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A general method for quantifying ligand binding to unmodified receptors using Gaussia luciferase
- Dr. András Tóth -
- Semmelweis University -
- Budapest, Hungary -
Abstract
Resonance energy transfer-based methods are useful approaches for the direct measurement of ligand binding to receptors, especially for kinetic ligand binding measurements. However, their major disadvantage is the necessity of the genetic modification of the receptor of interest, which may profoundly alter the receptor properties. Therefore, we established a tag-free system to measure ligand-receptor interactions in live cells using the Gaussia luciferase (GLuc). GLuc has exceptional brightness and low molecular weight, comparable to NanoLuc. In our assay, HEK 293 cells are transfected with the unmodified receptor and cell surface-targeted GLuc (GLuc‒PM), and bioluminescence resonance energy transfer is measured between GLuc‒PM and fluorescent ligands. Similar to traditional competitive radioligand binding measurements, our method allows the determination of the fundamental pharmacological parameters, such as KD, Kon, Koff, both for labeled and unlabeled ligands. The system can be adjusted for any cell surface receptors if a fluorescent ligand is available. We also demonstrate the applicability of the system for ligand screening.
About Dr. András Tóth
Dr. András Tóth received his MD degree in 2013, and his Ph.D. degree in 2018 at Semmelweis University, Budapest, Hungary. Currently, he works as a resident doctor in the Internal Medicine and Haematology Clinic and as a part-time research associate in the laboratory of Professor László Hunyady. With the aid of the Jellinek Harry student scholarship, he joined the laboratory of Professor Johannes Backs (Cardiology Clinic, Heidelberg, Germany) for a year in 2011. His main research interests include the understanding of G protein-coupled receptor functions and the therapeutic potential of biased signaling.
Author list and affiliations
András Dávid Tóth1,2,3, Dániel Garger1, Susanne Prokop1,4, Eszter Soltész-Katona1,2, Péter Várnai1,2, András Balla1,2, Gábor Turu1,2, László Hunyady1,2,*
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1. Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary
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2. MTA-SE Laboratory of Molecular Physiology, Budapest, Hungary
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3. Department of Internal Medicine and Hematology, Semmelweis University, Budapest, Hungary
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4. Szentágothai János Doctoral School of Neuroscience, Semmelweis University, Budapest, Hungary
Dr. András Tóth on the web
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