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Fine mapping of the antigen–antibody interaction of scFv215, a recombinant antibody inhibiting RNA polymerase II from Drosophila melanogaster
Author(s) -
Liu Zhihong,
Song Danying,
Kramer Achim,
Martin Andrew C. R.,
Dandekar Thomas,
SchneiderMergener Jens,
Bautz Ekkehard K. F.,
Dübel Stefan
Publication year - 1999
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/(sici)1099-1352(199903/04)12:2<103::aid-jmr447>3.0.co;2-b
Subject(s) - immunoglobulin light chain , mutagenesis , microbiology and biotechnology , alanine , site directed mutagenesis , epitope , alanine scanning , binding site , directed mutagenesis , biology , amino acid , biochemistry , epitope mapping , drosophila melanogaster , rna , polymerase , antibody , protein subunit , complementarity determining region , chemistry , mutation , genetics , dna , gene , mutant
A bacterially expressed single chain antibody (scFv215) directed against the largest subunit of drosophila RNA polymerase II was analysed. Structure and function of the antigen binding site in scFv215 were probed by chain shuffling and by site‐specific mutagenesis. The entire variable region of either the heavy or light chain was replaced by an unrelated heavy or light chain. Both replacements resulted in a total loss of binding activity suggesting that the antigen binding site is contributed by both chains. The functional contributions of each complementarity determining region (CDR) were investigated by site specific mutagenesis of each CDR separately. Mutations in two of the CDRs, CDR1 of light chain and CDR2 of heavy chain, reduced the binding activity significantly. Each of the amino acids in these two CDRs was replaced individually by alanine (alanine walking). Seven amino acid substitutions in the two CDRs were found to reduce the binding activity by more than 50%. The data support a computer model of scFv215 which fits an epitope model based on a mutational analysis of the epitope suggesting an alpha‐helical structure for the main contact area. Copyright © 1999 John Wiley & Sons, Ltd.