The selective infective phage (SIP) technology allows a rapid positive selection of interacting pairs of biological molecules that restore to non-infectious phages their ability to infect the bacterial host. After a successful infection, the phage is amplified and the DNA encoding the interacting ligand is isolated from the phage genome and sequenced. In our studies we have evaluated the usefulness of SIP for the identification and cloning of proteins interacting with a biotinylated target binding to a newly designed adapter molecule consisting of streptavidin fused to the C-terminus of the extracellular 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 non-infectious phage displaying a short peptide, which specifically binds the streptavidin, and the adapter molecule restore phage infectivity. To evaluate the potential of SIP as a general and generic tool for the screening of cDNA libraries that encode the ligands displayed at the surface of the phage 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 systems we show that the size of either the ligand or the target is a limiting factor for the technology.
Size of the ligand complex between the N-terminal domain of the gene III coat protein and the non-infectious phage strongly influences the usefulness of in vitro selective infective phage technology
- Views Icon Views
- Share Icon Share
Régis CÈBE, Martin GEISER; Size of the ligand complex between the N-terminal domain of the gene III coat protein and the non-infectious phage strongly influences the usefulness of in vitro selective infective phage technology. Biochem J 15 December 2000; 352 (3): 841–849. doi: https://doi.org/10.1042/bj3520841
Download citation file: