Size of the ligand complex between the N-terminal domain of the gene III coat protein and the non-infectious phage strongly influences the usefulnessof in vitro selective infective phage technology

The selective infective phage (SIP) technology allows a rapid positive sele ction of interacting pairs of biological molecules that restore to non-infe ctious phages their ability to infect the bacterial host. After a successfu l infection, the phage is amplified and the DNA encoding the interacting li gand is isolated from the phage genome and sequenced. In our studies we hav e evaluated the usefulness of SIP for the identification and cloning of pro teins interacting with a biotinylated target binding to a newly designed ad apter molecule consisting of streptavidin fused to the C-terminus of the ex tracellular domain of the phage minor coat protein III. The new adapter was expressed in Escherichia coli and refolded from inclusion bodies. The two different domains joined within the chimaera were found to be biologically functional. We also demonstrated that non-covalent interactions between a n on-infectious phage displaying a short peptide, which specifically binds th e streptavidin, and the adapter molecule restore phage infectivity. To eval uate the potential of SIP as a general and generic tool for the screening o f cDNA libraries that encode the ligands displayed at the surface of the ph age and binding to biotinylated targets, we have increased both the size of the displayed ligand on the phage and the size of the biotinylated target bound to the streptavidin domain of the adapter molecule. In our model syst ems we show that the size of either the ligand or the target is a limiting factor for the technology.