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Estrogen-related receptor beta mutant and application thereof

A technology of estrogen and mutants, applied in the fields of genetic engineering and protein engineering, can solve the problems that hinder the research of ERRβ target drugs, and it is difficult to obtain soluble ERRβLBD protein

Pending Publication Date: 2021-12-03
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, it is difficult to obtain highly soluble and stable ERRβ LBD protein, which has been one of the bottlenecks hindering the research of ERRβ target drugs

Method used

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  • Estrogen-related receptor beta mutant and application thereof
  • Estrogen-related receptor beta mutant and application thereof
  • Estrogen-related receptor beta mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1, bioinformatics analysis to explore and determine mutation sites.

[0041] In order to identify the key residues that may lead to the low solubility and poor stability of ERRβ LBD, bioinformatics analysis was performed on ERRβ and ERRα and ERRγ with high solubility and stability, including primary sequence alignment, 3D structure alignment and mutation model simulation . Such as figure 1 , based on the sequence alignment, it was found that the 215th amino acid of ERRβ LBD helix 1 (H1) is tyrosine (Y215), while the other two members of ERR, ERRα and ERRγ, are both histidine here, and histidine is more than tyrosine Amino acids are more hydrophilic and charged. Such as figure 2 , further analysis of the three-dimensional structure of ERRα (PDB: 2e2r) and ERRγ (PDB: 1kv6) found that the histidine in H1 here plays an important role in maintaining the stability of ERRα and ERRγ. In ERRα and ERRγ, the imidazole side chain of histidine here forms hydrogen bonds ...

Embodiment 2

[0042] Example 2, using point mutation technology to construct a mutant expression plasmid.

[0043] See SEQ ID NO.1 for the amino acid sequence of human ERRβ (UniProtKB identification number: O95718-3). In the present invention, firstly, the prokaryotic expression plasmid vector pET24a (Novagen, USA) is used, and the wild-type ERRβ sequence is used as a template, and the designed primers containing mutations are cloned into the ERRβ gene by overlapping extension PCR. Taking the Y215H mutation as an example, the primers used are:

[0044] F: 5'-cattgaccaagattgtctcacacctactggtggctgagccgga-3'

[0045] R: 5'-tccggctcagccaccagtaggtgtgagacaatcttggtcaatg-3'

[0046] The reaction conditions were pre-denaturation at 95°C for 5 min, followed by 26 cycles of the following program: denaturation at 95°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 6 min. The DNA after the PCR reaction was digested with ThermoFastDigest restriction endonuclease DPN1 to destroy the w...

Embodiment 3

[0059] Example 3, Determination of solubility and expression of ERRβ protein containing Y215H mutation.

[0060] The constructed mutant protein and wild-type protein were expressed and tested in vitro on a small scale. The constructed human pET24a ERRβLBD gene contains a 6 polyhistidine fusion tag. Transform the constructed plasmid into Escherichia coli BL21(DE3), culture and amplify at 30°C, cool down to 18°C ​​when the OD600 is about 0.8, and add a final concentration of 0.1mM isopropyl 1-thio-β -D-galactoside (IPTG) induced target protein expression overnight. The expressed E. coli cells were collected after low-speed centrifugation (4200 r.p.m.) at 4°C for 30 min, and resuspended in buffer (25 mM Tris pH7.5, 25 mM imidazole, 300 mM sodium chloride) on ice. Freeze at -70°C for 30 min, thaw in running water, and disrupt cells by ultrasonication. The lysed cell solution was centrifuged at 4°C for 30 minutes at high speed (20000 r.p.m.), and then the supernatant was taken a...

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Abstract

The invention discloses an estrogen-related receptor beta mutant and application thereof, and relates to gene engineering and protein engineering. The estrogen-related receptor beta mutant is obtained by comparing a human ERR beta amino acid sequence as shown in SEQ ID No.1 and mutating tyrosine at a corresponding position of a mutation site of a ligand binding domain helix 1 into histidine. The solubility and the stability of ERR beta in-vitro expression protein are improved by adopting a molecular biological technology, and the original protein functionality is kept; and a large number of stable mutant proteins can be obtained after the mutant plasmids are expressed and purified, and meanwhile, the mutant proteins have the functions of combining normal ligands and coregulators and transducing downstream signals, a protein tool is provided for researching the ERR beta, and the mutant proteins can be used in various fields of targeted drug screening, antibody preparation, protein-drug physiological pharmacology parameter detection, protein-protein interaction, structural biology research and the like.

Description

technical field [0001] The invention relates to genetic engineering and protein engineering, in particular to a class of estrogen-related receptor beta mutants and applications thereof. Background technique [0002] Protein is the material basis of life, and all important components and life activities of the body require the participation of protein. Separating and preparing proteins from biological materials and studying their structures and functions are of great significance for understanding the laws of life activities and clarifying the nature of life phenomena. The preparation of exogenous recombinant protein can use gene recombination technology, starting from obtaining the recombinant vector with the gene fragment of the target protein, and then transferring it into a host cell that can express the target protein, and then inducing the host cell to express a specific target protein under specific conditions. Recombinant protein. Recombinant proteins are widely use...

Claims

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Application Information

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IPC IPC(8): C07K14/72C12N15/10C12N15/70C12N15/62
CPCC07K14/721C12N15/1031C12N15/70C07K2319/21C12Q2531/113Y02A50/30
Inventor 李勇姚本强
Owner XIAMEN UNIV
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