Estrogen related receptor beta mutant and application thereof

文档序号：335740 发布日期：2021-12-03 浏览：29次中文

阅读说明：本技术 一类雌激素相关受体β突变体及其应用 (Estrogen related receptor beta mutant and application thereof ) 是由李勇姚本强于 2020-05-28 设计创作，主要内容包括：一类雌激素相关受体β突变体及其应用,涉及基因工程和蛋白质工程。所述一类雌激素相关受体β突变体由SEQ ID No.1所示的人源ERRβ氨基酸序列比对后,配体结合域螺旋1于突变位点相应位置的酪氨酸突变为组氨酸获得。采取基于分子生物学技术提高ERRβ体外表达蛋白的溶解性和稳定性,并保持原有蛋白功能性。突变体质粒经表达、纯化后可以得到大量且稳定的突变体蛋白,同时具备正常的配体和辅调节因子结合及转导下游信号的功能,为研究ERRβ提供了蛋白质工具,可用于靶向药物筛选、抗体制备、蛋白-药物生理药理学参数检测、蛋白-蛋白相互作用以及结构生物学研究等各个领域。(An estrogen related receptor beta mutant and application thereof, relating to gene engineering and protein engineering. The estrogen related receptor beta mutants are obtained by comparing human ERR beta amino acid sequences shown in SEQ ID No.1, and mutating tyrosine at the corresponding position of a mutation site of a ligand binding domain helix 1 into histidine. The solubility and stability of ERR beta in vitro expression protein are improved by adopting a molecular biology-based technology, and the original protein functionality is kept. The mutant plasmid can obtain a large amount of stable mutant protein after expression and purification, has the functions of normal ligand and co-regulatory factor combination and downstream signal transduction, provides a protein tool for researching ERR beta, and can be used in various fields of targeted drug screening, antibody preparation, protein-drug physiological and pharmacological parameter detection, protein-protein interaction, structural biology research and the like.)

1. An estrogen related receptor beta mutant is characterized in that after the amino acid sequences of human ERR beta shown in SEQ ID No.1 are compared, tyrosine at the corresponding position of a mutation site of a ligand binding domain spiral 1 is mutated into histidine to obtain the estrogen related receptor beta mutant.

2. The estrogen-related receptor beta mutant according to claim 1, wherein the mutants obtained by mutating tyrosine at the corresponding position to histidine comprise full-length or partial functional domain proteins containing the mutation site, and the full-length humanized amino acid sequence containing the mutation site is shown as SEQ ID No. 2.

3. The class of estrogen-related receptor beta mutants of claim 1, wherein the ERR beta amino acid sequence is of a different species or a homologous isomer.

4. The class of estrogen-related receptor beta mutants according to claim 1, wherein the mutation site includes but is not limited to at least one of Y215H, E234D, R382H, Y356H, Q416E.

5. The estrogen-related receptor beta mutant according to claim 1, wherein the mutant proteins are expressed in vitro by a prokaryotic expression system.

6. The class of estrogen-related receptor beta mutants according to claim 5, wherein the prokaryotic expression system includes, but is not limited to, an E.coli expression system.

7. The use of a class of estrogen-related receptor beta mutants according to claim 1, wherein said use includes, but is not limited to, ERR ligand and targeted drug screening, protein crystallization studies, antibody preparation, protein-drug physio-pharmacological parameter detection and protein-protein interaction, and structural biology studies.

Technical Field

The invention relates to gene engineering and protein engineering, in particular to estrogen related receptor beta mutants and application thereof.

Background

Proteins are the material basis of life, and all important components and life activities of the body need to be participated in by the proteins. The protein is separated and prepared from the biological material, and the structure and the function of the protein are researched, so that the protein has great significance for understanding the rule of life activities and explaining the essence of life phenomena. The preparation of the exogenous recombinant protein can be realized by applying a gene recombination technology, starting from obtaining a recombinant vector with a gene segment of a target protein, then transferring the recombinant vector into a host cell capable of expressing the target protein, and further inducing the host cell to express the specific recombinant protein under specific conditions. Recombinant proteins have wide applications in both industrial production and research: industrially produced protease preparations; the pig insulin is used for treating diabetes and diagnosing target spots in vitro in medical treatment; in the drug research, the target protein is subjected to in vitro drug screening and receptor-drug biology and pharmacological characteristic research; other studies such as the determination of protein structure and the preparation of protein antibodies.

Optimization of recombinant protein expression and purification processes requires improvement of protein stability and solubility while preserving protein activity. According to the analysis of the structure and the function of the protein, the modification can be designed and changed in a site-directed mutagenesis mode so as to improve the stability, the enzymatic activity, the solubility and the like of the protein. Site-directed mutagenesis refers to introducing required changes, including base addition, deletion, point mutation and the like, by methods such as Polymerase Chain Reaction (PCR) and the like, and finally changing the characters and characteristics of target proteins expressed by DNA.

