Engineering a novel endopeptidase based on SARS 3CLpro
Identifieur interne : 000770 ( new/Analysis ); précédent : 000769; suivant : 000771Engineering a novel endopeptidase based on SARS 3CLpro
Auteurs : Chih-Jung Kuo [Taïwan] ; Yan-Ping Shih [Taïwan] ; Daphne Kan [Taïwan] ; Po-Huang Liang [Taïwan]Source :
- Biotechniques [ 0736-6205 ] ; 2009.
Abstract
A 3C-like protease (3CLpro) from the severe acute respiratory syndrome–coronavirus (SARS-CoV) is required for viral replication, cleaving the replicase polyproteins at 11 sites with the conserved Gln↓(Ser, Ala, Gly) sequences. In this study, we developed a mutant 3CLpro (T25G) with an expanded S1′ space that demonstrates 43.5-fold better
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DOI: 10.2144/000113303
PubMed: 20041855
PubMed Central: 7081961
Affiliations:
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type="wicri:doubleKey">0736-6205:2009:Kuo C:engineering:a:novel</idno><idno type="wicri:Area/Main/Merge">001529</idno><idno type="wicri:Area/Main/Curation">001527</idno><idno type="wicri:Area/Main/Exploration">001527</idno><idno type="wicri:Area/new/Extraction">000770</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Engineering a novel endopeptidase based on SARS 3CL<sup>pro</sup></title><author><name sortKey="Kuo, Chih Jung" sort="Kuo, Chih Jung" uniqKey="Kuo C" first="Chih-Jung" last="Kuo">Chih-Jung Kuo</name><affiliation wicri:level="1"><nlm:aff id="AF0001">Institute of Biological Chemistry, Academia Sinica and Core Facility of Recombinant Protein Production, Taipei, Taiwan</nlm:aff><country xml:lang="fr">Taïwan</country><wicri:regionArea>Institute of Biological Chemistry, Academia Sinica and Core Facility of Recombinant Protein Production, Taipei</wicri:regionArea><wicri:noRegion>Taipei</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:aff id="AF0002">Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan</nlm:aff><country xml:lang="fr">Taïwan</country><wicri:regionArea>Institute of Biochemical Sciences, National Taiwan University, Taipei</wicri:regionArea><wicri:noRegion>Taipei</wicri:noRegion></affiliation></author><author><name sortKey="Shih, Yan Ping" sort="Shih, Yan Ping" uniqKey="Shih Y" first="Yan-Ping" last="Shih">Yan-Ping Shih</name><affiliation wicri:level="1"><nlm:aff id="AF0001">Institute of Biological Chemistry, Academia Sinica and Core Facility of Recombinant Protein Production, Taipei, Taiwan</nlm:aff><country xml:lang="fr">Taïwan</country><wicri:regionArea>Institute of Biological Chemistry, Academia Sinica and Core Facility of Recombinant Protein Production, Taipei</wicri:regionArea><wicri:noRegion>Taipei</wicri:noRegion></affiliation></author><author><name sortKey="Kan, Daphne" sort="Kan, Daphne" uniqKey="Kan D" first="Daphne" last="Kan">Daphne Kan</name><affiliation wicri:level="1"><nlm:aff id="AF0001">Institute of Biological Chemistry, Academia Sinica and Core Facility of Recombinant Protein Production, Taipei, Taiwan</nlm:aff><country xml:lang="fr">Taïwan</country><wicri:regionArea>Institute of Biological Chemistry, Academia Sinica and Core Facility of Recombinant Protein Production, Taipei</wicri:regionArea><wicri:noRegion>Taipei</wicri:noRegion></affiliation></author><author><name sortKey="Liang, Po Huang" sort="Liang, Po Huang" uniqKey="Liang P" first="Po-Huang" last="Liang">Po-Huang Liang</name><affiliation wicri:level="1"><nlm:aff id="AF0001">Institute of Biological Chemistry, Academia Sinica and Core Facility of Recombinant Protein Production, Taipei, Taiwan</nlm:aff><country xml:lang="fr">Taïwan</country><wicri:regionArea>Institute of Biological Chemistry, Academia Sinica and Core Facility of Recombinant Protein Production, Taipei</wicri:regionArea><wicri:noRegion>Taipei</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:aff id="AF0002">Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan</nlm:aff><country xml:lang="fr">Taïwan</country><wicri:regionArea>Institute of Biochemical