Molecular cloning, expression, and purification of SARS-CoV nsp13.
Identifieur interne : 002760 ( PubMed/Curation ); précédent : 002759; suivant : 002761Molecular cloning, expression, and purification of SARS-CoV nsp13.
Auteurs : Zheng Fan [République populaire de Chine] ; Kunpeng Peng ; Xinyu Tan ; Bin Yin ; Xiuhua Dong ; Feichan Qiu ; Yan Shen ; Heng Wang ; Jiangang Yuan ; Boqin Qiang ; Xiaozhong PengSource :
- 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 Escherichia coli BL21(DE3). The His-tag-fused protein was expressed by induction of 0.5mM IPTG and purified by a single Ni(2+) 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.
DOI: 10.1016/j.pep.2004.08.003
PubMed: 15866708
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<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>
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<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>
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<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>
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<term>Recombinant Fusion Proteins</term>
<term>Viral Proteins</term>
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<term>Protéines virales</term>
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<keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Virus du SRAS</term>
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</keywords>
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<term>Genetic Vectors</term>
<term>Methyltransferases</term>
<term>RNA, Viral</term>
<term>Recombinant Fusion Proteins</term>
<term>SARS Virus</term>
<term>Viral Proteins</term>
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<term>Protéines virales</term>
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<term>Protéines virales</term>
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<term>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</term>
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<term>Chromatography, Affinity</term>
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<term>Gene Expression Regulation, Viral</term>
<term>Molecular Sequence Data</term>
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<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>
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<front><div type="abstract" xml:lang="en">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 Escherichia coli BL21(DE3). The His-tag-fused protein was expressed by induction of 0.5mM IPTG and purified by a single Ni(2+) 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.</div>
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<Title>Protein expression and purification</Title>
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<ArticleTitle>Molecular cloning, expression, and purification of SARS-CoV nsp13.</ArticleTitle>
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<Abstract><AbstractText>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 Escherichia coli BL21(DE3). The His-tag-fused protein was expressed by induction of 0.5mM IPTG and purified by a single Ni(2+) 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.</AbstractText>
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<ForeName>Zheng</ForeName>
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<AffiliationInfo><Affiliation>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.</Affiliation>
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<ForeName>Boqin</ForeName>
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