Cloning, expression, purification, and characterisation of the HEAT-repeat domain of TOR from the thermophilic eukaryote Chaetomium thermophilum.
Identifieur interne : 000892 ( Main/Exploration ); précédent : 000891; suivant : 000893Cloning, expression, purification, and characterisation of the HEAT-repeat domain of TOR from the thermophilic eukaryote Chaetomium thermophilum.
Auteurs : Graham C. Robinson [Suisse] ; Yogesh Vegunta [Suisse] ; Caroline Gabus [Suisse] ; Christl Gaubitz [Suisse] ; Stéphane Thore [France]Source :
- Protein expression and purification [ 1096-0279 ] ; 2017.
Descripteurs français
- KwdFr :
- Chaetomium (enzymologie), Chaetomium (génétique), Clonage moléculaire (MeSH), Domaines protéiques (MeSH), Expression des gènes (MeSH), Protéines fongiques (biosynthèse), Protéines fongiques (composition chimique), Protéines fongiques (génétique), Protéines fongiques (isolement et purification), Protéines recombinantes (MeSH), Séquences répétées d'acides aminés (MeSH), Sérine-thréonine kinases TOR (biosynthèse), Sérine-thréonine kinases TOR (composition chimique), Sérine-thréonine kinases TOR (génétique), Sérine-thréonine kinases TOR (isolement et purification).
- MESH :
- biosynthèse : Protéines fongiques, Sérine-thréonine kinases TOR.
- composition chimique : Protéines fongiques, Sérine-thréonine kinases TOR.
- enzymologie : Chaetomium.
- génétique : Chaetomium, Protéines fongiques, Sérine-thréonine kinases TOR.
- isolement et purification : Protéines fongiques, Sérine-thréonine kinases TOR.
- Clonage moléculaire, Domaines protéiques, Expression des gènes, Protéines recombinantes, Séquences répétées d'acides aminés.
English descriptors
- KwdEn :
- Chaetomium (enzymology), Chaetomium (genetics), Cloning, Molecular (MeSH), Fungal Proteins (biosynthesis), Fungal Proteins (chemistry), Fungal Proteins (genetics), Fungal Proteins (isolation & purification), Gene Expression (MeSH), Protein Domains (MeSH), Recombinant Proteins (MeSH), Repetitive Sequences, Amino Acid (MeSH), TOR Serine-Threonine Kinases (biosynthesis), TOR Serine-Threonine Kinases (chemistry), TOR Serine-Threonine Kinases (genetics), TOR Serine-Threonine Kinases (isolation & purification).
- MESH :
- chemical , biosynthesis : Fungal Proteins, TOR Serine-Threonine Kinases.
- chemical , chemistry : Fungal Proteins, TOR Serine-Threonine Kinases.
- enzymology : Chaetomium.
- genetics : Chaetomium, Fungal Proteins, TOR Serine-Threonine Kinases.
- chemical , isolation & purification : Fungal Proteins, TOR Serine-Threonine Kinases.
- Cloning, Molecular, Gene Expression, Protein Domains, Recombinant Proteins, Repetitive Sequences, Amino Acid.
Abstract
The Target of Rapamycin Complex is a central controller of cell growth and differentiation in eukaryotes. Its global architecture has been described by cryoelectron microscopy, and regions of its central TOR protein have been described by X-ray crystallography. However, the N-terminal region of this protein, which consists of a series of HEAT repeats, remains uncharacterised at high resolution, most likely due to the absence of a suitable purification procedure. Here, we present a robust method for the preparation of the HEAT-repeat domain, utilizing the thermophilic fungus Chaetomium thermophilum as a source organism. We describe construct design and stable expression in insect cells. An efficient two-step purification procedure is presented, and the purified product is characterised by SEC and MALDI-TOF MS. The methods described pave the way for a complete high-resolution characterisation of this elusive region of the TOR protein.
DOI: 10.1016/j.pep.2017.03.007
PubMed: 28284995
Affiliations:
- France, Suisse
- Aquitaine, Canton de Genève, Nouvelle-Aquitaine
- Bordeaux, Genève
- Université de Genève
Links toward previous steps (curation, corpus...)
