CN113092782A - Method for screening chemical nuclear receptor activity based on Alpha technology high-throughput and multi-target - Google Patents
Method for screening chemical nuclear receptor activity based on Alpha technology high-throughput and multi-target Download PDFInfo
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Abstract
The invention discloses a method for screening chemical nuclear receptor activity with high flux and multiple targets based on Alpha technology, which relates to the technical field of detection of environmental compounds, and the method comprises the steps of firstly preparing GST-hNR-LBD protein and Biotin-SRC-RID polypeptide, then carrying out dark first incubation on a mixture of pre-incubated GST-hNR-LBD protein and receptor microbeads and a substance to be detected, then adding the pre-incubated Biotin-SRC-RID polypeptide and a streptavidin labeled donor microbead mixture for dark second incubation, and finally utilizing the Alpha method to rapidly determine hNR receptor activity of chemicals with different concentrations. The method has the advantages of high test flux, strong specificity, low detection limit and ultrahigh sensitivity.
Description
Technical Field
The invention relates to the technical field of detection of environmental compounds, in particular to a method for screening chemical nuclear receptor activity based on Alpha technology high-flux and multi-target.
Background
Environmental pollution is increasingly serious, food-borne diseases emerge endlessly, and the problem of environmental and food safety has become one of the focuses of global public attention. Among them, Environmental Endocrine Disruptors (EEDs) can be enriched in organisms and human bodies by the action of food chain amplification, interfere with normal Endocrine function, and cause permanent harm to human bodies. The pollutants have stable chemical properties, are not easy to decompose in vivo and in vitro, have the characteristics of wide distribution, low content, easy enrichment, various varieties, various expression forms and the like, and are environmental pollutants which have the greatest harm to organisms. A large body of experimental evidence, as well as epidemiological investigations, has shown that many exogenous chemical substances in the environment can interfere with the endocrine function of humans and animals, thereby affecting health and reproduction.
The research on endocrine disruptors has gradually become a research hotspot in the international health and environmental fields, and the development of an efficient environmental endocrine disruptor detection method has important significance. In recent years, chromatographic analysis, spectroscopic analysis, and the like are commonly used as a method for measuring endocrine disruptors in an environment. The chromatographic analysis method comprises high performance liquid chromatography, gas chromatography, liquid-mass spectrometry and gas-mass spectrometry, and the method for measuring the environmental endocrine disruptors by using the spectrometry mainly comprises spectrophotometry, fluorescence, chemiluminescence and the like. Although there have been many studies in this respect, due to the characteristics of the matrix complexity of the sample and the low concentration of the environmental endocrine disruptors, the existing environmental endocrine disruptor analysis and detection technologies all have the disadvantages of poor selectivity, long time consumption, high cost and the like, and the practical application thereof is greatly limited.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a method for screening the activity of a chemical nuclear receptor on the basis of Alpha technology with high flux and multiple targets, thereby realizing the high-efficiency, high-flux, multiple-target and high-sensitivity rapid detection of the activity of the nuclear receptor of environmental trace pollutants.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the invention provides a method for screening chemical nuclear receptor activity with high flux and multiple targets based on Alpha technology, which comprises the following steps:
(1) preparing a ligand binding domain (GST-hNR-LBD) protein of GST-recombinant human nuclear receptor, comprising the steps of:
