Molecular cloning, expression, purification, and mass spectrometric characterization of 3C-like protease of SARS coronavirus
Identifieur interne : 006418 ( Main/Merge ); précédent : 006417; suivant : 006419Molecular cloning, expression, purification, and mass spectrometric characterization of 3C-like protease of SARS coronavirus
Auteurs : Haifang Sun [République populaire de Chine] ; Haibin Luo [République populaire de Chine] ; Changying Yu [République populaire de Chine] ; Tao Sun [République populaire de Chine] ; Jing Chen [République populaire de Chine] ; Shuying Peng [République populaire de Chine] ; Jun Qin [République populaire de Chine] ; Jianhua Shen [République populaire de Chine] ; Yiming Yang [République populaire de Chine] ; Youhua Xie [République populaire de Chine] ; Kaixian Chen [République populaire de Chine] ; Yuan Wang [République populaire de Chine] ; Xu Shen [République populaire de Chine] ; Hualiang Jiang [République populaire de Chine]Source :
- Protein Expression and Purification [ 1046-5928 ] ; 2003.
Abstract
Severe acute respiratory syndrome (SARS) is an acute respiratory illness, which has broken out in China. It has been known that SARS coronavirus (SARS_CoV) is a novel human coronavirus and is responsible for SARS infection. Belonging to one of the major proteins associated with SARS_CoV, SARS 3C-like protease (SARS_3CLpro) functions as a cysteine protease engaging in the proteolytic cleavage of the viral precursor polyprotein to a series of functional proteins required for coronavirus replication and is considered as an appealing target for designing anti-SARS agents. To facilitate the studies regarding the functions and structures of SARS_3CLpro, in this report the synthetic genes encoding 3CLpro of SARS_CoV were assembled, and the plasmid was constructed using pQE30 as vector and expressed in
Url:
DOI: 10.1016/j.pep.2003.08.016
PubMed: 14965777
PubMed Central: 7129208
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Chine</country><wicri:regionArea>Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203</wicri:regionArea><wicri:noRegion>Shanghai 201203</wicri:noRegion></affiliation></author><author><name sortKey="Luo, Haibin" sort="Luo, Haibin" uniqKey="Luo H" first="Haibin" last="Luo">Haibin Luo</name><affiliation wicri:level="1"><nlm:aff id="AFF1">Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203</wicri:regionArea><wicri:noRegion>Shanghai 201203</wicri:noRegion></affiliation></author><author><name sortKey="Yu, Changying" sort="Yu, Changying" uniqKey="Yu C" first="Changying" last="Yu">Changying Yu</name><affiliation wicri:level="1"><nlm:aff id="AFF1">Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203</wicri:regionArea><wicri:noRegion>Shanghai 201203</wicri:noRegion></affiliation></author><author><name sortKey="Sun, Tao" sort="Sun, Tao" uniqKey="Sun T" first="Tao" last="Sun">Tao Sun</name><affiliation wicri:level="1"><nlm:aff id="AFF1">Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203</wicri:regionArea><wicri:noRegion>Shanghai 201203</wicri:noRegion></affiliation></author><author><name sortKey="Chen, Jing" sort="Chen, Jing" uniqKey="Chen J" first="Jing" last="Chen">Jing Chen</name><affiliation wicri:level="1"><nlm:aff id="AFF1">Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203</wicri:regionArea><wicri:noRegion>Shanghai 201203</wicri:noRegion></affiliation></author><author><name sortKey="Peng, Shuying" sort="Peng, Shuying" uniqKey="Peng S" first="Shuying" last="Peng">Shuying Peng</name><affiliation wicri:level="1"><nlm:aff id="AFF1">Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203</wicri:regionArea><wicri:noRegion>Shanghai 201203</wicri:noRegion></affiliation></author><author><name sortKey="Qin, Jun" sort="Qin, Jun" uniqKey="Qin J" first="Jun" last="Qin">Jun Qin</name><affiliation wicri:level="1"><nlm:aff id="AFF2">Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031</wicri:regionArea><wicri:noRegion>Shanghai 200031</wicri:noRegion></affiliation></author><author><name sortKey="Shen, Jianhua" sort="Shen, Jianhua" uniqKey="Shen J" first="Jianhua" last="Shen">Jianhua Shen</name><affiliation wicri:level="1"><nlm:aff id="AFF1">Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203</wicri:regionArea><wicri:noRegion>Shanghai 201203</wicri:noRegion></affiliation></author><author><name sortKey="Yang, Yiming" sort="Yang, Yiming" uniqKey="Yang Y" first="Yiming" last="Yang">Yiming Yang</name><affiliation wicri:level="1"><nlm:aff id="AFF1">Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203</wicri:regionArea><wicri:noRegion>Shanghai 201203</wicri:noRegion></affiliation></author><author><name sortKey="Xie, Youhua" sort="Xie, Youhua" uniqKey="Xie Y" first="Youhua" last="Xie">Youhua Xie</name><affiliation wicri:level="1"><nlm:aff id="AFF2">Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031</wicri:regionArea><wicri:noRegion>Shanghai 200031</wicri:noRegion></affiliation></author><author><name sortKey="Chen, Kaixian" sort="Chen, Kaixian" uniqKey="Chen K" first="Kaixian" last="Chen">Kaixian Chen</name><affiliation wicri:level="1"><nlm:aff id="AFF1">Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203</wicri:regionArea><wicri:noRegion>Shanghai 201203</wicri:noRegion></affiliation></author><author><name sortKey="Wang, Yuan" sort="Wang, Yuan" uniqKey="Wang Y" first="Yuan" last="Wang">Yuan Wang</name><affiliation wicri:level="1"><nlm:aff id="AFF2">Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031</wicri:regionArea><wicri:noRegion>Shanghai 200031</wicri:noRegion></affiliation></author><author><name sortKey="Shen, Xu" sort="Shen, Xu" uniqKey="Shen X" first="Xu" last="Shen">Xu Shen</name><affiliation wicri:level="1"><nlm:aff id="AFF1">Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203</wicri:regionArea><wicri:noRegion>Shanghai 201203</wicri:noRegion></affiliation></author><author><name sortKey="Jiang, Hualiang" sort="Jiang, Hualiang" uniqKey="Jiang H" first="Hualiang" last="Jiang">Hualiang Jiang</name><affiliation wicri:level="1"><nlm:aff id="AFF1">Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Drug Discovery and Design Center, State Key Lab of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203</wicri:regionArea><wicri:noRegion>Shanghai 201203</wicri:noRegion></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="2003">2003</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Severe acute respiratory syndrome (SARS) is an acute respiratory illness, which has broken out in China. It has been known that SARS coronavirus (SARS_CoV) is a novel human coronavirus and is responsible for SARS infection. Belonging to one of the major proteins associated with SARS_CoV, SARS 3C-like protease (SARS_3CL<sup>pro</sup>) functions as a cysteine protease engaging in the proteolytic cleavage of the viral precursor polyprotein to a series of functional proteins required for coronavirus replication and is considered as an appealing target for designing anti-SARS agents. To facilitate the studies regarding the functions and structures of SARS_3CL<sup>pro</sup>, in this report the synthetic genes encoding 3CL<sup>pro</sup> of SARS_CoV were assembled, and the plasmid was constructed using pQE30 as vector and expressed in <italic>Escherichia coli</italic> M15 cells. The highly yielded (∼15 mg/L) expressed protease was purified by use of NTA-Ni<sup>2+</sup> affinity chromatography and FPLC system, and its sequence was determined by LC/MS with the residue coverage of 46.4%.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Kathryn, V H" uniqKey="Kathryn V">V.H Kathryn</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Peiris, J S" uniqKey="Peiris J">J.S Peiris</name></author><author><name sortKey="Lai, S T" uniqKey="Lai S">S.T Lai</name></author><author><name sortKey="Poon, L L" uniqKey="Poon L">L.L Poon</name></author><author><name sortKey="Guan, Y" uniqKey="Guan Y">Y Guan</name></author><author><name sortKey="Yam, L Y" uniqKey="Yam L">L.Y Yam</name></author><author><name sortKey="Lim, W" uniqKey="Lim W">W Lim</name></author><author><name sortKey="Nicholls, J" uniqKey="Nicholls J">J Nicholls</name></author><author><name sortKey="Yee, W K" uniqKey="Yee W">W.K 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