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Improving the binding affinity of an antibody using molecular modeling and site‐directed mutagenesis
Author(s) -
Casipit Clayton L.,
Tal Rony,
Wittman Vaughan,
Chavaillaz PierreAndré,
Arbuthnott Kathy,
Weidanz Jon A.,
Jiao JinAn,
Wong Hing C.
Publication year - 1998
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560070802
Subject(s) - amino acid , homology modeling , alanine , biochemistry , fusion protein , epitope , peptide sequence , escherichia coli , peptide , mutagenesis , binding site , chemistry , microbiology and biotechnology , site directed mutagenesis , alanine scanning , protein engineering , antibody , biology , mutant , recombinant dna , gene , genetics , enzyme
Activated Factor X releases F1.2, a 271‐amino acid peptide, from the amino terminus of prothrombin during blood coagulation. A nine‐amino acid peptide, C9 (DSDRAIEGR), corresponding to the carboxyl terminus of F1.2 was synthesized and used to produce a monoclonal antibody, TA1 ( K D 1.22 × 10 −6 M). To model the TA1 antibody, we entered the sequence information of the cloned TA1 F v into the antibody modeling program, ABM, which combines homology methods, conformational search procedures, and energy screening and has proved to be a reliable and reproducible antibody modeling method. Using a novel protein fusion procedure, we expressed the C9 peptide fused to the carboxyl terminus of the PENI repressor protein from Bacillus licheniformis in Escherichia coli . We constructed fusion proteins containing alanine substitutions for each amino acid in the C9 epitope. Binding studies, using the C9 alanine mutants and TA1, and spatial constraints predicted by the modeled TA1 binding cleft enabled us to establish a plausible conformation for C9 complexed with TA1. Furthermore, based on binding results of conservative amino acid substitutions in C9 and mutations in the antibody, we were able to refine the complex model and identify antibody mutations that would improve binding affinity.