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 used in the Tox21 and ToxCast programs^4,5. These methods can rapidly identify potential toxicants and provide valuable information on the mechanisms of chemical toxicity. However, these HTS bioassays mainly rely on cell-based systems, which can be complex and expensive. Additionally, high-throughput sequencing methods have also been used for chemical toxicity assessment, but achieving high-throughput evaluation of chemicals remains challenging⁶. Previous studies have developed fluorescence polarization (FP)-based receptor-ligand competitive binding assays to determine the binding potency of several environmental pollutants, including per- and polyfluoroalkyl substances (PFAS)^7,8,9, bisphenol A (BPA)^10,11, and particulate matter (PM)¹², with nuclear receptors such as peroxisome proliferator-activated receptor (PPAR)^7,8,9,10,13, farnesoid X receptor (FXR)^11,12, and thyroid receptor (TR)^14,15. This approach is efficient, cost-effective, and provides mechanistic insights.

In this study, the protocol for the receptor-ligand binding assay is described based on detecting the fluorescence polarization (FP) of a small fluorescent probe. The principle of the FP-based receptor-ligand binding assay is illustrated in Figure 1. When a small fluorescent molecule is excited by plane-polarized light, the emitted light becomes highly depolarized due to rapid molecular rotation. However, when the tracer binds to a larger receptor, its rotation is slowed. A high FP value is detected when the tracer is bound to the large receptor, whereas a low FP value is observed when the tracer is free. Peroxisome proliferator-activated receptor gamma (PPARγ) was purified for the binding of the probe to the receptor. Rosiglitazone (Rosi), perfluorooctanesulfonic acid (PFOS), and triphenyl phosphate (TPHP) were used to compete for the binding of the probe with the receptor. Rosi, a specific agonist of PPARγ, was used as a positive control in the receptor competitive binding assays. Additionally, PFOS and TPHP have been previously identified as weak agonists of PPARγ in past studies^{8,9,10,11,12,13,14,15,16,17}. Furthermore, they belong to different structural categories of compounds known for environmental exposure and are notable for their relatively high detection rates in human populations. These compounds were used to further validate the broad applicability of the competition binding assay. The procedure consists of four steps: construction and transformation of recombinant vectors, expression and purification of the receptor protein (ligand-binding domain), receptor-probe binding, and competitive binding of chemicals with the receptor.

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 receptor, retinoid X receptor (RXR), and binds to response elements of PPARγ, thereby regulating the transcription of downstream target genes^9,16.

1. Design primers for the PPARγ-LBD (see Table 1) and amplify the PPARγ-LBD DNA segment (see Table 2 and Table 3).
2. Linearize the His×6-tagged pET28a vector by digesting it with restriction endonucleases XhoI and BamHI¹⁸.
3. Clone the PPARγ-LBD DNA segment into the His×6-tagged pET28a vector using the commercially available cloning kit, resulting in the recombinant plasmid pET28a-PPARγ-LBD-6×His¹⁸.
4. Transfect the recombinant expression plasmid pET28a-PPARγ-LBD-6×His into BL21 (DE3) Escherichia coli cells for protein expression¹⁸.
5. Add 5 µL of the recombinant vector to competent BL21(DE3) cells, incubate on ice for 30 min, perform a heat shock at 42 °C for 45 s, then immediately return to the ice.
6. Add 900 µL of LB medium, shake at 37 °C for 1 h (160-200 rpm), then centrifuge at ~3000 x g for 5 min at room temperature.
7. Discard the supernatant, resuspend the bacterial pellet in 100 µL of LB medium, and spread onto a solid medium. Invert the plates and culture at 37 °C for 12-16 h.
8. Select individual colonies for sequencing identification and subsequent protein expression and purification.

