WO2023165108A1 - Modified gpr75 and uses thereof - Google Patents

Abstract

Provided are a modified GPR75 and the uses thereof. Provided is the modified GPR75, the modified GPR75 comprising: a first domain, the first domain comprising an amino acid sequence derived from a β2 adrenoceptor; and a second domain, the second domain being a domain in which an irregular sequence between the fifth transmembrane helix and the sixth transmembrane helix and irregular sequences at the N-terminus and the C-terminus are deleted from a wild-type GPR75, and an amino acid sequence derived from BRIL fusion protein is connected between the fifth transmembrane helix and the sixth transmembrane helix. The provided modified GPR75 can be used for GPR75 structure analysis, fluorescent molecular labeling, fusion of phosphorylated polypeptides or signal proteins, GPR75 activity analysis, nucleic acid encoded small molecule library screening, computer-aided drug design and drug screening.

Description

Modified GPR75 and its application

Priority and related applications

This disclosure claims the priority of the Chinese patent application 202210205737.1 filed on March 4, 2022, entitled "Modified GPR75 and Its Uses", and the entire content of this application including the appendix is incorporated into this application by reference.

technical field

The disclosure belongs to the field of biotechnology, and more specifically, the disclosure relates to a modified GPR75 and its use.

Background technique

G protein-coupled receptors are the largest class of cell membrane receptors in the human body. The completion of the Human Genome Project provides a basis for analyzing the distribution, sequence and function of the family members ¹ . There are more than 800 G protein-coupled receptor members in the human body, including about 370 non-olfactory G protein-coupled receptors and more than 400 olfactory receptors. These G protein-coupled receptors are divided into six subfamilies, rhodopsin family, adhesion family, secretin receptor family, glutamate receptor family family), Frizzled family, and Tasted family. Different G protein-coupled receptors are involved in mediating a series of important biological functions of organisms, from chemosensory recognition (vision, smell, taste) to the regulation of endocrine molecules, etc. ² . The importance of function stems from the fact that the receptors of this family can recognize a variety of ligands, common ligands include monoamines (dopamine, norepinephrine, serotonin, histamine), amino acid transmitters (glutamate , γ-aminobutyric acid), polypeptides (tchykinins, neurotensin, somatostatin, trypsin secretin, glucagon-like peptide-1, endocrine releasing factor), lipid derivatives ( Lysophosphatidic acid, sphingosine phosphate, eicosanoid) and odor etc. Statistics show that about 35% of the clinical drugs approved by the FDA target about 135 different G protein-coupled receptors ³ . In ongoing clinical studies, more than 20% of the drugs tested target G protein-coupled receptors ⁴ . Nevertheless, about 50% of non-olfactory G protein-coupled receptors may be potential targets for disease treatment, and there is still no relevant new drug clinical test, which needs further development and research. This shows the value and potential of G protein-coupled receptor family members in the field of new drug research and development.

GPR75 (G protein-coupled receptor 75, G protein-coupled receptor 75) is a member of the G protein-coupled receptor family, and its endogenous agonist ligands include metabolite 20-HETE ⁵ and chemokine CCL5/RANTES ⁶ . GPR75 has an expression distribution in a large number of cell types, among which GPR75 expressed in pancreatic islets regulates insulin release through the activation of CCL5, and participates in the regulation of glucose homeostasis in the human ^body6 . In addition, big data studies published in scientific journals have shown that the GPR75 gene is involved in the regulation of obesity in mice and is a clinically important target for obesity treatment ⁷ . The human GPR75 gene contains 540 amino acids and has the typical 7 transmembrane characteristics of members of the G protein-coupled receptor family ⁸ . The C-terminus of the receptor has a random coil sequence of about 140 amino acids in length. Due to their longer random coils and lack of an aspartate/arginine/tyrosine (DRY) motif, they are classified as atypical chemokine receptors (ACRs) ⁹ . CCL5 activates the GPR75 receptor on the cell membrane, resulting in the up-regulation of intracellular phospholipase C-mediated IP3 (inositol triphosphate, inositol triphosphate) and Ca ²⁺ concentrations ¹⁰ .

In 2017, ^the Nobel Prize in Chemistry was awarded to three scientists who made outstanding contributions to the development of cryo-electron microscopy11, which marked a new era in ^the field of structural biology12. In the same year, cryo-electron microscopy was applied to the structural analysis of the receptor-G protein complex. Over the ensuing 4-year period, approximately 45 independent high-resolution structures of receptor-G protein complexes were successively ^resolved13 .