extracting total RNA of human breast cancer cells MCF-7 by using Trizol reagent, inverting the total RNA into cDNA and using the cDNA as a template;
referring to a coding sequence of a ligand binding Domain hNR-LBD (hNR-LBD) of a Human nuclear receptor published by NCBI, designing primers and amplifying to obtain a hNR-LBD DNA fragment;
cloning Escherichia coli according to hNR-LBD DNA fragment to obtain GST-hNR-LBD protein;
(2) preparing a Biotin-nuclear receptor interaction domain of transcription co-activator of nuclear receptor (Biotin-SRC-RID) polypeptide, comprising the steps of:
synthesizing a nuclear receptor interaction domain (SRC-RID) polypeptide of a nuclear receptor transcription co-activator by using a polypeptide Solid Phase Synthesis method (SPPS);
after the synthesis is finished, cracking the SRC-RID polypeptide, precipitating by using a cold ether centrifugal precipitation method, further purifying by using HPLC, and labeling by using a Biotin labeling kit to obtain a Biotin-SRC-RID polypeptide;
(3) hNR receptor activity is rapidly measured at different concentrations of chemicals using the Alpha method, comprising the steps of:
mixing GST-hNR-LBD protein and Anti-GST Alpha receptor microbead uniformly according to volume ratio by using analysis buffer solution, and pre-incubating to make GST-hNR-LBD protein labeled on the receptor microbead;
uniformly mixing the Biotin-SRC-RID polypeptide and streptavidin-labeled donor microbeads by using an analysis buffer solution according to a volume ratio, and pre-incubating to label the Biotin-SRC-RID polypeptide on the donor microbeads;
adding a pre-incubated GST-hNR-LBD protein and receptor microbead mixture into a pore plate hole, then adding an equal volume of analysis buffer solution and a chemical to be detected, shaking the pore plate to fully and uniformly mix the components, and carrying out first incubation in the dark at room temperature;
adding a mixture of pre-incubated Biotin-SRC-RID polypeptide and streptavidin-labeled donor microbeads into a pore plate hole, oscillating the pore plate to fully and uniformly mix the components, and performing secondary incubation at room temperature in the dark;
reading the pore plate signal by using a multifunctional microplate reader provided with an AlphaLisa or AlphaScreen cartridge, drawing a sigmoidal dose-effect curve of each test chemical according to the obtained reading, and judging the strength of the receptor binding activity of the substance according to the half-maximal effect concentration EC50 obtained from the curve.
Further, the human nuclear receptor includes at least one of human estrogen receptor (hER α and hER β), thyroid hormone receptor (hTR α and hTR β), vitamin D receptor (hVDR α), retinoic acid receptor (hRAR), estrogen-related receptor (hERR γ).
Further, the step of obtaining GST-hNR-LBD protein using E.coli clone based on hNR-LBD DNA fragment in step (1) comprises:
connecting hNR-LBD DNA fragment to an expression vector of Escherichia coli with a GST tag by using a double-enzyme cutting method, transforming Escherichia coli DH5 alpha competent cells, screening positive clone sequencing, and obtaining a prokaryotic recombinant expression vector;
extracting recombinant plasmids, and transforming escherichia coli BL21 competent cells to obtain hNR-LBD recombinant expression bacteria;
carrying out amplification culture on hNR-LBD recombinant expression bacteria (at 37 ℃), adding IPTG (isopropyl-beta-thiogalactoside), and inducing hNR-LBD recombinant protein expression;
hNR-LBD recombinant protein is purified by ultrasonic disruption and affinity chromatography to obtain GST-hNR-LBD protein.
Further, the SRC-RID polypeptide is one or more mixture of polypeptides with a length of 10-60 amino acids containing LXLL domain, wherein L represents leucine and X represents any amino acid.
Further, the molar ratio of GST-hNR-LBD protein to Biotin-SRC-RID polypeptide in step (3) is 1:1-1:10, preferably 1: 5.
Further, the volume ratio of the GST-hNR-LBD protein to the Anti-GST Alpha receptor microbeads in the step (3) is 2:1-10:1, preferably 3: 1.
Further, the volume ratio of the Biotin-SRC-RID polypeptide to the streptavidin-labeled donor microbead in the step (3) is 2:1-10:1, preferably 5: 1.
Further, the aperture plate in the step (3) is a 384-aperture plate.
