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Journal of Biomolecular Screening
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A Dual Luciferase Multiplexed High-Throughput Screening Platform for Protein-Protein Interactions

Bart W. Nieuwenhuijsen

Neuroscience Discovery Research, Wyeth Research, Princeton, NJ

Youping Huang

Biometrics Research, Wyeth Research, Princeton, NJ

Yuren Wang

Neuroscience Discovery Research, Wyeth Research, Princeton, NJ

Fernando Ramirez

Gary Kalgaonkar

Biological Chemistry/Screening, Wyeth Research, Princeton, NJ

Kathleen H. Young

Neuroscience Discovery Research, Wyeth Research, Princeton, NJyoungk3{at}wyeth.com

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 G{alpha}Zand 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.

Key Words: dual luciferase • high throughput • yeast • yeast 2-hybrid • RGS protein

Journal of Biomolecular Screening, Vol. 8, No. 6, 676-684 (2003)
DOI: 10.1177/1087057103258287
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 J Biomol ScreenHome page
D. L. Roman, S. Ota, and R. R. Neubig
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[Abstract] [PDF]