Expression, purification, and characterization of SARS coronavirus RNA polymerase.
Identifieur interne : 004832 ( Main/Exploration ); précédent : 004831; suivant : 004833Expression, purification, and characterization of SARS coronavirus RNA polymerase.
Auteurs : Ao Cheng [République populaire de Chine] ; Wei Zhang ; Youhua Xie ; Weihong Jiang ; Eddy Arnold ; Stefan G. Sarafianos ; Jianping DingSource :
- Virology [ 0042-6822 ] ; 2005.
Descripteurs français
- KwdFr :
- Agents antiVIH (pharmacologie), Clonage moléculaire, Domaine catalytique, Escherichia coli (génétique), Expression des gènes, Fragments peptidiques (), Fragments peptidiques (génétique), Fragments peptidiques (isolement et purification), Fragments peptidiques (métabolisme), Inhibiteurs de la transcriptase inverse (pharmacologie), Protéines recombinantes (), Protéines recombinantes (génétique), Protéines recombinantes (isolement et purification), Protéines recombinantes (métabolisme), RNA replicase (), RNA replicase (génétique), RNA replicase (isolement et purification), RNA replicase (métabolisme), Syndrome respiratoire aigu sévère (virologie), Virus du SRAS (enzymologie).
- MESH :
- enzymologie : Virus du SRAS.
- génétique : Escherichia coli, Fragments peptidiques, Protéines recombinantes, RNA replicase.
- isolement et purification : Fragments peptidiques, Protéines recombinantes, RNA replicase.
- métabolisme : Fragments peptidiques, Protéines recombinantes, RNA replicase.
- pharmacologie : Agents antiVIH, Inhibiteurs de la transcriptase inverse.
- virologie : Syndrome respiratoire aigu sévère.
- Clonage moléculaire, Domaine catalytique, Expression des gènes, Fragments peptidiques, Protéines recombinantes, RNA replicase.
English descriptors
- KwdEn :
- Anti-HIV Agents (pharmacology), Catalytic Domain, Cloning, Molecular, Escherichia coli (genetics), Gene Expression, Peptide Fragments (chemistry), Peptide Fragments (genetics), Peptide Fragments (isolation & purification), Peptide Fragments (metabolism), RNA Replicase (chemistry), RNA Replicase (genetics), RNA Replicase (isolation & purification), RNA Replicase (metabolism), Recombinant Proteins (chemistry), Recombinant Proteins (genetics), Recombinant Proteins (isolation & purification), Recombinant Proteins (metabolism), Reverse Transcriptase Inhibitors (pharmacology), SARS Virus (enzymology), Severe Acute Respiratory Syndrome (virology).
- MESH :
- chemical , chemistry : Peptide Fragments, RNA Replicase, Recombinant Proteins.
- chemical , genetics : Peptide Fragments, RNA Replicase, Recombinant Proteins.
- chemical , isolation & purification : Peptide Fragments, RNA Replicase, Recombinant Proteins.
- chemical , metabolism : Peptide Fragments, RNA Replicase, Recombinant Proteins.
- chemical , pharmacology : Anti-HIV Agents, Reverse Transcriptase Inhibitors.
- enzymology : SARS Virus.
- genetics : Escherichia coli.
- virology : Severe Acute Respiratory Syndrome.
- Catalytic Domain, Cloning, Molecular, Gene Expression.
Abstract
The RNA-dependent RNA polymerase (RdRp) of SARS coronavirus (SARS-CoV) is essential for viral replication and a potential target for anti-SARS drugs. We report here the cloning, expression, and purification of the N-terminal GST-fused SARS-CoV RdRp and its polymerase catalytic domain in Escherichia coli. During purification, the full-length GST-RdRp was found to cleave into three main fragments: an N-terminal p12 fragment, a middle p30 fragment, and a C-terminal p64 fragment comprising the catalytic domain, presumably due to bacterial proteases. Biochemical assays show that the full-length GST-RdRp has RdRp activity and the p64 and p12 fragments form a complex that exhibits comparable RdRp activity, whereas the GST-p64 protein has no activity, suggesting that the p12 domain is required for polymerase activity possibly via involvement in template-primer binding. Nonnucleoside HIV-1 RT inhibitors are shown to have no evident inhibitory effect on SARS-CoV RdRp activity. This work provides a basis for biochemical and structural studies of SARS-CoV RdRp and for development of anti-SARS drugs.
DOI: 10.1016/j.virol.2005.02.017
PubMed: 15840516
Affiliations:
Links toward previous steps (curation, corpus...)
