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Effect of pressure on refolding of recombinant pentameric cholera toxin B.
- Source :
-
Journal of biotechnology [J Biotechnol] 2014 Mar 10; Vol. 173, pp. 98-105. Date of Electronic Publication: 2014 Jan 17. - Publication Year :
- 2014
-
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 (1mg/ml) is necessary for the association of the monomers. Pentameric CTB was obtained when suspensions of CTB IB were compressed at 2.4kbar for 16h in the presence of Tween 20 and incubated at 1bar for 120h. Soluble and biologically active pentameric CTB was obtained, with a yield of 213mg CTB/liter of culture. The experience gained in this study can be important to improve the refolding of proteins with quaternary structure.<br /> (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Subjects :
- Cholera Toxin genetics
Circular Dichroism
Escherichia coli metabolism
Hydrostatic Pressure adverse effects
Models, Molecular
Protein Conformation
Protein Multimerization
Protein Structure, Quaternary
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins metabolism
Vibrio cholerae metabolism
Cholera Toxin chemistry
Cholera Toxin metabolism
Protein Refolding
Vibrio cholerae genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1873-4863
- Volume :
- 173
- Database :
- MEDLINE
- Journal :
- Journal of biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 24445168
- Full Text :
- https://doi.org/10.1016/j.jbiotec.2013.12.006