Further, the assay buffer composition in step (3) is 5-50mM HEPES pH 7.5, 50-300mM NaCl and 0.005-0.05% Tween-20, preferably 15mM HEPES pH 7.5, 150mM NaCl and 0.01% Tween-20.
Further, in the step (3), the first incubation time of GST-hNR-LBD protein, the mixture of receptor microbeads and the substance to be tested at room temperature is 10-60 minutes, preferably 30 minutes.
Further, in the step (3), the second incubation time of the GST-hNR-LBD protein and acceptor bead mixture, the substance to be tested and the Biotin-SRC-RID polypeptide and streptavidin labeled donor bead mixture at room temperature is 0.5-2 hours, preferably 1 hour.
Further, the sigmoidal dose-effect curve for each test chemical was plotted in step (3) using Graphpad Prism software based on the readings obtained.
The invention is based on Alpha technology to detect the activity of a chemical nuclear receptor in high flux, and the main reaction components of the invention are GST-hNR-LBD protein, Biotin-SRC-RID polypeptide, donor beads and acceptor beads. The principle of the invention is as follows: the Alpha technique is based on two microbeads to detect interactions between biomolecules, and includes two detection methods, AlphaLisa and AlphaScreen, both using the same donor microbeads (Donner Beads) but different Acceptor Beads (Acceptor Beads). The present invention firstly labels GST-hNR-LBD protein on acceptor micro-beads, and simultaneously labels Biotin-SRC-RID polypeptide on donor micro-beads. The SRC-RID polypeptide is a conserved alpha-helical structure which is widely existed in the interaction between a nuclear Receptor transcription helper activator (SRC) family and a Human nuclear Receptor ligand binding Domain hNR-LBD (hNR-LBD), is also called a nuclear Receptor binding cassette and plays an important role in the interaction between a nuclear Receptor and a coregulator. When the tested substances in the sample have nuclear receptor activity, they can be combined with GST-hNR-LBD protein on a solid phase carrier (receptor microbead), so as to affect the conformation of the receptor protein and change the recruitment of the Biotin-SRC-RID polypeptide on another solid phase carrier (donor microbead). When the interaction between the biomolecules is changed, the distance between the donor bead and the acceptor bead is shortened or separated, and cascade amplification chemical reaction is excited, at the moment, if 680nm exciting light is used for irradiating the donor bead, activated oxygen molecules are initiated to act on adjacent acceptor beads to generate luminescence reaction, so that 615nm or 520-620nm fluorescence signals are generated, and the fluorescence signals can represent the strength of the combination of different substances to be detected and the nuclear acceptor.
Compared with the prior art, the invention has the beneficial effects that:
the method for measuring the nuclear receptor activity of the environmental endocrine disruptors by chromatography has good separation effect, can simultaneously measure various pollutant compounds, and has the advantages of high sensitivity and the like, but instruments used by the method are expensive, complex in operation, long in time consumption, and not beneficial to popularization and application. In addition, in the environment, especially endocrine disruptors in industrial wastewater, due to the complexity of the water matrix, the direct determination of the matrix interference of the contaminants in the sample is large, and generally sample pretreatment, separation, enrichment and derivatization are required, so that the determination process is complicated and time-consuming, and new environmental contaminants are easily introduced during sample treatment. The biological detection is based on the interaction between the SRC protein and a nuclear receptor, but the SRC protein has large molecular weight and is far away from the nuclear receptor protein, so that the detection is difficult to carry out by utilizing the principle of fluorescence energy transfer. At present, conventional field and laboratory detection technologies have advantages and disadvantages, but cannot effectively meet the requirements of high-throughput, rapid and sensitive detection. The method for screening the nuclear receptor activity of the chemical with high flux and multiple targets based on the Alpha technology can realize the detection of 6 nuclear receptor activities of 1 substance by a 384-well plate at one time and the detection of the activity of the same 1 nuclear receptor of more than 20 chemical substances by the 384-well plate at one time, and simultaneously each substance has not less than 5 concentrations and 2 repeated wells, and the total test time length is not more than 3 hours. Compared with the traditional method, the method provided by the invention has the advantages of low detection limit, ultrahigh sensitivity, strong specificity, quick and simple analysis, high test flux, easiness in miniaturization and automation and the like, realizes high-flux, multi-target and quick screening of the nuclear receptor activity of chemicals, and can be widely applied to quantitative analysis of the nuclear receptor activity of trace compounds in the fields of environmental science, food science, drug residues, clinical medicine, animal and plant quarantine, complex matrix sample analysis and the like.