- to stream PubMed, to step Corpus: 002780
- to stream PubMed, to step Curation: 002780
- to stream PubMed, to step Checkpoint: 002709
- to stream Ncbi, to step Merge: 000F06
- to stream Ncbi, to step Curation: 000F06
- to stream Ncbi, to step Checkpoint: 000F06
- to stream Main, to step Merge: 004B27
- to stream Main, to step Curation: 004832
Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Expression, purification, and characterization of SARS coronavirus RNA polymerase.</title><author><name sortKey="Cheng, Ao" sort="Cheng, Ao" uniqKey="Cheng A" first="Ao" last="Cheng">Ao Cheng</name><affiliation wicri:level="1"><nlm:affiliation>Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031</wicri:regionArea><wicri:noRegion>Shanghai 200031</wicri:noRegion></affiliation></author><author><name sortKey="Zhang, Wei" sort="Zhang, Wei" uniqKey="Zhang W" first="Wei" last="Zhang">Wei Zhang</name></author><author><name sortKey="Xie, Youhua" sort="Xie, Youhua" uniqKey="Xie Y" first="Youhua" last="Xie">Youhua Xie</name></author><author><name sortKey="Jiang, Weihong" sort="Jiang, Weihong" uniqKey="Jiang W" first="Weihong" last="Jiang">Weihong Jiang</name></author><author><name sortKey="Arnold, Eddy" sort="Arnold, Eddy" uniqKey="Arnold E" first="Eddy" last="Arnold">Eddy Arnold</name></author><author><name sortKey="Sarafianos, Stefan G" sort="Sarafianos, Stefan G" uniqKey="Sarafianos S" first="Stefan G" last="Sarafianos">Stefan G. Sarafianos</name></author><author><name sortKey="Ding, Jianping" sort="Ding, Jianping" uniqKey="Ding J" first="Jianping" last="Ding">Jianping Ding</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2005">2005</date><idno type="RBID">pubmed:15840516</idno><idno type="pmid">15840516</idno><idno type="doi">10.1016/j.virol.2005.02.017</idno><idno type="wicri:Area/PubMed/Corpus">002780</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002780</idno><idno type="wicri:Area/PubMed/Curation">002780</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">002780</idno><idno type="wicri:Area/PubMed/Checkpoint">002709</idno><idno type="wicri:explorRef" wicri:stream="Checkpoint" wicri:step="PubMed">002709</idno><idno type="wicri:Area/Ncbi/Merge">000F06</idno><idno type="wicri:Area/Ncbi/Curation">000F06</idno><idno type="wicri:Area/Ncbi/Checkpoint">000F06</idno><idno type="wicri:doubleKey">0042-6822:2005:Cheng A:expression:purification:and</idno><idno type="wicri:Area/Main/Merge">004B27</idno><idno type="wicri:Area/Main/Curation">004832</idno><idno type="wicri:Area/Main/Exploration">004832</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Expression, purification, and characterization of SARS coronavirus RNA polymerase.</title><author><name sortKey="Cheng, Ao" sort="Cheng, Ao" uniqKey="Cheng A" first="Ao" last="Cheng">Ao Cheng</name><affiliation wicri:level="1"><nlm:affiliation>Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031</wicri:regionArea><wicri:noRegion>Shanghai 200031</wicri:noRegion></affiliation></author><author><name sortKey="Zhang, Wei" sort="Zhang, Wei" uniqKey="Zhang W" first="Wei" last="Zhang">Wei Zhang</name></author><author><name sortKey="Xie, Youhua" sort="Xie, Youhua" uniqKey="Xie Y" first="Youhua" last="Xie">Youhua Xie</name></author><author><name sortKey="Jiang, Weihong" sort="Jiang, Weihong" uniqKey="Jiang W" first="Weihong" last="Jiang">Weihong Jiang</name></author><author><name sortKey="Arnold, Eddy" sort="Arnold, Eddy" uniqKey="Arnold E" first="Eddy" last="Arnold">Eddy Arnold</name></author><author><name sortKey="Sarafianos, Stefan G" sort="Sarafianos, Stefan G" uniqKey="Sarafianos S" first="Stefan G" last="Sarafianos">Stefan G. Sarafianos</name></author><author><name sortKey="Ding, Jianping" sort="Ding, Jianping" uniqKey="Ding J" first="Jianping" last="Ding">Jianping Ding</name></author></analytic><series><title level="j">Virology</title><idno type="ISSN">0042-6822</idno><imprint><date when="2005" type="published">2005</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Anti-HIV Agents (pharmacology)</term><term>Catalytic Domain</term><term>Cloning, Molecular</term><term>Escherichia coli (genetics)</term><term>Gene Expression</term><term>Peptide Fragments (chemistry)</term><term>Peptide Fragments (genetics)</term><term>Peptide Fragments (isolation & purification)</term><term>Peptide Fragments (metabolism)</term><term>RNA Replicase (chemistry)</term><term>RNA Replicase (genetics)</term><term>RNA Replicase (isolation & purification)</term><term>RNA Replicase (metabolism)</term><term>Recombinant Proteins (chemistry)</term><term>Recombinant Proteins (genetics)</term><term>Recombinant Proteins (isolation & purification)</term><term>Recombinant Proteins (metabolism)</term><term>Reverse Transcriptase Inhibitors (pharmacology)</term><term>SARS Virus (enzymology)</term><term>Severe Acute Respiratory Syndrome (virology)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Agents antiVIH (pharmacologie)</term><term>Clonage moléculaire</term><term>Domaine catalytique</term><term>Escherichia coli (génétique)</term><term>Expression des gènes</term><term>Fragments peptidiques ()</term><term>Fragments peptidiques (génétique)</term><term>Fragments peptidiques (isolement et purification)</term><term>Fragments