High-level expression of fully active human glutaredoxin (thioltransferase) in E. coli and characterization of Cys7 to Ser mutant protein.
Identifieur interne : 001203 ( Main/Exploration ); précédent : 001202; suivant : 001204High-level expression of fully active human glutaredoxin (thioltransferase) in E. coli and characterization of Cys7 to Ser mutant protein.
Auteurs : C A Padilla [Suède] ; G. Spyrou ; A. HolmgrenSource :
- FEBS letters [ 0014-5793 ] ; 1996.
Descripteurs français
- KwdFr :
- Cystéine (MeSH), Données de séquences moléculaires (MeSH), Escherichia coli (génétique), Expression des gènes (MeSH), Glutarédoxines (MeSH), Humains (MeSH), Mutagenèse dirigée (MeSH), Oxidoreductases (MeSH), Oxydoréduction (MeSH), Protéines (composition chimique), Protéines (génétique), Protéines (métabolisme), Protéines recombinantes (isolement et purification), Protéines recombinantes (métabolisme), Relation structure-activité (MeSH), Structures macromoléculaires (MeSH), Séquence d'acides aminés (MeSH), Séquence nucléotidique (MeSH), Sérine (MeSH), Transfection (MeSH), Vecteurs génétiques (MeSH).
- MESH :
- composition chimique : Protéines.
- génétique : Escherichia coli, Protéines.
- isolement et purification : Protéines recombinantes.
- métabolisme : Protéines, Protéines recombinantes.
- Cystéine, Données de séquences moléculaires, Expression des gènes, Glutarédoxines, Humains, Mutagenèse dirigée, Oxidoreductases, Oxydoréduction, Relation structure-activité, Structures macromoléculaires, Séquence d'acides aminés, Séquence nucléotidique, Sérine, Transfection, Vecteurs génétiques.
English descriptors
- KwdEn :
- Amino Acid Sequence (MeSH), Base Sequence (MeSH), Cysteine (MeSH), Escherichia coli (genetics), Gene Expression (MeSH), Genetic Vectors (MeSH), Glutaredoxins (MeSH), Humans (MeSH), Macromolecular Substances (MeSH), Molecular Sequence Data (MeSH), Mutagenesis, Site-Directed (MeSH), Oxidation-Reduction (MeSH), Oxidoreductases (MeSH), Proteins (chemistry), Proteins (genetics), Proteins (metabolism), Recombinant Proteins (isolation & purification), Recombinant Proteins (metabolism), Serine (MeSH), Structure-Activity Relationship (MeSH), Transfection (MeSH).
- MESH :
- chemical , chemistry : Proteins.
- chemical , genetics : Proteins.
- chemical , isolation & purification : Recombinant Proteins.
- chemical , metabolism : Proteins, Recombinant Proteins.
- chemical : Cysteine, Glutaredoxins, Macromolecular Substances, Oxidoreductases, Serine.
- genetics : Escherichia coli.
- Amino Acid Sequence, Base Sequence, Gene Expression, Genetic Vectors, Humans, Molecular Sequence Data, Mutagenesis, Site-Directed, Oxidation-Reduction, Structure-Activity Relationship, Transfection.
Abstract
Glutaredoxin (Grx) (12 kDa) is a hydrogen donor for ribonucleotide reductase and also a general GSH-disulfide reductase of importance for redox regulation. To overexpress human glutaredoxin in Escherichia coli, a cDNA encoding human Grx was modified and cloned into the vector pET-3d and expressed in E. coli BL21 (DE3) by IPTG induction. High-level expression of Grx was verified by GSH-disulfide oxidoreductase activity, SDS-PAGE and immunoblotting analysis. The recombinant human Grx in its reduced form was purified to homogenity with 50% yield and exhibited the same dehydroascorbate reductase and hydrogen donor activity for ribonucleotide reductase (Km approximately 0.2 microM) as the human placenta protein. Human Grx contains a total of 5 half-cystine residues including a non-conserved Cys7 residue and is easily oxidized to form dimers during storage. A Grx mutant Cys7 to Ser was generated by site-directed mutagenesis and the protein was purified to homogeneity. The mutant protein showed full activity and exhibited a much reduced tendency to form dimers compared with the wild type protein. Peptide sequencing confirmed the mutation and removal of the N-terminal Met residue in both wild type and mutant proteins. Fluorescence spectra demonstrated only tyrosine fluorescence in human Grx with a peak at 310 nm which increased 20% upon reduction and decreased by addition of GSSG demonstrating that glutathione-containing disulfides are excellent substrates.
