Molecular cloning, expression, and purification of SARS-CoV nsp13
Identifieur interne : 004754 ( Main/Exploration ); précédent : 004753; suivant : 004755Molecular cloning, expression, and purification of SARS-CoV nsp13
Auteurs : Zheng Fan [République populaire de Chine] ; Kunpeng Peng [République populaire de Chine] ; Xinyu Tan [République populaire de Chine] ; Bin Yin [République populaire de Chine] ; Xiuhua Dong [République populaire de Chine] ; Feichan Qiu [République populaire de Chine] ; Yan Shen [République populaire de Chine] ; Heng Wang [République populaire de Chine] ; Jiangang Yuan [République populaire de Chine] ; Boqin Qiang [République populaire de Chine] ; Xiaozhong Peng [République populaire de Chine]Source :
- Protein Expression and Purification [ 1046-5928 ] ; 2005.
Descripteurs français
- KwdFr :
- ARN viral (génétique), Chromatographie d'affinité, Clonage moléculaire, Données de séquences moléculaires, Escherichia coli (génétique), Methyltransferases (biosynthèse), Methyltransferases (génétique), Methyltransferases (isolement et purification), Protéines de fusion recombinantes (biosynthèse), Protéines de fusion recombinantes (génétique), Protéines virales (biosynthèse), Protéines virales (génétique), Protéines virales (isolement et purification), RT-PCR (), Régulation de l'expression des gènes viraux, Spectrométrie de masse MALDI (), Séquence d'acides aminés, Vecteurs génétiques (génétique), Virus du SRAS (enzymologie), Virus du SRAS (génétique).
- MESH :
- biosynthèse : Methyltransferases, Protéines de fusion recombinantes, Protéines virales.
- enzymologie : Virus du SRAS.
- génétique : ARN viral, Escherichia coli, Methyltransferases, Protéines de fusion recombinantes, Protéines virales, Vecteurs génétiques, Virus du SRAS.
- isolement et purification : Methyltransferases, Protéines virales.
- Chromatographie d'affinité, Clonage moléculaire, Données de séquences moléculaires, RT-PCR, Régulation de l'expression des gènes viraux, Spectrométrie de masse MALDI, Séquence d'acides aminés.
English descriptors
- KwdEn :
- Amino Acid Sequence, Chromatography, Affinity, Cloning, Molecular, Escherichia coli (genetics), Gene Expression Regulation, Viral, Genetic Vectors (genetics), Methyltransferases (biosynthesis), Methyltransferases (genetics), Methyltransferases (isolation & purification), Molecular Sequence Data, RNA, Viral (genetics), Recombinant Fusion Proteins (biosynthesis), Recombinant Fusion Proteins (genetics), Reverse Transcriptase Polymerase Chain Reaction (methods), SARS Virus (enzymology), SARS Virus (genetics), Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization (methods), Viral Proteins (biosynthesis), Viral Proteins (genetics), Viral Proteins (isolation & purification).
- MESH :
- chemical , biosynthesis : Methyltransferases, Recombinant Fusion Proteins, Viral Proteins.
- enzymology : SARS Virus.
- genetics : Escherichia coli, Genetic Vectors, Methyltransferases, RNA, Viral, Recombinant Fusion Proteins, SARS Virus, Viral Proteins.
- chemical , isolation & purification : Methyltransferases, Viral Proteins.
- methods : Reverse Transcriptase Polymerase Chain Reaction, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization.
- Amino Acid Sequence, Chromatography, Affinity, Cloning, Molecular, Gene Expression Regulation, Viral, Molecular Sequence Data.