Structure-based drug design is a revolution in traditional new drug development ^{approaches14,15} . In the past 20 years, the field of G protein-coupled receptor structure research has undergone tremendous changes. In 2000, the first high ^- resolution structure of G protein-coupled receptor-rhodopsin was resolved, laying the foundation for structural and functional studies in this field16. In 2007, Professor Brian Kobilka and his collaborators successfully resolved the high-resolution crystal structure of the β2 adrenoceptor by using antibodies to stabilize the conformation of the receptor or using the method of fusing ^T4 lysozyme17,18 . In 2011, Professor Brian Kobilka's laboratory further analyzed the crystal structure of the agonist-β2 adrenergic receptor-Gs protein ternary ^complex19 , and he shared the 2012 Nobel Prize in Chemistry with his mentor Robert J. Lefkowitz . In the era of traditional protein crystallography, about 50 or so independent crystal structures of G protein-coupled receptors have been solved. High-resolution structural information lays the foundation for structure-based drug design. One of the most famous examples is that Professor Brian Kobilka and his collaborators used the high-resolution crystal structure of the μ opioid receptor to carry out molecular docking of more than 3 million small molecule compounds, and finally obtained an opioid drug with lower side effects. pain drug PZM21 ²⁰ .

In the era of AI-enabled innovative drug research and development, how to use protein sequence information for structural analysis, and how to use computer-aided drug design (CADD) or molecular structure-based drug design (structure-based drug design) , SBDD) ideas, the design and screening of active small molecule drugs urgently need innovative new thinking and new solutions.

In order to develop targeted drug molecules for the GPR75 receptor, we need to analyze the structure of the GPR75 receptor. In recent years, the mainstream structural research method in the field is to use agonist-G protein-coupled receptor-G protein to form a ternary complex to analyze the activated state structure of the receptor. It has been reported that the GPR75 receptor in the human body plays an important role in controlling obesity, and human GPR75 receptor truncates show a lower proportion of obesity. Knocking out GPR75 in mice can significantly suppress obesity and enhance blood sugar control in high-fat diet model mice ⁷ . Inhibition of the activity of the GPR75 receptor has been suggested as a strategy to control obesity clinically. In order to be able to target the GPR75 receptor and develop clinically useful molecules, inhibitors are required. Therefore, it is extremely challenging to obtain the structure of the inactive receptor and develop specific inhibitors of receptor activity.

Contents of the invention

The problem to be solved by the invention

Because GPR75 receptors are clinically valuable molecules that require inhibitors, and currently lack the receptor structure in its inactive state, the present disclosure provides a modified GPR75.

solutions to problems

A first aspect of the present disclosure provides a modified GPR75 comprising:

a first domain comprising an amino acid sequence derived from a β2 adrenoceptor; and,

The second structural domain, the second structural domain is the random sequence between the fifth transmembrane helix and the sixth transmembrane helix and the random sequence at the N-terminal and C-terminal in the wild-type GPR75, and is derived from BRIL The amino acid sequence of the fusion protein connects the domain between the fifth transmembrane helix and the sixth transmembrane helix.

In some embodiments of the present disclosure, the first domain comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 80% homology to the amino acid sequence shown in SEQ ID NO: 4.

In some embodiments of the present disclosure, the second domain comprises the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 80% homology to the amino acid sequence shown in SEQ ID NO: 3.

In some embodiments of the present disclosure, the engineered GPR75 comprises one or more of the following sequences:

(i) amino acid sequence as shown in SEQ ID NO:5;

(ii) at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO:5 The amino acid sequence, and it retains the function of binding the specific ligand of the amino acid sequence shown in SEQ ID NO:5;

(iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO: 5, and it retains the combination of the amino acid sequence shown in SEQ ID NO: 5 function of the specific ligand; or,

(iv) an amino acid sequence encoded by a nucleotide sequence that hybridizes to a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 5 under stringent conditions, and the amino acid sequence remains in The amino acid sequence shown in SEQ ID NO:5 binds to the function of a specific ligand, and the stringent conditions are medium stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.

In some embodiments of the present disclosure, the engineered GPR75 further comprises a tag, a protease cleavage site, a signal peptide, a peptide linker, or any combination thereof.

In some embodiments of the present disclosure, the engineered GPR75 comprises a tag at its N-terminus and/or C-terminus.

In some embodiments of the present disclosure, the engineered GPR75 comprises a signal peptide at its N-terminus.

In some embodiments of the present disclosure, the protease cleavage site is located between two adjacent elements; the elements are selected from the group consisting of a first domain, a second domain, a tag, a signal peptide and a peptide linker.

