IgE reactivity of tandem repeats derived from cockroach allergen, Bla g 1.
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 approximately 48 mg x 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.
Pomés
Anna
A
Asthma and Allergic Diseases Center, Department of Medicine, University of Virginia, Charlottesville, VA, USA. apomes@inbio.com
Vailes
Lisa D
LD
Helm
Ricki M
RM
Chapman
Martin D
MD
D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000936:Antigen-Antibody Complex / Q000276:immunology / Q000378:metabolism; D052179:Antigens, Plant; D003058:Cockroaches / Q000737:chemistry* / Q000276:immunology; D000939:Epitopes / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007073:Immunoglobulin E / Q000276:immunology*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D020449:Repetitive Sequences, Amino Acid / Q000276:immunology*
D000485:Allergens; D000936:Antigen-Antibody Complex; D052179:Antigens, Plant; D000939:Epitopes; D011994:Recombinant Proteins; C105482:allergen Bla g 1; D007073:Immunoglobulin E