Drawings
FIG. 1 is a dose-response graph of the test substance PhOP versus the activity of the tested human nuclear receptor hNR;
figure 2A is a graph of hERR γ activity dose-effect of bisphenols;
FIG. 2B is a graph of hERR γ activity dose-effect curves for ether species;
figure 2C is a hERR γ activity dose-effect curve for DDT and its metabolites;
fig. 2D is a dose-response curve for hERR γ activity for common drugs.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
The embodiment discloses a method for screening the nuclear receptor activity of a chemical by high flux and multiple targets based on Alpha technology, which comprises the following three steps:
preparation of GST-hERR gamma-LBD protein
Extracting total RNA of human breast cancer cell MCF-7 by using Trizol reagent, and reversing the total RNA into cDNA as a template. Primers were designed and amplified to obtain hERR gamma-LBD DNA fragment with reference to the coding sequence of the human estrogen related receptor gamma hERR gamma published by NCBI (NCBI GenBank: NP-001230435.1). The encoding gene of hERR gamma-LBD (52..271aa) is connected to an escherichia coli GST tag expression vector pGEX-4T-1 by a double-enzyme cutting method, escherichia coli DH5 alpha competent cells are transformed, and positive clone sequencing is screened to obtain a recombinant expression vector pGEX-4T-hERR gamma-LBD. And (3) extracting recombinant plasmids, and transforming escherichia coli BL21 competent cells to obtain hERR gamma-LBD recombinant expression bacteria. The recombinant strain is subjected to amplification culture at 37 ℃, and IPTG is added for induction expression. Then purifying the recombinant protein by ultrasonic disruption and affinity chromatography to obtain hERR gamma-LBD protein, detecting the expression and purification condition of the target protein by SDS-PAGE electrophoresis, determining the concentration of the purified protein, and analyzing the enzymatic property of the recombinant protein.
Preparation of II, Biotin-SRC-RID polypeptide
The SRC-RID synthesized in this example has the sequence of SLLLHLLKSQT, the length of 11 amino acids, the theoretical molecular weight of 1252.52Da and the theoretical isoelectric point of 8.49, and is synthesized by Fmoc (9-fluorenylmethyloxycarbonyl) solid phase method. Firstly swelling the king resin, performing Fmoc protection removal on Fmoc amino acid connected to the king resin, and coupling the second position activated amino acid at the C end of a peptide chain. Then sequentially coupling secondary Fmoc protective amino acids, performing deprotection on free end amino acids after each Fmoc protective amino acid is coupled, and then coupling the free end amino acids with the next Fmoc protective amino acid. In the process, in order to ensure the correctness of polypeptide synthesis, the repeated synthesis procedure coupling is carried out on Fmoc protective amino acids which are difficult to carry out acylation reaction. Removing Fmoc protection from N-terminal peptide chain amino acid, cracking the synthesized polypeptide after the synthesis is finished, precipitating the synthesized polypeptide by using a cold ether centrifugal precipitation method, and purifying the polypeptide by using HPLC. The polypeptide is marked by Sulfo-NHS-Biotin to obtain the Biotin-SRC-RID polypeptide.