Le document en format XML
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Robinson</name><affiliation wicri:level="4"><nlm:affiliation>Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva, Switzerland.</nlm:affiliation><country xml:lang="fr">Suisse</country><wicri:regionArea>Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva</wicri:regionArea><orgName type="university">Université de Genève</orgName><placeName><settlement type="city">Genève</settlement><region nuts="3" type="region">Canton de Genève</region></placeName></affiliation></author><author><name sortKey="Vegunta, Yogesh" sort="Vegunta, Yogesh" uniqKey="Vegunta Y" first="Yogesh" last="Vegunta">Yogesh Vegunta</name><affiliation wicri:level="4"><nlm:affiliation>Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva, Switzerland.</nlm:affiliation><country xml:lang="fr">Suisse</country><wicri:regionArea>Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva</wicri:regionArea><orgName type="university">Université de Genève</orgName><placeName><settlement type="city">Genève</settlement><region nuts="3" type="region">Canton de Genève</region></placeName></affiliation></author><author><name sortKey="Gabus, Caroline" sort="Gabus, Caroline" uniqKey="Gabus C" first="Caroline" last="Gabus">Caroline Gabus</name><affiliation wicri:level="4"><nlm:affiliation>Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva, Switzerland.</nlm:affiliation><country xml:lang="fr">Suisse</country><wicri:regionArea>Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva</wicri:regionArea><orgName type="university">Université de Genève</orgName><placeName><settlement type="city">Genève</settlement><region nuts="3" type="region">Canton de Genève</region></placeName></affiliation></author><author><name sortKey="Gaubitz, Christl" sort="Gaubitz, Christl" uniqKey="Gaubitz C" first="Christl" last="Gaubitz">Christl Gaubitz</name><affiliation wicri:level="4"><nlm:affiliation>Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva, Switzerland.</nlm:affiliation><country xml:lang="fr">Suisse</country><wicri:regionArea>Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva</wicri:regionArea><orgName type="university">Université de Genève</orgName><placeName><settlement type="city">Genève</settlement><region nuts="3" type="region">Canton de Genève</region></placeName></affiliation></author><author><name sortKey="Thore, Stephane" sort="Thore, Stephane" uniqKey="Thore S" first="Stéphane" last="Thore">Stéphane Thore</name><affiliation wicri:level="4"><nlm:affiliation>Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva, Switzerland; INSERM U-1212, CNRS UMR-5320, Université de Bordeaux, 146 rue Léo Saignat, 33000 Bordeaux, France. Electronic address: stephane.thore@inserm.fr.</nlm:affiliation><country xml:lang="fr">France</country><wicri:regionArea>Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva, Switzerland; INSERM U-1212, CNRS UMR-5320, Université de Bordeaux, 146 rue Léo Saignat, 33000 Bordeaux</wicri:regionArea><placeName><region type="region" nuts="2">Nouvelle-Aquitaine</region><region type="old region" nuts="2">Aquitaine</region><settlement type="city">Bordeaux</settlement></placeName><orgName type="university">Université de Genève</orgName></affiliation></author></analytic><series><title level="j">Protein expression and purification</title><idno type="eISSN">1096-0279</idno><imprint><date when="2017" type="published">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Chaetomium (enzymology)</term><term>Chaetomium (genetics)</term><term>Cloning, Molecular (MeSH)</term><term>Fungal Proteins (biosynthesis)</term><term>Fungal Proteins (chemistry)</term><term>Fungal Proteins (genetics)</term><term>Fungal Proteins (isolation & purification)</term><term>Gene Expression (MeSH)</term><term>Protein Domains (MeSH)</term><term>Recombinant Proteins (MeSH)</term><term>Repetitive Sequences, Amino Acid (MeSH)</term><term>TOR Serine-Threonine Kinases (biosynthesis)</term><term>TOR Serine-Threonine Kinases (chemistry)</term><term>TOR Serine-Threonine Kinases (genetics)</term><term>TOR Serine-Threonine Kinases (isolation & purification)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Chaetomium (enzymologie)</term><term>Chaetomium (génétique)</term><term>Clonage moléculaire (MeSH)</term><term>Domaines protéiques (MeSH)</term><term>Expression des gènes (MeSH)</term><term>Protéines fongiques (biosynthèse)</term><term>Protéines fongiques (composition chimique)</term><term>Protéines fongiques (génétique)</term><term>Protéines fongiques (isolement et purification)</term><term>Protéines recombinantes (MeSH)</term><term>Séquences répétées d'acides aminés (MeSH)</term><term>Sérine-thréonine kinases TOR (biosynthèse)</term><term>Sérine-thréonine kinases TOR (composition chimique)</term><term>Sérine-thréonine kinases TOR (génétique)</term><term>Sérine-thréonine