Changes in the specificity of antibodies by site-specific mutagenesis followed by random mutagenesis

• Published in: Protein engineering Vol. 12; no. 5; pp. 407 - 415
• Main Authors: Miyazaki, Chie, Iba, Yoshitaka, Yamada, Yukio, Takahashi, Haruo, Sawada, Jun-ichi, Kurosawa, Yoshikazu
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01-05-1999
• Subjects: Amino Acid Sequence; antibodies; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - immunology; Antibody Specificity - genetics; Base Sequence; Computer Simulation; Cortodoxone - immunology; DNA Primers - genetics; Enzyme-Linked Immunosorbent Assay; error-prone PCR; Gene Library; Hydrocortisone - immunology; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - immunology; Kinetics; Molecular Sequence Data; Mutagenesis - genetics; Mutagenesis - immunology; Mutagenesis, Site-Directed; Mutation; phage-display antibody; Polymerase Chain Reaction; steroid; structural models; Surface Plasmon Resonance; antibodies; phage-display antibody; structural models; error-prone PCR; steroid
• ISSN: 0269-2139; 1741-0126; 1460-213X; 1741-0134
• DOI: 10.1093/protein/12.5.407

The specificity for 11-deoxycortisol (11-DOC) of a monoclonal antibody (mAb), designated SCET, was changed to specificity for cortisol (CS) by site-specific mutagenesis followed by random mutagenesis. The Fab form of SCET was expressed on the surface of a phage. During the first step, mutations were introduced at 14 amino acid positions in three complementarity-determining regions (CDRs) of the VH domain that seemed likely to form the steroid-binding pocket. A clone, DcC16, was isolated from the resultant library with multiple mutations and this clone was shown to have CS-binding activity but also to retain high 11-DOC-binding activity. During the second step, mutations were introduced randomly into the entire VH-coding region of the DcC16 clone by an error-prone polymerase chain reaction, and CS-specific mutant antibodies were selected in the presence of 11-DOC as a competitor. Three representative clones were analyzed with the BIAcore instrument, and each revealed a large increase in the binding constant for CS and a decrease in that for 11-DOC. Structural models, constructed by computer simulation, indicated the probable molecular basis for these changes in specificity.