Thirdly, hNR receptor activity of chemicals with different concentrations is rapidly determined by using AlphaScreen method
One) utilizing AlphaScreen method to rapidly determine different nuclear receptor activities of 4-phenoxyphenol
GST-hER α -LBD, GST-hER β -LBD, GST-hTR α -LBD, GST-hTR β -LBD, GST-hDR α -LBD, GST-hRAR-LBD and GST-hERR γ -LBD proteins were prepared, respectively, according to the method of the first example of this patent. And dissolving the chemical substances to be detected by using DMSO (dimethyl sulfoxide) to prepare a concentration gradient, and reducing the concentration of the DMSO to be below 5% as much as possible when preparing an experimental solution, wherein the concentration of the DMSO in each concentration group is the same. 1nM purified GST-hNR-LBD protein was mixed with Anti-GST AlphaScreen receptor beads in a volume ratio of 3:1 with assay buffer (15mM HEPES pH 7.5, 150mM NaCl, 0.01% Tween-20) and pre-incubated to label GST-hNR-LBD protein on the receptor beads. And (3) uniformly mixing 5nM Biotin-SRC-RID polypeptide and streptavidin-labeled donor microbeads with an analysis buffer solution according to the volume ratio of 5:1, and pre-incubating to label the Biotin-SRC-RID polypeptide on the donor microbeads. The pre-incubated GST-hNR-LBD protein and receptor bead mixture was added to the 384 well plate followed by an equal volume of assay buffer and 1. mu.L of test substance. The 384 well plate was gently shaken to mix the components well and incubated in the dark at room temperature for 30 min. Then adding the pre-incubated Biotin-SRC-RID polypeptide and streptavidin labeled donor bead mixture into a 384-well plate, slightly shaking the 384-well plate to fully mix the components, and incubating for 1 hour in the dark at room temperature. And (3) reading a microplate signal by using an MD SpectraMax i3x multifunctional microplate reader provided with an AlphaScreen cartridge, drawing a sigmoidal dose-effect curve of each test chemical by using Graphpad Prism software according to the obtained reading, and judging the strength of the receptor binding activity of the substance according to EC50 obtained by fitting the curve. Wells with DMSO alone added were used as negative controls and wells with the corresponding receptor standard added as positive controls. The compound tested was 4-phenoxyphenol (PhOP) and the human nuclear receptors tested hNR included human estrogen receptors (hER α and hER β), thyroid hormone receptors (hTR α and hTR β), vitamin D receptors (hVDR α), retinoic acid receptors (hRAR) and estrogen related receptors (hERR γ). The comparison of the strength of the binding activity of the test substance PhOP to the tested human nuclear receptor hNR is shown in fig. 1. EC50 from the fitted curve judged that PhOP had binding activity to hER α, hER β and hER γ, but not to hTR α, hTR β, hVDR α and hRAR.
II) rapid determination of hERR gamma receptor activity of chemicals of different concentrations by AlphaScreen method
And dissolving the chemical substances to be detected by using DMSO (dimethyl sulfoxide) to prepare a concentration gradient, and reducing the concentration of the DMSO to be below 5% as much as possible when preparing an experimental solution, wherein the concentration of the DMSO in each concentration group is the same. GST-hERR γ -LBD protein purified at 1nM was mixed with Anti-GST AlphaScreen receptor beads in an assay buffer (15mM HEPES pH 7.5, 150mM NaCl, 0.01% Tween-20) at 3:1 by volume, and pre-incubated to label GST-hERR γ -LBD protein on the receptor beads. And (3) uniformly mixing 5nM Biotin-SRC-RID polypeptide and streptavidin-labeled donor microbeads with an analysis buffer solution according to the volume ratio of 5:1, and pre-incubating to label the Biotin-SRC-RID polypeptide on the donor microbeads. The pre-incubated GST-hERR γ -LBD protein and receptor bead mixture was added to 384 well plates followed by an equal volume of assay buffer and 1 μ L of test substance. The 384 well plate was gently shaken to mix the components well and incubated in the dark at room temperature for 30 min. Then adding the pre-incubated Biotin-SRC-RID polypeptide and streptavidin labeled donor bead mixture into a 384-well plate, slightly shaking the 384-well plate to fully mix the components, and incubating for 1 hour in the dark at room temperature. And (3) reading a microplate signal by using an MD SpectraMax i3x multifunctional microplate reader provided with an AlphaScreen cartridge, drawing a sigmoidal dose effect curve of each test chemical by using Graphpad Prism software according to the obtained reading, and judging the hERR gamma