Nuclear receptors are important target proteins for Drug development worldwide, and play a wide and profound role in the regulation of basic vital activities such as human developmental differentiation and metabolic homeostasis (Santos et al, Nat Rev Drug Discov,2017,16(1), 19-34); members of the superfamily include receptors for endocrine-mediated actions of steroid hormones, thyroid hormones, lipid soluble vitamins a and D, and a number of orphan nuclear receptors for which the endogenous ligand is unknown.

As an orphan nuclear receptor, estrogen-related receptors ERRs (estrogen-related receptors) exist in the human genome in three subtypes, including ERR α, ERR β and ERR γ (Eudy JD et al, Genomic,1998,50(3): 382-384). ERRs participate in important physiological activities of organism such as metabolic regulation, cell cycle and homeostasis, are closely related to various diseases such as alcoholic fatty liver, inflammatory osteoporosis, insulin resistance and cancer, and are a valuable drug research target. Similar to the structural composition of classical nuclear receptors, ERRs have five major functional domains: variable N-terminal domain (NTD), highly conserved DNA Binding Domain (DBD), Ligand Binding Domain (LBD), Hinge domain (Hinge) between DBD and LBD, and C-terminal extension domain (CTD).

Estrogen-related receptors ERRs are self-activating proteins, i.e., in the absence of ligand binding, ERRs are in an activated conformation, bind to co-activators, and promote transcription of downstream genes. In addition, the Ligand Binding Domains (LBD) of ERRs can bind with different ligands, thereby generating conformational change, recruiting various co-regulatory factors including co-activating factors and co-inhibiting factors, and finally achieving the purpose of regulating the transcription level of target genes. The activator ligand can make ERRs recruit various coactivators such as SRC1, SRC2, SRC3, PGC-1 alpha, Trap220 and the like, while the antagonist ligand can make ERRs bind to the cosuppressors such as NCoR and the like. By differential recruitment of co-regulatory factors, different ligands are caused to differentially regulate the expression level of target genes and ultimately regulate the physiological functions of the body.

Research shows that ERR beta is widely distributed and participates in regulating and controlling various important physiological functions. During the embryonic period, ERR beta high-abundance expression can promote embryonic development, and after birth, ERR beta is widely distributed in heart, kidney, prostate, central nerve and other parts to regulate cell cycle and homeostasis. ERR beta gene knockout mice lose weight, reduce fat mass, increase metabolic activity and energy consumption, and suggest that ERR beta is closely related to the maintenance of energy homeostasis. Furthermore, like ERR γ, ERR β also has the ability to regulate the cell proliferation cycle, induce reprogramming of embryonic and pluripotent stem cells. This particular ability makes it a new thermal target for the treatment of cancer-related diseases. Meanwhile, ERR beta also plays an important role in the adverse effect of environmental pollutants such as polychlorinated biphenyl (PCB), bisphenol A (BPA) and the like on animals and human beings.

However, it is difficult to obtain ERR β LBD protein with high solubility and stability, which has been one of the bottlenecks hindering ERR β target drug research. Among the three subtypes of ERRs, the LBD of ERR γ and ERR α successfully achieves in vitro purification as early as over ten years ago because of good in vitro expression solubility and stability and has been extensively studied for pharmaceuticals by means of a highly efficient, inexpensive biochemical platform (Audet-Walsh et al, acta pharmacogenetic sinica,2015,36(1): 51-61). Because of the difficulty in obtaining high purity proteins in vitro, the research progress of protein biochemical research, large-scale in vitro drug screening, receptor-drug biology and pharmacological characteristics involving ERR β is significantly behind that of ERR α and γ.

With the development of protein engineering and molecular biology techniques, the artificial modification of protein molecules by rational design and directed mutagenesis has become a fundamental approach to solve the problems in the field of protein engineering. By sequence and structural alignment with highly homologous soluble proteins, it can help identify key residues that lead to poor solubility or aggregation problems, such as the F602S mutation that recognizes glucocorticoid receptor and the C808S mutation that recognizes mineralocorticoid receptor (Bledsoe et al, Cell,2002,110, 93-105; Li, Y.et al, Mol Cell,2015,19, 367-. Cysteine residues are often preferred for systematic mutations to avoid misfolding and aggregation of the protein due to disulfide bond formation. It should be noted that the mutations are strictly ensured not to alter the original function of the protein. In particular for the nuclear receptors of the present invention, it is desirable to ensure that the ligand binding pocket and the co-regulatory factor binding site are not disturbed by mutations in the event that mutated residues are introduced into the LBDs.

Disclosure of Invention

The invention aims to provide an estrogen related receptor beta mutant constructed by using rational design and directed mutation means and application thereof.