DOI: 10.1016/0014-5793(95)01413-6
PubMed: 8549805
Affiliations:
Links toward previous steps (curation, corpus...)
Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">High-level expression of fully active human glutaredoxin (thioltransferase) in E. coli and characterization of Cys7 to Ser mutant protein.</title><author><name sortKey="Padilla, C A" sort="Padilla, C A" uniqKey="Padilla C" first="C A" last="Padilla">C A Padilla</name><affiliation wicri:level="3"><nlm:affiliation>Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.</nlm:affiliation><country xml:lang="fr">Suède</country><wicri:regionArea>Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm</wicri:regionArea><placeName><settlement type="city">Stockholm</settlement><region nuts="2">Svealand</region></placeName></affiliation></author><author><name sortKey="Spyrou, G" sort="Spyrou, G" uniqKey="Spyrou G" first="G" last="Spyrou">G. Spyrou</name></author><author><name sortKey="Holmgren, A" sort="Holmgren, A" uniqKey="Holmgren A" first="A" last="Holmgren">A. Holmgren</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="1996">1996</date><idno type="RBID">pubmed:8549805</idno><idno type="pmid">8549805</idno><idno type="doi">10.1016/0014-5793(95)01413-6</idno><idno type="wicri:Area/Main/Corpus">001205</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Corpus" wicri:corpus="PubMed">001205</idno><idno type="wicri:Area/Main/Curation">001205</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Curation">001205</idno><idno type="wicri:Area/Main/Exploration">001205</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">High-level expression of fully active human glutaredoxin (thioltransferase) in E. coli and characterization of Cys7 to Ser mutant protein.</title><author><name sortKey="Padilla, C A" sort="Padilla, C A" uniqKey="Padilla C" first="C A" last="Padilla">C A Padilla</name><affiliation wicri:level="3"><nlm:affiliation>Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.</nlm:affiliation><country xml:lang="fr">Suède</country><wicri:regionArea>Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm</wicri:regionArea><placeName><settlement type="city">Stockholm</settlement><region nuts="2">Svealand</region></placeName></affiliation></author><author><name sortKey="Spyrou, G" sort="Spyrou, G" uniqKey="Spyrou G" first="G" last="Spyrou">G. Spyrou</name></author><author><name sortKey="Holmgren, A" sort="Holmgren, A" uniqKey="Holmgren A" first="A" last="Holmgren">A. Holmgren</name></author></analytic><series><title level="j">FEBS letters</title><idno type="ISSN">0014-5793</idno><imprint><date when="1996" type="published">1996</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Amino Acid Sequence (MeSH)</term><term>Base Sequence (MeSH)</term><term>Cysteine (MeSH)</term><term>Escherichia coli (genetics)</term><term>Gene Expression (MeSH)</term><term>Genetic Vectors (MeSH)</term><term>Glutaredoxins (MeSH)</term><term>Humans (MeSH)</term><term>Macromolecular Substances (MeSH)</term><term>Molecular Sequence Data (MeSH)</term><term>Mutagenesis, Site-Directed (MeSH)</term><term>Oxidation-Reduction (MeSH)</term><term>Oxidoreductases (MeSH)</term><term>Proteins (chemistry)</term><term>Proteins (genetics)</term><term>Proteins (metabolism)</term><term>Recombinant Proteins (isolation & purification)</term><term>Recombinant Proteins (metabolism)</term><term>Serine (MeSH)</term><term>Structure-Activity Relationship (MeSH)</term><term>Transfection (MeSH)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Cystéine (MeSH)</term><term>Données de séquences moléculaires (MeSH)</term><term>Escherichia coli (génétique)</term><term>Expression des gènes (MeSH)</term><term>Glutarédoxines (MeSH)</term><term>Humains (MeSH)</term><term>Mutagenèse dirigée (MeSH)</term><term>Oxidoreductases (MeSH)</term><term>Oxydoréduction (MeSH)</term><term>Protéines (composition chimique)</term><term>Protéines (génétique)</term><term>Protéines (métabolisme)</term><term>Protéines recombinantes (isolement et purification)</term><term>Protéines recombinantes (métabolisme)</term><term>Relation structure-activité (MeSH)</term><term>Structures macromoléculaires (MeSH)</term><term>Séquence d'acides aminés (MeSH)</term><term>Séquence nucléotidique (MeSH)</term><term>Sérine (MeSH)</term><term>Transfection (MeSH)</term><term>Vecteurs génétiques (MeSH)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Proteins</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>Cysteine</term><term>Glutaredoxins</term><term>Macromolecular Substances</term><term>Oxidoreductases</term><term>Serine</term></keywords><keywords scheme="MESH" qualifier="composition chimique" xml:lang="fr"><term>Protéines</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Escherichia coli</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Escherichia