The Brassica pollen allergen Bra r 1 belongs to a new family of Ca2+-binding proteins, characterized by the presence of two potential EF-hand calcium-binding domains. Disruption of these EF-hand motifs by amino acid substitutions demonstrated that both domains of Bra r 1 constitute functional Ca2+-binding sites. Calcium-binding deficient mutants displayed significantly reduced IgE-binding activity. Injection of these mutated Bra r 1 variants into a murine model system showed that mouse IgG raised against the mutants recognized native Bra r 1 in Brassica pollen extracts suggesting the potential use of the engineered allergens for effective immunotherapy.