SrasV1, PubMed, Checkpoint, bibRecord, 002624

Molecular cloning, expression, and purification of SARS-CoV nsp13.

Identifieur interne : 002624 ( PubMed/Checkpoint ); précédent : 002623; suivant : 002625

Molecular 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 Peng

Source :

Protein expression and purification [ 1046-5928 ] ; 2005.

RBID : pubmed:15866708

Descripteurs français

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

Affiliations:

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pubmed:15866708

Le document en format XML

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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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<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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<tree><noCountry><name sortKey="Dong, Xiuhua" sort="Dong, Xiuhua" uniqKey="Dong X" first="Xiuhua" last="Dong">Xiuhua Dong</name>
<name sortKey="Peng, Kunpeng" sort="Peng, Kunpeng" uniqKey="Peng K" first="Kunpeng" last="Peng">Kunpeng Peng</name>
<name sortKey="Peng, Xiaozhong" sort="Peng, Xiaozhong" uniqKey="Peng X" first="Xiaozhong" last="Peng">Xiaozhong Peng</name>
<name sortKey="Qiang, Boqin" sort="Qiang, Boqin" uniqKey="Qiang B" first="Boqin" last="Qiang">Boqin Qiang</name>
<name sortKey="Qiu, Feichan" sort="Qiu, Feichan" uniqKey="Qiu F" first="Feichan" last="Qiu">Feichan Qiu</name>
<name sortKey="Shen, Yan" sort="Shen, Yan" uniqKey="Shen Y" first="Yan" last="Shen">Yan Shen</name>
<name sortKey="Tan, Xinyu" sort="Tan, Xinyu" uniqKey="Tan X" first="Xinyu" last="Tan">Xinyu Tan</name>
<name sortKey="Wang, Heng" sort="Wang, Heng" uniqKey="Wang H" first="Heng" last="Wang">Heng Wang</name>
<name sortKey="Yin, Bin" sort="Yin, Bin" uniqKey="Yin B" first="Bin" last="Yin">Bin Yin</name>
<name sortKey="Yuan, Jiangang" sort="Yuan, Jiangang" uniqKey="Yuan J" first="Jiangang" last="Yuan">Jiangang Yuan</name>
</noCountry>
<country name="République populaire de Chine"><noRegion><name sortKey="Fan, Zheng" sort="Fan, Zheng" uniqKey="Fan Z" first="Zheng" last="Fan">Zheng Fan</name>
</noRegion>
</country>
</tree>
</affiliations>
</record>

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   |texte=   Molecular cloning, expression, and purification of SARS-CoV nsp13.
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Molecular cloning, expression, and purification of SARS-CoV nsp13.

Molecular cloning, expression, and purification of SARS-CoV nsp13.

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