High-level expression of pig liver thioltransferase (glutaredoxin) in Escherichia coli.
Identifieur interne : 001318 ( Main/Exploration ); précédent : 001317; suivant : 001319High-level expression of pig liver thioltransferase (glutaredoxin) in Escherichia coli.
Auteurs : Y F Yang [États-Unis] ; W W WellsSource :
- The Journal of biological chemistry [ 0021-9258 ] ; 1990.
Descripteurs français
- KwdFr :
- ADN (génétique), Acides aminés (analyse), Animaux (MeSH), Cinétique (MeSH), Clonage moléculaire (MeSH), Concentration en ions d'hydrogène (MeSH), Escherichia coli (génétique), Expression des gènes (MeSH), Focalisation isoélectrique (MeSH), Foie (enzymologie), Glutarédoxines (MeSH), Immunotransfert (MeSH), Mutation (MeSH), Oxidoreductases (MeSH), Plasmides (MeSH), Protéines (analyse), Protéines (génétique), Protéines (métabolisme), Protéines recombinantes (analyse), Protéines recombinantes (métabolisme), Suidae (MeSH), Séquence nucléotidique (MeSH), Transformation bactérienne (MeSH).
- MESH :
- analyse : Acides aminés, Protéines, Protéines recombinantes.
- enzymologie : Foie.
- génétique : ADN, Escherichia coli, Protéines.
- métabolisme : Protéines, Protéines recombinantes.
- Animaux, Cinétique, Clonage moléculaire, Concentration en ions d'hydrogène, Expression des gènes, Focalisation isoélectrique, Glutarédoxines, Immunotransfert, Mutation, Oxidoreductases, Plasmides, Suidae, Séquence nucléotidique, Transformation bactérienne.
English descriptors
- KwdEn :
- Amino Acids (analysis), Animals (MeSH), Base Sequence (MeSH), Cloning, Molecular (MeSH), DNA (genetics), Escherichia coli (genetics), Gene Expression (MeSH), Glutaredoxins (MeSH), Hydrogen-Ion Concentration (MeSH), Immunoblotting (MeSH), Isoelectric Focusing (MeSH), Kinetics (MeSH), Liver (enzymology), Mutation (MeSH), Oxidoreductases (MeSH), Plasmids (MeSH), Proteins (analysis), Proteins (genetics), Proteins (metabolism), Recombinant Proteins (analysis), Recombinant Proteins (metabolism), Swine (MeSH), Transformation, Bacterial (MeSH).
- MESH :
- chemical , analysis : Amino Acids, Proteins, Recombinant Proteins.
- chemical , genetics : DNA, Proteins.
- enzymology : Liver.
- genetics : Escherichia coli.
- chemical , metabolism : Proteins, Recombinant Proteins.
- Animals, Base Sequence, Cloning, Molecular, Gene Expression, Glutaredoxins, Hydrogen-Ion Concentration, Immunoblotting, Isoelectric Focusing, Kinetics, Mutation, Oxidoreductases, Plasmids, Swine, Transformation, Bacterial.
Abstract
We report the first high-level expression of a mammalian thioltransferase (glutaredoxin) in Escherichia coli. A NcoI site (CCATGG) was introduced into the cDNA encoding pig liver thioltransferase (glutaredoxin) by site-directed mutagenesis, in which the first G of the original sequence, GCATGG, was replaced by a C. The altered cDNA was cloned into an expression vector, plasmid pKK233-2, between the unique NcoI and HindIII sites and expressed in E. coli JM105 at a high level (8% of total soluble protein) after 6 h of isopropyl-beta-D-thiogalactopyranoside induction. The soluble and unfused product was measured by the thiol-transferase thiol-disulfide exchange assay and immunoblotting analysis. The recombinant enzyme was purified to a single band as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The amino acid composition of the expressed enzyme agreed with that of the known sequence of pig liver thioltransferase (glutaredoxin). N-terminal sequence analysis revealed that unlike the native pig liver protein which is N-acetylated, the recombinant enzyme was unblocked at the N terminus (alanine). Various kinetic properties of the recombinant enzyme with regard to the exchange reaction were identical with those of the native enzyme.