The estrogen related receptor beta mutants are obtained by comparing human ERR beta amino acid sequences shown in SEQ ID No.1, and mutating tyrosine at the corresponding position of a mutation site of a ligand binding domain helix 1 into histidine.

The mutant with histidine mutated from tyrosine at the corresponding position of ERR beta comprises full-length or partial functional domain protein containing the mutation site, wherein the full-length humanized amino acid sequence containing the mutation site is shown as SEQIDNo.2, and the ERR beta amino acid sequence is different species or homologous isomers.

The mutation site may be a combination of multiple sites including, but not limited to, at least one of Y215H, E234D, R382H, Y356H, Q416E, and the like.

The ERR β protein includes a full-length or partial domain protein containing tyrosine on helix 1.

The estrogen related receptor beta mutant protein is expressed in vitro by a prokaryotic expression system, including but not limited to an escherichia coli expression system.

The estrogen related receptor beta mutant protein can be applied to ERR ligand and targeted drug screening, protein crystallization research, antibody preparation, protein-drug physiological and pharmacological parameter detection, protein-protein interaction, structural biology research and the like.

The estrogen-related receptor beta mutant plasmids can obtain a large amount of stable mutant proteins after expression and purification, simultaneously have the functions of normal ligand and co-regulatory factor combination and downstream signal transduction, provide a protein tool for researching ERR beta, and can be used in various fields of targeted drug screening, antibody preparation, protein-drug physiological and pharmacological parameter detection, protein-protein interaction, structural biology research and the like.

Experiments prove that compared with wild type, the protein expressed by ERR beta mutant plasmid has the characteristics of improved solubility or stability or both improved solubility and stability. Protein expression and purification results show that the solubility of wild ERR beta LBD is extremely low, only 0.01mg/mL, and a lot of impurities exist, so that the protein cannot be obtained by purification; and the single mutation of Y215H and the three mutations of R382H/Y356H/Y215H containing Y215H both obviously improve the protein solubility, and the soluble protein amount reaches 1mg/mL and is improved by 100 times compared with the wild type. Similar other experiment results show that the single mutation or multiple mutations containing Y215H have the obvious effect of increasing the solubility and expression amount of the ERR beta protein expressed in vitro.

ERR beta protein mutant has the characteristic of keeping similar functions of the original wild protein. Cell-based reporter gene experiments showed that ERR beta Y215H mutant and wild-type full-length protein have similar sustained activation characteristics and are regulated by exogenous ligands. The functional experiment shows that the introduction of mutation not only can not change the binding of ERR beta with a co-regulatory factor, but also can show that a ligand binding pocket is not interfered by the mutation and can still bind a related ligand to regulate the activity and downstream signals of ERR beta.

The ERR beta LBD protein mutant fills the blank of ERR beta LBD protein after being separated, purified and prepared, and has important application in scientific research, medicine industry and other aspects. The applications include, but are not limited to, the applications of the mutant protein in targeted drug screening, antibody preparation, protein-drug physiological and pharmacological parameter detection, protein-protein interaction, structural biological research and the like. For example, ERR β Y215H mutant proteins were used to determine the binding function of ERR β to various ligands and co-regulators.

The orphan nuclear receptor ERRs family protein has been discovered for nearly thirty years, and related researches also prove that ERR beta is closely related to cell metabolism, tumorigenesis and other physiological and pathological conditions. However, unlike ERR α or ERR γ, ERR β is difficult to obtain large and stable proteins in vitro, and the speed of drug studies targeting ERR β is delayed until the relevant in vitro studies are in the way. The invention provides a mutant residue site which does not affect the function and has the function of remarkably improving the solubility and the stability of in vitro expressed protein, so that a large amount of stable protein can be easily obtained in vitro, a protein tool is provided for researching ERR beta, and the invention has creativity. Meanwhile, the ERR beta mutant provided by the invention has important social value and economic value and has practicability.

An obvious aim of the invention is to improve the solubility and stability of ERR beta in vitro expression protein based on molecular biology technology and keep the original protein functionality. Successful purification and acquisition of ERR beta protein can promote targeted ERR beta related basic research, and meet the research requirements of high-efficiency, low-cost and large-scale in-vitro drug screening and medicines for treating ERRs related diseases.

The invention provides a mutant residue site which can obviously improve the solubility and stability of an estrogen related receptor beta ligand binding domain (ERR beta RR binding domain). The ERR beta RR5H containing Y215H mutation can obtain a large amount of stable protein after being purified, has the functions of normal ligand and co-regulatory factor combination and downstream signal transduction, provides a protein tool for researching the ERR beta, and can be used in various fields of targeted drug screening, antibody preparation, protein-drug physiological and pharmacological parameter detection, protein-protein interaction, structural biology research and the like.