In some embodiments of the present disclosure, the modified GPR75 comprises one or more of the following sequences:

(i) amino acid sequence as shown in SEQ ID NO:13;

(ii) at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 13 The amino acid sequence, and it retains the function of binding the specific ligand of the amino acid sequence shown in SEQ ID NO:13;

(iii) an amino acid sequence in which 1 or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO: 13, and which retains the combination of the amino acid sequence shown in SEQ ID NO: 13 function of the specific ligand; or,

(iv) an amino acid sequence encoded by a nucleotide sequence that hybridizes to a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 13 under stringent conditions, and the amino acid sequence is retained by The amino acid sequence shown in SEQ ID No: 13 binds to the function of a specific ligand, and the stringent conditions are medium stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.

The second aspect of the present disclosure provides a polynucleotide encoding the modified GPR75 described in the first aspect of the present disclosure.

The third aspect of the present disclosure provides an expression vector comprising the polynucleotide described in the second aspect of the present disclosure.

The fourth aspect of the present disclosure provides a host cell comprising the expression vector described in the third aspect of the present disclosure.

The fifth aspect of the present disclosure provides the modified GPR75 as described in the first aspect of the present disclosure, the polynucleotide as described in the second aspect of the present disclosure, the expression vector as described in the third aspect of the present disclosure, or the expression vector as described in the third aspect of the present disclosure. Use of the host cell described in the four aspects for GPR75 structural analysis, fluorescent molecular markers, fusion of phosphorylated polypeptides or signal proteins, GPR75 activity analysis, nucleic acid encoding small molecule library screening, computer-aided drug design and drug screening.

The effect of the invention

In this disclosure, innovatively combining ideas such as molecular structure prediction and molecular design, the protein sequence that was originally not suitable for the study of the inactive state structure was truncated, the fusion protein was transformed, and a β2 adrenoceptor with 24 amino acids was fused at the N-terminus The sequence method can improve the stability and expression of the receptor. In addition, we designed the Flag tag sequence, Strep tag sequence and 6×His tag sequence at the N-terminal and C-terminal of the receptor, respectively, to facilitate the purification of heterologously expressed receptors. Finally, this disclosure designs the sequence of the protein with multiple enzyme cutting sites (3C/TEV) and the fusion sequence of Sortase A, which can facilitate the reverse purification, matrix immobilization, and positioning of the purified protein in different schemes later as needed. Spot-specific fluorescent labels, etc., the modified sequences will be conveniently applied to nucleic acid-encoded small molecule drug libraries, drug binding experiments, etc. Moreover, the modified GPR75 provided by the present disclosure has better stability and higher expression than wild-type GPR75.

Description of drawings

Figure 1A and Figure 1B are the prediction models of GPR75 protein structure. Figure 1A shows the AlphaFold2 and RoseTTAFold prediction models for full-length GPR75, and Figure 1B shows the predicted transmembrane region of GPR75. It can be seen from Figure 1A that GPR75 has a long random coil structure, which is not suitable for direct structural biology research.

Figure 2 shows the idea of GPR75 protein transformation. First, we truncated the transmembrane region of GPR75 and inserted the BRIL fusion protein at the third intracellular helical position by using the predicted results of AlphaFold2. Subsequently, we gradually optimized the fusion site and selected the best fusion site as the final version of the fusion protein.

Fig. 3 is the idea of optimizing the fusion site of GPR75 protein.

Figure 4 is a graph showing the difference in effect of GPR75 protein fusion site optimization.

Figure 5 shows the difference in expression between wild-type GPR75 and modified GPR75 detected by western blot. The figure shows the Western blot results of GPR75 wild type and modified type. Lane 1 is GPR75 wild type, and lane 2 is GPR75 modified type. From the Western blot results, the expression level of modified GPR75 is significantly increased. The modified GPR75 has a higher protein expression level. The expression level of wild-type GPR75 is extremely low, and can hardly be detected under the same expression conditions.

Fig. 6 is a gel filtration chromatography UV-280 absorption peak comparison between modified GPR75 fused with BRIL and truncated GPR75 (no BRIL fusion and no N-terminal random sequence deletion of wild-type GPR75) in an embodiment of the present disclosure. The solid UV-280 absorption peak in the figure is the modified GPR75 fused with BRIL, and the dotted UV-280 absorption peak is the truncated GPR75.

Figure 7A and Figure 7B are the SDS-PAGE gel image (Figure 7A) and the photograph of frozen data (Figure 7B) of the modified GPR75 in the embodiment of the present disclosure. The purified modified GPR75 has higher purity, and the data particle dispersion of frozen sample preparation is good.

8A and 8B are background activity analysis of modified GPR75 in test cases of the present disclosure. The modified GPR75 without ligand binding can accelerate the GTP hydrolysis activity of Gq protein, while the ligand 20-HETE showed the effect of inhibiting the activity of 75 protein (Fig. 8A). In the experiment, the _IC50 of 20-HETE was measured to be about 2nM (Fig. 8B).

