Engineering of Immunoglobulin Fc and Single-Chain Fv Proteins in Escherichia coli

Abstract

The field of antibody engineering encompasses the investigation and redesign of constant domain associated effector functions, as well as the intensive current research on variable domain associated antigen-binding sites. We have investigated the potential of E. coli expression of single-chain Fv and Fc proteins to both provide model systems and practical reagents for antibody effector region interactions. We shall first review the current status of single-chain Fv protein engineering. A selected single-chain Fv protein with human IgM Cμ specificity has been constructed and characterized in our laboratory. The variable region genes of anti-μ monoclonal antibody DA4.4 were isolated and an engineered sFv version of this antibody expressed in E. coli was shown to be an IgM-specific binding protein. A simple screening method was developed for protein-protein interactions between recombinant CH-producing E. coli and CH-binding proteins displayed on the surface of bacteriophage M13. This method, which allows facile engineering and recovery of recombinant gene products, provides a model system for the characterization and designed modification of antibody effector domains.