| Full text | |
| Author(s): |
Malavasi, N. V.
[1]
;
Cordeiro, Y.
[2]
;
Rodrigues, D.
[1]
;
Chura-Chambi, R. M.
[1]
;
Lemke, L. S.
[1]
;
Morganti, L.
[1]
Total Authors: 6
|
| Affiliation: | [1] CNENSP, Ctr Biotecnol, IPEN, Sao Paulo - Brazil
[2] Univ Fed Rio de Janeiro, Fac Farm, Rio De Janeiro - Brazil
Total Affiliations: 2
|
| Document type: | Journal article |
| Source: | Process Biochemistry; v. 49, n. 1, p. 54-60, JAN 2014. |
| Web of Science Citations: | 0 |
| Abstract | |
The application of high hydrostatic pressure (HHP) impairs electrostatic and hydrophobic intermolecular interactions, promoting the dissociation of recombinant inclusion bodies (IBs) under mild conditions that favor subsequent protein refolding. We demonstrated that IBs of a mutant version of green fluorescent protein (eGFP F64L/S65T), produced at 37 C, present native-like secondary and tertiary structures that are progressively lost with an increase in bacterial cultivation temperature. The IBs produced at 37 degrees C are more efficiently dissociated at 2.4 kbar than those produced at 47 degrees C, yielding 25 times more soluble, functional eGFP after the lower pressure (0.69 kbar) refolding step. The association of a negative temperature (-9 degrees C) with HHP enhances the efficiency of solubilization of IBs and of eGFP refolding. The rate of refolding of eGFP as temperature increases from 10 C to 50 degrees C is proportional to the temperature, and a higher yield was obtained at 20 C. High level refolding yield (92%) was obtained by adjusting the temperatures of expression of IBs (37 C), of their dissociation at HHP (-9 degrees C) and of eGFP refolding (20 degrees C). Our data highlight new prospects for the refolding of proteins, a process of fundamental interest in modern biotechnology. (C) 2013 Elsevier Ltd. 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 |