Expression, purification and sublocalization of SARS-CoV nucleocapsid protein in insect cells.
Identifieur interne : 002A74 ( PubMed/Curation ); précédent : 002A73; suivant : 002A75Expression, purification and sublocalization of SARS-CoV nucleocapsid protein in insect cells.
Auteurs : Ai-Xia Ren [République populaire de Chine] ; You-Hua Xie ; Yu-Ying Kong ; Guan-Zhen Yang ; Yao-Zhou Zhang ; Yuan Wang ; Xiang-Fu WuSource :
- Acta biochimica et biophysica Sinica [ 1672-9145 ] ; 2004.
Descripteurs français
- KwdFr :
- Animaux, Conformation des protéines, Cytoplasme (), Escherichia coli (métabolisme), Humains, Lignée cellulaire, Microscopie confocale, Microscopie de fluorescence, Plasmides (métabolisme), Protéines nucléocapside (), Protéines nucléocapside (isolement et purification), Protéines nucléocapside (métabolisme), Protéines recombinantes (), Protéines recombinantes (métabolisme), Technique de Western, Virus du SRAS (métabolisme), Électrophorèse sur gel de polyacrylamide.
- MESH :
- isolement et purification : Protéines nucléocapside.
- métabolisme : Escherichia coli, Plasmides, Protéines nucléocapside, Protéines recombinantes, Virus du SRAS.
- Animaux, Conformation des protéines, Cytoplasme, Humains, Lignée cellulaire, Microscopie confocale, Microscopie de fluorescence, Protéines nucléocapside, Protéines recombinantes, Technique de Western, Électrophorèse sur gel de polyacrylamide.
English descriptors
- KwdEn :
- Animals, Blotting, Western, Cell Line, Cytoplasm (chemistry), Electrophoresis, Polyacrylamide Gel, Escherichia coli (metabolism), Humans, Insecta, Microscopy, Confocal, Microscopy, Fluorescence, Nucleocapsid Proteins (chemistry), Nucleocapsid Proteins (isolation & purification), Nucleocapsid Proteins (metabolism), Plasmids (metabolism), Protein Conformation, Recombinant Proteins (chemistry), Recombinant Proteins (metabolism), SARS Virus (metabolism).
- MESH :
- chemical , chemistry : Nucleocapsid Proteins, Recombinant Proteins.
- chemistry : Cytoplasm.
- chemical , isolation & purification : Nucleocapsid Proteins.
- metabolism : Escherichia coli, Nucleocapsid Proteins, Plasmids, Recombinant Proteins, SARS Virus.
- Animals, Blotting, Western, Cell Line, Electrophoresis, Polyacrylamide Gel, Humans, Insecta, Microscopy, Confocal, Microscopy, Fluorescence, Protein Conformation.
Abstract
The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is a major viral protein recognized by acute and early convalescent sera from SARS patients. To facilitate the studies on the function and structure of the N protein, this report describe the expression and purification of recombinant SARS-CoV N protein using the baculovirus expression system. Recombinant hexa-histidine-tagged N protein with a molecular mass of 47 kD was produced in insect cells. Recombinant N protein was purified to near homogeneity by Ni2+-NTA affinity chromatography. In addition, we examined the subcellular localization of the N protein by confocal microscopy in Trichoplusia ni BT1 Tn 5B1-4 cells infected with recombinant baculovirus. The N protein was found localized in the cytoplasm as well as in the nucleolus. The purified recombinant N protein can be used in further functional study of SARS-CoV.
