A High-Throughput Screening Approach for Evaluating the Binding Affinity of Environmental Pollutants to Nuclear Receptors

Summary

This protocol describes a high-throughput screening system that uses fluorescence polarization of a specific fluorescent probe binding to a nuclear receptor as a readout for screening environmental pollutants.

Abstract

Increasing levels of compounds have been detected in the environment, causing widespread pollution and posing risks to human health. However, despite their high environmental occurrence, there is very limited information regarding their toxicological effects. It is urgent to develop high-throughput screening (HTS) methods to guide toxicological studies. In this study, a receptor-ligand binding assay using an HTS system was developed to determine the binding potency of environmental pollutants on nuclear receptors. The test is conducted using a microplate reader (i.e., a 96-well plate containing various chemicals) by measuring the fluorescence polarization (FP) of a specific fluorescent probe. This assay consists of four parts: the construction and transformation of recombinant vectors, the expression and purification of the receptor protein (ligand-binding domain), receptor-probe binding, and competitive binding of chemicals with the receptor. The binding potency of two environmental pollutants, perfluorooctanesulfonic acid (PFOS) and triphenyl phosphate (TPHP), with peroxisome proliferator-activated receptor gamma (PPARγ) was determined to illustrate the assay procedure. Finally, the advantages and disadvantages of this method and its potential applications were also discussed.

Introduction

A large number of chemicals have been widely detected in the environment and human bodies, raising significant concerns about their impact on the ecological environment and human health^1,2,3. Despite their high environmental occurrence, information regarding their toxicological effects is scarce. Therefore, it is urgent to develop high-throughput screening (HTS) methods to facilitate the assessment of chemical toxicity.

Several high-throughput screening (HTS) methods have been reported for chemical toxicity assessment, such as the HTS bioassays use....

Protocol

The details of the reagents and the equipment are listed in the Table of Materials.

1. Construction and transformation of recombinant vectors

NOTE: PPARγ is a ligand-dependent transcription factor with a classical nuclear receptor structure, comprising a DNA-binding domain that regulates target genes and a ligand-binding domain activated by ligands. Upon ligand activation, PPARγ forms a heterodimer with another nuclear recep.......

Discussion

Fluorescence polarization (FP), surface plasmon resonance (SPR), and nuclear magnetic resonance (NMR) are common techniques used for assessing direct binding interactions between proteins and compounds^19,20. FP has been widely employed in the investigation of molecular interactions for drug discovery and chemical screening^21,22,23. In comparison, SPR and NMR assays are e.......

Materials

Name	Company	Catalog Number	Comments
C1-BODIPY-C12 Probe	Thermo Fisher Scientific, China	102209-82-3	Binds to PPARγ-LBD and emits fluorescence.
Coomassie Brilliant Blue R-250	Solarbio, China	6104-59-2	Stain the protein bands.
GraphPad prism	Dotmatics		https://www.graphpad.com/features
imidazole	Solarbio, China	I8090	Prepare buffers for the protein purification process.
Isopropyl β-D-1-thiogalactopyranoside	Solarbio, China	367-93-1	Induce the expression of PPARγ-LBD
Microplate reader	Biotek , USA	Synergy H1	Detecting FP value
NaCl	Shanghai Reagent	7647-15-5	Prepare buffers for the protein purification process.
NaH2PO4 · 2H2O	Shanghai Reagent	13472-35-0	Prepare buffers for the protein purification process.
Ni NTA Beads 6FF	Smart-Lifesciences, China	SA005005	Protein purification.
Origin 8.5	OriginLab, Northampton, MA, U.S.A.
Perfluorooctanesulfonic acid (PFOS)	J&K Scientific Ltd, China	1763-23-1	The detected environmental pollutants
Phenylmethylsulfonyl fluoride (PMSF)	Solarbio, China	P0100	Inhibit protein degradation.
PPARγ-Competitor Assay Kit	Thermo Fisher Scientific	PV6136	https://www.thermofisher.com/order/catalog/product/PV6136
PPARγ-LBD Ligand Screening Assay Kit	Cayman	600616	https://www.caymanchem.com/product/600616
Rosiglitazone (Rosi)	aladdin, China	122320-73-4	The agonists of PPARγ
Shaker	ZHICHENG, China	ZWY-211C	Bacterial culture expansion and induction of protein expression
Triphenyl phosphate (TPHP)	Macklin, China	T819317	The detected environmental pollutants
Tris	Solarbio, China	T8230	Prepare buffers for the protein purification process.
Tryptone	OXOID Limited, China	LP0042B	Prepare Lysogeny Broth (LB) medium.
Ultrasonic Cleaner	Kimberly, China	LHO-1	Disrupt the bacteria to achieve complete lysis
Urea	Solarbio, China	U8020	Prepare buffers for the protein purification process.
Yeast extract	OXOID Limited, China	LP0021B	Prepare Lysogeny Broth (LB) medium.