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Hypoallergenic Variant of the Major Egg White Allergen Gal d 1 Produced by Disruption of Cysteine Bridges
Background: Gal d 1 (ovomucoid) is the dominant allergen in the chicken egg white. Hypoallergenic variants of this allergen can be used in immunotherapy as an egg allergy treatment approach. We hypothesised that disruption of two of the nine cysteine-cysteine bridges by site-directed mutagenesis wil...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331602/ https://www.ncbi.nlm.nih.gov/pubmed/28230769 http://dx.doi.org/10.3390/nu9020171 |
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author | Dhanapala, Pathum Withanage-Dona, Dulashi Tang, Mimi L. K. Doran, Tim Suphioglu, Cenk |
author_facet | Dhanapala, Pathum Withanage-Dona, Dulashi Tang, Mimi L. K. Doran, Tim Suphioglu, Cenk |
author_sort | Dhanapala, Pathum |
collection | PubMed |
description | Background: Gal d 1 (ovomucoid) is the dominant allergen in the chicken egg white. Hypoallergenic variants of this allergen can be used in immunotherapy as an egg allergy treatment approach. We hypothesised that disruption of two of the nine cysteine-cysteine bridges by site-directed mutagenesis will allow the production of a hypoallergenic variant of the protein; Methods: Two cysteine residues at C192 and C210 in domain III of the protein were mutated to alanine using site-directed mutagenesis, to disrupt two separate cysteine-cysteine bridges. The mutated and non-mutated proteins were expressed in Escherichia coli (E. coli) by induction with isopropyl β-d-1-thiogalactopyranoside (IPTG). The expressed proteins were analysed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting to confirm expression. Immunoglobulin E (IgE) reactivity of the two proteins was analysed, by immunoblotting, against a pool of egg-allergic patients’ sera. A pool of non-allergic patients’ sera was also used in a separate blot as a negative control; Results: Mutant Gal d 1 showed diminished IgE reactivity in the immunoblot by showing lighter bands when compared to the non-mutated version, although there was more of the mutant protein immobilised on the membrane when compared to the wild-type protein. The non-allergic negative control showed no bands, indicating an absence of non-specific binding of secondary antibody to the proteins; Conclusion: Disruption of two cysteine bridges in domain III of Gal d 1 reduces IgE reactivity. Following downstream laboratory and clinical testing, this mutant protein can be used in immunotherapy to induce tolerance to Gal d 1 and in egg allergy diagnosis. |
format | Online Article Text |
id | pubmed-5331602 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-53316022017-03-13 Hypoallergenic Variant of the Major Egg White Allergen Gal d 1 Produced by Disruption of Cysteine Bridges Dhanapala, Pathum Withanage-Dona, Dulashi Tang, Mimi L. K. Doran, Tim Suphioglu, Cenk Nutrients Article Background: Gal d 1 (ovomucoid) is the dominant allergen in the chicken egg white. Hypoallergenic variants of this allergen can be used in immunotherapy as an egg allergy treatment approach. We hypothesised that disruption of two of the nine cysteine-cysteine bridges by site-directed mutagenesis will allow the production of a hypoallergenic variant of the protein; Methods: Two cysteine residues at C192 and C210 in domain III of the protein were mutated to alanine using site-directed mutagenesis, to disrupt two separate cysteine-cysteine bridges. The mutated and non-mutated proteins were expressed in Escherichia coli (E. coli) by induction with isopropyl β-d-1-thiogalactopyranoside (IPTG). The expressed proteins were analysed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting to confirm expression. Immunoglobulin E (IgE) reactivity of the two proteins was analysed, by immunoblotting, against a pool of egg-allergic patients’ sera. A pool of non-allergic patients’ sera was also used in a separate blot as a negative control; Results: Mutant Gal d 1 showed diminished IgE reactivity in the immunoblot by showing lighter bands when compared to the non-mutated version, although there was more of the mutant protein immobilised on the membrane when compared to the wild-type protein. The non-allergic negative control showed no bands, indicating an absence of non-specific binding of secondary antibody to the proteins; Conclusion: Disruption of two cysteine bridges in domain III of Gal d 1 reduces IgE reactivity. Following downstream laboratory and clinical testing, this mutant protein can be used in immunotherapy to induce tolerance to Gal d 1 and in egg allergy diagnosis. MDPI 2017-02-21 /pmc/articles/PMC5331602/ /pubmed/28230769 http://dx.doi.org/10.3390/nu9020171 Text en © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Dhanapala, Pathum Withanage-Dona, Dulashi Tang, Mimi L. K. Doran, Tim Suphioglu, Cenk Hypoallergenic Variant of the Major Egg White Allergen Gal d 1 Produced by Disruption of Cysteine Bridges |
title | Hypoallergenic Variant of the Major Egg White Allergen Gal d 1 Produced by Disruption of Cysteine Bridges |
title_full | Hypoallergenic Variant of the Major Egg White Allergen Gal d 1 Produced by Disruption of Cysteine Bridges |
title_fullStr | Hypoallergenic Variant of the Major Egg White Allergen Gal d 1 Produced by Disruption of Cysteine Bridges |
title_full_unstemmed | Hypoallergenic Variant of the Major Egg White Allergen Gal d 1 Produced by Disruption of Cysteine Bridges |
title_short | Hypoallergenic Variant of the Major Egg White Allergen Gal d 1 Produced by Disruption of Cysteine Bridges |
title_sort | hypoallergenic variant of the major egg white allergen gal d 1 produced by disruption of cysteine bridges |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331602/ https://www.ncbi.nlm.nih.gov/pubmed/28230769 http://dx.doi.org/10.3390/nu9020171 |
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