Drawings

FIG. 1 is a chart of an alignment of human ERR β, ERR α and ERR γ LBD sequences. Secondary structural elements are boxed above the sequence and the residues are colored according to their side chain properties. The bases of the residues containing acidic side chains are underlined, the basic side chains are filled circles, the hydroxyl or amino side chains are filled triangles, and the residues of the smaller or hydrophobic side chains are not labeled. Asterisks indicate residues in the ligand binding pockets of these three ERRs. The position of the ERR β Y215H mutation and its residue interacting with helix H3 are indicated by arrows.

FIG. 2 is a graph showing the structural effect of ERR β Y215H mutation on protein stability. Structural alignment ERR α (PDB code: 1xb7) with ERR γ LBD (PDB code: 2e2r) and ERR β LBD models. Y215 and H260 in ERR beta are marked as figures and indicated by thin lines, residues at corresponding positions of ERR alpha and ERR gamma are marked as figures and indicated by sticks, and involved hydrogen bonds are indicated by arrows.

FIG. 3 shows the results of the purification and detection of ERR β LBD protein. The purification results of wild protein ERR beta, ERR beta Y215H single mutant, ERR beta R382H/Y356H/Y215H triple mutant and positive control ERR gamma LBD expression protein with high expression level. The protein loading species for each lane are labeled as in the figure and the loading amount is the same.

FIG. 4 is a cell-based full-length wild-type and mutant ERR β transcriptional function assay. Results are the average of three replicates and error bars indicate standard deviation within the sampled population. Denotes p < 0.01.

FIG. 5 is a graph of the determination of the ability of ERRs LBD protein expressed in vitro to recruit co-activators of the LXXLL motif core as regulated by BPA. ERR β LBD protein is a single mutant of Y215H. Dose-response curves of three ERR subtypes recruiting the coactivator SRC2 motif ability versus BPA in the AlphaScreen assay. Results are the average of three replicates and error bars indicate standard deviation within the sampled population.

FIG. 6 is a graph of the determination of the ability of ERRs LBD protein expressed in vitro to recruit co-activators of the LXLL motif core under the control of 4-OHT. ERR β LBD protein is a single mutant of Y215H. Dose-response curves for three ERR subtypes recruiting the coactivator SRC2 motif ability versus 4-OHT in the AlphaScreen assay. Results are the average of three replicates and error bars indicate standard deviation within the sampled population.

FIG. 7 is a graph of the determination of the ability of ERRs, SF-1, LRH-1, and ROR γ proteins expressed in vitro to recruit co-activators of the core of the LLXYL motif. SRC2 of the LXLL motif core and PGC-1 alpha of the LLXYL motif core were tested for differential binding to ERRs or other orphan nuclear receptors. ERR β LBD protein is a single mutant of Y215H. The coactivator core sequence in the experiment is as follows: biotin-SRC1-2: TERHKI LHRLL QESS, biotin-SRC2-3: KKENAL LRYLL DKDD,

biotin-SRC3-3:KENNAL LRYLL DRDD，biotin-PGC1-α-1:AEEPSL LKKLL LAPA，

biotin-PGC1-α-2:RRPCSE LLKYL TTND。

Detailed Description

The present invention will be further described with reference to the following examples, which are provided for illustration only and are not intended to limit the scope of the present invention.

The embodiment of the invention adopts rational design and directed mutation to construct a class of estrogen-related receptor beta mutants. The rational design is that the sequence and structure between soluble proteins with high homology to ERR beta are analyzed by bioinformatics method to identify key residues causing low solubility or denaturation precipitation of ERR beta, and hydrogen bond stable protein structure is introduced into ERR beta protein by mutation. The targeted mutation, such as the Y215H mutation, introduces a new hydrogen bond between helix 1(H1) and helix 3(H3) in the ERR β protein to stabilize the protein, because there is a hydrogen bond at this site in both ERR α and ERR γ wild-type proteins with high solubility and stability, which can significantly improve the structural stability of both, whereas there is no such a hydrogen bond in ERR β. Molecular dynamics analysis also shows that the introduction of hydrogen bonds into the part plays an important role in improving the overall stability.

The construction mode includes, but is not limited to, introducing base primers corresponding to the designed mutant amino acids into ERR beta gene (ESRRB) through Polymerase Chain Reaction (PCR) by using a molecular cloning technology, and determining the correct construction of the mutant genes through gene complete sequence sequencing.

The mutant is not limited to single mutation, is a mutant combination comprising Y215H, is characterized in that at least Y215H mutation exists in ERR beta mutant, and can also comprise mutual or multiple combinations of Y215H and E234D, R382H, Y356H, Q416E and the like.

The expression system comprises but is not limited to the expression construction of ERR beta mutant protein by using an escherichia coli expression system.

