Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)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.462512518Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [07/54624-4]CNPq [479816/2007-7]