Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

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dc.contributor.authorMarcondes, Marcelo F. [UNIFESP]
dc.contributor.authorTorquato, Ricardo J. S. [UNIFESP]
dc.contributor.authorAssis, Diego M. [UNIFESP]
dc.contributor.authorJuliano, Maria A. [UNIFESP]
dc.contributor.authorHayashi, Mirian A. F. [UNIFESP]
dc.contributor.authorOliveira, Vitor [UNIFESP]
dc.contributor.institutionUniversidade Federal de São Paulo (UNIFESP)
dc.date.accessioned2016-01-24T13:59:05Z
dc.date.available2016-01-24T13:59:05Z
dc.date.issued2010-01-01
dc.description.abstractIn the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (INIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system the IMP fusion protein, with a poly-l-lis-tag (6x His). was obtained by cloning the coding region of INIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia call BL21 (DE3) pLysS After isolation and purification of the fusion protein by affinity chromatography Using Ni-Sepharose resin, the protein was Purified further using ion exchange chromatography with a Hi-trap resource Q column the recombinant IMP was characterized by Western blotting using three distinct antibodies, circular dichioism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. the successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning. (C) 2009 Elsevier Inc All rights reserved.en
dc.description.affiliationUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, Brazil
dc.description.affiliationUniversidade Federal de São Paulo, Dept Biochem, BR-04044020 São Paulo, Brazil
dc.description.affiliationUniversidade Federal de São Paulo, Dept Pharmacol, BR-04044020 São Paulo, Brazil
dc.description.affiliationUnifespUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, Brazil
dc.description.affiliationUnifespUniversidade Federal de São Paulo, Dept Biochem, BR-04044020 São Paulo, Brazil
dc.description.affiliationUnifespUniversidade Federal de São Paulo, Dept Pharmacol, BR-04044020 São Paulo, Brazil
dc.description.sourceWeb of Science
dc.description.sponsorshipFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
dc.description.sponsorshipConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
dc.format.extent123-128
dc.identifierhttp://dx.doi.org/10.1016/j.bbrc.2009.11.014
dc.identifier.citationBiochemical and Biophysical Research Communications. San Diego: Academic Press Inc Elsevier Science, v. 391, n. 1, p. 123-128, 2010.
dc.identifier.doi10.1016/j.bbrc.2009.11.014
dc.identifier.issn0006-291X
dc.identifier.urihttp://repositorio.unifesp.br/handle/11600/32086
dc.identifier.wosWOS:000273624500023
dc.language.isoeng
dc.publisherElsevier B.V.
dc.relation.ispartofBiochemical and Biophysical Research Communications
dc.rightsinfo:eu-repo/semantics/restrictedAccess
dc.rights.licensehttp://www.elsevier.com/about/open-access/open-access-policies/article-posting-policy
dc.subjectPeptidaseen
dc.subjectMitochondriaen
dc.subjectBacterial expressionen
dc.subjectEnzymatic activityen
dc.subjectFRET substrateen
dc.titleMitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substratesen
dc.typeinfo:eu-repo/semantics/article
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