kinases TOR (isolement et purification)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>Fungal Proteins</term><term>TOR Serine-Threonine Kinases</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Fungal Proteins</term><term>TOR Serine-Threonine Kinases</term></keywords><keywords scheme="MESH" qualifier="biosynthèse" xml:lang="fr"><term>Protéines fongiques</term><term>Sérine-thréonine kinases TOR</term></keywords><keywords scheme="MESH" qualifier="composition chimique" xml:lang="fr"><term>Protéines fongiques</term><term>Sérine-thréonine kinases TOR</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Chaetomium</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Chaetomium</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Chaetomium</term><term>Fungal Proteins</term><term>TOR Serine-Threonine Kinases</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Chaetomium</term><term>Protéines fongiques</term><term>Sérine-thréonine kinases TOR</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Fungal Proteins</term><term>TOR Serine-Threonine Kinases</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Protéines fongiques</term><term>Sérine-thréonine kinases TOR</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Cloning, Molecular</term><term>Gene Expression</term><term>Protein Domains</term><term>Recombinant Proteins</term><term>Repetitive Sequences, Amino Acid</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Clonage moléculaire</term><term>Domaines protéiques</term><term>Expression des gènes</term><term>Protéines recombinantes</term><term>Séquences répétées d'acides aminés</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The Target of Rapamycin Complex is a central controller of cell growth and differentiation in eukaryotes. Its global architecture has been described by cryoelectron microscopy, and regions of its central TOR protein have been described by X-ray crystallography. However, the N-terminal region of this protein, which consists of a series of HEAT repeats, remains uncharacterised at high resolution, most likely due to the absence of a suitable purification procedure. Here, we present a robust method for the preparation of the HEAT-repeat domain, utilizing the thermophilic fungus Chaetomium thermophilum as a source organism. We describe construct design and stable expression in insect cells. An efficient two-step purification procedure is presented, and the purified product is characterised by SEC and MALDI-TOF MS. The methods described pave the way for a complete high-resolution characterisation of this elusive region of the TOR protein.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">28284995</PMID><DateCompleted><Year>2017</Year><Month>07</Month><Day>17</Day></DateCompleted><DateRevised><Year>2018</Year><Month>11</Month><Day>01</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">1096-0279</ISSN><JournalIssue CitedMedium="Internet"><Volume>133</Volume><PubDate><Year>2017</Year><Month>05</Month></PubDate></JournalIssue><Title>Protein expression and purification</Title><ISOAbbreviation>Protein Expr Purif</ISOAbbreviation></Journal><ArticleTitle>Cloning, expression, purification, and characterisation of the HEAT-repeat domain of TOR from the thermophilic eukaryote Chaetomium thermophilum.</ArticleTitle><Pagination><MedlinePgn>90-95</MedlinePgn></Pagination><ELocationID EIdType="pii" ValidYN="Y">S1046-5928(16)30311-4</ELocationID><ELocationID EIdType="doi" ValidYN="Y">10.1016/j.pep.2017.03.007</ELocationID><Abstract><AbstractText>The Target of Rapamycin Complex is a central controller of cell growth and differentiation in eukaryotes. Its global architecture has been described by cryoelectron microscopy, and regions of its central TOR protein have been described by X-ray crystallography. However, the N-terminal region of this protein, which consists of a series of HEAT repeats, remains uncharacterised at high resolution, most likely due to the absence of a suitable purification procedure. Here, we present a robust method for the preparation of the HEAT-repeat domain, utilizing the thermophilic fungus Chaetomium thermophilum as a source organism. We describe construct design and stable expression in insect cells. An efficient two-step purification procedure is presented, and the purified product is characterised by SEC and MALDI-TOF MS. The methods described pave the way for a complete high-resolution characterisation of this elusive region of the TOR protein.</AbstractText><CopyrightInformation>Copyright © 2017 Elsevier Inc. All rights reserved.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Robinson</LastName><ForeName>Graham C</ForeName><Initials>GC</Initials><AffiliationInfo><Affiliation>Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva, Switzerland.