coli</term><term>Protéines</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Protéines recombinantes</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>Amino Acid Sequence</term><term>Base Sequence</term><term>Gene Expression</term><term>Genetic Vectors</term><term>Humans</term><term>Molecular Sequence Data</term><term>Mutagenesis, Site-Directed</term><term>Oxidation-Reduction</term><term>Structure-Activity Relationship</term><term>Transfection</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Cystéine</term><term>Données de séquences moléculaires</term><term>Expression des gènes</term><term>Glutarédoxines</term><term>Humains</term><term>Mutagenèse dirigée</term><term>Oxidoreductases</term><term>Oxydoréduction</term><term>Relation structure-activité</term><term>Structures macromoléculaires</term><term>Séquence d'acides aminés</term><term>Séquence nucléotidique</term><term>Sérine</term><term>Transfection</term><term>Vecteurs génétiques</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Glutaredoxin (Grx) (12 kDa) is a hydrogen donor for ribonucleotide reductase and also a general GSH-disulfide reductase of importance for redox regulation. To overexpress human glutaredoxin in Escherichia coli, a cDNA encoding human Grx was modified and cloned into the vector pET-3d and expressed in E. coli BL21 (DE3) by IPTG induction. High-level expression of Grx was verified by GSH-disulfide oxidoreductase activity, SDS-PAGE and immunoblotting analysis. The recombinant human Grx in its reduced form was purified to homogenity with 50% yield and exhibited the same dehydroascorbate reductase and hydrogen donor activity for ribonucleotide reductase (Km approximately 0.2 microM) as the human placenta protein. Human Grx contains a total of 5 half-cystine residues including a non-conserved Cys7 residue and is easily oxidized to form dimers during storage. A Grx mutant Cys7 to Ser was generated by site-directed mutagenesis and the protein was purified to homogeneity. The mutant protein showed full activity and exhibited a much reduced tendency to form dimers compared with the wild type protein. Peptide sequencing confirmed the mutation and removal of the N-terminal Met residue in both wild type and mutant proteins. Fluorescence spectra demonstrated only tyrosine fluorescence in human Grx with a peak at 310 nm which increased 20% upon reduction and decreased by addition of GSSG demonstrating that glutathione-containing disulfides are excellent substrates.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">8549805</PMID><DateCompleted><Year>1996</Year><Month>02</Month><Day>22</Day></DateCompleted><DateRevised><Year>2019</Year><Month>06</Month><Day>21</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0014-5793</ISSN><JournalIssue CitedMedium="Print"><Volume>378</Volume><Issue>1</Issue><PubDate><Year>1996</Year><Month>Jan</Month><Day>02</Day></PubDate></JournalIssue><Title>FEBS letters</Title><ISOAbbreviation>FEBS Lett</ISOAbbreviation></Journal><ArticleTitle>High-level expression of fully active human glutaredoxin (thioltransferase) in E. coli and characterization of Cys7 to Ser mutant protein.</ArticleTitle><Pagination><MedlinePgn>69-73</MedlinePgn></Pagination><Abstract><AbstractText>Glutaredoxin (Grx) (12 kDa) is a hydrogen donor for ribonucleotide reductase and also a general GSH-disulfide reductase of importance for redox regulation. To overexpress human glutaredoxin in Escherichia coli, a cDNA encoding human Grx was modified and cloned into the vector pET-3d and expressed in E. coli BL21 (DE3) by IPTG induction. High-level expression of Grx was verified by GSH-disulfide oxidoreductase activity, SDS-PAGE and immunoblotting analysis. The recombinant human Grx in its reduced form was purified to homogenity with 50% yield and exhibited the same dehydroascorbate reductase and hydrogen donor activity for ribonucleotide reductase (Km approximately 0.2 microM) as the human placenta protein. Human Grx contains a total of 5 half-cystine residues including a non-conserved Cys7 residue and is easily oxidized to form dimers during storage. A Grx mutant Cys7 to Ser was generated by site-directed mutagenesis and the protein was purified to homogeneity. The mutant protein showed full activity and exhibited a much reduced tendency to form dimers compared with the wild type protein. Peptide sequencing confirmed the mutation and removal of the N-terminal Met residue in both wild type and mutant proteins. Fluorescence spectra demonstrated only tyrosine fluorescence in human Grx with a peak at 310 nm which increased 20% upon reduction and decreased by addition of GSSG demonstrating that glutathione-containing disulfides are excellent substrates.