Abstract
The SARS-nsp13 protein was identified as an mRNA cap1 methyltransferase. In this study, the nsp13 gene was cloned from the SARS-CoV PUMC02 strain viral RNA by RT-PCR, and inserted into the expression plasmid pET30a(+). The recombinant plasmid pET30a(+)-nsp13 was confirmed by restriction enzymes and sequencing analysis, and transformed into
Url:
DOI: 10.1016/j.pep.2004.08.003
PubMed: 15866708
PubMed Central: 7129550
Affiliations:
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Le document en format XML
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Medical College, National Human Genome Center, Beijing 100005</wicri:regionArea><placeName><settlement type="city">Pékin</settlement></placeName></affiliation></author><author><name sortKey="Peng, Kunpeng" sort="Peng, Kunpeng" uniqKey="Peng K" first="Kunpeng" last="Peng">Kunpeng Peng</name><affiliation wicri:level="1"><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005</wicri:regionArea><placeName><settlement type="city">Pékin</settlement></placeName></affiliation></author><author><name sortKey="Tan, Xinyu" sort="Tan, Xinyu" uniqKey="Tan X" first="Xinyu" last="Tan">Xinyu Tan</name><affiliation wicri:level="1"><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005</wicri:regionArea><placeName><settlement type="city">Pékin</settlement></placeName></affiliation></author><author><name sortKey="Yin, Bin" sort="Yin, Bin" uniqKey="Yin B" first="Bin" last="Yin">Bin Yin</name><affiliation wicri:level="1"><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005</wicri:regionArea><placeName><settlement type="city">Pékin</settlement></placeName></affiliation></author><author><name sortKey="Dong, Xiuhua" sort="Dong, Xiuhua" uniqKey="Dong X" first="Xiuhua" last="Dong">Xiuhua Dong</name><affiliation wicri:level="1"><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005</wicri:regionArea><placeName><settlement type="city">Pékin</settlement></placeName></affiliation></author><author><name sortKey="Qiu, Feichan" sort="Qiu, Feichan" uniqKey="Qiu F" first="Feichan" last="Qiu">Feichan Qiu</name><affiliation wicri:level="1"><nlm:aff id="aff2">Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730</wicri:regionArea><placeName><settlement type="city">Pékin</settlement></placeName></affiliation></author><author><name sortKey="Shen, Yan" sort="Shen, Yan" uniqKey="Shen Y" first="Yan" last="Shen">Yan Shen</name><affiliation wicri:level="1"><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005</wicri:regionArea><placeName><settlement type="city">Pékin</settlement></placeName></affiliation></author><author><name sortKey="Wang, Heng" sort="Wang, Heng" uniqKey="Wang H" first="Heng" last="Wang">Heng Wang</name><affiliation wicri:level="1"><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005</wicri:regionArea><placeName><settlement type="city">Pékin</settlement></placeName></affiliation></author><author><name sortKey="Yuan, Jiangang" sort="Yuan, Jiangang" uniqKey="Yuan J" first="Jiangang" last="Yuan">Jiangang Yuan</name><affiliation wicri:level="1"><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005</wicri:regionArea><placeName><settlement type="city">Pékin</settlement></placeName></affiliation></author><author><name sortKey="Qiang, Boqin" sort="Qiang, Boqin" uniqKey="Qiang B" first="Boqin" last="Qiang">Boqin Qiang</name><affiliation wicri:level="1"><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005</wicri:regionArea><placeName><settlement type="city">Pékin</settlement></placeName></affiliation></author><author><name sortKey="Peng, Xiaozhong" sort="Peng, Xiaozhong" uniqKey="Peng X" first="Xiaozhong" last="Peng">Xiaozhong Peng</name><affiliation wicri:level="1"><nlm:aff id="aff1">National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005</wicri:regionArea><placeName><settlement type="city">Pékin</settlement></placeName></affiliation></author></analytic><series><title level="j">Protein Expression and Purification</title><idno type="ISSN">1046-5928</idno><idno type="eISSN">1096-0279</idno><imprint><date when="2005">2005</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Amino Acid Sequence</term><term>Chromatography, Affinity</term><term>Cloning, Molecular</term><term>Escherichia coli (genetics)</term><term>Gene Expression Regulation, Viral</term><term>Genetic