Characterization of intermediate states of protein folding and obtainment of biofu...
Functional and structural characterization of YbbN protein of Xylella fastidiosa
| Full text | |
| Author(s): |
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]
Total Authors: 8
|
| Affiliation: | [1] IPEN CNEN SP, Ctr Biotecnol, Sao Paulo - Brazil
[2] Univ Sao Paulo, Inst Biociencias, Dept Genet & Biol Evolut, Sao Paulo - Brazil
Total Affiliations: 2
|
| Document type: | Journal article |
| Source: | Protein Expression and Purification; v. 106, p. 72-77, FEB 2015. |
| Web of Science Citations: | 2 |
| 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, 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) | |
| FAPESP's process: | 10/13353-0 - Characterization of intermediate states of protein folding and obtainment of biofunctional proteins by high hydrostatic pressure |
| Grantee: | Ligia Ely Morganti Ferreira Dias |
| Support Opportunities: | Regular Research Grants |