Molecular cloning, expression, and purification of SARS-CoV nsp13
Identifieur interne : 001296 ( Pmc/Checkpoint ); précédent : 001295; suivant : 001297Molecular 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.
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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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, 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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 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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></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">15866708</idno><idno type="pmc">7129550</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7129550</idno><idno type="RBID">PMC:7129550</idno><idno type="doi">10.1016/j.pep.2004.08.003</idno><date when="2005">2005</date><idno type="wicri:Area/Pmc/Corpus">001444</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" 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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, 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></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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<front><journal-meta><journal-id journal-id-type="nlm-ta">Protein Expr Purif</journal-id><journal-id journal-id-type="iso-abbrev">Protein Expr. Purif</journal-id><journal-title-group><journal-title>Protein Expression and Purification</journal-title></journal-title-group><issn pub-type="ppub">1046-5928</issn><issn pub-type="epub">1096-0279</issn><publisher><publisher-name>Elsevier Inc.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">15866708</article-id><article-id pub-id-type="pmc">7129550</article-id><article-id pub-id-type="publisher-id">S1046-5928(04)00278-5</article-id><article-id pub-id-type="doi">10.1016/j.pep.2004.08.003</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Molecular cloning, expression, and purification of SARS-CoV nsp13</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Fan</surname><given-names>Zheng</given-names></name><xref rid="aff1" ref-type="aff">a</xref><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author"><name><surname>Peng</surname><given-names>Kunpeng</given-names></name><xref rid="aff1" ref-type="aff">a</xref><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author"><name><surname>Tan</surname><given-names>Xinyu</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Yin</surname><given-names>Bin</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Dong</surname><given-names>Xiuhua</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Qiu</surname><given-names>Feichan</given-names></name><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Shen</surname><given-names>Yan</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Heng</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Yuan</surname><given-names>Jiangang</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Qiang</surname><given-names>Boqin</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Peng</surname><given-names>Xiaozhong</given-names></name><email>pengxiaozhong@pumc.edu.cn</email><xref rid="aff1" ref-type="aff">a</xref><xref rid="cor1" ref-type="corresp">⁎</xref></contrib></contrib-group><aff id="aff1"><label>a</label>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</aff><aff id="aff2"><label>b</label>Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China</aff><author-notes><corresp id="cor1"><label>⁎</label>Corresponding author. Fax: +86 10 65240529 <email>pengxiaozhong@pumc.edu.cn</email></corresp><fn id="fn1"><label>1</label><p>These two authors contributed equally to this work.</p></fn></author-notes><pub-date pub-type="pmc-release"><day>11</day><month>4</month><year>2005</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><month>6</month><year>2005</year></pub-date><pub-date pub-type="epub"><day>11</day><month>4</month><year>2005</year></pub-date><volume>41</volume><issue>2</issue><fpage>235</fpage><lpage>240</lpage><history><date date-type="received"><day>28</day><month>4</month><year>2004</year></date><date date-type="rev-recd"><day>29</day><month>7</month><year>2004</year></date></history><permissions><copyright-statement>Copyright © 2005 Elsevier Inc. All rights reserved.</copyright-statement><copyright-year>2005</copyright-year><copyright-holder>Elsevier Inc.</copyright-holder><license><license-p>Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.</license-p></license></permissions><abstract><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></abstract><kwd-group><title>Keywords</title><kwd>Severe acute respiratory syndrome (SARS)</kwd><kwd>nsp13 (SARS-CoV methyltransferase)</kwd><kwd>Molecular cloning</kwd><kwd>Expression</kwd><kwd>Purification</kwd><kwd>Mass spectrometric identification</kwd></kwd-group></article-meta></front></pmc><affiliations><list><country><li>République populaire de Chine</li></country><settlement><li>Pékin</li></settlement></list><tree><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><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></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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