Vectors (genetics)</term><term>Methyltransferases (biosynthesis)</term><term>Methyltransferases (genetics)</term><term>Methyltransferases (isolation & purification)</term><term>Molecular Sequence Data</term><term>RNA, Viral (genetics)</term><term>Recombinant Fusion Proteins (biosynthesis)</term><term>Recombinant Fusion Proteins (genetics)</term><term>Reverse Transcriptase Polymerase Chain Reaction (methods)</term><term>SARS Virus (enzymology)</term><term>SARS Virus (genetics)</term><term>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization (methods)</term><term>Viral Proteins (biosynthesis)</term><term>Viral Proteins (genetics)</term><term>Viral Proteins (isolation & purification)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ARN viral (génétique)</term><term>Chromatographie d'affinité</term><term>Clonage moléculaire</term><term>Données de séquences moléculaires</term><term>Escherichia coli (génétique)</term><term>Methyltransferases (biosynthèse)</term><term>Methyltransferases (génétique)</term><term>Methyltransferases (isolement et purification)</term><term>Protéines de fusion recombinantes (biosynthèse)</term><term>Protéines de fusion recombinantes (génétique)</term><term>Protéines virales (biosynthèse)</term><term>Protéines virales (génétique)</term><term>Protéines virales (isolement et purification)</term><term>RT-PCR ()</term><term>Régulation de l'expression des gènes viraux</term><term>Spectrométrie de masse MALDI ()</term><term>Séquence d'acides aminés</term><term>Vecteurs génétiques (génétique)</term><term>Virus du SRAS (enzymologie)</term><term>Virus du SRAS (génétique)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>Methyltransferases</term><term>Recombinant Fusion Proteins</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" qualifier="biosynthèse" xml:lang="fr"><term>Methyltransferases</term><term>Protéines de fusion recombinantes</term><term>Protéines virales</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><term>Genetic Vectors</term><term>Methyltransferases</term><term>RNA, Viral</term><term>Recombinant Fusion Proteins</term><term>SARS Virus</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>ARN viral</term><term>Escherichia coli</term><term>Methyltransferases</term><term>Protéines de fusion recombinantes</term><term>Protéines virales</term><term>Vecteurs génétiques</term><term>Virus du SRAS</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Methyltransferases</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Methyltransferases</term><term>Protéines virales</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Reverse Transcriptase Polymerase Chain Reaction</term><term>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Amino Acid Sequence</term><term>Chromatography, Affinity</term><term>Cloning, Molecular</term><term>Gene Expression Regulation, Viral</term><term>Molecular Sequence Data</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Chromatographie d'affinité</term><term>Clonage moléculaire</term><term>Données de séquences moléculaires</term><term>RT-PCR</term><term>Régulation de l'expression des gènes viraux</term><term>Spectrométrie de masse MALDI</term><term>Séquence d'acides aminés</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The SARS-nsp13 protein was identified as an mRNA cap1 methyltransferase. In this study, the nsp13 gene was cloned from the SARS-CoV PUMC02 strain viral RNA by RT-PCR, and inserted into the expression plasmid pET30a(+). The recombinant plasmid pET30a(+)-nsp13 was confirmed by restriction enzymes and sequencing analysis, and transformed into <italic>Escherichia coli</italic> BL21(DE3). The His-tag-fused protein was expressed by induction of 0.5 mM IPTG and purified by a single Ni<sup>2+</sup> affinity chromatography. The protein was validated by western blot and MS analysis. A large quantity of the nsp13 protein obtained with this method may be useful for further study of its structure and function.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Ksiazek, T G" uniqKey="Ksiazek T">T.G. Ksiazek</name></author><author><name sortKey="Erdman, D" uniqKey="Erdman D">D. Erdman</name></author><author><name sortKey="Goldsmith, C S" uniqKey="Goldsmith C">C.S. 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