Sciences, National Taiwan University, Taipei</wicri:regionArea><wicri:noRegion>Taipei</wicri:noRegion></affiliation></author></analytic><series><title level="j">Biotechniques</title><idno type="ISSN">0736-6205</idno><idno type="eISSN">1940-9818</idno><imprint><date when="2009">2009</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>A 3C-like protease (3CL<sup>pro</sup>) from the severe acute respiratory syndrome–coronavirus (SARS-CoV) is required for viral replication, cleaving the replicase polyproteins at 11 sites with the conserved Gln↓(Ser, Ala, Gly) sequences. In this study, we developed a mutant 3CL<sup>pro</sup> (T25G) with an expanded S1′ space that demonstrates 43.5-fold better <italic>k</italic><sub>cat</sub>/K<sub><italic>m</italic></sub> compared with wild-type in cleaving substrates with a larger Met at P1′ and is suitable for tag removal from recombinant fusion proteins. Two vectors for expressing fusion proteins with the T25G recognition site (Ala-Val-Leu-Gln↓Met) in <italic>Escherichia coli</italic> and yeast were constructed. Identical cloning sites were used in these vectors for parallel cloning. <italic>Pst</italic>I was chosen as a 5′ cloning site because it overlapped the nucleotide sequence encoding the protease site and avoided addition of extra amino acids at the N terminus of recombinant proteins. 3CL<sup>pro</sup> (T25G) was found to have a 3-fold improvement over TEV<sup>pro</sup> in tag cleavage at each respective preferred cleavage site.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Liang, P H" uniqKey="Liang P">P.H. Liang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ruan, Y J" uniqKey="Ruan Y">Y.J. Ruan</name></author><author><name sortKey="Wei, C L" uniqKey="Wei C">C.L. Wei</name></author><author><name sortKey="Ee, A L" uniqKey="Ee A">A.L. Ee</name></author><author><name sortKey="Vega, V B" uniqKey="Vega V">V.B. Vega</name></author><author><name sortKey="Thoreau, H" uniqKey="Thoreau H">H. 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Chou</name></author><author><name sortKey="Liu, Y Y" uniqKey="Liu Y">Y.Y. Liu</name></author><author><name sortKey="Yeh, Y H" uniqKey="Yeh Y">Y.H. Yeh</name></author><author><name sortKey="Chang, F Y" uniqKey="Chang F">F.Y. Chang</name></author><author><name sortKey="Lin, S C" uniqKey="Lin S">S.C. Lin</name></author><author><name sortKey="Qiu, Z L" uniqKey="Qiu Z">Z.L. Qiu</name></author><author><name sortKey="Sun, Y J" uniqKey="Sun Y">Y.J. Sun</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Emsley, P" uniqKey="Emsley P">P. Emsley</name></author><author><name sortKey="Cowtan, K" uniqKey="Cowtan K">K. Cowtan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kapust, R B" uniqKey="Kapust R">R.B. Kapust</name></author><author><name sortKey="Tozser, J" uniqKey="Tozser J">J. Tözsér</name></author><author><name sortKey="Copeland, T D" uniqKey="Copeland T">T.D. Copeland</name></author><author><name sortKey="Waugh, D S" uniqKey="Waugh D">D.S. Waugh</name></author></analytic></biblStruct></listBibl></div1></back></TEI><affiliations><list><country><li>Taïwan</li></country></list><tree><country name="Taïwan"><noRegion><name sortKey="Kuo, Chih Jung" sort="Kuo, Chih Jung" uniqKey="Kuo C" first="Chih-Jung" last="Kuo">Chih-Jung Kuo</name></noRegion><name sortKey="Kan, Daphne" sort="Kan, Daphne" uniqKey="Kan D" first="Daphne" last="Kan">Daphne Kan</name><name sortKey="Kuo, Chih Jung" sort="Kuo, Chih Jung" uniqKey="Kuo C" first="Chih-Jung" last="Kuo">Chih-Jung Kuo</name><name sortKey="Liang, Po Huang" sort="Liang, Po Huang" uniqKey="Liang P" first="Po-Huang" last="Liang">Po-Huang Liang</name><name sortKey="Liang, Po Huang" sort="Liang, Po Huang" uniqKey="Liang P" first="Po-Huang" last="Liang">Po-Huang Liang</name><name sortKey="Shih, Yan Ping" sort="Shih, Yan Ping" uniqKey="Shih Y" first="Yan-Ping" last="Shih">Yan-Ping Shih</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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