Recombinant expression of glycerol-3-phosphate dehydrogenase using the Pichia pastoris system

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dc.contributor.authorSanches Peres, Maristela de Freitas [UNESP]
dc.contributor.authorSilva, Viviane Cristina [UNESP]
dc.contributor.authorValentini, Sandro Roberto [UNESP]
dc.contributor.authorGattas, Edwil Aparecida de Lucca [UNESP]
dc.contributor.institutionUniversidade Estadual Paulista (Unesp)
dc.date.accessioned2014-05-20T13:24:16Z
dc.date.available2014-05-20T13:24:16Z
dc.date.issued2010-08-01
dc.description.abstractIn the present study, the GPD2 gene from Saccharomyces cerevisiae, which codifies for the enzyme glycerol-3-phosphate dehydrogenase (GPDH), was cloned from the pPICZ-alpha expression vector and used with the purpose of inducing the extracellular expression of the glycerol-3-phosphate dehydrogenase under the control of the methanol-regulated AOX promoter. The presence of the GPD2 insert was confirmed by PCR analysis. Pichia pastoris X-33 (Mut(+)) was transformed with linearized plasmids by electroporation and transformants were selected on YPDS plates containing 100 mu g/mL of zeocin. Several clones were selected and the functionality of this enzyme obtained in a culture medium was assayed. Among the mutants tested, one exhibited 3.1 x 10(-2) U/mg of maximal activity. Maximal enzyme activity was achieved at 6 days of growth. Medium composition and pre-induction osmotic stress influenced protein production. Pre-induction osmotic stress (culturing cells in medium with either 0.35 M sodium chloride or 1.0 M sorbitol for 4h prior to induction) led to an increase in cell growth with sorbitol and resulted in a significant increase in GPDH productivity with sodium chloride in 24h of induction approximately fivefold greater than under standard conditions (without pre-induction). (C) 2010 Elsevier B.V. All rights reserved.en
dc.description.affiliationSão Paulo State Univ, UNESP, Sch Pharmaceut Sci, Dept Food & Nutr, BR-14801902 São Paulo, Brazil
dc.description.affiliationSão Paulo State Univ, UNESP, Sch Pharmaceut Sci, Dept Biol Sci, BR-14801902 São Paulo, Brazil
dc.description.affiliationUnespSão Paulo State Univ, UNESP, Sch Pharmaceut Sci, Dept Food & Nutr, BR-14801902 São Paulo, Brazil
dc.description.affiliationUnespSão Paulo State Univ, UNESP, Sch Pharmaceut Sci, Dept Biol Sci, BR-14801902 São Paulo, Brazil
dc.format.extent128-132
dc.identifierhttp://dx.doi.org/10.1016/j.molcatb.2010.01.008
dc.identifier.citationJournal of Molecular Catalysis B-enzymatic. Amsterdam: Elsevier B.V., v. 65, n. 1-4, p. 128-132, 2010.
dc.identifier.doi10.1016/j.molcatb.2010.01.008
dc.identifier.issn1381-1177
dc.identifier.lattes5333250355049814
dc.identifier.lattes4006598610021833
dc.identifier.urihttp://hdl.handle.net/11449/7482
dc.identifier.wosWOS:000278926300022
dc.language.isoeng
dc.publisherElsevier B.V.
dc.relation.ispartofJournal of Molecular Catalysis B: Enzymatic
dc.relation.ispartofsjr0,522
dc.rights.accessRightsAcesso restrito
dc.sourceWeb of Science
dc.subjectGlycerol-3-phosphate dehydrogenase (GPDH)en
dc.subjectPichia pastorisen
dc.subjectHeterologous protein expressionen
dc.titleRecombinant expression of glycerol-3-phosphate dehydrogenase using the Pichia pastoris systemen
dc.typeArtigo
dcterms.licensehttp://www.elsevier.com/about/open-access/open-access-policies/article-posting-policy
dcterms.rightsHolderElsevier B.V.
unesp.author.lattes5333250355049814
unesp.author.lattes4006598610021833[4]
unesp.campusUniversidade Estadual Paulista (Unesp), Faculdade de Ciências Farmacêuticas, Araraquarapt

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