Refolding by High Pressure of a Toxin Containing Seven Disulfide Bonds: Bothropstoxin-1 from Bothrops jararacussu

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 t...

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Bibliographic Details
Published in:Molecular biotechnology 2011-07, Vol.48 (3), p.228-234
Main Authors: Balduino, Keli N., Spencer, Patrick J., Malavasi, Natalia V., Chura-Chambi, Rosa M., Lemke, Laura S., Morganti, Ligia
Format: Article
Language:English
Subjects:
Animals
Bacteria
Biochemistry
Biological and medical sciences
Biological Techniques
Biotechnology
Bothrops - metabolism
Bothrops jararacussu
Cell Biology
Cell Death - drug effects
Cell Line
Chemistry
Chemistry and Materials Science
Crotalid Venoms - chemistry
Crotalid Venoms - metabolism
Crotalid Venoms - pharmacology
Deoxyribonucleic acid
Disulfides - chemistry
Disulfides - metabolism
DNA
Electrophoresis, Polyacrylamide Gel
Escherichia coli
Escherichia coli - genetics
Fundamental and applied biological sciences. Psychology
Glutathione
Guanidine
Human Genetics
Hydrogen-Ion Concentration
Hydrostatic Pressure
Inclusion Bodies - chemistry
Mice
Microscopy, Electron, Scanning
Protein Refolding
Protein Science
Protein Stability
Proteins
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Recombinant Proteins - pharmacology
Toxins
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Summary: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.
ISSN:1073-6085
1559-0305
DOI:10.1007/s12033-010-9363-5