Cloning, Expression, and Mapping of Allergenic Determinants of αS1-Casein, a Major Cow's Milk Allergen

Milk is one of the first components introduced into human diet. It also represents one of the first allergen sources, which induces IgE-mediated allergies in childhood ranging from gastrointestinal, skin, and respiratory manifestations to severe life-threatening manifestations, such as anaphylaxis. Here we isolated a cDNA coding for a major cow's milk allergen, alpha S1-casein, from a bovine mammary gland cDNA library with allergic patients' IgE Abs. Recombinant alpha S1-casein was expressed in Escherichia coli, purified, and characterized by circular dichroism as a folded protein. IgE epitopes of alpha S1-casein were determined with recombinant fragments and synthetic peptides spanning the alpha S1-casein sequence using microarrayed components and sera from 66 cow's milk-sensitized patients. The allergenic activity of r alpha S1-casein and the alpha S1-casein-derived peptides was determined using rat basophil leukemia cells transfected with human Fc epsilon RI, which had been loaded with the patients' serum IgE. Our results demonstrate that r alpha S1-casein as well as alpha S1-casein-derived peptides exhibit IgE reactivity, but mainly the intact r alpha S1-casein induced strong basophil degranulation. These results suggest that primarily intact alpha S1-casein or larger IgE-reactive portions thereof are responsible for IgE-mediated symptoms of food allergy. Recombinant alpha S1-casein as well as alpha S1-casein-derived peptides may be used in clinical studies to further explore pathomechanisms of food allergy as well as for the development of new diagnostic and therapeutic strategies for milk allergy. The Journal of Immunology, 2009, 182: 7019-7029.