Application of the split-protein sensor Trp1 to protein interaction discovery in the yeast Saccharomyces cerevisiae.
Yeast two-hybrid based systems are powerful tools for the detection and characterization of protein-protein interactions (PPIs). However, some important protein classes, e.g., integral membrane proteins and transcription factors, are difficult to study using these technologies. To overcome these limitations, we have employed a novel protein complementation screening platform. Protein interactions are detected by reconstitution of the split-protein sensor TRP1, enabling trp1 cells to grow on medium lacking tryptophan. Since the interaction readout is direct and independent of transcriptional reporter activation the rate of false positives is lowered. Furthermore, the technology allows for detection of protein interactions in their natural setting, e.g., the cytosol, the nucleus, and at cellular or organellar membranes. The protocols used for screening are explained in detail and as an example we describe the isolation of novel binding partners found with APP screened against a human cDNA library.
Dualsystems Biotech Inc, Zurich, Switzerland.
Mandana
M
Rezwan
Nicolas
N
Lentze
Lukas
L
Baumann
Daniel
D
Auerbach
D019747:Aldose-Ketose Isomerases / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D010957:Plasmids / Q000235:genetics; D025941:Protein Interaction Mapping / Q000379:methods*; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014170:Transformation, Genetic
D029701:Saccharomyces cerevisiae Proteins; D019747:Aldose-Ketose Isomerases; C490353:TRP1 protein, S cerevisiae