2. Expression and purification of the receptor protein

1. Incubate the transformed BL21 (DE3) cells in 200 mL LB medium supplemented with 100 µg/mL ampicillin on an orbital shaker (230 rpm) for 1-2 h at 37 °C.
2. Induce the cells when the OD₆₀₀ reaches 0.4-0.6 absorbance units by adding 10 µM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubate at 16 °C for 16 h.
3. Collect the bacterial suspension and centrifuge at 8000 × g, 4 °C, for 10 min.
4. Lyse the cells in 20 mL soluble lysis buffer (50 mM of NaH₂PO₄, 300 mM of NaCl, 10 mM of imidazole, pH 8.0), adding 200 µL of phenylmethylsulfonyl fluoride (PMSF) and 200 µL of lysozyme. Resuspend the bacterial pellet using a 5 mL pipette, then proceed with sonication at 30% power for 20 min.
5. Add lysis buffer equal to 5 times the column volume to equilibrate the nickel column. Repeat this process twice and set aside.
6. Centrifuge the sonicated bacterial suspension at 8000 × g for 15 min at 4 °C to obtain the bacterial supernatant lysate (CL).
7. Load the CL onto the nickel column (for protein adsorption) to obtain the flow-through (FT).
8. Wash the column with 5 times the column volume of wash buffer (50 mM of NaH₂PO₄, 300 mM of NaCl, 20 mM of imidazole, pH 8.0) to obtain the wash eluate (W1-W6).
9. Wash the column with 1 mL of elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0) to elute the target protein and obtain the protein fractions (E1-E5).
10. Take a 20 µL aliquot of each fraction (CL, FT, W1-W6, and E1-E5) and analyze using SDS-PAGE¹⁸ with Coomassie Brilliant Blue staining. PPARγ-LBD runs as a 34.9 kDa protein on a denaturing gel.

3. Receptor binding assay

NOTE: In this assay, C1-BODIPY-C12 was used as a site-specific fluorescent probe to establish the receptor-ligand binding system. C1-BODIPY-C12, a specific ligand for PPARγ, is a fluorescent analog of fatty acid with the BODIPY fluorescent group incorporated into the fatty acid at the C1 position.

1. Dilute the purified human PPARγ-LBD in Tris-HCl buffer (20 mM of Tris, 100 mM of NaCl, pH 8.0) to a concentration range of 1 nM to 6400 nM. Also, dilute the C1-BODIPY-C12 probe in Tris-HCl buffer to a concentration of 50 nM.
2. Mix the diluted PPARγ-LBD solution (55 µL per well) and the C1-BODIPY-C12 probe solution (55 µL per well) in a 96-well black plate. Incubate at room temperature for 5 min.
3. Measure fluorescence polarization (FP) with the microplate reader.
4. Plot the FP values against the receptor concentration, fit the curve using the specific binding with the Hill slope equation using statistical and graphing software, and calculate the K_d value.

4. Competitive binding assay

NOTE: In this assay, 800 nM of human PPARγ-LBD and 50 nM C1-BODIPY-C12 probe were used for receptor binding. Rosiglitazone (Rosi), triphenyl phosphate (TPHP), and perfluorooctanesulfonic acid (PFOS) were used to compete with the binding of the probe to PPARγ.

1. Dilute the three compounds in Tris-HCl buffer within a concentration range from 0-200 µM.
2. Prepare the receptor-probe binding solution with a final concentration of 800 nM of human PPARγ-LBD and 50 nM of C1-BODIPY-C12 probe.
3. Mix the receptor-probe binding solution (55 µL per well) and compound solution (55 µL per well) in a 96-well black plate. Incubate at room temperature for 5 min.
4. Measure fluorescence polarization (FP) with the microplate reader.
5. Plot the FP values as a function of the ligand concentration. Obtain the half-maximal inhibitory concentration (IC₅₀) of each ligand from the competition curve using the sigmoidal model processed by a graphing and analysis software.

Results

Protein expression and purification of PPARγ-LBD
PPARγ-LBD was heterologously expressed in BL21 (DE3) as a histidine-tagged protein. The protein was detected in the soluble fractions, and the purified PPARγ-LBD showed a single band on SDS-PAGE with an apparent molecular weight of approximately 34.9 kDa (Figure 2), consistent with the predicted molecular weight of the protein.

The binding of C₁-BODIPY-C_1...

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.