Please use this identifier to cite or link to this item: http://repositorio.unicamp.br/jspui/handle/REPOSIP/58331
Type: Artigo de periódico
Title: Protein refolding at high pressure: Optimization using eGFP as a model
Author: Malavasi, NV
Foguel, D
Bonafe, CFS
Braga, CACA
Chura-Chambi, RM
Vieira, JM
Morganti, L
Abstract: Refolding 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.
Subject: High hydrostatic pressure
Refolding
Green fluorescent protein
Inclusion body
Aggregation
Country: Inglaterra
Editor: Elsevier Sci Ltd
Rights: fechado
Identifier DOI: 10.1016/j.procbio.2010.10.002
Date Issue: 2011
Appears in Collections:Unicamp - Artigos e Outros Documentos

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