Figures 9A to 9C show the complex preparation and frozen data analysis of the engineered GPR75 and the anti-BRIL fab fragment in the test example of the present disclosure. The engineered GPR75 and the anti-BRIL fab fragment co-migrate on molecular sieves (Fig. 9A), and SDS-PAGE further demonstrates that they can form a stable complex (Fig. 9B). Two-dimensional sorting of single particles by cryo-EM revealed features of the receptor and complexes with the anti-BRIL fab fragment (Fig. 9C).

Detailed ways

For easier understanding of the present disclosure, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

In this specification, the numerical range represented by "numerical value A - numerical value B" means the range which includes numerical value A and B of an end point.

In this specification, the use of "substantially" or "substantially" means that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, more preferably within 1%.

In this specification, the meaning expressed by "may" includes the meaning of performing certain processing and not performing certain processing.

In this specification, "optional" or "optionally" means that the next described event or situation may or may not occur, and that the description includes situations where the event occurs and situations where the event does not occur.

In this specification, references to "some specific/preferred embodiments", "other specific/preferred embodiments", "embodiments" and the like refer to specific elements described in relation to the embodiments (for example, A feature, structure, property, and/or characteristic) is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.

According to the present disclosure, the terms "polypeptide", "protein", and "peptide" are used interchangeably herein to refer to a polymeric form of amino acids of any length, which may include encoded and non-encoded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides with similar peptide backbones.

According to this disclosure, the terms "nucleic acid molecule", "polynucleotide", "polynucleic acid", and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or Ribonucleotides, or analogs thereof. A polynucleotide can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids , vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes and primers. Nucleic acid molecules can be linear or circular.

According to the present disclosure, the term "G protein-coupled receptor" or "GPCR" or "GPR" refers to a transmembrane receptor capable of transmitting a signal from the outside of the cell to the inside of the cell through the G protein pathway and/or the arrestin pathway. Hundreds of such receptors are known in the art; see, for example, Fredriksson et al., Mol. Pharmacol. 63:1256-1272, 2003, and Vassilatis, D.K., Proc Natl Acad Sci USA 100:4903-4908 ( 2003), each of which is incorporated herein by reference. G protein-coupled receptors are polypeptides sharing a common structural motif with seven regions of between 22 and 24 hydrophobic amino acids that form seven alpha helices, each spanning the cell membrane. Each span is identified by a number, ie, transmembrane-1 (TM1), transmembrane-2 (TM2), etc., which in the invention may also be referred to as first transmembrane helix, second transmembrane helix, etc. The transmembrane is also linked by amino acid regions between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the outer or "extracellular" side of the cell membrane helix, the regions are referred to as "extracellular" regions 1, 2 and 3 (EC1, EC2 and EC3), respectively. The transmembrane is also linked by amino acid regions between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the inner or "intracellular" side of the cell membrane helix, the regions are referred to as "intracellular" regions 1, 2 and 3 (IC1, IC2 and IC3), respectively. The "carboxy" ("C") terminus of the receptor is located in the intracellular space within the cell, and the "amino" ("N") terminus of the receptor is located in the extracellular space outside the cell. Any of the above regions can be readily identified by analysis of the primary amino acid sequence of the GPCR.

According to the present disclosure, the term "ligand" or "receptor ligand" means a molecule that specifically binds to a GPCR either intracellularly or extracellularly. Without limiting purpose, ligands may be proteins, (poly)peptides, lipids, small molecules, protein scaffolds, antibodies, antibody fragments, nucleic acids, carbohydrates. Ligands can be synthetic or naturally occurring. The term "ligand" includes "natural ligands", which are endogenous, natural ligands of native GPCRs. In most cases, a ligand is a "modulator" that increases or decreases the intracellular response upon contact with (eg, binds to) a GPCR expressed by the cell. Examples of ligands that act as modulators include agonists, partial agonists, inverse agonists, and antagonists. Herein, "agonist" refers to a ligand that increases the signaling activity of a receptor by binding to the receptor. Full agonists stimulate the receptor maximally; partial agonists do not elicit full activity even at saturating concentrations. Partial agonists can also function as "blockers" by preventing the binding of more potent agonists. "Antagonist" refers to a ligand that binds to a receptor without stimulating any activity. An "antagonist" is also called a "blocker" because of its ability to prevent the binding of other ligands and thus block agonist-induced activity. Furthermore, "inverse agonist" refers to an antagonist that, in addition to blocking the effects of the agonist, reduces the basal or constitutive activity of the receptor below that of the receptor without ligand bound.

According to the present disclosure, the three-letter and one-letter codes for amino acids are used as described in J.biol.chem, 243, p3558 (1968).

