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Stimulus Controlled Infectivity of Phage: A Novel Method for the Selection of Stimulus-Responsive Peptides

Mark Blenner and Scott Banta. Chemical Engineering, Columbia University, 500 W 120th St, New York, NY 10027

Here we present the development of a technique called Stimulus Controlled Infectivity of Phage (SCIP) and its application in the selection of ligand induced peptide conformational changes. Filamentous phage infect E. Coli expressing the F' pilus via interaction between the gene III coat protein and the TolA receptor on the bacterial surface. The gene III coat protein is comprised of three domains that have functional roles in the infection process: N1, which binds to the TolA receptor, N2, which binds to the F' pilus and CT, which is connected to the phage coat. Once the N2 domain binds, the pilus retracts and brings the N1 domain closer to the TolA receptor, allowing phage infection of E. Coli.. A flexible 19 amino acid linker (EGGGSEGGGSEGGGSEGGG) connects N1 and N2 and is believed to provide sufficient flexibility to allow a pilus bound phage to simultaneously bind the TolA receptor. Infectivity of phage with a flexible linker without glutamic acid residues is explored as well as phage with no linker, a cysteine constrained linker, and a structured tryptophan zipper linker. This system allows for the selection of peptide linkers exhibiting stimulus controlled conformational changes upon ligand binding from both a na´ve and GPG constrained libraries using tandem positive and negative selections. During the positive selection, the phage population is allowed to infect E. Coli and propagate normally, removing any non-infective phage from the library. Phage are then recovered and incubated with ligand. Random peptide linkers that are able to bind the ligand and undergo a conformational change will lose their infective ability. The non-binders are removed by allowing them to infect E. Coli over a short time scale, and removing the E. Coli by centrifugation. The remaining phage are recovered, the ligand removed, and infectivity is once again restored. This technique can be used to develop allosteric ligand binders, protease resistant ligand binders, and stimulus-responsive elements for bionanotechnology applications.