Production of authentic SARS-CoV M(pro) with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction.
Identifieur interne : 003629 ( Main/Exploration ); précédent : 003628; suivant : 003630Production of authentic SARS-CoV M(pro) with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction.
Auteurs : Xiaoyu Xue [République populaire de Chine] ; Haitao Yang ; Wei Shen ; Qi Zhao ; Jun Li ; Kailin Yang ; Cheng Chen ; Yinghua Jin ; Mark Bartlam ; Zihe RaoSource :
- Journal of molecular biology [ 0022-2836 ] ; 2007.
Descripteurs français
- KwdFr :
- Calbindines, Endopeptidases (métabolisme), Expression des gènes, Glutathione transferase (métabolisme), Protéine G liant le calcium S100 (isolement et purification), Protéines de fusion recombinantes (métabolisme), Protéines de la matrice virale (), Protéines de la matrice virale (antagonistes et inhibiteurs), Protéines de la matrice virale (biosynthèse), Protéines de la matrice virale (métabolisme), Protéines virales (biosynthèse), Vecteurs génétiques, Virus du SRAS.
- MESH :
- antagonistes et inhibiteurs : Protéines de la matrice virale.
- biosynthèse : Protéines de la matrice virale, Protéines virales.
- isolement et purification : Protéine G liant le calcium S100.
- métabolisme : Endopeptidases, Glutathione transferase, Protéines de fusion recombinantes, Protéines de la matrice virale.
- Calbindines, Expression des gènes, Protéines de la matrice virale, Vecteurs génétiques, Virus du SRAS.
English descriptors
- KwdEn :
- Calbindins, Endopeptidases (metabolism), Gene Expression, Genetic Vectors, Glutathione Transferase (metabolism), Recombinant Fusion Proteins (metabolism), S100 Calcium Binding Protein G (isolation & purification), SARS Virus, Viral Matrix Proteins (antagonists & inhibitors), Viral Matrix Proteins (biosynthesis), Viral Matrix Proteins (chemistry), Viral Matrix Proteins (metabolism), Viral Proteins (biosynthesis).
- MESH :
- chemical , antagonists & inhibitors : Viral Matrix Proteins.
- chemical , biosynthesis : Viral Matrix Proteins, Viral Proteins.
- chemical , chemistry : Viral Matrix Proteins.
- chemical , isolation & purification : S100 Calcium Binding Protein G.
- chemical , metabolism : Endopeptidases, Glutathione Transferase, Recombinant Fusion Proteins, Viral Matrix Proteins.
- chemical : Calbindins.
- Gene Expression, Genetic Vectors, SARS Virus.
Abstract
The viral proteases have proven to be the most selective and useful for removing the fusion tags in fusion protein expression systems. As a key enzyme in the viral life-cycle, the main protease (M(pro)) is most attractive for drug design targeting the SARS coronavirus (SARS-CoV), the etiological agent responsible for the outbreak of severe acute respiratory syndrome (SARS) in 2003. In this study, SARS-CoV M(pro) was used to specifically remove the GST tag in a new fusion protein expression system. We report a new method to produce wild-type (WT) SARS-CoV M(pro) with authentic N and C termini, and compare the activity of WT protease with those of three different types of SARS-CoV M(pro) with additional residues at the N or C terminus. Our results show that additional residues at the N terminus, but not at the C terminus, of M(pro) are detrimental to enzyme activity. To explain this, the crystal structures of WT SARS-CoV M(pro) and its complex with a Michael acceptor inhibitor were determined to 1.6 Angstroms and 1.95 Angstroms resolution respectively. These crystal structures reveal that the first residue of this protease is important for sustaining the substrate-binding pocket and inhibitor binding. This study suggests that SARS-CoV M(pro) could serve as a new tag-cleavage endopeptidase for protein overproduction, and the WT SARS-CoV M(pro) is more appropriate for mechanistic characterization and inhibitor design.
DOI: 10.1016/j.jmb.2006.11.073
PubMed: 17189639
Affiliations:
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Shen</name></author><author><name sortKey="Zhao, Qi" sort="Zhao, Qi" uniqKey="Zhao Q" first="Qi" last="Zhao">Qi Zhao</name></author><author><name sortKey="Li, Jun" sort="Li, Jun" uniqKey="Li J" first="Jun" last="Li">Jun Li</name></author><author><name sortKey="Yang, Kailin" sort="Yang, Kailin" uniqKey="Yang K" first="Kailin" last="Yang">Kailin Yang</name></author><author><name sortKey="Chen, Cheng" sort="Chen, Cheng" uniqKey="Chen C" first="Cheng" last="Chen">Cheng Chen</name></author><author><name sortKey="Jin, Yinghua" sort="Jin, Yinghua" uniqKey="Jin Y" first="Yinghua" last="Jin">Yinghua Jin</name></author><author><name sortKey="Bartlam, Mark" sort="Bartlam, Mark" uniqKey="Bartlam M" first="Mark" last="Bartlam">Mark Bartlam</name></author><author><name sortKey="Rao, Zihe" sort="Rao, Zihe" uniqKey="Rao Z" first="Zihe" last="Rao">Zihe Rao</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2007">2007</date><idno 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sort="Zhao, Qi" uniqKey="Zhao Q" first="Qi" last="Zhao">Qi Zhao</name></author><author><name sortKey="Li, Jun" sort="Li, Jun" uniqKey="Li J" first="Jun" last="Li">Jun Li</name></author><author><name sortKey="Yang, Kailin" sort="Yang, Kailin" uniqKey="Yang K" first="Kailin" last="Yang">Kailin Yang</name></author><author><name sortKey="Chen, Cheng" sort="Chen, Cheng" uniqKey="Chen C" first="Cheng" last="Chen">Cheng Chen</name></author><author><name sortKey="Jin, Yinghua" sort="Jin, Yinghua" uniqKey="Jin Y" first="Yinghua" last="Jin">Yinghua Jin</name></author><author><name sortKey="Bartlam, Mark" sort="Bartlam, Mark" uniqKey="Bartlam M" first="Mark" last="Bartlam">Mark Bartlam</name></author><author><name sortKey="Rao, Zihe" sort="Rao, Zihe" uniqKey="Rao Z" first="Zihe" last="Rao">Zihe Rao</name></author></analytic><series><title level="j">Journal of molecular biology</title><idno type="ISSN">0022-2836</idno><imprint><date when="2007" type="published">2007</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Calbindins</term><term>Endopeptidases (metabolism)</term><term>Gene Expression</term><term>Genetic Vectors</term><term>Glutathione Transferase (metabolism)</term><term>Recombinant Fusion Proteins (metabolism)</term><term>S100 Calcium Binding Protein G (isolation & purification)</term><term>SARS Virus</term><term>Viral Matrix Proteins (antagonists & inhibitors)</term><term>Viral Matrix Proteins (biosynthesis)</term><term>Viral Matrix Proteins (chemistry)</term><term>Viral Matrix Proteins (metabolism)</term><term>Viral Proteins (biosynthesis)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Calbindines</term><term>Endopeptidases (métabolisme)</term><term>Expression des gènes</term><term>Glutathione transferase (métabolisme)</term><term>Protéine G liant le calcium S100 (isolement et purification)</term><term>Protéines de fusion recombinantes (métabolisme)</term><term>Protéines de la matrice virale ()</term><term>Protéines de la matrice virale (antagonistes et inhibiteurs)</term><term>Protéines de la matrice virale (biosynthèse)</term><term>Protéines de la matrice virale (métabolisme)</term><term>Protéines virales (biosynthèse)</term><term>Vecteurs génétiques</term><term>Virus du SRAS</term></keywords><keywords scheme="MESH" type="chemical" qualifier="antagonists & inhibitors" xml:lang="en"><term>Viral Matrix Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>Viral Matrix Proteins</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Viral Matrix Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>S100 Calcium Binding Protein G</term></keywords><keywords scheme="MESH" type="chemical" 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Vectors</term><term>SARS Virus</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Calbindines</term><term>Expression des gènes</term><term>Protéines de la matrice virale</term><term>Vecteurs génétiques</term><term>Virus du SRAS</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The viral proteases have proven to be the most selective and useful for removing the fusion tags in fusion protein expression systems. As a key enzyme in the viral life-cycle, the main protease (M(pro)) is most attractive for drug design targeting the SARS coronavirus (SARS-CoV), the etiological agent responsible for the outbreak of severe acute respiratory syndrome (SARS) in 2003. In this study, SARS-CoV M(pro) was used to specifically remove the GST tag in a new fusion protein expression system. We report a new method to produce wild-type (WT) SARS-CoV M(pro) with authentic N and C termini, and compare the activity of WT protease with those of three different types of SARS-CoV M(pro) with additional residues at the N or C terminus. Our results show that additional residues at the N terminus, but not at the C terminus, of M(pro) are detrimental to enzyme activity. To explain this, the crystal structures of WT SARS-CoV M(pro) and its complex with a Michael acceptor inhibitor were determined to 1.6 Angstroms and 1.95 Angstroms resolution respectively. These crystal structures reveal that the first residue of this protease is important for sustaining the substrate-binding pocket and inhibitor binding. This study suggests that SARS-CoV M(pro) could serve as a new tag-cleavage endopeptidase for protein overproduction, and the WT SARS-CoV M(pro) is more appropriate for mechanistic characterization and inhibitor design.</div></front></TEI><affiliations><list><country><li>République populaire de Chine</li></country><settlement><li>Pékin</li></settlement></list><tree><noCountry><name sortKey="Bartlam, Mark" sort="Bartlam, Mark" uniqKey="Bartlam M" first="Mark" last="Bartlam">Mark Bartlam</name><name sortKey="Chen, Cheng" sort="Chen, Cheng" uniqKey="Chen C" first="Cheng" last="Chen">Cheng Chen</name><name sortKey="Jin, Yinghua" sort="Jin, Yinghua" uniqKey="Jin Y" first="Yinghua" last="Jin">Yinghua Jin</name><name sortKey="Li, Jun" sort="Li, Jun" uniqKey="Li J" first="Jun" last="Li">Jun Li</name><name sortKey="Rao, Zihe" sort="Rao, Zihe" uniqKey="Rao Z" first="Zihe" last="Rao">Zihe Rao</name><name sortKey="Shen, Wei" sort="Shen, Wei" uniqKey="Shen W" first="Wei" last="Shen">Wei Shen</name><name sortKey="Yang, Haitao" sort="Yang, Haitao" uniqKey="Yang H" first="Haitao" last="Yang">Haitao Yang</name><name sortKey="Yang, Kailin" sort="Yang, Kailin" uniqKey="Yang K" first="Kailin" last="Yang">Kailin Yang</name><name sortKey="Zhao, Qi" sort="Zhao, Qi" uniqKey="Zhao Q" first="Qi" last="Zhao">Qi Zhao</name></noCountry><country name="République populaire de Chine"><noRegion><name sortKey="Xue, Xiaoyu" sort="Xue, Xiaoyu" uniqKey="Xue X" first="Xiaoyu" last="Xue">Xiaoyu Xue</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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