Recombinant fel d I: expression, purification, IgE binding and reaction with cat-allergic human T cells

Abstract

This study describes the properties of the two recombinantly expressed polypeptide chains of Fel d I, the major allergen produced by the domestic 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 Ni 2+ ion affinity chromatography. This method provides high yields in a single step of rchain 1 and rchain 2 of Fel d I with a > 90% level of purity. Polymerase chain reaction (PCR) methods were used to introduce a thrombin cleavage site (LVPR↓GS) at the N -terminus of both chains. Thrombin cleavage of rchain 1 and rchain 2 followed by HPLC purification of the cleavage products allowed the isolation of each recombinant chain with only two additional residuals (GS) at the N-terminus of the native sequence. Amino acid sequencing analysis of the N-terminus and mass spectrometry of these polypeptides demonstrated that they are highly pure and full-length. Direct ELISA assays showed that IgE from cat-allergic patients binds to both rchain 1 and rchain 2 of Fel d I, demonstrating that both these chains contribute to the allergenicity of this heterodimeric protein. An examination of the reactivity of T cells derived from cat-allergic patients revealed that both polypeptide chains contribute to the T cell response to this allergen. Consequently, it is concluded that the immunological response to Fel d I is composed of a reaction at both the B and T cell level to each of the two chains that constitute the native allergen.