According to the present disclosure, the term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant or animal cells. Bacteria that are readily transformed include members of the enterobacteriaceae such as strains of Escherichia coli or Salmonella; the Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line) and NSO cells.

According to the present disclosure, amino acid "addition" refers to the addition of amino acids at the C-terminus or N-terminus of an amino acid sequence. According to the present disclosure, an amino acid "deletion" means that 1, 2 or more than 3 amino acids can be deleted from the amino acid sequence. According to the present disclosure, amino acid "insertion" refers to inserting amino acid residues at appropriate positions in the amino acid sequence, and the inserted amino acid residues may be all or partly adjacent to each other, or the inserted amino acids may not be adjacent to each other.

According to the present disclosure, amino acid "substitution" refers to the replacement of a certain amino acid residue at a certain position in the amino acid sequence by other amino acid residues; wherein, the "substitution" may be a conservative amino acid substitution.

According to the present disclosure, "conservative modification", "conservative substitution" or "conservative substitution" refers to other amino acid replacement proteins with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) Amino acids in , so that frequent changes can be made without altering the biological activity of the protein. Those skilled in the art are aware that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224, (4th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to destroy biological activity. Exemplary conservative substitutions are set forth below under "Exemplary Amino Acid Conservative Substitutions".

Exemplary Amino Acid Conservative Substitutions

original residue	conservative substitution
Ala(A)	Gly; Ser
Arg(R)	Lys; His
Asn(N)	Gln; His; Asp
Asp(D)	Glu;Asn
Cys(C)	Ser; Ala; Val
Gln(Q)	Asn; Glu
Glu(E)	Asp; Gln
Gly(G)	Ala
His(H)	Asn; Gln
Ile (I)	Leu; Val
Leu(L)	Ile;
Lys(K)	Arg; His
Met(M)	Leu; Ile; Tyr
Phe(F)	Tyr; Met; Leu
Pro(P)	Ala
Ser(S)	Thr
Thr(T)	Ser
Trp(W)	Tyr; Phe
Tyr(Y)	Trp; Phe
Val(V)	Ile; Leu

According to the present disclosure, "moderate to very high stringency conditions" include "moderate stringency conditions", "medium-high stringency conditions", "high stringency conditions" or "very high stringency conditions", which describe the conditions for nucleic acid hybridization and washing. Guidance on performing hybridization reactions is found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated herein by reference. Aqueous and non-aqueous methods are described in this document and either can be used. For example, specific hybridization conditions are as follows: (1) low stringency hybridization conditions in 6× sodium chloride/sodium citrate (SSC), at about 45° C., then at least 50° C., in 0.2× SSC, 0.1% SDS Medium wash 2 times (for low stringency conditions, the washing temperature can be increased to 55°C); (2) medium stringency hybridization conditions at 6×SSC, at about 45°C, then at 60°C, at 0.2×SSC, Wash 1 time or multiple times in 0.1% SDS; (3) high stringency hybridization conditions are at 6×SSC, at about 45° C., then at 65° C., wash 1 time or more times in 0.2×SSC, 0.1% SDS and Preferably; (4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS, at 65°C, and then at 65°C, wash once or more in 0.2×SSC, 1%SDS.

According to the present disclosure, "exogenous" refers to a substance produced outside an organism, a cell, or a human body as the case may be. "Endogenous" refers to a substance produced in a cell, organism, or human body, as the case may be.

According to the present disclosure, "homology" or "identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both compared sequences is occupied by the same base or subunit of an amino acid monomer, for example if every position in two DNA molecules is occupied by an adenine, then the molecules are homologous at that position . The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of compared positions x 100. For example, when the sequences are optimally aligned, if 6 of the 10 positions in the two sequences match or are homologous, then the two sequences are 60% homologous; if 95 of the 100 positions in the two sequences match or homology, then the two sequences are 95% homologous. Generally, when aligning two sequences, comparisons are made to give the greatest percent homology. For example, comparison can be performed by the BLAST algorithm, the parameters of which are chosen to give the largest match between the respective sequences over the entire length of the respective reference sequences. The following references relate to the BLAST algorithm that is often used in sequence analysis: BLAST ALGORITHMS: Altschul, S.F. et al., (1990) J.Mol.Biol.215:403-410; Gish, W. et al., (1993 ) Nature Genet.3:266-272; Madden, T.L. et al., (1996) Meth.Enzymol.266:131-141; Altschul, S.F. et al., (1997) Nucleic Acids Res.25:3389-3402; Zhang, J. et al., (1997) Genome Res. 7:649-656. Other conventional BLAST algorithms, such as those provided by NCBI BLAST, are also well known to those skilled in the art.