binding activity of the substance according to EC50 obtained by fitting the curve. Wells with DMSO alone were used as negative controls and wells with bisphenol a (bpa) added were used as positive controls. The compounds tested were (1) bisphenols: bisphenol a (bpa), bisphenol b (bpb), bisphenol e (bpe), bisphenol fluorene (BHPF), bisphenol f (bpf), bisphenol ap (bpap); (2) ethers: 4-Benzyloxyphenol (BiOP), 4-heptyloxyphenol (HepOP), 4-pentyloxyphenol (PeOP), 4-Butoxyphenol (BOP), 4-phenoxyphenol (PhOP); (3) DDT and its metabolites: o, p ' -DDT, p ' -DDD, o, p ' -DDE, p ' -DDMU, p ' -DDT; (4) common drugs: ibuprofen, aspirin and resveratrol. The strength of the test substance in comparison with the hERR γ binding activity is shown in fig. 2A-2D. The results of the EC50 obtained from the fitted curve indicate that the binding activity of 6 bisphenols hERR gamma is ranked as follows: BPE > BPA > BPF > BPB > BPAP > BHPF; the binding activity of 5 ether substances hERR gamma is judged to be ranked according to EC50 obtained by a fitted curve as follows: BOP > PeOP > PhOP > BiOP > HepOP; among 7 DDT and metabolites thereof tested, DDD substances of two conformations showed stronger hERR gamma binding activity, o, p ' -DDT, o, p ' -DDE, p ' -DDMU also had obvious activity, and p, p ' -DDT and p, p ' -DDE were inactive; tests on 3 common drugs which may have a structural hERR gamma binding activity or natural chemicals which are popular in the market recently and are beneficial to human bodies show that none of the substances has substantially no hERR gamma binding activity.
The above embodiments are only intended to illustrate the technical solution of the present invention, but not to limit it, and a person skilled in the art can modify the technical solution of the present invention or substitute it with an equivalent, and the protection scope of the present invention is subject to the claims.
Claims (10)
1. A method for high-throughput, multi-target screening of chemical nuclear receptor activity based on Alpha technology, comprising the steps of:
(1) preparing GST-recombinant human nuclear receptor ligand binding domain GST-hNR-LBD protein, comprising the steps of:
extracting total RNA of human breast cancer cells MCF-7 by using Trizol reagent, inverting the total RNA into cDNA and using the cDNA as a template;
designing primer amplification to obtain hNR-LBD DNA fragment by referring to the coding sequence of the ligand binding domain hNR-LBD of the human nuclear receptor published by NCBI;
cloning Escherichia coli according to hNR-LBD DNA fragment to obtain GST-hNR-LBD protein;
(2) preparing Biotin-nuclear receptor interaction structural domain Biotin-SRC-RID polypeptide of a transcription co-activator of a nuclear receptor, comprising the following steps:
synthesizing a nuclear receptor interaction domain SRC-RID polypeptide of a nuclear receptor transcription co-activator by using a polypeptide solid phase synthesis method;
after the synthesis is finished, cracking the SRC-RID polypeptide, precipitating by using a cold ether centrifugal precipitation method, purifying by using HPLC, and labeling by using a Biotin labeling kit to obtain a Biotin-SRC-RID polypeptide;
(3) hNR receptor activity is rapidly measured at different concentrations of chemicals using the Alpha method, comprising the steps of:
mixing GST-hNR-LBD protein and Anti-GST Alpha receptor microbead with analysis buffer solution, pre-incubating to make GST-hNR-LBD protein labeled on receptor microbead;
uniformly mixing the Biotin-SRC-RID polypeptide and streptavidin-labeled donor microbeads with an analysis buffer solution, and pre-incubating to label the Biotin-SRC-RID polypeptide on the donor microbeads;
adding a pre-incubated GST-hNR-LBD protein and receptor microbead mixture into a pore plate hole, then adding an analysis buffer solution and a chemical to be detected which are equal in volume, oscillating the pore plate to uniformly mix the components, and carrying out first incubation in the dark at room temperature;
adding a mixture of pre-incubated Biotin-SRC-RID polypeptide and streptavidin-labeled donor microbeads into a pore plate hole, oscillating the pore plate to uniformly mix the components, and performing secondary incubation at room temperature in the dark;
reading the pore plate signal by using a multifunctional microplate reader provided with an AlphaLisa or AlphaScreen cartridge, drawing a sigmoidal dose-effect curve of each test chemical according to the obtained reading to obtain half-maximal effect concentration EC50, and judging the strength of the receptor binding activity of the substance according to EC 50.
