Protein Engineering, Vol. 12, No. 5, 407-415,
May 1999
© 1999 Oxford University Press
Changes in the specificity of antibodies by site-specific mutagenesis followed by random mutagenesis
Toyota Central R&D Laboratories Inc., Nagakute, Aichi 480-1192, 1 Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192 and 2 Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, Kamiyogo, Setagaya, Tokyo 158, Japan
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.
Keywords: antibodies/error-prone PCR/phage-display antibody/steroid/structural models
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