The project is to develop a monovalent phage display vector that can be used for studying the interactions of antibody-antigen, protein-ligand, and protein- receptors. The availability of a vector that can display a single fusion protein will have an advantage over the current M13-phage based vectors. The new vector may ultimately lead to the development of an in vitro system for studying high and low affinity antibodies. The proposal will address a reported weakness in the existing Ml3 fusion display vectors, in which fusions are made with the gene III protein (5 copies per viral particle) or gene VIII product (2700 copies per phage particle). Because of the multiple copies of these fusion proteins, phage with low affinity epitopes are generally isolated, instead of the presumably more rare high affinity epitopes. The proposal is to use the Bacillus subtilis phage phi29 instead of M13. Peptide and protein fusions will be made with the gp3 protein, which is covalently bound to each 5' end of mature phage double-stranded DNA and is required for phage replication. The DNA will be isolated from phage particles or cell lysates and cut with an appropriate restriction endonuclease to remove one end, but retaining the end containing the protein-gp3 fusion protein and the gene encoding it (in the Phase II form). This system, in which DNA has an attached monovalent protein probe, can be used for construction of peptide libraries and for generation of antibody-gp3 fusions.
