In vivo selectively infective phage as a tool to detect protein interactions: evaluation of a novel vector system with yeast Ste7p-Fus3p interacting proteins
Abstract
The selectively infective phage (SIP) approach allows rapid identification of interacting proteins by linking protein-protein interaction to phage infectivity. Infection of E. coli by filamentous phage depends on viral g3p. This protein consists of three domains, N1, N2 and CT. Phages lacking the N1 domain are non-infective unless a bait (X)-prey (Y) interaction links it to phage anchored N2-CT domains. We have developed all the vectors required for an in vivo selectively infective phage strategy (SIP). This includes a bait vector, pG3N1, a prey vector, pHOS41, and a gene III deletion helper phage, HPd3. The bait vector pG3N1 allows expression of a bait protein (X) fused to the C-terminus of the N1 domain. The prey vector pHOS41 allows expression of prey (Y) proteins, fused to the N-terminus of the N2-CT domains. The gene III deletion helper phage delivers all phage proteins necessary for phage production, except g3p. Escherichia coli transformed with these three vectors produces non-infective phages unless a bait-prey interaction links the g3p domains. Fus3p and Ste7p, two proteins from the Saccharomyces cerevisiae pheromone-responsive pathway have been cloned to evaluate the SIP strategy. The presence of the interacting N1-Fu...Continue Reading
References
Direct interaction rescue, a novel filamentous phage technique to study protein-protein interactions
Citations
Identification of Gal80p-interacting proteins by Saccharomyces cerevisiae whole genome phage display
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