Investigation on solubilization protocols in the refolding of the thioredoxin TsnC from Xylella fastidiosa by high hydrostatic pressure approach

Autor(es)

Lemke, Laura Simoni ^[1] ; Chura-Chambi, Rosa Maria ^[1] ; Rodrigues, Daniella ^[1] ; Rosa Cussiol, Jose Renato ^[2] ; Malavasi, Natalia Vallejo ^[1] ; Pires Alegria, Thiago Geronimo ^[2] ; Soares Netto, Luis Eduardo ^[2] ; Morganti, Ligia ^[1]

Afiliação do(s) autor(es)

^[1] IPEN CNEN SP, Ctr Biotecnol, Sao Paulo - Brazil

^[2] Univ Sao Paulo, Inst Biociencias, Dept Genet & Biol Evolut, Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Protein Expression and Purification; v. 106, p. 72-77, FEB 2015.
• Citações Web of Science: 2

Resumo

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, 1 mM GSSG, 0.75 M guanidine hydrochloride, and 0.5 M 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. (C) 2014 Elsevier Inc. All rights reserved. (AU)

• Processo FAPESP: 10/13353-0 - Caracterização de estados intermediários do enovelamento e obtenção de proteínas biofuncionais pela utilização de altas pressões hidrostáticas
• Beneficiário: Ligia Ely Morganti Ferreira Dias
• Modalidade de apoio: Auxílio à Pesquisa - Regular