The improved solubility and stability are the characteristics that compared with the wild type, the protein expressed by the ERR beta mutant has the improved solubility or the improved stability or both the solubility and the stability. Protein expression and purification results show that the solubility of wild ERR beta LBD is extremely low, only 0.01mg/ml, and a large number of impurities exist, so that the protein cannot be obtained by purification; and the single mutation of Y215H and the three mutations of R382H/Y356H/Y215H containing Y215H both obviously improve the protein solubility, and the soluble protein amount reaches 1mg/ml, which is improved by 100 times compared with the wild type. Similar other experiment results show that the single mutation or multiple mutations containing Y215H have the obvious effect of increasing the solubility and expression amount of the ERR beta protein expressed in vitro.

Specific examples are given below.

Example 1 bioinformatic analysis searched for and identified mutation sites.

To find key residues that may lead to low solubility and poor stability of ERR β LBD, bioinformatics analysis including primary sequence alignment, three-dimensional structure alignment and mutation model simulation was performed on ERR β and ERR α and ERR γ with high solubility stability. As shown in fig. 1, the 215 th amino acid of ERR β LBD helix 1(H1) was found to be tyrosine (γ 215) on the basis of sequence alignment, while the other two members of ERR α and ERR γ were both histidine here, whereas histidine has stronger hydrophilicity and chargeability than tyrosine. As shown in FIG. 2, further analysis of the three-dimensional structures of ERR α (PDB:2e2r) and ERR γ (PDB:1kv6) revealed that here the histidine of H1 plays an important role in maintaining the stability of ERR α and ERR γ. In ERR α and ERR γ, the imidazole side chain of histidine here forms hydrogen bonds with the serine on ERR α helix 3(H3) and the histidine backbone carbonyl oxygen on ERR γ helix 3, respectively, stabilizing the ERR α and ERR γ protein three-dimensional structure. Whereas the hydrophobic tyrosine (Y215) benzene ring nucleus in ERR β is not involved in the formation of such hydrogen bonds. Thus, the mutant ERR β Y215H is expected to introduce hydrogen bonds that are effective in enhancing the stability of ERR β proteins. The mutation model of FIG. 2 also shows that mutations at this site have an important role in improving overall stability. Importantly, the Y215H mutant residue is located outside the ligand binding pocket, away from the co-regulatory factor binding site, and thus Y215H may serve as a potential mutation site that can improve overall stability without affecting ERR β protein function. Using similar analytical methods, E234D, R382H, Y356H, Q416E, etc. were also identified as potential mutation sites, and analysis led to the theoretical merit of mutating two or more sites simultaneously.

Example 2, a mutant expression plasmid was constructed using the point mutation technique.

The amino acid sequence of human ERR beta (UniProtKB identification number: O95718-3) is shown in SEQ ID NO. 1. The invention firstly adopts a prokaryotic expression plasmid vector pET24a (Novagen, USA) and takes a wild type ERR beta sequence as a template, and clones a designed primer containing mutation into the ERR beta gene by a method of overlapping extension PCR. Taking the Y215H mutation as an example, the primers used were:

F:5’-cattgaccaagattgtctcacacctactggtggctgagccgga-3’

R:5’-tccggctcagccaccagtaggtgtgagacaatcttggtcaatg-3’

the reaction conditions were pre-denaturation at 95 ℃ for 5min, followed by 26 cycles of the following procedure: denaturation at 95 ℃ for 1min, annealing at 60 ℃ for 1min, and extension at 72 ℃ for 6 min. The DNA after the PCR reaction was digested with ThermoFastdigest restriction enzyme DPN1 to destroy the wild-type ERR β as a template. And after 30min of enzyme digestion at 37 ℃, transferring the enzyme digestion product into escherichia coli, repairing the gap of the mutant plasmid in the escherichia coli, carrying out replication amplification, screening by using a resistant culture medium, selecting the monoclonal bacterial colony for further amplification, then sending to a sequencing company for sequence identification, and determining that the mutant gene is correctly constructed according to a sequencing result. Finally constructing to obtain the mutant gene shown as the amino acid sequence SEQ ID NO. 2.

The construction of the two combined mutation sites in the summary of the invention is different from the construction of single mutation clones. After the single mutation is introduced into ERR beta gene, the single mutation plasmid is used as new cloning template, and the primer containing new mutation site is cloned into ERR beta gene. Taking Y215H and R382H double mutation as examples, the constructed ERR beta Y215H is used as a template, and the following primers are used for introducing a new R382H mutation site. The specific experimental steps are the same as those of the construction of a single mutation experiment.

F:5’-aggactacgagctgagccagcaccatgaggagccctggaggac-3’

R:5’-gtcctccagggctcctcatggtgctggctcagctcgtagtcct-3’

Similarly, more than two combined sites are needed to clone a primer designed to contain a new mutation into the ERR beta gene after the double mutation is introduced into the ERR beta gene. Taking the three mutations of Y215H, R382H and Y356H as examples, the successfully constructed Y215H and R382H are used as templates, and the following primers are used to introduce a new mutation site of Y356H. The specific experimental steps are the same as those of the construction of a single mutation experiment.

