In vivo selectively infective phage as a tool to detect protein interactions: evaluation of a novel vector system with yeast Ste7p-Fus3p interacting proteins.
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-Fus3p adapter increased the infectivity of Ste7p-N2-CT phages approximately 1400-fold, which makes SIP a promising technology for the detection and further investigation of interacting proteins.
Hertveldt
Kirsten
K
Laboratorium voor Gentechnologie, Kasteelpark Arenberg 21, B-3001 Leuven, Belgium. kirsten.Hertveldt@agr.kuleuven.ac.be
Robben
Johan
J
Volckaert
Guido
G
D036022:Capsid Proteins; D003090:Coliphages; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000821:virology*; D005656:Fungal Proteins / Q000378:metabolism; D005822:Genetic Vectors; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014760:Viral Fusion Proteins / Q000235:genetics; D014779:Virus Replication
D036022:Capsid Proteins; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C089181:RVS161 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014760:Viral Fusion Proteins; D011494:Protein Kinases; C075580:FUS3 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; C119844:STE7 protein, S cerevisiae