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Vegunta</LastName><ForeName>Yogesh</ForeName><Initials>Y</Initials><AffiliationInfo><Affiliation>Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva, Switzerland.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Gabus</LastName><ForeName>Caroline</ForeName><Initials>C</Initials><AffiliationInfo><Affiliation>Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva, Switzerland.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Gaubitz</LastName><ForeName>Christl</ForeName><Initials>C</Initials><AffiliationInfo><Affiliation>Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva, Switzerland.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Thore</LastName><ForeName>Stéphane</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva, Switzerland; INSERM U-1212, CNRS UMR-5320, Université de Bordeaux, 146 rue Léo Saignat, 33000 Bordeaux, France. Electronic address: stephane.thore@inserm.fr.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2017</Year><Month>03</Month><Day>08</Day></ArticleDate></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="D005656">Fungal Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D011994">Recombinant Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 2.7.1.1</RegistryNumber><NameOfSubstance UI="D058570">TOR Serine-Threonine Kinases</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D002597" MajorTopicYN="Y">Chaetomium</DescriptorName><QualifierName UI="Q000201" MajorTopicYN="N">enzymology</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D003001" MajorTopicYN="Y">Cloning, Molecular</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D005656" MajorTopicYN="Y">Fungal Proteins</DescriptorName><QualifierName UI="Q000096" MajorTopicYN="N">biosynthesis</QualifierName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000302" MajorTopicYN="N">isolation & purification</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D015870" MajorTopicYN="Y">Gene Expression</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D000072417" MajorTopicYN="N">Protein Domains</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D011994" MajorTopicYN="N">Recombinant Proteins</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D020449" MajorTopicYN="N">Repetitive Sequences, Amino Acid</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D058570" MajorTopicYN="N">TOR Serine-Threonine Kinases</DescriptorName><QualifierName UI="Q000096" MajorTopicYN="N">biosynthesis</QualifierName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000302" MajorTopicYN="N">isolation & purification</QualifierName></MeshHeading></MeshHeadingList><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="Y">Chaetomium thermophilum</Keyword><Keyword MajorTopicYN="Y">Mass spectrometry</Keyword><Keyword MajorTopicYN="Y">MultiBac expression system</Keyword><Keyword MajorTopicYN="Y">Purification</Keyword><Keyword MajorTopicYN="Y">TOR</Keyword><Keyword MajorTopicYN="Y">Target of rapamycin signalling</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2016</Year><Month>10</Month><Day>17</Day></PubMedPubDate><PubMedPubDate PubStatus="revised"><Year>2017</Year><Month>03</Month><Day>02</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2017</Year><Month>03</Month><Day>06</Day></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2017</Year><Month>3</Month><Day>13</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2017</Year><Month>7</Month><Day>18</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2017</Year><Month>3</Month><Day>13</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">28284995</ArticleId><ArticleId IdType="pii">S1046-5928(16)30311-4</ArticleId><ArticleId IdType="doi">10.1016/j.pep.2017.03.007</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>France</li><li>Suisse</li></country><region><li>Aquitaine</li><li>Canton de Genève</li><li>Nouvelle-Aquitaine</li></region><settlement><li>Bordeaux</li><li>Genève</li></settlement><orgName><li>Université de Genève</li></orgName></list><tree><country name="Suisse"><region name="Canton de Genève"><name sortKey="Robinson, Graham C" sort="Robinson, Graham C" uniqKey="Robinson G" first="Graham C" last="Robinson">Graham C. Robinson</name></region><name sortKey="Gabus, Caroline" sort="Gabus, Caroline" uniqKey="Gabus C" first="Caroline" last="Gabus">Caroline Gabus</name><name sortKey="Gaubitz, Christl" sort="Gaubitz, Christl" uniqKey="Gaubitz C" first="Christl" last="Gaubitz">Christl Gaubitz</name><name sortKey="Vegunta, Yogesh" sort="Vegunta, Yogesh" uniqKey="Vegunta Y" first="Yogesh" last="Vegunta">Yogesh Vegunta</name></country><country name="France"><region name="Nouvelle-Aquitaine"><name sortKey="Thore, Stephane" sort="Thore, Stephane" uniqKey="Thore S" first="Stéphane" last="Thore">Stéphane Thore</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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