title: Investigation on solubilization protocols in the refolding of the thioredoxin TsnC from Xylella fastidiosa by high hydrostatic pressure approach.
abstract: The lack of efficient refolding methodologies must be overcome to take full advantage of the fact that bacteria express high levels of aggregated recombinant proteins. High hydrostatic pressure (HHP) impairs intermolecular hydrophobic and electrostatic interactions, dissociating aggregates, which makes HHP a useful tool to solubilize proteins for subsequent refolding. A process of refolding was set up by using as a model TsnC, a thioredoxin that catalyzes the disulfide reduction to a dithiol, a useful indication of biological activity. The inclusion bodies (IB) were dissociated at 2.4 kbar. The effect of incubation of IB suspensions at 1-800 bar, the guanidine hydrochloride concentration, the oxidized/reduced glutathione (GSH/GSSG) ratios, and the additives in the refolding buffer were analyzed. To assess the yields of fully biologically active protein obtained for each tested condition, it was crucial to analyze both the TsnC solubilization yield and its enzymatic activity. Application of 2.4 kbar to the IB suspension in the presence of 9 mM GSH, 1mM GSSG, 0.75 M guanidine hydrochloride, and 0.5M arginine with subsequent incubation at 1 bar furnished high refolding yield (81%). The experience gained in this study shall help to establish efficient HHP-based protein refolding processes for other proteins.
authors.affiliation: Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, Centro de Biotecnologia, São Paulo, Brazil.
authors.forename: Laura Simoni
authors.initials: LS
authors.lastname: Lemke
authors.affiliation: Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, Centro de Biotecnologia, São Paulo, Brazil.
authors.forename: Rosa Maria
authors.initials: RM
authors.lastname: Chura-Chambi
authors.affiliation: Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, Centro de Biotecnologia, São Paulo, Brazil.
authors.forename: Daniella
authors.initials: D
authors.lastname: Rodrigues
authors.affiliation: Instituto de Biociências, Departamento de Genética e Biologia Evolutiva, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Jose Renato Rosa
authors.initials: JR
authors.lastname: Cussiol
authors.affiliation: Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, Centro de Biotecnologia, São Paulo, Brazil.
authors.forename: Natalia Vallejo
authors.initials: NV
authors.lastname: Malavasi
authors.affiliation: Instituto de Biociências, Departamento de Genética e Biologia Evolutiva, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Thiago Geronimo Pires
authors.initials: TG
authors.lastname: Alegria
authors.affiliation: Instituto de Biociências, Departamento de Genética e Biologia Evolutiva, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Luis Eduardo Soares
authors.initials: LE
authors.lastname: Netto
authors.affiliation: Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, Centro de Biotecnologia, São Paulo, Brazil. Electronic address: lmorganti@ipen.br.
authors.forename: Ligia
authors.initials: L
authors.lastname: Morganti
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D001671:Biochemistry / Q000379:methods*; D002942:Circular Dichroism; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology; D006874:Hydrostatic Pressure; D058849:Protein Refolding / Q000187:drug effects; D017433:Protein Structure, Secondary; D012995:Solubility; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D044167:Xylella / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D013879:Thioredoxins; D019791:Guanidine; D019803:Glutathione Disulfide
keywords: High hydrostatic pressure; Refolding; Thioredoxin; TsnC