Co-Directors: Dr. Jill M Weimer and Dr. Sergio Padilla-Lopez
Staff: Tiffin Duffy (RA)
Mission Statement:
The goal of our core is to screen for novel protein-protein interactors using a yeast two-hybrid system.
Current Services:
1. Cloning of bait plasmids for two-hybrid screening
2. Cytotrap Yeast Two-Hybrid screening
Utility of the system:
For our screening, we use a unique yeast two-hybrid system manufactured by Aligent called the Cytotrap Yeast Two-Hybrid. This system has many advantages over the traditional transcriptionally activated system including:
• Screen of cytoplasmic interactions, rather than nuclear, allows posttranslational processing of target and bait proteins;
• Allows for screening of transcriptional activators and inhibitors;
• And, because the system is inducible, one can screen and/or discover toxic proteins
How the system works:
• Protein-protein interactions in the cytoplasm are detected by recruitment of the human Sos gene product (hSos) to the membrane of the cell where it activates the Ras pathway (growth at 37° instead of only 25°).
• This system uses the yeast strain cdc25H, which contains a temperature-sensitive mutation in the cdc25 gene; the yeast homologue for hSos.
• This protein, a guanyl nucleotide exchange factor, is essential for activation of the Ras pathway and ultimately for the survival and growth of the cell.
• The mutation in the cdc25 protein is temperature sensitive; the cells can grow at 25°C but not at 37°C. This cdc25 mutation can be complemented by the hSos gene product to allow growth at 37°, providing that the hSos protein is localized to the membrane via a protein-protein interaction.
Schematic of Cytotrap system (courtesy of Aligent/Stratagene):
For more information on how this system works, click here.
