Reconstruction of an epitope capable of binding murine monoclonal antibodies NK2 within the sequence of human leukocyte interferon αF by site-directed mutagenesis

Using site-directed mutagenesis, an epitope of human leukocyte IFN-A capable of binding murine monoclonal antibodies NK2 has been reconstructed within the sequence of IFN-F, which lacked ability to bind these particular type of antibodies. It was found by the analysis of the recombinant IFN-A1–92F93–100 encoding by a hybrid gene that N-terminal part of IFN-A played no significant role in NK2 binding. Then by site-directed mutagenesis of IFN-F gene three residues of IFN-F polypeptide chain (Asn113, Val114 and Lys121) were substituted for the residues of IFN-A sequence (Lys113, Glu114 and Arg121), and it was shown that the mutated IFN-F(A 113,114) and IFN-F(A 113,114,121) restored the partial and full ability, respectively, to bind NK2 as compared with unmodified IFN-A, while IFN-F(A 121) lacked NK2 binding completely, as well as parental IFN-F. It shows that the residues Lys112 and Glu114 are crucial for NK2 binding, whereas Arg121, presumably, supports the necessary spatial arrangement of the epitope, being, nevertheless, essential for the binding of NK2.