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Padilla</LastName><ForeName>C A</ForeName><Initials>CA</Initials><AffiliationInfo><Affiliation>Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Spyrou</LastName><ForeName>G</ForeName><Initials>G</Initials></Author><Author ValidYN="Y"><LastName>Holmgren</LastName><ForeName>A</ForeName><Initials>A</Initials></Author></AuthorList><Language>eng</Language><DataBankList CompleteYN="Y"><DataBank><DataBankName>GENBANK</DataBankName><AccessionNumberList><AccessionNumber>X76648</AccessionNumber></AccessionNumberList></DataBank></DataBankList><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>FEBS Lett</MedlineTA><NlmUniqueID>0155157</NlmUniqueID><ISSNLinking>0014-5793</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="C516005">GLRX protein, human</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D054477">Glutaredoxins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D046911">Macromolecular Substances</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>452VLY9402</RegistryNumber><NameOfSubstance UI="D012694">Serine</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 1.-</RegistryNumber><NameOfSubstance UI="D010088">Oxidoreductases</NameOfSubstance></Chemical><Chemical><RegistryNumber>K848JZ4886</RegistryNumber><NameOfSubstance UI="D003545">Cysteine</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="D003545" MajorTopicYN="Y">Cysteine</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D004926" MajorTopicYN="N">Escherichia coli</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D015870" MajorTopicYN="Y">Gene Expression</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D005822" MajorTopicYN="N">Genetic Vectors</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D054477" MajorTopicYN="N">Glutaredoxins</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D006801" MajorTopicYN="N">Humans</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D046911" MajorTopicYN="N">Macromolecular Substances</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D008969" MajorTopicYN="N">Molecular Sequence Data</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D016297" MajorTopicYN="Y">Mutagenesis, Site-Directed</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010084" MajorTopicYN="N">Oxidation-Reduction</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010088" MajorTopicYN="Y">Oxidoreductases</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D011506" MajorTopicYN="N">Proteins</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</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="Q000302" MajorTopicYN="N">isolation & purification</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D012694" MajorTopicYN="Y">Serine</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D013329" MajorTopicYN="N">Structure-Activity Relationship</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D014162" MajorTopicYN="N">Transfection</DescriptorName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>1996</Year><Month>1</Month><Day>2</Day></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>1996</Year><Month>1</Month><Day>2</Day><Hour>0</Hour><Minute>1</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>1996</Year><Month>1</Month><Day>2</Day><Hour>0</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">8549805</ArticleId><ArticleId IdType="pii">0014579395014136</ArticleId><ArticleId IdType="doi">10.1016/0014-5793(95)01413-6</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>Suède</li></country><region><li>Svealand</li></region><settlement><li>Stockholm</li></settlement></list><tree><noCountry><name sortKey="Holmgren, A" sort="Holmgren, A" uniqKey="Holmgren A" first="A" last="Holmgren">A. Holmgren</name><name sortKey="Spyrou, G" sort="Spyrou, G" uniqKey="Spyrou G" first="G" last="Spyrou">G. Spyrou</name></noCountry><country name="Suède"><region name="Svealand"><name sortKey="Padilla, C A" sort="Padilla, C A" uniqKey="Padilla C" first="C A" last="Padilla">C A Padilla</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Bois/explor/GlutaredoxinV1/Data/Main/Exploration
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 001203 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Main/Exploration/biblio.hfd -nk 001203 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Bois
|area= GlutaredoxinV1
|flux= Main
|étape= Exploration
|type= RBID
|clé= pubmed:8549805
|texte= High-level expression of fully active human glutaredoxin (thioltransferase) in E. coli and characterization of Cys7 to Ser mutant protein.
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Main/Exploration/RBID.i -Sk "pubmed:8549805" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Main/Exploration/biblio.hfd \
| NlmPubMed2Wicri -a GlutaredoxinV1
| This area was generated with Dilib version V0.6.37. |  |