization of the cDNA sequence.
Identifieur interne : 004594 ( Main/Exploration ); précédent : 004593; suivant : 004595Enhancement of poplar glutaredoxin expression by optimization of the cDNA sequence.
Auteurs : Nicolas Rouhier [France] ; Eric Gelhaye ; Pierre-Eric Sautière ; Jean-Pierre JacquotSource :
- Protein expression and purification [ 1046-5928 ] ; 2002.
Descripteurs français
- KwdFr :
- ADN complémentaire (génétique), Analyse de séquence d'ADN (MeSH), Arbres (MeSH), Clonage moléculaire (MeSH), Données de séquences moléculaires (MeSH), Escherichia coli (MeSH), Glutarédoxines (MeSH), Oxidoreductases (biosynthèse), Oxidoreductases (génétique), Protéines (génétique), Protéines (isolement et purification), Protéines (physiologie), Séquence d'acides aminés (MeSH), Séquence nucléotidique (MeSH).
- MESH :
- biosynthèse : Oxidoreductases.
- génétique : ADN complémentaire, Oxidoreductases, Protéines.
- isolement et purification : Protéines.
- physiologie : Protéines.
- Analyse de séquence d'ADN, Arbres, Clonage moléculaire, Données de séquences moléculaires, Escherichia coli, Glutarédoxines, Séquence d'acides aminés, Séquence nucléotidique.
English descriptors
- KwdEn :
- Amino Acid Sequence (MeSH), Base Sequence (MeSH), Cloning, Molecular (MeSH), DNA, Complementary (genetics), Escherichia coli (MeSH), Glutaredoxins (MeSH), Molecular Sequence Data (MeSH), Oxidoreductases (biosynthesis), Oxidoreductases (genetics), Proteins (genetics), Proteins (isolation & purification), Proteins (physiology), Sequence Analysis, DNA (MeSH), Trees (MeSH).
- MESH :
- chemical , biosynthesis : Oxidoreductases.
- chemical , genetics : DNA, Complementary, Oxidoreductases, Proteins.
- chemical , isolation & purification : Proteins.
- chemical , physiology : Proteins.
- Amino Acid Sequence, Base Sequence, Cloning, Molecular, Escherichia coli, Glutaredoxins, Molecular Sequence Data, Sequence Analysis, DNA, Trees.
Abstract
Glutaredoxins are low-molecular-weight oxidore ductases that play an important role in redox regulation in eukaryotic and prokaryotic cells. Because of their low abundance, these proteins are poorly characterized in plants. Furthermore, very poor yields have been obtained with the expression systems prepared so far, and in addition, the recombinant products contain a His-tag which can interfere with the biochemical characterization. In order to obtain more information about those important regulatory proteins in plants, a cDNA coding for an extended glutaredoxin has been introduced into the expression plasmid pET-3d and the resulting construction has been used to transform Escherichia coli strain BL21(DE3) in the presence of plasmid helper pSBET or not. Initially poor or ineffective protein expression has been improved by successively cloning a N-terminus truncated form of the protein, introducing silent mutations both at the 5' and at the 3' ends of the nucleotide sequence, and finally altering the 3' end in order to change the C-terminus amino acid sequence of the protein. The first modifications have allowed us to produce the protein in large amounts but essentially in an insoluble form which could be resolubilized and purified. On the other hand, changing the C-terminus sequence resulted in protein preparations of high purity and in a soluble form. The recombinant proteins were biochemically active and the yield varied between 6 and 14 mg of homogeneous protein per liter of culture.
DOI: 10.1006/prep.2001.1574
PubMed: 11858718
Affiliations:
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Université Henri Poincaré, Vandoeuvre, France.</nlm:affiliation><country xml:lang="fr">France</country><wicri:regionArea>Unité Mixte de Recherche 1136 IaM (Interaction Arbres Microorganismes), INRA-UHP Nancy I. 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Because of their low abundance, these proteins are poorly characterized in plants. Furthermore, very poor yields have been obtained with the expression systems prepared so far, and in addition, the recombinant products contain a His-tag which can interfere with the biochemical characterization. In order to obtain more information about those important regulatory proteins in plants, a cDNA coding for an extended glutaredoxin has been introduced into the expression plasmid pET-3d and the resulting construction has been used to transform Escherichia coli strain BL21(DE3) in the presence of plasmid helper pSBET or not. Initially poor or ineffective protein expression has been improved by successively cloning a N-terminus truncated form of the protein, introducing silent mutations both at the 5' and at the 3' ends of the nucleotide sequence, and finally altering the 3' end in order to change the C-terminus amino acid sequence of the protein. The first modifications have allowed us to produce the protein in large amounts but essentially in an insoluble form which could be resolubilized and purified. On the other hand, changing the C-terminus sequence resulted in protein preparations of high purity and in a soluble form. The recombinant proteins were biochemically active and the yield varied between 6 and 14 mg of homogeneous protein per liter of culture.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">11858718</PMID><DateCompleted><Year>2002</Year><Month>06</Month><Day>05</Day></DateCompleted><DateRevised><Year>2007</Year><Month>11</Month><Day>15</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">1046-5928</ISSN><JournalIssue CitedMedium="Print"><Volume>24</Volume><Issue>2</Issue><PubDate><Year>2002</Year><Month>Mar</Month></PubDate></JournalIssue><Title>Protein expression and purification</Title><ISOAbbreviation>Protein Expr Purif</ISOAbbreviation></Journal><ArticleTitle>Enhancement of poplar glutaredoxin expression by optimization of the cDNA sequence.