Identification of Gal80p-interacting proteins by Saccharomyces cerevisiae whole genome phage display.
Networks of interacting proteins and protein interaction maps can help in functional annotation in genome analysis projects. We present the application of genomic phage display as a tool to identify interacting proteins in Saccharomyces cerevisiae. We have developed a large phagemid display library (approximately 7.7x10(7) independent clones) of sheared S. cerevisiae genomic DNA (12.1 Mbp genome size) fused to gene III (lacking the N1 domain) of the filamentous phage M13. Baits tagged with an N-terminal E-tag and a C-terminal His(6)-tag are prepared in a novel Escherichia coli expression system. Using E-Gal80-His(6) as bait, biopanning of the library resulted in the isolation of two different clones containing fragments of the known interacting partner Gal4p. In addition, three new ligands (Ubr1p, YCL045c and Prp8p) with potential physiological relevance were isolated. Interactions were confirmed by ELISA. These results demonstrate the accessibility of the S. cerevisiae genome to display technology for protein-protein interaction screening.
Hertveldt
Kirsten
K
Laboratorium voor Gentechnologie, Katholieke Universiteit Leuven, Belgium. kirsten.hertveldt@agr.kuleuven.ac.be
Dechassa
Mekonnen Lemma
ML
Robben
Johan
J
Volckaert
Guido
G
D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000235:genetics; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D004268:DNA-Binding Proteins; D005822:Genetic Vectors / Q000235:genetics; D016681:Genome, Fungal; D015698:Genomic Library; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; C055002:GAL80 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors