| Full text | |
| Author(s): |
Rodrigues, D.
[1]
;
Farinha-Arcieri, L. E.
[2]
;
Ventura, A. M.
[2]
;
Chura-Chambi, R. M.
[1]
;
Malavasi, N. V.
[1]
;
Lemke, L. S.
[1]
;
Guimaraes, J. S.
[1]
;
Ho, P. L.
[3]
;
Morganti, L.
[1]
Total Authors: 9
|
| Affiliation: | [1] Inst Pesquisas Energet & Nucl, Ctr Biotecnol, IPEN, CNEN SP, BR-05508000 Sao Paulo - Brazil
[2] Univ Sao Paulo, Dept Microbiol, Inst Ciencias Biomed, Sao Paulo - Brazil
[3] Inst Butantan, Ctr Biotecnol, Sao Paulo - Brazil
Total Affiliations: 3
|
| Document type: | Journal article |
| Source: | Journal of Biotechnology; v. 173, p. 98-105, MAR 10 2014. |
| Web of Science Citations: | 6 |
| Abstract | |
The production of recombinant proteins is an essential tool for the expansion of modern biological research and biotechnology. The expression of heterologous proteins in Escherichia coli often results in an incomplete folding process that leads to the accumulation of inclusion bodies (IB), aggregates that hold a certain degree of native-like secondary structure. High hydrostatic pressure (HHP) impairs intermolecular hydrophobic and electrostatic interactions, leading to dissociation of aggregates under non-denaturing conditions and is therefore a useful tool to solubilize proteins for posterior refolding. Cholera toxin (CT) is composed of a non-toxic pentamer of B subunits (CTB), a useful adjuvant in vaccines, and a toxic subunit A (CTA). We studied the process of refolding of CTB using HHP. HHP was shown to be effective for dissociation of CTB monomers from IB. Posterior incubation at atmospheric pressure of concentrated CTB (1 mg/ml) is necessary for the association of the monomers. Pentameric CTB was obtained when suspensions of CTB IB were compressed at 2.4 kbar for 16 h in the presence of Tween 20 and incubated at 1 bar for 120 h. Soluble and biologically active pentameric CTB was obtained, with a yield of 213 mg CTB/liter of culture. The experience gained in this study can be important to improve the refolding of proteins with quaternary structure. (c) 2013 Elsevier B. V. 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 |