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Endotoxin-free purification for the isolation of Bovine Viral Diarrhoea Virus E2 protein from insoluble inclusion body aggregates
BACKGROUND: Protein expression in Escherichia coli may result in the recombinant protein being expressed as insoluble inclusion bodies. In addition, proteins purified from E. coli contain endotoxins which need to be removed for in vivo applications. The structural protein, E2, from Bovine Viral Diar...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3160874/ https://www.ncbi.nlm.nih.gov/pubmed/21787435 http://dx.doi.org/10.1186/1475-2859-10-57 |
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author | Cavallaro, Antonino S Mahony, Donna Commins, Margaret Mahony, Timothy J Mitter, Neena |
author_facet | Cavallaro, Antonino S Mahony, Donna Commins, Margaret Mahony, Timothy J Mitter, Neena |
author_sort | Cavallaro, Antonino S |
collection | PubMed |
description | BACKGROUND: Protein expression in Escherichia coli may result in the recombinant protein being expressed as insoluble inclusion bodies. In addition, proteins purified from E. coli contain endotoxins which need to be removed for in vivo applications. The structural protein, E2, from Bovine Viral Diarrhoea Virus (BVDV) is a major immunogenic determinant, and is an ideal candidate as a subunit vaccine. The E2 protein contains 17 cysteine residues creating difficulties in E. coli expression. In this report we outline a procedure for successfully producing soluble and endotoxin-free BVDV E2 protein from inclusion bodies (IB). RESULTS: The expression of a truncated form of BVDV-E2 protein (E2-T1) in E. coli resulted in predominantly aggregated insoluble IB. Solubilisation of E2-T1 with high purity and stability from IB aggregates was achieved using a strong reducing buffer containing 100 mM Dithiothreitol. Refolding by dialysis into 50 mM Tris (pH 7.0) containing 0.2% Igepal CA630 resulted in a soluble but aggregated protein solution. The novel application of a two-phase extraction of inclusion body preparations with Triton X-114 reduced endotoxin in solubilised E2-T1 to levels suitable for in vivo use without affecting protein yields. Dynamic light scattering analyses showed 37.5% of the protein was monomeric, the remaining comprised of soluble aggregates. Mice immunised with E2-T1 developed a high titre antibody response by ELISA. Western hybridisation analysis showed E2-T1 was recognised by sera from immunised mice and also by several BVDV-E2 polyclonal and monoclonal antibodies. CONCLUSION: We have developed a procedure using E. coli to produce soluble E2-T1 protein from IB, and due to their insoluble nature we utilised a novel approach using Triton X-114 to efficiently remove endotoxin. The resultant protein is immunogenic and detectable by BVDV-E2 specific antibodies indicating its usefulness for diagnostic applications and as a subunit vaccine. The optimised E. coli expression system for E2-T1 combined with methodologies for solubilisation, refolding and integrated endotoxin removal presented in this study should prove useful for other vaccine applications. |
format | Online Article Text |
id | pubmed-3160874 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-31608742011-08-25 Endotoxin-free purification for the isolation of Bovine Viral Diarrhoea Virus E2 protein from insoluble inclusion body aggregates Cavallaro, Antonino S Mahony, Donna Commins, Margaret Mahony, Timothy J Mitter, Neena Microb Cell Fact Research BACKGROUND: Protein expression in Escherichia coli may result in the recombinant protein being expressed as insoluble inclusion bodies. In addition, proteins purified from E. coli contain endotoxins which need to be removed for in vivo applications. The structural protein, E2, from Bovine Viral Diarrhoea Virus (BVDV) is a major immunogenic determinant, and is an ideal candidate as a subunit vaccine. The E2 protein contains 17 cysteine residues creating difficulties in E. coli expression. In this report we outline a procedure for successfully producing soluble and endotoxin-free BVDV E2 protein from inclusion bodies (IB). RESULTS: The expression of a truncated form of BVDV-E2 protein (E2-T1) in E. coli resulted in predominantly aggregated insoluble IB. Solubilisation of E2-T1 with high purity and stability from IB aggregates was achieved using a strong reducing buffer containing 100 mM Dithiothreitol. Refolding by dialysis into 50 mM Tris (pH 7.0) containing 0.2% Igepal CA630 resulted in a soluble but aggregated protein solution. The novel application of a two-phase extraction of inclusion body preparations with Triton X-114 reduced endotoxin in solubilised E2-T1 to levels suitable for in vivo use without affecting protein yields. Dynamic light scattering analyses showed 37.5% of the protein was monomeric, the remaining comprised of soluble aggregates. Mice immunised with E2-T1 developed a high titre antibody response by ELISA. Western hybridisation analysis showed E2-T1 was recognised by sera from immunised mice and also by several BVDV-E2 polyclonal and monoclonal antibodies. CONCLUSION: We have developed a procedure using E. coli to produce soluble E2-T1 protein from IB, and due to their insoluble nature we utilised a novel approach using Triton X-114 to efficiently remove endotoxin. The resultant protein is immunogenic and detectable by BVDV-E2 specific antibodies indicating its usefulness for diagnostic applications and as a subunit vaccine. The optimised E. coli expression system for E2-T1 combined with methodologies for solubilisation, refolding and integrated endotoxin removal presented in this study should prove useful for other vaccine applications. BioMed Central 2011-07-26 /pmc/articles/PMC3160874/ /pubmed/21787435 http://dx.doi.org/10.1186/1475-2859-10-57 Text en Copyright ©2011 Cavallaro et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Cavallaro, Antonino S Mahony, Donna Commins, Margaret Mahony, Timothy J Mitter, Neena Endotoxin-free purification for the isolation of Bovine Viral Diarrhoea Virus E2 protein from insoluble inclusion body aggregates |
title | Endotoxin-free purification for the isolation of Bovine Viral Diarrhoea Virus E2 protein from insoluble inclusion body aggregates |
title_full | Endotoxin-free purification for the isolation of Bovine Viral Diarrhoea Virus E2 protein from insoluble inclusion body aggregates |
title_fullStr | Endotoxin-free purification for the isolation of Bovine Viral Diarrhoea Virus E2 protein from insoluble inclusion body aggregates |
title_full_unstemmed | Endotoxin-free purification for the isolation of Bovine Viral Diarrhoea Virus E2 protein from insoluble inclusion body aggregates |
title_short | Endotoxin-free purification for the isolation of Bovine Viral Diarrhoea Virus E2 protein from insoluble inclusion body aggregates |
title_sort | endotoxin-free purification for the isolation of bovine viral diarrhoea virus e2 protein from insoluble inclusion body aggregates |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3160874/ https://www.ncbi.nlm.nih.gov/pubmed/21787435 http://dx.doi.org/10.1186/1475-2859-10-57 |
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