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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 and Suphioglu, Cenk 2017, Hypoallergenic variant of the major egg white allergen Gal d 1 produced by disruption of cysteine bridges, Nutrients, vol. 9, no. 2, pp. 1-11, doi: 10.3390/nu9020171.

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Title Hypoallergenic variant of the major egg white allergen Gal d 1 produced by disruption of cysteine bridges
Author(s) Dhanapala, Pathum
Withanage-Dona, Dulashi
Tang, Mimi L. K.
Doran, Tim
Suphioglu, Cenk
Journal name Nutrients
Volume number 9
Issue number 2
Start page 1
End page 11
Total pages 11
Publisher MDPI
Place of publication Basel, Switzerland
Publication date 2017-02
ISSN 2072-6643
Keyword(s) allergens
egg allergy
hypoallergens
immunotherapy
Animals
Chickens
Cysteine
Egg Hypersensitivity
Egg White
Escherichia coli
Female
Genetic Techniques
Humans
Immune Tolerance
Immunoglobulin E
Mutagenesis
Mutation
Ovomucin
Science & Technology
Life Sciences & Biomedicine
Nutrition & Dietetics
ORAL IMMUNOTHERAPY
FOOD ALLERGIES
IGE
RECOMBINANT
VACCINATION
EPITOPES
SAFETY
Summary 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.
Language eng
DOI 10.3390/nu9020171
Field of Research 090805 Food Processing
1111 Nutrition And Dietetics
HERDC Research category C1 Refereed article in a scholarly journal
ERA Research output type C Journal article
Copyright notice ©2017, The Authors
Free to Read? Yes
Use Rights Creative Commons Attribution licence
Persistent URL http://hdl.handle.net/10536/DRO/DU:30093808

Document type: Journal Article
Collections: School of Life and Environmental Sciences
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Every reasonable effort has been made to ensure that permission has been obtained for items included in DRO. If you believe that your rights have been infringed by this repository, please contact drosupport@deakin.edu.au.