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Updated: Jul 14, 2026
06:21
An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness
Published on: May 7, 2018
Ligand binding by recombinant domains from insect ecdysone receptors
L D Graham1, W M Johnson, A Pawlak-Skrzecz
1CSIRO Molecular and Health Technologies, Sydney Laboratory, North Ryde, NSW, Australia. lloyd.graham@csiro.au
Insect Biochemistry and Molecular Biology
|May 23, 2007
Summary
Researchers purified insect ligand binding domains (LBDs) to study ecdysone receptor interactions. A novel fluorescence polarization assay was developed for high-throughput screening of potential insecticides targeting insect pests.
Area of Science:
- Biochemistry
- Molecular Biology
- Insect Physiology
Background:
- The ecdysone receptor (EcR) and ultraspiracle (USP) proteins form heterodimers crucial for insect development.
- Understanding ligand interactions with these receptors is key to developing targeted pest control strategies.
Purpose of the Study:
- To characterize the binding affinities of various ligands to recombinant EcR/USP heterodimers from four major insect pests.
- To develop and validate a novel, high-throughput fluorescence polarization assay for screening ecdysone receptor agonists.
Main Methods:
- Purification of recombinant EcR/USP ligand binding domain (LBD) heterodimers from four insect species.
- Ligand binding assays using radiolabeled ponasterone A and determination of K(d) and K(i) values.
- Development of a fluorescence polarization assay using a fluorescein-inokosterone conjugate for high-throughput screening.
Main Results:
- Recombinant LBD heterodimers exhibited high affinity for ponasterone A (K(d) 0.7-2.5 nM).
- Ligand binding affinities correlated with the ability to induce gene expression in mammalian cells.
- The novel fluorescence polarization assay demonstrated high throughput and ranked ligand affinities similarly to traditional assays.
Conclusions:
- In vitro binding data using recombinant LBD heterodimers accurately predict in vivo activity of ecdysone receptor agonists.
- The developed fluorescence polarization assay is suitable for high-throughput screening of potential insecticides targeting insect pests.
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