According to the present disclosure, the term "codon optimized" means that the nucleotide sequence encoding a polypeptide has been configured to contain codons preferred by the host cell or organism in order to improve gene expression and increase translation efficiency in the host cell or organism.

According to the present disclosure, the term "tag" refers to a short peptide that is fused or linked to a protein of interest (such as the modified GPR75 of the present disclosure) and thereby facilitates soluble expression, detection and/or purification of the recombinant protein. Tags can be fused or linked to the N- and/or C-terminus of the protein of interest (optionally via a linker or protease cleavage site). Such tags are well known to those skilled in the art and have been described in detail in the prior art literature. For example, such tags include, but are not limited to, histidine tags (Sockolosky, J.T. and F.C. Szoka (2013). Protein Expr Purif 87(2):129-135), glutathione transferase (GST) tags (Hayashi , K.and C.Kojima(2008).Protein Expr Purif 62(1):120-127), maltose binding protein (MBP) tag (Bataille, L., W.Dieryck, A.Hocquellet, C.Cabanne, K .Bathany, S.Lecommandoux, B.Garbay and E.Garanger, Protein Expression and Purification Volume 110, June 2015, Pages 165-171), Thioredoxin (Trx) tag (Tomala, M., A.Lavrentieva, P .Moretti, U.Rinas, C.Kasper, F.Stahl, A.Schambach, E.Warlich, U.Martin, T.Cantz and T.Scheper, 2010, Protein Expr Purif 73(1):51-57), NusA tag (Li, K., T.Jiang, B.Yu, L.Wang, C.Gao, C.Ma, P.Xu and Y.Ma(2013).Sci Rep 3:2347), disulfide bond Constructase DsbA tag (Zhang, Y., D.R.Olsen, K.B.Nguyen, P.S.Olson, E.T.Rhodes and D.Mascarenhas (1998). Protein Expr Purif 12 (2): 159-165), DsbC tag (Kurokawa, Y., H.Yanagi and T.Yura(2001).J Biol Chem 276(17):14393-14399), SUMO tag (Marblestone, J.G., S.C.Edavettal, Y.Lim, P.Lim, X.Zuo and T.R.Butt(2006 ). Protein Sci15 (1): 182-189), msyB tag (Zou, Z., L. Cao, P. Zhou, Y. Su, Y. Sun and W. Li (2008). J Biotechnol 135 (4) :333-339), TF tag, elicitor tag (Kim, E.K., J.C.Moon, J.M.Lee, M.S.Jeong, C.Oh, S.M.Ahn, Y.J.Yoo and H.H.Jang(2012). Protein Expr Purif 86(1): 53-57), ubiquitin tag (Sabin, E.A., Lee-Ng, Chun Ting, Shuster, Jeffrey R., Barr, Philip J. (1989). Nature Biotechnology 7(7):705-709), Myc tag, Flag tags, fluorescent protein (such as GFP) tags (Pedelacq, J.D., S. Cabantous, T. Tran, T. C. Terwilliger and G. S. Waldo (2006). Nat Biotechnol 24 (1): 79-88), biotin tags, and affinity and vegetarian labels.

According to the present disclosure, the terms "signal peptide", "signal sequence" or "signal peptide sequence" refer to short peptides that, when fused to a protein of interest (such as the engineered GPR75 of the present disclosure), are capable of promoting the expression of The target protein is secreted to the cell membrane or extracellularly. The signal peptide is usually located at the N-terminal of the protein of interest, and various signal peptides are known to those skilled in the art, such as but not limited to hemagglutinin signal sequence, human insulin signal sequence, human interleukin 2 signal sequence, albumin signal sequence, etc. .

According to the present disclosure, the term "protease cleavage site" refers to a site that can be specifically recognized and cleaved by a protease. Various specific proteases and their recognition sites are well known to those skilled in the art and can be found in many prior art documents. Those skilled in the art can use a suitable protease cleavage site in the fusion protein according to the actual situation, and use the corresponding protease to perform cleavage. The use of a protease cleavage site may be advantageous, for example, to cleave a signal peptide and/or tag from a fusion protein, thereby obtaining a mature protein with the activity of interest.

According to the present disclosure, the term "peptide linker" refers to a short peptide used to link two molecules such as proteins. Usually, the fusion protein is obtained by introducing (for example, by PCR amplification or ligase) the polynucleotide sequence encoding the short peptide between two DNA fragments respectively encoding the two target proteins to be linked, and performing protein expression , such as target protein 1-peptide linker-target protein 2.

According to the present disclosure, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector is capable of expressing the protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids, phages, cosmids and the like.

According to the present disclosure, the terms "cell", "cell line" and "cell culture" are used interchangeably and all such designations include progeny. Thus, the terms "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all progeny may not be precisely identical in DNA content due to deliberate or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cells are included. Where a different name is intended, it is clear from the context.

