Eukaryotic Expression System Pichia pastoris Affects the Lipase Catalytic Properties: A Monolayer Study
Identifieur interne : 000029 ( Pmc/Curation ); précédent : 000028; suivant : 000030Eukaryotic Expression System Pichia pastoris Affects the Lipase Catalytic Properties: A Monolayer Study
Auteurs : Madiha Bou Ali ; Yassine Ben Ali ; Imen Aissa ; Youssef GargouriSource :
- PLoS ONE [ 1932-6203 ] ; 2014.
Abstract
Recombinant DNA methods are being widely used to express proteins in both prokaryotic and eukaryotic cells for both fundamental and applied research purposes. Expressed protein must be well characterized to be sure that it retains the same properties as the native one, especially when expressed protein will be used in the pharmaceutical field. In this aim, interfacial and kinetic properties of native, untagged recombinant and tagged recombinant forms of a pancreatic lipase were compared using the monomolecular film technique. Turkey pancreatic lipase (TPL) was chosen as model. A kinetic study on the dependence of the stereoselectivity of these three forms on the surface pressure was performed using three dicaprin isomers spread in the form of monomolecular films at the air-water interface. The heterologous expression and the N-His-tag extension were found to modify the pressure preference and decrease the catalytic hydrolysis rate of three dicaprin isomers. Besides, the heterologous expression was found to change the TPL regioselectivity without affecting its stereospecificity contrary to the N-tag extension which retained that regioselectivity and changed the stereospecificity at high surface pressures. The study of parameters, termed Recombinant expression Effects on Catalysis (REC), N-Tag Effects on Catalysis (TEC), and N-Tag and Recombinant expression Effects on Catalysis (TREC) showed that the heterologous expression effects on the catalytic properties of the TPL were more deleterious than the presence of an N-terminal tag extension.
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DOI: 10.1371/journal.pone.0104221
PubMed: 25133585
PubMed Central: 4136768
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Expressed protein must be well characterized to be sure that it retains the same properties as the native one, especially when expressed protein will be used in the pharmaceutical field. In this aim, interfacial and kinetic properties of native, untagged recombinant and tagged recombinant forms of a pancreatic lipase were compared using the monomolecular film technique. Turkey pancreatic lipase (TPL) was chosen as model. A kinetic study on the dependence of the stereoselectivity of these three forms on the surface pressure was performed using three dicaprin isomers spread in the form of monomolecular films at the air-water interface. The heterologous expression and the N-His-tag extension were found to modify the pressure preference and decrease the catalytic hydrolysis rate of three dicaprin isomers. Besides, the heterologous expression was found to change the TPL regioselectivity without affecting its stereospecificity contrary to the N-tag extension which retained that regioselectivity and changed the stereospecificity at high surface pressures. The study of parameters, termed Recombinant expression Effects on Catalysis (REC), N-Tag Effects on Catalysis (TEC), and N-Tag and Recombinant expression Effects on Catalysis (TREC) showed that the heterologous expression effects on the catalytic properties of the TPL were more deleterious than the presence of an N-terminal tag extension.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Martinelle, M" uniqKey="Martinelle M">M Martinelle</name></author><author><name sortKey="Holmquist, M" uniqKey="Holmquist M">M Holmquist</name></author><author><name sortKey="Hult, K" uniqKey="Hult K">K Hult</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jaeger, Ke" uniqKey="Jaeger K">KE Jaeger</name></author><author><name sortKey="Eggert, T" uniqKey="Eggert T">T Eggert</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sassenfeld, Hm" uniqKey="Sassenfeld H">HM 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<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id><journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title-group><journal-title>PLoS ONE</journal-title></journal-title-group><issn pub-type="epub">1932-6203</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25133585</article-id><article-id pub-id-type="pmc">4136768</article-id><article-id pub-id-type="publisher-id">PONE-D-14-14607</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0104221</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Biology and Life Sciences</subject><subj-group><subject>Biochemistry</subject><subj-group><subject>Enzymology</subject><subject>Lipids</subject></subj-group></subj-group></subj-group></article-categories><title-group><article-title>Eukaryotic Expression System <italic>Pichia pastoris</italic> Affects the Lipase Catalytic Properties: A Monolayer Study</article-title><alt-title alt-title-type="running-head">Yeast Expression System Affects the Lipase Catalytic Properties</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Bou Ali</surname><given-names>Madiha</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Ben Ali</surname><given-names>Yassine</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Aissa</surname><given-names>Imen</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Gargouri</surname><given-names>Youssef</given-names></name><xref ref-type="aff" rid="aff1"></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib></contrib-group><aff id="aff1"><addr-line>Laboratory of Biochemistry, National Engineering School of Sfax (ENIS), University of Sfax, Sfax, Tunisia</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Deschenes</surname><given-names>Robert J.</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1"><addr-line>College of Medicine, University of South Florida, United States of America</addr-line></aff><author-notes><corresp id="cor1">* E-mail: <email>ytgargouri@yahoo.fr</email></corresp><fn fn-type="conflict"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type="con"><p>Conceived and designed the experiments: YG YBA. Performed the experiments: MB IA. Analyzed the data: YBA YG MB IA. Contributed reagents/materials/analysis tools: YBA YG MB IA. Contributed to the writing of the manuscript: MB YBA YG.</p></fn></author-notes><pub-date pub-type="collection"><year>2014</year></pub-date><pub-date pub-type="epub"><day>18</day><month>8</month><year>2014</year></pub-date><volume>9</volume><issue>8</issue><elocation-id>e104221</elocation-id><history><date date-type="received"><day>1</day><month>4</month><year>2014</year></date><date date-type="accepted"><day>10</day><month>7</month><year>2014</year></date></history><permissions><copyright-year>2014</copyright-year><copyright-holder>Bou Ali et al</copyright-holder><license><license-p>This is an open-access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><abstract><p>Recombinant DNA methods are being widely used to express proteins in both prokaryotic and eukaryotic cells for both fundamental and applied research purposes. Expressed protein must be well characterized to be sure that it retains the same properties as the native one, especially when expressed protein will be used in the pharmaceutical field. In this aim, interfacial and kinetic properties of native, untagged recombinant and tagged recombinant forms of a pancreatic lipase were compared using the monomolecular film technique. Turkey pancreatic lipase (TPL) was chosen as model. A kinetic study on the dependence of the stereoselectivity of these three forms on the surface pressure was performed using three dicaprin isomers spread in the form of monomolecular films at the air-water interface. The heterologous expression and the N-His-tag extension were found to modify the pressure preference and decrease the catalytic hydrolysis rate of three dicaprin isomers. Besides, the heterologous expression was found to change the TPL regioselectivity without affecting its stereospecificity contrary to the N-tag extension which retained that regioselectivity and changed the stereospecificity at high surface pressures. The study of parameters, termed Recombinant expression Effects on Catalysis (REC), N-Tag Effects on Catalysis (TEC), and N-Tag and Recombinant expression Effects on Catalysis (TREC) showed that the heterologous expression effects on the catalytic properties of the TPL were more deleterious than the presence of an N-terminal tag extension.</p></abstract><funding-group><funding-statement>The authors have no support or funding to report.</funding-statement></funding-group><counts><page-count count="8"></page-count></counts><custom-meta-group><custom-meta id="data-availability"><meta-name>Data Availability</meta-name><meta-value>The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.</meta-value></custom-meta></custom-meta-group></article-meta><notes><title>Data Availability</title><p>The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.</p></notes></front></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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