Improving the binding affinity of an antibody using molecular modeling and site‐directed mutagenesis

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 (KD 1.22 × 10−6 M). To...

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Bibliographic Details
Published in:Protein science 1998-08, Vol.7 (8), p.1671-1680
Main Authors: Casipit, Clayton L., Tal, Rony, Wittman, Vaughan, Chavaillaz, Pierre‐André, Arbuthnott, Kathy, Weidanz, Jon A., Jiao, Jin‐An, Wong, Hing C.
Format: Article
Language:English
Subjects:
affinity improvement
Alanine - chemistry
Amino Acids - chemistry
Antibodies - chemistry
Bacillus - metabolism
Binding, Competitive
Blotting, Western
computer modeling
Computer Simulation
Escherichia coli - metabolism
Hydrogen Bonding
Membrane Proteins - immunology
Models, Molecular
Mutagenesis, Site-Directed
Oligonucleotides
peptide‐antibody complex
Protein Binding
Recombinant Fusion Proteins
site‐directed mutagenesis
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Summary: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 (KD 1.22 × 10−6 M). To model the TA1 antibody, we entered the sequence information of the cloned TA1 Fv 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.
ISSN:0961-8368
1469-896X
DOI:10.1002/pro.5560070802