</ArticleTitle><Pagination><MedlinePgn>234-41</MedlinePgn></Pagination><Abstract><AbstractText>Glutaredoxins are low-molecular-weight oxidore ductases that play an important role in redox regulation in eukaryotic and prokaryotic cells. Because of their low abundance, these proteins are poorly characterized in plants. Furthermore, very poor yields have been obtained with the expression systems prepared so far, and in addition, the recombinant products contain a His-tag which can interfere with the biochemical characterization. In order to obtain more information about those important regulatory proteins in plants, a cDNA coding for an extended glutaredoxin has been introduced into the expression plasmid pET-3d and the resulting construction has been used to transform Escherichia coli strain BL21(DE3) in the presence of plasmid helper pSBET or not. Initially poor or ineffective protein expression has been improved by successively cloning a N-terminus truncated form of the protein, introducing silent mutations both at the 5' and at the 3' ends of the nucleotide sequence, and finally altering the 3' end in order to change the C-terminus amino acid sequence of the protein. The first modifications have allowed us to produce the protein in large amounts but essentially in an insoluble form which could be resolubilized and purified. On the other hand, changing the C-terminus sequence resulted in protein preparations of high purity and in a soluble form. The recombinant proteins were biochemically active and the yield varied between 6 and 14 mg of homogeneous protein per liter of culture.</AbstractText><CopyrightInformation>Copyright 2002 Elsevier Science (USA).</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Rouhier</LastName><ForeName>Nicolas</ForeName><Initials>N</Initials><AffiliationInfo><Affiliation>Unité Mixte de Recherche 1136 IaM (Interaction Arbres Microorganismes), INRA-UHP Nancy I. Université Henri Poincaré, Vandoeuvre, France.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Gelhaye</LastName><ForeName>Eric</ForeName><Initials>E</Initials></Author><Author ValidYN="Y"><LastName>Sautière</LastName><ForeName>Pierre-Eric</ForeName><Initials>PE</Initials></Author><Author ValidYN="Y"><LastName>Jacquot</LastName><ForeName>Jean-Pierre</ForeName><Initials>JP</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Protein Expr Purif</MedlineTA><NlmUniqueID>9101496</NlmUniqueID><ISSNLinking>1046-5928</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D018076">DNA, Complementary</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D054477">Glutaredoxins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D011506">Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 1.-</RegistryNumber><NameOfSubstance UI="D010088">Oxidoreductases</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000595" MajorTopicYN="N">Amino Acid Sequence</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D001483" MajorTopicYN="N">Base Sequence</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D003001" MajorTopicYN="N">Cloning, Molecular</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D018076" MajorTopicYN="N">DNA, Complementary</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004926" MajorTopicYN="N">Escherichia coli</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D054477" MajorTopicYN="N">Glutaredoxins</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D008969" MajorTopicYN="N">Molecular Sequence Data</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010088" MajorTopicYN="N">Oxidoreductases</DescriptorName><QualifierName UI="Q000096" MajorTopicYN="N">biosynthesis</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D011506" MajorTopicYN="N">Proteins</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName><QualifierName UI="Q000302" MajorTopicYN="N">isolation & purification</QualifierName><QualifierName UI="Q000502" MajorTopicYN="N">physiology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D017422" MajorTopicYN="N">Sequence Analysis, DNA</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D014197" MajorTopicYN="N">Trees</DescriptorName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>2002</Year><Month>2</Month><Day>23</Day><Hour>10</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2002</Year><Month>6</Month><Day>6</Day><Hour>10</Hour><Minute>1</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2002</Year><Month>2</Month><Day>23</Day><Hour>10</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">11858718</ArticleId><ArticleId IdType="doi">10.1006/prep.2001.1574</ArticleId><ArticleId IdType="pii">S1046592801915748</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>France</li></country><region><li>Grand Est</li><li>Lorraine (région)</li></region><settlement><li>Nancy</li></settlement><orgName><li>Université Henri Poincaré</li></orgName></list><tree><noCountry><name sortKey="Gelhaye, Eric" sort="Gelhaye, Eric" uniqKey="Gelhaye E" first="Eric" last="Gelhaye">Eric Gelhaye</name><name sortKey="Jacquot, Jean Pierre" sort="Jacquot, Jean Pierre" uniqKey="Jacquot J" first="Jean-Pierre" last="Jacquot">Jean-Pierre Jacquot</name><name sortKey="Sautiere, Pierre Eric" sort="Sautiere, Pierre Eric" uniqKey="Sautiere P" first="Pierre-Eric" last="Sautière">Pierre-Eric Sautière</name></noCountry><country name="France"><region name="Grand Est"><name sortKey="Rouhier, Nicolas" sort="Rouhier, Nicolas" uniqKey="Rouhier N" first="Nicolas" last="Rouhier">Nicolas Rouhier</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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