F:5’-tcgccaactccgattccatgcacatcgaggatctagaggctgt-3’

R:5’-acagcctctagatcctcgatgtgcatggaatcggagttggcga-3’

It is noted that the mutations at two or more of the combined sites do not have strict mutation sequence requirements. Taking the three mutations of Y215H, R382H and Y356H as examples, to obtain the three mutations, ERR β Y215H may be constructed first and then R382H and Y356H are introduced, or R382H or Y356H may be constructed first and then the remaining two mutation sites are introduced.

Eukaryotic mutant plasmids are similarly constructed using eukaryotic expression vectors with wild-type ERR β genes, such as CMX-human ERR β and pBind ERR β LBD.

SEQ ID No.1

MSSDDRHLGSSCGSFIKTEPSSPSSGIDALSHHSPSGSSDASGGFGLALGTHANGLDSPPMFAGAGLGGTPCRKSYEDCASGIMEDSAIKCEYMLNAIPKRLCLVCGDIASGYHYGVASCEACKAFFKRTIQGNIEYSCPATNECEITKRRRKSCQACRFMKCLKVGMLKEGVRLDRVRGGRQKYKRRLDSESSPYLSLQISPPAKKPLTKIVSYLLVAEPDKLYAMPPPGMPEGDIKALTTLCDLADRELVVIIGWAKHIPGFSSLSLGDQMSLLQSAWMEILILGIVYRSLPYDDKLVYAEDYIMDEEHSRLAGLLELYRAILQLVRRYKKLKVEKEEFVTLKALALANSDSMYIEDLEAVQKLQDLLHEALQDYELSQRHEEPWRTGKLLLTLPLLRQTAAKAVQHFYSVKLQGKVPMHKLFLEMLEAKV

SEQ ID No.2

MSSDDRHLGSSCGSFIKTEPSSPSSGIDALSHHSPSGSSDASGGFGLALGTHANGLDSPPMFAGAGLGGTPCRKSYEDCASGIMEDSAIKCEYMLNAIPKRLCLVCGDIASGYHYGVASCEACKAFFKRTIQGNIEYSCPATNECEITKRRRKSCQACRFMKCLKVGMLKEGVRLDRVRGGRQKYKRRLDSESSPYLSLQISPPAKKPLTKIVSHLLVAEPDKLYAMPPPGMPEGDIKALTTLCDLADRELVVIIGWAKHIPGFSSLSLGDQMSLLQSAWMEILILGIVYRSLPYDDKLVYAEDYIMDEEHSRLAGLLELYRAILQLVRRYKKLKVEKEEFVTLKALALANSDSMYIEDLEAVQKLQDLLHEALQDYELSQRHEEPWRTGKLLLTLPLLRQTAAKAVQHFYSVKLQGKVPMHKLFLEMLEAKV

Example 3, ERR β protein solubility and expression level assay containing the Y215H mutation.

And carrying out small-scale in vitro expression and detection on the constructed mutant protein and wild protein. The constructed human pET24a ERR beta LBD gene contains a 6 polyhistidine fusion tag. The constructed plasmid was transformed into E.coli BL21(DE3) and cultured and amplified at 30 ℃ to 18 ℃ when OD600 was about 0.8, and isopropyl 1-thio-beta-D-galactoside (IPTG) was added to induce target protein expression overnight at a final concentration of 0.1 mM. The expressed E.coli cells were harvested after 30min at 4 ℃ by low speed centrifugation (4200r.p.m.) and resuspended in buffer (25mM Tris pH7.5,25mM imidazole, 300mM sodium chloride) on ice. Freezing at-70 deg.C for 30min, thawing in flowing water, and disrupting cells by ultrasound. The lysed cell fluid was centrifuged at 4 ℃ at high speed (20000r.p.m.) for 30min, after which the supernatant was incubated with nickel metal-chelated beads for 10 min. The supernatant was then discarded by centrifugation (about 7000 r.p.m). After washing free protein with a buffer containing 25mM imidazole, protein was eluted in one run with an elution buffer containing 500mM imidazole. The eluted protein was assayed for soluble ERR β LBD protein content and purity using polyacrylamide gel electrophoresis (SDS-PAGE). As shown in fig. 3, SDS protein gel showed that wild-type ERR β LBD was very low in solubility, only 0.01mg/ml, and numerous impurities, which failed to purify to obtain protein;

and the single mutation of Y215H and the triple mutation of R382H/Y356H/Y215H have the obvious capacity of improving the protein expression and solubility, and the soluble protein concentration is 1mg/ml, which is improved by 100 times compared with the wild type. ERR γ LBD is the positive control for stable expression of the experiment. Similar other experiment results show that the single mutation or multiple mutations containing Y215H have the obvious effect of increasing the solubility and expression amount of the ERR beta protein expressed in vitro.

