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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 Ni2+ 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 decreases 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.