2. The method of claim 1, wherein the human nuclear receptors comprise at least one of human estrogen receptors hER α and hER β, thyroid hormone receptors hTR α and hTR β, vitamin D receptor hVDR α, retinoic acid receptor hRAR, and estrogen-related receptor hERR γ.
3. The method of claim 1, wherein the step of obtaining GST-hNR-LBD protein from hNR-LBD DNA fragment using e.coli cloning in step (1) comprises:
connecting hNR-LBD DNA fragment to an expression vector of escherichia coli with a GST label by using a double-enzyme cutting method, transforming escherichia coli DH5 alpha competent cells, screening positive clone sequencing, and obtaining a prokaryotic recombinant expression vector;
extracting recombinant plasmids, and transforming escherichia coli BL21 competent cells to obtain hNR-LBD recombinant expression bacteria;
carrying out amplification culture on hNR-LBD recombinant expression bacteria, adding IPTG (isopropyl-beta-thiogalactoside), and inducing hNR-LBD recombinant protein expression;
hNR-LBD recombinant protein is purified through ultrasonic disruption and affinity chromatography to obtain GST-hNR-LBD protein.
4. The method of claim 1, wherein the SRC-RID polypeptide is one or more mixture of polypeptides 10-60 amino acids in length comprising a LXXLL domain, wherein L represents leucine and X represents any amino acid.
5. The method of claim 1, wherein in step (3) the molar ratio of GST-hNR-LBD protein to Biotin-SRC-RID polypeptide is 1:1 to 1:10, the volume ratio of GST-hNR-LBD protein to Anti-GST Alpha acceptor microbead is 2:1 to 10:1, and the volume ratio of Biotin-SRC-RID polypeptide to streptavidin-labeled donor microbead is 2:1 to 10: 1.
6. The method of claim 5, wherein the molar ratio of GST-hNR-LBD protein to Biotin-SRC-RID polypeptide in step (3) is preferably 1:5, the volume ratio of GST-hNR-LBD protein to Anti-GST Alpha acceptor beads is preferably 3:1, and the volume ratio of Biotin-SRC-RID polypeptide to streptavidin-labeled donor beads is preferably 5: 1.
7. The method of claim 1, wherein the well plate in step (3) is a 384-well plate.
8. The method of claim 1, wherein the assay buffer components in step (3) are 5-50mM HEPES at pH 7.5, 50-300mM NaCl, and 0.005-0.05% Tween-20.
9. The method of claim 1, wherein the first incubation time in step (3) is 10 to 60 minutes and the second incubation time is 0.5 to 2 hours.
10. The method of claim 1, wherein the sigmoidal dose-effect curve for each test chemical is plotted in step (3) using Graphpad Prism software based on the readings obtained.
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