Example 4, the Y215H mutation retained wild-type ERR β protein function.

The position of Y215H residue is far away from the functional center of ERR beta protein, and the mutation at the position is predicted not to change the function of the protein. To further verify the function of the mutant protein, luciferase reporter gene assay experiments were performed to compare the mutant protein to wild-type protein for differences in transcriptional activation and ligand-regulated ability. 293T cells were cultured in DMEM containing 10% fetal bovine serum and seeded at a density of 5X 10 in 24-well plates one day before transfection⁴Cells/well, transfection was performed the next day. Transient transfection was performed using Lipofectamine 2000 (Invitrogen). The CMX-human ERR β Y215H or wild-type CMX-human ERR β plasmid constructed in example 2 was transfected into 293T cells with ERR β response element delta-MTV-ERE plasmid and Renilla-Luc plasmid containing internal control, respectively. Cells were treated 5h after transfection with bisphenol A (BPA) or 4-hydroxyttamoxifen (4-OHT) ligand. Cells were harvested 24h after transfection and luciferase assays were performed using the dual luciferase reporter assay kit (Promega). As shown in fig. 4, the experimental results showed that Y215H mutant ERR β and wild-type ERR β showed similar sustained self-activation characteristics, and that the activation ability showed similar response after being treated with the ligand compound. The functional experiment shows that the introduction of mutation not only does not change the binding of ERR beta with a co-regulatory factor, but also shows that the ligand binding pocket is not interfered by the mutation and can still bind with related ligand to further regulate the transcriptional activity of ERR beta.

Example 5, purified ERR β Y215H protein can be used to determine the binding capacity of various ligands and co-regulators.

The separation and preparation of protein has wide application in scientific research and pharmaceutical industry. Purified ERR β proteins were first used for their respective ligand activity and affinity assays. pET24a ERR β LBD Y215H, pET24a ERR α LBD and pET24a ERR γ LBD plasmids were scale-up cultured and expressed as in example 3. The lysed cell liquid was centrifuged at 4 ℃ and the supernatant was applied to a nickel ion affinity column (NiSO4-loaded HiTrapHPcolumn, GEHealthcare) from GE. Subsequently, the sample-loaded nickel column was subjected to gradient elution in AKTA pure purifier of GE using 25 to 500mM imidazole in elution buffer. The eluted protein was further purified using an anion exchange column (Q-Sepharose column), and the purification efficiency was checked by SDS protein gel during protein purification.

The AlphaScreen technology is now widely used in drug development based on the interaction of nuclear receptors with ligands. (Jin et al, Nature communications 2013,4, 1937). The principle is that the ERRs LBD protein with His6 label will be combined with acceptor bead labeled with nickel, and the biotin-labeled co-regulatory factor polypeptide will be combined with donor bead labeled with streptavidin. If the ligand induces a conformational change in the nuclear receptor such that it binds to the cofactor polypeptide, then the donor bead is brought into proximity with the acceptor bead and excited with 680nm light, a 520-620 nm fluorescence signal is detected. The reaction system for this experiment was 20-80nM of the receptor LBD protein, 20nM of biotinylated co-regulator polypeptide, 5. mu.g/ml of donor and acceptor glass beads, buffer (25mM hepes,100mM NaCl and 0.1mg/ml of bone serum album, pH 7.0). The AlphaScreen kit (Perkins-Elmer) is used for detecting the ligand-regulated binding capacity of three subtype proteins of ERRs and co-activator and a ligand dose-activity curve. The result shows that the result of the ERR alpha of the experimental negative control group is similar to the current research theory and is not regulated and controlled by two ligands. Whereas the EC50 of BPA promoting ERR beta mutant activity with SRC2 was about 300nM and the IC50 of 4-OHT inhibition of ERR beta was about 280nM, similar to the experimental positive control group. EC50 for BPA in ERR γ was about 160nM (as in FIG. 5), while IC50 for 4-OHT was about 50nM (as in FIG. 6). Thus, AlphaScreen experiments demonstrated that ERR β LBD mutants purified in vitro could have the property of binding to the coactivator SRC2 containing the classical LXLL motif and have ligand dose-dependent regulatory properties like ERR α or ERR γ LBD (see FIGS. 5 and 6).

Most coactivators contain the classical LXXLL motif, as does coactivator SRC 2. Some co-activators contain a non-canonical motif, e.g., PGC1- α the second motif is LLKYL, an inverted form of classical LXLL. To further understand whether the in vitro ERR β LBD-expressing mutants can maintain similar abilities, the ability of three ERRs members and three other orphan receptors LRH1, SF1 and ROR γ to recruit SRCs and PGC1- α, respectively, was identified. The AlphaScreen experiment shows that six orphan nuclear receptors exhibit a potent ability to recruit SRCs containing the classical LXXLL motif in the absence of ligand, which is consistent with the general properties of nuclear receptors. In particular, all three ERRs showed the ability to recruit PGC1- α -2 of the classical LXLL motif SRCs and the non-classical LLXYL motif, in contrast to LRH1, SF1 and ROR γ which had little interaction with PGC1- α -2 (FIG. 7). These experiments demonstrate that ERR β mutant proteins expressed in vitro, similar to wild-type ERR α and ERR γ proteins, can be used to determine the binding function of various ligands and co-regulators.

