Open AccessIgE reactivity of tandem repeats derived from cockroach allergen, Bla g 1
Author(s) -
Pomés Anna,
Vailes Lisa D.,
Helm Ricki M.,
Chapman Martin D.
Publication year - 2002
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2002.02990.x
Subject(s) - pichia pastoris , immunoglobulin e , recombinant dna , german cockroach , allergen , chemistry , tandem repeat , microbiology and biotechnology , molecular mass , cockroach , antibody , biology , allergy , biochemistry , immunology , gene , enzyme , genome , ecology
Sensitization to cockroach allergens is associated with the development of asthma. Bla g 1 is a German cockroach allergen that shows allergenic cross‐reactivity with American cockroach allergen, Per a 1, and has a molecular structure composed of multiple tandem amino‐acid repeats. Two consecutive repeats are not identical but form a duplex that constitutes a basic molecular unit of Bla g 1. By molecular mass, purified natural Bla g 1 would contain approximately two duplexes. We investigated the pattern of IgE antibody binding to this repeated structure, and whether one or two duplexes are sufficient for IgE binding. Recombinant (r)Bla g 1 duplexes were expressed in Escherichia coli and in Pichia pastoris , and analyzed for monoclonal antibody and IgE antibody binding by ELISA and/or immunoblotting. Optimal rBla g 1 expression was obtained using methanol‐inducible P. pastoris (> 95% pure protein, yield ≈ 48 mg·L −1 ), and rBla g 1 was produced as multiple molecular forms of molecular mass 43, 32, 21 and 6 kDa, that were the result of proteolytic cleavage. There was an excellent correlation between IgE antibody binding to natural and recombinant Bla g 1 ( r = 0.91, n = 29, P < 0.001), and immunoblot analysis showed that a single Bla g 1 duplex was sufficient for IgE antibody binding. The rBla g 1 is suitable for structural studies and a candidate for clinical use in diagnosis of cockroach allergy and development of new forms of immunotherapy.