DOI: 10.1093/abbs/36.11.754
PubMed: 15514849
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pubmed:15514849Le document en format XML
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<author><name sortKey="Ren, Ai Xia" sort="Ren, Ai Xia" uniqKey="Ren A" first="Ai-Xia" last="Ren">Ai-Xia Ren</name>
<affiliation wicri:level="1"><nlm:affiliation>Institute of Biochemistry and Biotechnology, College of Life Science, Zhejiang University, Hangzhou 310029, China.</nlm:affiliation>
<country xml:lang="fr">République populaire de Chine</country>
<wicri:regionArea>Institute of Biochemistry and Biotechnology, College of Life Science, Zhejiang University, Hangzhou 310029</wicri:regionArea>
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<author><name sortKey="Xie, You Hua" sort="Xie, You Hua" uniqKey="Xie Y" first="You-Hua" last="Xie">You-Hua Xie</name>
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<author><name sortKey="Kong, Yu Ying" sort="Kong, Yu Ying" uniqKey="Kong Y" first="Yu-Ying" last="Kong">Yu-Ying Kong</name>
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<author><name sortKey="Wang, Yuan" sort="Wang, Yuan" uniqKey="Wang Y" first="Yuan" last="Wang">Yuan Wang</name>
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<term>Electrophoresis, Polyacrylamide Gel</term>
<term>Escherichia coli (metabolism)</term>
<term>Humans</term>
<term>Insecta</term>
<term>Microscopy, Confocal</term>
<term>Microscopy, Fluorescence</term>
<term>Nucleocapsid Proteins (chemistry)</term>
<term>Nucleocapsid Proteins (isolation & purification)</term>
<term>Nucleocapsid Proteins (metabolism)</term>
<term>Plasmids (metabolism)</term>
<term>Protein Conformation</term>
<term>Recombinant Proteins (chemistry)</term>
<term>Recombinant Proteins (metabolism)</term>
<term>SARS Virus (metabolism)</term>
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<term>Conformation des protéines</term>
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<term>Escherichia coli (métabolisme)</term>
<term>Humains</term>
<term>Lignée cellulaire</term>
<term>Microscopie confocale</term>
<term>Microscopie de fluorescence</term>
<term>Plasmides (métabolisme)</term>
<term>Protéines nucléocapside ()</term>
<term>Protéines nucléocapside (isolement et purification)</term>
<term>Protéines nucléocapside (métabolisme)</term>
<term>Protéines recombinantes ()</term>
<term>Protéines recombinantes (métabolisme)</term>
<term>Technique de Western</term>
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<term>Plasmids</term>
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<term>SARS Virus</term>
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<term>Cell Line</term>
<term>Electrophoresis, Polyacrylamide Gel</term>
<term>Humans</term>
<term>Insecta</term>
<term>Microscopy, Confocal</term>
<term>Microscopy, Fluorescence</term>
<term>Protein Conformation</term>
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<keywords scheme="MESH" xml:lang="fr"><term>Animaux</term>
<term>Conformation des protéines</term>
<term>Cytoplasme</term>
<term>Humains</term>
<term>Lignée cellulaire</term>
<term>Microscopie confocale</term>
<term>Microscopie de fluorescence</term>
<term>Protéines nucléocapside</term>
<term>Protéines recombinantes</term>
<term>Technique de Western</term>
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<front><div type="abstract" xml:lang="en">The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is a major viral protein recognized by acute and early convalescent sera from SARS patients. To facilitate the studies on the function and structure of the N protein, this report describe the expression and purification of recombinant SARS-CoV N protein using the baculovirus expression system. Recombinant hexa-histidine-tagged N protein with a molecular mass of 47 kD was produced in insect cells. Recombinant N protein was purified to near homogeneity by Ni2+-NTA affinity chromatography. In addition, we examined the subcellular localization of the N protein by confocal microscopy in Trichoplusia ni BT1 Tn 5B1-4 cells infected with recombinant baculovirus. The N protein was found localized in the cytoplasm as well as in the nucleolus. The purified recombinant N protein can be used in further functional study of SARS-CoV.</div>
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<Abstract><AbstractText>The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is a major viral protein recognized by acute and early convalescent sera from SARS patients. To facilitate the studies on the function and structure of the N protein, this report describe the expression and purification of recombinant SARS-CoV N protein using the baculovirus expression system. Recombinant hexa-histidine-tagged N protein with a molecular mass of 47 kD was produced in insect cells. Recombinant N protein was purified to near homogeneity by Ni2+-NTA affinity chromatography. In addition, we examined the subcellular localization of the N protein by confocal microscopy in Trichoplusia ni BT1 Tn 5B1-4 cells infected with recombinant baculovirus. The N protein was found localized in the cytoplasm as well as in the nucleolus. The purified recombinant N protein can be used in further functional study of SARS-CoV.</AbstractText>
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