Alexenko A P, Izotova L
Institute of Genetics and Selection of Industrial Microorganisms, Moscow, USSR.
Biochem Biophys Res Commun. 1990 Jun 29;169(3):1061-7. doi: 10.1016/0006-291x(90)92002-h.
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-A1-92/F93-100 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 Lys113 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.
利用定点诱变技术，在缺乏结合这些特定类型抗体能力的IFN-F序列中重建了人白细胞IFN-A的一个能够结合鼠单克隆抗体NK2的表位。通过对杂合基因编码的重组IFN-A1-92/F93-100进行分析发现，IFN-A的N端部分在NK2结合中不起重要作用。然后通过对IFN-F基因进行定点诱变，将IFN-F多肽链的三个残基（Asn113、Val114和Lys121）替换为IFN-A序列的残基（Lys113、Glu114和Arg121），结果表明，与未修饰的IFN-A相比，突变后的IFN-F(A 113, 114)和IFN-F(A 113, 114, 121)分别恢复了部分和全部结合NK2的能力，而IFN-F(A 121)以及亲本IFN-F完全缺乏NK2结合能力。这表明Lys113和Glu114残基对于NK2结合至关重要，而Arg121大概支持表位的必要空间排列，尽管如此，它对于NK2的结合也是必不可少的。
