Fine mapping of the antigen-antibody interaction of scFv215, a recombinant antibody inhibiting RNA polymerase II from Drosophila melanogaster.
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.
Universität Heidelberg, Molekulare Genetik, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany.
Z
Z
Liu
D
D
Song
A
A
Kramer
A C
AC
Martin
T
T
Dandekar
J
J
Schneider-Mergener
E K
EK
Bautz
S
S
Dübel
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000937:Antigen-Antibody Reactions; D003198:Computer Simulation; D004331:Drosophila melanogaster / Q000276:immunology*; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000276:immunology; D019476:Insect Proteins / Q000037:antagonists & inhibitors / Q000276:immunology*; D018448:Models, Immunological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology
D000939:Epitopes; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D012319:RNA Polymerase II; D000409:Alanine