Protein refolding at high pressure: Optimization using eGFP as a model

dc.creatorMalavasi, NV
dc.creatorFoguel, D
dc.creatorBonafe, CFS
dc.creatorBraga, CACA
dc.creatorChura-Chambi, RM
dc.creatorVieira, JM
dc.creatorMorganti, L
dc.date2011
dc.dateFEB
dc.date2014-07-30T14:18:27Z
dc.date2015-11-26T16:37:54Z
dc.date2014-07-30T14:18:27Z
dc.date2015-11-26T16:37:54Z
dc.date.accessioned2018-03-28T23:21:05Z
dc.date.available2018-03-28T23:21:05Z
dc.identifierProcess Biochemistry. Elsevier Sci Ltd, v. 46, n. 2, n. 512, n. 518, 2011.
dc.identifier1359-5113
dc.identifierWOS:000287274000013
dc.identifier10.1016/j.procbio.2010.10.002
dc.identifierhttp://www.repositorio.unicamp.br/jspui/handle/REPOSIP/58331
dc.identifierhttp://repositorio.unicamp.br/jspui/handle/REPOSIP/58331
dc.identifier.urihttp://repositorioslatinoamericanos.uchile.cl/handle/2250/1272146
dc.descriptionFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
dc.descriptionConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
dc.descriptionRefolding of a mutant form of green fluorescent protein (eGFP), which only emits characteristic fluorescence when in the natively folded state, was accomplished under high hydrostatic pressure (HHP). Compression of eGFP inclusion bodies (IB) at 2.40 kbar for 30 min dissociated most of the aggregates and reduced the quantity of IBs. However, fluorescence at 509 nm indicated that eGFP did not refold under this condition. The refolding process was evaluated under various decompression conditions, following IB dissociation at 2.40 kbar. During stepwise decompression, increases in fluorescence were obtained at pressures ranging between 1.38 kbar and atmospheric pressure. The highest levels of eGFP refolding were achieved by incubation at pressure levels between 0.35 and 0.69 kbar in the absence of chaotropic reagents. The refolding was abolished when HHP was applied in the presence of 0.5-1.5 M GdnHCl. Our approach focused on monitoring the bioactivity of the recombinant protein, i.e., fluorescence, instead of solubility, which is not an ideal indicator of proper refolding. The higher yields of a bioactive product by incubation at pressure levels of 0.35-0.69 kbar without using chaotropic salts improve upon the HHP-refolding methods that have been previously described. (C) 2010 Elsevier Ltd. All rights reserved.
dc.description46
dc.description2
dc.description512
dc.description518
dc.descriptionFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
dc.descriptionConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
dc.descriptionFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
dc.descriptionConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
dc.descriptionFAPESP [07/54624-4]
dc.descriptionCNPq [479816/2007-7]
dc.languageen
dc.publisherElsevier Sci Ltd
dc.publisherOxford
dc.publisherInglaterra
dc.relationProcess Biochemistry
dc.relationProcess Biochem.
dc.rightsfechado
dc.rightshttp://www.elsevier.com/about/open-access/open-access-policies/article-posting-policy
dc.sourceWeb of Science
dc.subjectHigh hydrostatic pressure
dc.subjectRefolding
dc.subjectGreen fluorescent protein
dc.subjectInclusion body
dc.subjectAggregation
dc.subjectGreen Fluorescent Protein
dc.subjectHigh Hydrostatic-pressure
dc.subjectHuman Growth-hormone
dc.subjectBacterial Inclusion-bodies
dc.subjectEscherichia-coli
dc.subjectCrystal-structure
dc.subjectNative Protein
dc.subjectDissociation
dc.subjectAggregation
dc.subjectStability
dc.titleProtein refolding at high pressure: Optimization using eGFP as a model
dc.typeArtículos de revistas


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