peptidiques (métabolisme)</term><term>Inhibiteurs de la transcriptase inverse (pharmacologie)</term><term>Protéines recombinantes ()</term><term>Protéines recombinantes (génétique)</term><term>Protéines recombinantes (isolement et purification)</term><term>Protéines recombinantes (métabolisme)</term><term>RNA replicase ()</term><term>RNA replicase (génétique)</term><term>RNA replicase (isolement et purification)</term><term>RNA replicase (métabolisme)</term><term>Syndrome respiratoire aigu sévère (virologie)</term><term>Virus du SRAS (enzymologie)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Peptide Fragments</term><term>RNA Replicase</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Peptide Fragments</term><term>RNA Replicase</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Peptide Fragments</term><term>RNA Replicase</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Peptide Fragments</term><term>RNA Replicase</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="pharmacology" xml:lang="en"><term>Anti-HIV Agents</term><term>Reverse Transcriptase Inhibitors</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>SARS Virus</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>Fragments peptidiques</term><term>Protéines recombinantes</term><term>RNA replicase</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Fragments peptidiques</term><term>Protéines recombinantes</term><term>RNA replicase</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Fragments peptidiques</term><term>Protéines recombinantes</term><term>RNA replicase</term></keywords><keywords scheme="MESH" qualifier="pharmacologie" xml:lang="fr"><term>Agents antiVIH</term><term>Inhibiteurs de la transcriptase inverse</term></keywords><keywords scheme="MESH" qualifier="virologie" xml:lang="fr"><term>Syndrome respiratoire aigu sévère</term></keywords><keywords scheme="MESH" qualifier="virology" xml:lang="en"><term>Severe Acute Respiratory Syndrome</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Catalytic Domain</term><term>Cloning, Molecular</term><term>Gene Expression</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Clonage moléculaire</term><term>Domaine catalytique</term><term>Expression des gènes</term><term>Fragments peptidiques</term><term>Protéines recombinantes</term><term>RNA replicase</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The RNA-dependent RNA polymerase (RdRp) of SARS coronavirus (SARS-CoV) is essential for viral replication and a potential target for anti-SARS drugs. We report here the cloning, expression, and purification of the N-terminal GST-fused SARS-CoV RdRp and its polymerase catalytic domain in Escherichia coli. During purification, the full-length GST-RdRp was found to cleave into three main fragments: an N-terminal p12 fragment, a middle p30 fragment, and a C-terminal p64 fragment comprising the catalytic domain, presumably due to bacterial proteases. Biochemical assays show that the full-length GST-RdRp has RdRp activity and the p64 and p12 fragments form a complex that exhibits comparable RdRp activity, whereas the GST-p64 protein has no activity, suggesting that the p12 domain is required for polymerase activity possibly via involvement in template-primer binding. Nonnucleoside HIV-1 RT inhibitors are shown to have no evident inhibitory effect on SARS-CoV RdRp activity. This work provides a basis for biochemical and structural studies of SARS-CoV RdRp and for development of anti-SARS drugs.</div></front></TEI><affiliations><list><country><li>République populaire de Chine</li></country></list><tree><noCountry><name sortKey="Arnold, Eddy" sort="Arnold, Eddy" uniqKey="Arnold E" first="Eddy" last="Arnold">Eddy Arnold</name><name sortKey="Ding, Jianping" sort="Ding, Jianping" uniqKey="Ding J" first="Jianping" last="Ding">Jianping Ding</name><name sortKey="Jiang, Weihong" sort="Jiang, Weihong" uniqKey="Jiang W" first="Weihong" last="Jiang">Weihong Jiang</name><name sortKey="Sarafianos, Stefan G" sort="Sarafianos, Stefan G" uniqKey="Sarafianos S" first="Stefan G" last="Sarafianos">Stefan G. Sarafianos</name><name sortKey="Xie, Youhua" sort="Xie, Youhua" uniqKey="Xie Y" first="Youhua" last="Xie">Youhua Xie</name><name sortKey="Zhang, Wei" sort="Zhang, Wei" uniqKey="Zhang W" first="Wei" last="Zhang">Wei Zhang</name></noCountry><country name="République populaire de Chine"><noRegion><name sortKey="Cheng, Ao" sort="Cheng, Ao" uniqKey="Cheng A" first="Ao" last="Cheng">Ao Cheng</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/SrasV1/Data/Main/Exploration
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 004832 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Main/Exploration/biblio.hfd -nk 004832 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Sante
|area= SrasV1
|flux= Main
|étape= Exploration
|type= RBID
|clé= pubmed:15840516
|texte= Expression, purification, and characterization of SARS coronavirus RNA polymerase.
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Main/Exploration/RBID.i -Sk "pubmed:15840516" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Main/Exploration/biblio.hfd \
| NlmPubMed2Wicri -a SrasV1
| This area was generated with Dilib version V0.6.33. |  |