Hypoallergenic Variant of the Major Egg White Allergen Gal d 1 Produced by Disruption of Cysteine Bridges
| Autor: | Mimi L.K. Tang, Cenk Suphioglu, Dulashi Withanage-Dona, Pathum Dhanapala, Timothy J. Doran |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Mutagenesis (molecular biology technique) hypoallergens immunotherapy egg allergy allergens lcsh:TX341-641 Biology Ovomucin Article 03 medical and health sciences 0302 clinical medicine Egg White Mutant protein Escherichia coli Immune Tolerance Animals Humans Cysteine Egg Hypersensitivity Polyacrylamide gel electrophoresis Nutrition and Dietetics Hypoallergenic Allergens Immunoglobulin E Molecular biology Blot 030104 developmental biology 030228 respiratory system Genetic Techniques Mutagenesis Mutation Female lcsh:Nutrition. Foods and food supply Chickens Food Science Egg white |
| Zdroj: | Nutrients Nutrients, 9(2):171 Nutrients; Volume 9; Issue 2; Pages: 171 Nutrients, Vol 9, Iss 2, p 171 (2017) |
| ISSN: | 2072-6643 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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