PubMed: 2403567
Affiliations:
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last="Wells">W W Wells</name></author></analytic><series><title level="j">The Journal of biological chemistry</title><idno type="ISSN">0021-9258</idno><imprint><date when="1990" type="published">1990</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Amino Acids (analysis)</term><term>Animals (MeSH)</term><term>Base Sequence (MeSH)</term><term>Cloning, Molecular (MeSH)</term><term>DNA (genetics)</term><term>Escherichia coli (genetics)</term><term>Gene Expression (MeSH)</term><term>Glutaredoxins (MeSH)</term><term>Hydrogen-Ion Concentration (MeSH)</term><term>Immunoblotting (MeSH)</term><term>Isoelectric Focusing (MeSH)</term><term>Kinetics (MeSH)</term><term>Liver (enzymology)</term><term>Mutation (MeSH)</term><term>Oxidoreductases (MeSH)</term><term>Plasmids (MeSH)</term><term>Proteins (analysis)</term><term>Proteins (genetics)</term><term>Proteins (metabolism)</term><term>Recombinant Proteins (analysis)</term><term>Recombinant Proteins (metabolism)</term><term>Swine (MeSH)</term><term>Transformation, Bacterial (MeSH)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ADN (génétique)</term><term>Acides aminés (analyse)</term><term>Animaux (MeSH)</term><term>Cinétique (MeSH)</term><term>Clonage moléculaire (MeSH)</term><term>Concentration en ions d'hydrogène (MeSH)</term><term>Escherichia coli (génétique)</term><term>Expression des gènes (MeSH)</term><term>Focalisation isoélectrique (MeSH)</term><term>Foie (enzymologie)</term><term>Glutarédoxines (MeSH)</term><term>Immunotransfert (MeSH)</term><term>Mutation (MeSH)</term><term>Oxidoreductases (MeSH)</term><term>Plasmides (MeSH)</term><term>Protéines (analyse)</term><term>Protéines (génétique)</term><term>Protéines (métabolisme)</term><term>Protéines recombinantes (analyse)</term><term>Protéines recombinantes (métabolisme)</term><term>Suidae (MeSH)</term><term>Séquence nucléotidique (MeSH)</term><term>Transformation bactérienne (MeSH)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="analysis" xml:lang="en"><term>Amino Acids</term><term>Proteins</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>DNA</term><term>Proteins</term></keywords><keywords scheme="MESH" qualifier="analyse" xml:lang="fr"><term>Acides aminés</term><term>Protéines</term><term>Protéines recombinantes</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Foie</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Liver</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Escherichia coli</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>ADN</term><term>Escherichia coli</term><term>Protéines</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Proteins</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Protéines</term><term>Protéines recombinantes</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Base Sequence</term><term>Cloning, Molecular</term><term>Gene Expression</term><term>Glutaredoxins</term><term>Hydrogen-Ion Concentration</term><term>Immunoblotting</term><term>Isoelectric Focusing</term><term>Kinetics</term><term>Mutation</term><term>Oxidoreductases</term><term>Plasmids</term><term>Swine</term><term>Transformation, Bacterial</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Cinétique</term><term>Clonage moléculaire</term><term>Concentration en ions d'hydrogène</term><term>Expression des gènes</term><term>Focalisation isoélectrique</term><term>Glutarédoxines</term><term>Immunotransfert</term><term>Mutation</term><term>Oxidoreductases</term><term>Plasmides</term><term>Suidae</term><term>Séquence nucléotidique</term><term>Transformation bactérienne</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">We report the first high-level expression of a mammalian thioltransferase (glutaredoxin) in Escherichia coli. A NcoI site (CCATGG) was introduced into the cDNA encoding pig liver thioltransferase (glutaredoxin) by site-directed mutagenesis, in which the first G of the original sequence, GCATGG, was replaced by a C. The altered cDNA was cloned into an expression vector, plasmid pKK233-2, between the unique NcoI and HindIII sites and expressed in E. coli JM105 at a high level (8% of total soluble protein) after 6 h of isopropyl-beta-D-thiogalactopyranoside induction. The soluble and unfused product was measured by the thiol-transferase thiol-disulfide exchange assay and immunoblotting analysis. The recombinant enzyme was purified to a single band as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The amino acid composition of the expressed enzyme agreed with that of the known sequence of pig liver thioltransferase (glutaredoxin). N-terminal sequence analysis revealed that unlike the native pig liver protein which is N-acetylated, the recombinant enzyme was unblocked at the N terminus (alanine). Various kinetic properties of the recombinant enzyme with regard to the exchange reaction were identical with those of the native enzyme.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">2403567</PMID><DateCompleted><Year>1990</Year><Month>02</Month><Day>02</Day></DateCompleted><DateRevised><Year>2007</Year><Month>11</Month><Day>15</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0021-9258</ISSN><JournalIssue CitedMedium="Print"><Volume>265</Volume><Issue>1</Issue><PubDate><Year>1990</Year><Month>Jan</Month><Day>05</Day></PubDate></JournalIssue><Title>The Journal of biological chemistry</Title><ISOAbbreviation>J Biol Chem</ISOAbbreviation></Journal><ArticleTitle>High-level expression of pig liver thioltransferase (glutaredoxin) in Escherichia coli.