The present disclosure is further described through embodiments below in conjunction with the accompanying drawings, but it is not intended to limit the present disclosure. Specific materials and their sources used in embodiments of the present disclosure are provided below. However, it should be understood that these are only exemplary and not intended to limit the present disclosure, and materials with the same or similar type, model, quality, property or function as the following reagents and instruments can be used to implement the present disclosure. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment: the preparation of transformation type GPR75

1. Sequence optimization

In this example, the protein structure prediction software used is AlphaFold local version (v2.1.0) and RoseTTAFold.

1.1. Truncation and transformation of wild-type GPR75

Wild-type GPR75 has a long random sequence region, which is not suitable for protein structure analysis. In this example, according to AlphaFold2 and RoseTTAFold two models for the prediction of GPR75 secondary structure ( FIG. 1A ), the prediction results of the transmembrane region are very similar. The reliability of the random sequence region is very low, so we truncated the original random sequence region of the GPR75 receptor, leaving the key seven transmembrane regions of the receptor (Fig. 1B). The wild-type and its truncated sequences are shown below (see SEQ ID NO:1 for details), truncated between the fifth transmembrane helix and the sixth transmembrane helix of wild-type GPR75, and delete (delete) the wild-type GPR75 N-terminal and The random sequence at the C-terminal and the random sequence between the fifth transmembrane helix and the sixth transmembrane helix of wild-type GPR75.

Wild-type GPR75 (GPR75-WT) amino acid sequence (SEQ ID NO: 1):

Remarks: In the amino acid sequence of wild-type GPR75 above, the single underline is the amino acid sequence retained in the modified GPR75, which correspond to the first to fifth transmembrane helices from the N-terminus of wild-type GPR75, and the N-terminus of wild-type GPR75. The sixth to seventh transmembrane helices from the end; the parts in italics are the sequences retained in the truncated GPR75 and deleted in the modified GPR75.

1.2. Linkage of BRIL fusion protein to truncated GPR75

Consider the use of the classic BRIL fusion protein derived from bacterial soluble cytochrome b562 to perform fusion junctions at the fifth and sixth transmembrane helices (Figure 2), which have four transmembrane helices and have been used in multiple GPCR Crystal (such as PDB ID: 7F83, 7VOD, 6LPJ, 6KO5, 6OS0, etc.) and electron microscope structure (such as PDB ID: 7S8O, 6WW2, 6USF, etc.) are used in the analysis. Using the BRIL fusion protein can increase the size of the extracellular region of the remodeled GPR75, which can be used as a marker in electron microscope structure analysis.

The fusion site of BRIL fusion protein needs to be optimized. In this example, 16 sites were screened for the fusion site of the BRIL fusion protein (Figure 3). According to the prediction of the AlphaFold2 protein structure, this example selected the transformation that can form a stable helix with the fifth and sixth transmembrane helices sequence, which fuses the fifth and sixth transmembrane helices.

The BRIL fusion protein sequence (SEQ ID NO: 2) adopted in the transformed GPR75 in the present embodiment:

In order to further optimize the location of the fusion site, we distributed single amino acid floats to the fifth and sixth transmembrane helix-BRIL junction regions, and used AlphaFold2 to predict the secondary structure of the fusion protein. According to the prediction effect map of AlphaFold2 (Figure 4), we compared the effects of the selection of 16 fusion sites. The sequence obtained by fusion connection of the fifth and sixth transmembrane helix truncations above through the BRIL fusion protein sequence is (SEQ ID NO: 3):

1.3, N-terminal fusion β2 adrenoceptor sequence

In this example, in order to improve the stability and expression of the receptor, a sequence of 24 amino acids of human β2 adrenoceptor (UNIPORT: P07550) was fused to the N-terminus.

The sequence of the β2 adrenergic receptor adopted in the modified GPR75 in this embodiment is (SEQ ID NO:4):

In the present embodiment, the sequence of the transformed GPR75 with the β2 adrenoreceptor sequence added is as follows (SEQ ID NO: 5)

1.4. Addition of signal peptide, enzyme cleavage site and tag sequence

In this example, the N-terminal and C-terminal of the modified GPR75 were designed with Flag tag sequence, Strep tag sequence and 6×His tag sequence, so as to facilitate the purification of the heterologously expressed modified GPR75. Finally, in this example, the sequence of the protein is designed with multiple restriction sites (3C/TEV) and the fusion sequence of Sortase A, which can facilitate the reverse purification of the purified protein in different schemes, matrix immobilization, and Site-specific fluorescent labels, etc., the modified sequence will be conveniently applied to nucleic acid-encoded small molecule drug libraries, drug binding experiments, etc.

