This project will utilize a T7 expression vector for the production of anti-dansyl VH protein in E. coli. The VH proteins associated with two anti-dansyl hybridomas have been purified from bacterial extracts and recombined with homologous light chains to yield fully active VH-L products. These VH genes will be subjected to site-directed mutagenesis with single or paired primers to provide mutated VH proteins with replacements in the hypervariable regions. The mutant VH proteins will be recombined with L chains and the purified products characterized with respect to affinity by resonance energy transfer, a method established in this laboratory. In addition, the CDR3 segments of high affinity and low affinity VDJ genes will be interchanged to evaluate the role of CDR3 in specific reactivity, particularly with germ-line (IgM) antibodies. The goal of this research is development of a general method to manipulate the affinity of monoclonal antibodies for their ligands using techniques of genetic engineering. If this project succeeds the method will have many practical as well as basic, scientific applications.
