| Full text | |
| Author(s): |
Balduino, Keli N.
[1]
;
Spencer, Patrick J.
[1]
;
Malavasi, Natalia V.
[1]
;
Chura-Chambi, Rosa M.
[1]
;
Lemke, Laura S.
[1]
;
Morganti, Ligia
[1]
Total Authors: 6
|
| Affiliation: | [1] IPEN CNEN SP, Ctr Biotecnol, BR-05508000 Sao Paulo - Brazil
Total Affiliations: 1
|
| Document type: | Journal article |
| Source: | MOLECULAR BIOTECHNOLOGY; v. 48, n. 3, p. 228-234, JUL 2011. |
| Web of Science Citations: | 8 |
| Abstract | |
Aggregation is a serious obstacle for recovery of biologically active heterologous proteins from inclusion bodies (IBs) produced by recombinant bacteria. E. coli transformed with a vector containing the cDNA for Bothropstoxin-1 (BthTx-1) expressed the recombinant product as IBs. In order to obtain the native toxin, insoluble and aggregated protein was refolded using high hydrostatic pressure (HHP). IBs were dissolved and refolded (2 kbar, 16 h), and the effects of protein concentration, as well as changes in ratio and concentration of oxido-shuffling reagents, guanidine hydrochloride (GdnHCl), and pH in the refolding buffer, were assayed. A 32% yield (7.6 mg per liter of bacterial culture) in refolding of the native BthTx-1 was obtained using optimal conditions of the refolding buffer (Tris-HCl buffer, pH 7.5, containing 3 mM of a 2:3 ratio of GSH/GSSG, and 1 M GdnHCl). Scanning electron microscopy (SEM) showed that that disaggregation of part of IBs particles occurred upon compression and that the morphology of the remaining IBs, spherical particles, was not substantially altered. Dose-dependent cytotoxic activity of high-pressure refolded BthTx-1 was shown in C2C12 muscle cells. (AU) | |