RECOMBINANT FEL D I - EXPRESSION, PURIFICATION, IGE BINDING AND REACTION WITH CAT-ALLERGIC HUMAN T-CELLS

This study describes the properties of the two recombinantly expressed polypeptide chains of Fel d I, the major allergen produced by the dom estic cat (Felis domesticus). An inframe linker encoding polyhistidine has been added to the 5' ends of the Fel d I chains 1 and 2 cDNAs to facilitate purification using Ni2+ ion affinity chromatography. This m ethod provides high yields in a single step of rchain 1 and rchain 2 o f Fel d I with a > 90 % level of purity. Polymerase chain reaction (PC R) methods were used to introduce a thrombin cleavage site (LVPR down GS) at the N-terminus of both chains. Thrombin cleavage of rchain 1 an d rchain 2 followed by HPLC purification of the cleavage products allo wed the isolation of each recombinant chain with only two additional r esiduals (GS) at the N-terminus of the native sequence. Amino acid seq uencing analysis of the N-terminus and mass spectrometry of these poly peptides demonstrated that they are highly pure and full-length. Direc t ELISA assays showed that IgE from cat-allergic patients binds to bot h rchain 1 and rchain 2 of Fel d I, demonstrating that both these chai ns contribute to the allergenicity of this heterodimeric protein. An e xamination of the reactivity of T cells derived from cat-allergic pati ents revealed that both polypeptide chains contribute to the T cell re sponse to this allergen. Consequently, it is concluded that the immuno logical response to Fel d I is composed of a reaction at both the B an d T cell level to each of the two chains that constitute the native al lergen.