Identifying protein-protein interactions with the ecdysone receptor ligand binding domain
Disciplines
Biochemistry | Chemicals and Drugs | Hormones, Hormone Substitutes, and Hormone Antagonists
Abstract (300 words maximum)
Understanding protein-protein interactions (PPIs) are important in many biological processes. In the advanced biochemistry CURE lab, we focus on protein interactions with the ecdysone receptor (EcR) ligand binding domain (LBD) to better understand how the EcR functions to control development in drosophila. In our research, we use the yeast two-hybrid assay to screen for novel proteins that physically intertact with the LBD of the Ecdysone receptor. This assay uses the modular nature of the transcription factor Gal4. The DNA binding domain of Gal4 is fused with the LBD to form the “bait” protein and it’s DNA activation domain (AD) is fused to proteins from a drosophila cDNA library to form “prey” proteins, If the bait and a prey interact, the BD and AD domains will come together to reconstitute Gal4 and turn on reporter genes like alpha galactosidase which will turn the yeast colonies blue in presence of X-alpha gal. Control experiments were conducted to ensure that the assay is functioning optimally. Yeast two hybrid assay with bait LBD alone did not autoactivate the reporter genes. Known interactors p53 and Tantigen gave blue colonies indicating a true interaction while non-interacting proteins gave no blue colonies as expected. A library screen with the bait will be conducted and positive interactors will be identified. This research will add to the understanding of gene regulation by the ecdysone receptor.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Rajnish Singh
Identifying protein-protein interactions with the ecdysone receptor ligand binding domain
Understanding protein-protein interactions (PPIs) are important in many biological processes. In the advanced biochemistry CURE lab, we focus on protein interactions with the ecdysone receptor (EcR) ligand binding domain (LBD) to better understand how the EcR functions to control development in drosophila. In our research, we use the yeast two-hybrid assay to screen for novel proteins that physically intertact with the LBD of the Ecdysone receptor. This assay uses the modular nature of the transcription factor Gal4. The DNA binding domain of Gal4 is fused with the LBD to form the “bait” protein and it’s DNA activation domain (AD) is fused to proteins from a drosophila cDNA library to form “prey” proteins, If the bait and a prey interact, the BD and AD domains will come together to reconstitute Gal4 and turn on reporter genes like alpha galactosidase which will turn the yeast colonies blue in presence of X-alpha gal. Control experiments were conducted to ensure that the assay is functioning optimally. Yeast two hybrid assay with bait LBD alone did not autoactivate the reporter genes. Known interactors p53 and Tantigen gave blue colonies indicating a true interaction while non-interacting proteins gave no blue colonies as expected. A library screen with the bait will be conducted and positive interactors will be identified. This research will add to the understanding of gene regulation by the ecdysone receptor.