title: Hypoallergenic Variant of the Major Egg White Allergen Gal d 1 Produced by Disruption of Cysteine Bridges.
abstract: 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 inEscherichia 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.
authors.affiliation: Neuro Allergy Research Laboratory (NARL), School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Deakin University, 75 Pigdons Road, Geelong 3216 VIC, Australia. dpathum@deakin.edu.au.|Australian Animal Health Laboratory (AAHL), Biosecurity Flagship, Commonwealth Scientific and Industrial Research Organisation (CSIRO), 5 Portarlington Road, East Geelong 3219 VIC, Australia. dpathum@deakin.edu.au.|Poultry CRC, P.O. Box U242, University of New England, Armidale 2351 NSW, Australia. dpathum@deakin.edu.au.|Department of Orthopedic Surgery, Brigham and Women's Hospital, Harvard Medical School, 60 Fenwood Road, Boston, 02115 MA, USA. dpathum@deakin.edu.au.
authors.forename: Pathum
authors.initials: P
authors.lastname: Dhanapala
authors.affiliation: Neuro Allergy Research Laboratory (NARL), School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Deakin University, 75 Pigdons Road, Geelong 3216 VIC, Australia. pdhanapala@bwh.harvard.edu.
authors.forename: Dulashi
authors.initials: D
authors.lastname: Withanage-Dona
authors.affiliation: Department of Allergy and Immunology, Royal Children's Hospital, 50 Flemington Road, Parkville 3052 VIC, Australia. Mimi.Tang@rch.org.au.|Allergy and Immune Disorders, Murdoch Children's Research Institute, 50 Flemington Road, Parkville 3052 VIC, Australia. Mimi.Tang@rch.org.au.|The University of Melbourne, Parkville 3010 VIC, Australia. Mimi.Tang@rch.org.au.
authors.forename: Mimi L K
authors.initials: ML
authors.lastname: Tang
authors.affiliation: Australian Animal Health Laboratory (AAHL), Biosecurity Flagship, Commonwealth Scientific and Industrial Research Organisation (CSIRO), 5 Portarlington Road, East Geelong 3219 VIC, Australia. Timothy.Doran@csiro.au.|Poultry CRC, P.O. Box U242, University of New England, Armidale 2351 NSW, Australia. Timothy.Doran@csiro.au.
authors.forename: Tim
authors.initials: T
authors.lastname: Doran
authors.affiliation: Neuro Allergy Research Laboratory (NARL), School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Deakin University, 75 Pigdons Road, Geelong 3216 VIC, Australia. cenk.suphioglu@deakin.edu.au.|Poultry CRC, P.O. Box U242, University of New England, Armidale 2351 NSW, Australia. cenk.suphioglu@deakin.edu.au.
authors.forename: Cenk
authors.initials: C
authors.lastname: Suphioglu
mesh_terms: D000485:Allergens / Q000235:genetics*; D000818:Animals; D002645:Chickens; D003545:Cysteine / Q000378:metabolism*; D021181:Egg Hypersensitivity / Q000276:immunology / Q000517:prevention & control; D004529:Egg White / Q000737:chemistry*; D004926:Escherichia coli; D005260:Female; D005821:Genetic Techniques; D006801:Humans; D007108:Immune Tolerance; D007073:Immunoglobulin E / Q000378:metabolism; D007167:Immunotherapy; D016296:Mutagenesis; D009154:Mutation; D010059:Ovomucin / Q000235:genetics* / Q000276:immunology
chemical_list: D000485:Allergens; D010059:Ovomucin; D007073:Immunoglobulin E; D003545:Cysteine
keywords: allergens; egg allergy; hypoallergens; immunotherapy