The ERR beta protein mutant has the characteristic of keeping similar functions of the original wild protein. Cell-based reporter gene experiments showed that ERR beta Y215H mutant and wild-type full-length protein have similar sustained activation characteristics and are regulated by exogenous ligands. The functional experiment shows that the introduction of mutation not only can not change the binding of ERR beta with a co-regulatory factor, but also can show that a ligand binding pocket is not interfered by the mutation and can still bind a related ligand to regulate the activity and downstream signals of ERR beta.

Sequence listing

<110> university of mansion

<120> estrogen related receptor beta mutants and application thereof

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<170> SIPOSequenceListing 1.0

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<212> PRT

<213> human source (human)

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Met Ser Ser Asp Asp Arg His Leu Gly Ser Ser Cys Gly Ser Phe Ile

1 5 10 15

Lys Thr Glu Pro Ser Ser Pro Ser Ser Gly Ile Asp Ala Leu Ser His

20 25 30

His Ser Pro Ser Gly Ser Ser Asp Ala Ser Gly Gly Phe Gly Leu Ala

35 40 45

Leu Gly Thr His Ala Asn Gly Leu Asp Ser Pro Pro Met Phe Ala Gly

50 55 60

Ala Gly Leu Gly Gly Thr Pro Cys Arg Lys Ser Tyr Glu Asp Cys Ala

65 70 75 80

Ser Gly Ile Met Glu Asp Ser Ala Ile Lys Cys Glu Tyr Met Leu Asn

85 90 95

Ala Ile Pro Lys Arg Leu Cys Leu Val Cys Gly Asp Ile Ala Ser Gly

100 105 110

Tyr His Tyr Gly Val Ala Ser Cys Glu Ala Cys Lys Ala Phe Phe Lys

115 120 125

Arg Thr Ile Gln Gly Asn Ile Glu Tyr Ser Cys Pro Ala Thr Asn Glu

130 135 140

Cys Glu Ile Thr Lys Arg Arg Arg Lys Ser Cys Gln Ala Cys Arg Phe

145 150 155 160

Met Lys Cys Leu Lys Val Gly Met Leu Lys Glu Gly Val Arg Leu Asp

165 170 175

Arg Val Arg Gly Gly Arg Gln Lys Tyr Lys Arg Arg Leu Asp Ser Glu

180 185 190

Ser Ser Pro Tyr Leu Ser Leu Gln Ile Ser Pro Pro Ala Lys Lys Pro

195 200 205

Leu Thr Lys Ile Val Ser Tyr Leu Leu Val Ala Glu Pro Asp Lys Leu

210 215 220

Tyr Ala Met Pro Pro Pro Gly Met Pro Glu Gly Asp Ile Lys Ala Leu

225 230 235 240

Thr Thr Leu Cys Asp Leu Ala Asp Arg Glu Leu Val Val Ile Ile Gly

245 250 255

Trp Ala Lys His Ile Pro Gly Phe Ser Ser Leu Ser Leu Gly Asp Gln

260 265 270

Met Ser Leu Leu Gln Ser Ala Trp Met Glu Ile Leu Ile Leu Gly Ile

275 280 285

Val Tyr Arg Ser Leu Pro Tyr Asp Asp Lys Leu Val Tyr Ala Glu Asp

290 295 300

Tyr Ile Met Asp Glu Glu His Ser Arg Leu Ala Gly Leu Leu Glu Leu

305 310 315 320

Tyr Arg Ala Ile Leu Gln Leu Val Arg Arg Tyr Lys Lys Leu Lys Val

325 330 335

Glu Lys Glu Glu Phe Val Thr Leu Lys Ala Leu Ala Leu Ala Asn Ser

340 345 350

Asp Ser Met Tyr Ile Glu Asp Leu Glu Ala Val Gln Lys Leu Gln Asp

355 360 365

Leu Leu His Glu Ala Leu Gln Asp Tyr Glu Leu Ser Gln Arg His Glu

370 375 380

Glu Pro Trp Arg Thr Gly Lys Leu Leu Leu Thr Leu Pro Leu Leu Arg

385 390 395 400

Gln Thr Ala Ala Lys Ala Val Gln His Phe Tyr Ser Val Lys Leu Gln

405 410 415

Gly Lys Val Pro Met His Lys Leu Phe Leu Glu Met Leu Glu Ala Lys

420 425 430

Val

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