</ArticleTitle><Pagination><MedlinePgn>589-93</MedlinePgn></Pagination><Abstract><AbstractText>We report the first high-level expression of a mammalian thioltransferase (glutaredoxin) in Escherichia coli. A NcoI site (CCATGG) was introduced into the cDNA encoding pig liver thioltransferase (glutaredoxin) by site-directed mutagenesis, in which the first G of the original sequence, GCATGG, was replaced by a C. The altered cDNA was cloned into an expression vector, plasmid pKK233-2, between the unique NcoI and HindIII sites and expressed in E. coli JM105 at a high level (8% of total soluble protein) after 6 h of isopropyl-beta-D-thiogalactopyranoside induction. The soluble and unfused product was measured by the thiol-transferase thiol-disulfide exchange assay and immunoblotting analysis. The recombinant enzyme was purified to a single band as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The amino acid composition of the expressed enzyme agreed with that of the known sequence of pig liver thioltransferase (glutaredoxin). N-terminal sequence analysis revealed that unlike the native pig liver protein which is N-acetylated, the recombinant enzyme was unblocked at the N terminus (alanine). Various kinetic properties of the recombinant enzyme with regard to the exchange reaction were identical with those of the native enzyme.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Yang</LastName><ForeName>Y F</ForeName><Initials>YF</Initials><AffiliationInfo><Affiliation>Department of Biochemistry, Michigan State University, East Lansing 48824.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Wells</LastName><ForeName>W W</ForeName><Initials>WW</Initials></Author></AuthorList><Language>eng</Language><GrantList CompleteYN="Y"><Grant><GrantID>GM-38634</GrantID><Acronym>GM</Acronym><Agency>NIGMS NIH HHS</Agency><Country>United States</Country></Grant></GrantList><PublicationTypeList><PublicationType UI="D003160">Comparative Study</PublicationType><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013487">Research Support, U.S. Gov't, P.H.S.</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>J Biol Chem</MedlineTA><NlmUniqueID>2985121R</NlmUniqueID><ISSNLinking>0021-9258</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D000596">Amino Acids</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D054477">Glutaredoxins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D011506">Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D011994">Recombinant Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>9007-49-2</RegistryNumber><NameOfSubstance UI="D004247">DNA</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 1.-</RegistryNumber><NameOfSubstance UI="D010088">Oxidoreductases</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000596" MajorTopicYN="N">Amino Acids</DescriptorName><QualifierName UI="Q000032" MajorTopicYN="N">analysis</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D000818" MajorTopicYN="N">Animals</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="D004247" MajorTopicYN="N">DNA</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004926" MajorTopicYN="N">Escherichia coli</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D015870" MajorTopicYN="Y">Gene Expression</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D054477" MajorTopicYN="N">Glutaredoxins</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D006863" MajorTopicYN="N">Hydrogen-Ion Concentration</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D015151" MajorTopicYN="N">Immunoblotting</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D007525" MajorTopicYN="N">Isoelectric Focusing</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D007700" MajorTopicYN="N">Kinetics</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D008099" MajorTopicYN="N">Liver</DescriptorName><QualifierName UI="Q000201" MajorTopicYN="Y">enzymology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D009154" MajorTopicYN="N">Mutation</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010088" MajorTopicYN="Y">Oxidoreductases</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010957" MajorTopicYN="N">Plasmids</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D011506" MajorTopicYN="N">Proteins</DescriptorName><QualifierName UI="Q000032" MajorTopicYN="N">analysis</QualifierName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D011994" MajorTopicYN="N">Recombinant Proteins</DescriptorName><QualifierName UI="Q000032" MajorTopicYN="N">analysis</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D013552" MajorTopicYN="N">Swine</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D014169" MajorTopicYN="N">Transformation, Bacterial</DescriptorName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>1990</Year><Month>1</Month><Day>5</Day></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>1990</Year><Month>1</Month><Day>5</Day><Hour>0</Hour><Minute>1</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>1990</Year><Month>1</Month><Day>5</Day><Hour>0</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">2403567</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>États-Unis</li></country><region><li>Michigan</li></region><settlement><li>East Lansing</li></settlement><orgName><li>Université d'État du Michigan</li></orgName></list><tree><noCountry><name sortKey="Wells, W W" sort="Wells, W W" uniqKey="Wells W" first="W W" last="Wells">W W Wells</name></noCountry><country name="États-Unis"><region name="Michigan"><name sortKey="Yang, Y F" sort="Yang, Y F" uniqKey="Yang Y" first="Y F" last="Yang">Y F 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