A dual luciferase multiplexed high-throughput screening platform for protein-protein interactions.
To study the biology of regulators of G-protein signaling (RGS) proteins and to facilitate the identification of small molecule modulators of RGS proteins, the authors recently developed an advanced yeast 2-hybrid (YTH) assay format for GalphaZ and RGS-Z1. Moreover, they describe the development of a multiplexed luciferase-based assay that has been successfully adapted to screen large numbers of small molecule modulators of protein-protein interactions. They generated and evaluated 2 different luciferase reporter gene systems for YTH interactions, a Gal4 responsive firefly luciferase reporter gene and a Gal4 responsive Renilla luciferase reporter gene. Both the firefly and Renilla luciferase reporter genes demonstrated a 40- to 50-fold increase in luminescence in strains expressing interacting YTH fusion proteins versus negative control strains. Because the firefly and Renilla luciferase proteins have different substrate specificity, the assays were multiplexed. The multiplexed luciferase-based YTH platform adds speed, sensitivity, simplicity, quantification, and efficiency to YTH high-throughput applications and therefore greatly facilitates the identification of small molecule modulators of protein-protein interactions as tools or potential leads for drug discovery efforts.
Nieuwenhuijsen
Bart W
BW
Neuroscience Discovery Research, Wyeth Research, Princeton, NJ 08543, USA.
Huang
Youping
Y
Wang
Yuren
Y
Ramirez
Fernando
F
Kalgaonkar
Gary
G
Young
Kathleen H
KH
D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism; D008156:Luciferases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques / Q000295:instrumentation
D004791:Enzyme Inhibitors; D011506:Proteins; D006160:Guanosine Triphosphate; D008156:Luciferases; D020558:GTP Phosphohydrolases