Specifically, the modified GPR75 in this example is as follows from the N-terminal to the C-terminal:

Hemagglutinin signal sequence (MKTIIALSYIFCLVFA; SEQ ID NO:6);

Flag tag sequence (DYKDDDDA; SEQ ID NO:7);

β2 adrenergic receptor sequence (MGQPGNGSAFLLAPNRSHAPDHDV; SEQ ID NO: 4);

TEV protease cleavage site (ENLYFQG; SEQ ID NO:8);

Sequence obtained by truncated GPR75 linked by BRIL fusion protein sequence (SEQ ID NO:3);

Sortase A fusion sequence (LPETG; SEQ ID NO:9); it is a "USB-like interface", the attachment site of Sortase A enzyme.

Strep tag sequence (SAWSHPQFEK; SEQ ID NO: 10);

HRV 3C protease cleavage site (LEVLFQGP; SEQ ID NO: 11);

6×His tag sequence (HHHHHH; SEQ ID NO: 12).

The HRV 3C protease cleavage site and the 6×His tag sequence are connected by GS.

The complete amino acid sequence of the modified GPR75 (GPR75-M) is (SEQ ID NO: 13):

Remarks: The end of the C-terminal of the sequence shown in SEQ ID NO: 3 shares the amino acid L with the N-terminal of the Sortase A fusion sequence.

Nucleic acid sequence (SEQ ID NO: 14) after GPR75-M codon optimization (based on sf9 insect cells):

1.5. Gene synthesis and plasmid construction

We sent the sequence-optimized genes, including wild-type GPR75, truncated GPR75, and modified GPR75 genes, to Beijing Xianghong Biotechnology Co., Ltd. for gene synthesis, carrying NotI and HindIII restriction sites, ligated and recombined into the pFastbac-1 plasmid .

Wherein, the amino acid sequence (SEQ ID NO: 15) of truncated GPR75:

In the truncated GPR75, there is no BRIL fusion protein sequence, and the random sequence at the N-terminus of the wild-type GPR75 is not deleted, and other signal peptides, enzyme cleavage sites, and tag sequences are the same as those of the modified GPR75.

2. Preparation of recombinant baculovirus

2.1. Through heat-shock transformation, the recombinant pFastbac plasmid containing the gene of interest was introduced into Escherichia coli DH10Bac competent cells (Bomei De Biology), in the presence of 50 μg/mL kanamycin (BioBomei), 7 μg/mL gentamicin In LB solid medium of 10 μg/mL tetracycline (BioBomei), 200 μg/mL X-gal (inalco), 40 μg/mL IPTG (inalco), cultured at 37° C. for 48 hours. Pick uniform leukoplakia into 3 mL LB liquid medium containing three kinds of antibiotics (50 μg/mL kanamycin, 7 μg/mL gentamicin, 10 μg/mL tetracycline), and culture overnight at 37°C and 200 rpm , when the OD ₆₀₀ of the bacterial solution was about 0.6, the recombinant bacmid was extracted.

2.2. Take 1 mL of insect Medium (Graces), add 15 μL of transfection reagent (FuGENE), and incubate at room temperature for 15 minutes with 5 μg of recombinant baculovirus plasmid, and use this mixture to resuspend 10-12×10 ⁶ sf9 insect cells (the insect cells produced by The cell culture solution was centrifuged at room temperature and 500rpm for 10 minutes), cultivated at 27°C and 200rpm for 4 hours, and then added 5mL of ESF921 insect cell culture medium (Expression Systems), at 27°C and 200rpm Continue culturing for 48 hours. Transfer the sf9 cells that have been cultured for 48 hours to a 100mL Erlenmeyer flask, culture at 27°C and 110rpm until the cell density reaches 2-4×10 ⁶ /mL, centrifuge at 2500rpm for 10 minutes at room temperature, and the supernatant is the P1 recombinant rod virus.

2.3. Take the recombinant baculovirus of the P1 generation and transfect 100 mL of sf9 insect cells with a cell density of 1.5×10 ⁶ /mL at a ratio of 1:10,000. Culture at 27°C and 110 rpm until the cell density reaches 6×10 ⁶ /mL About mL, when the cell volume is enlarged and relatively uniform, centrifuge at 2500 rpm for 10 minutes at room temperature, and filter the supernatant with a 0.22 μm syringe filter to obtain the recombinant baculovirus of the P2 generation.

3. Protein purification small test

The P2-generation recombinant baculovirus was used to transfect 20 mL of sf9 insect cells at a density of 4×10 ⁶ /mL at a ratio of 1:50, and the cells were cultured at 27°C and 110 rpm for 48 hours. After culturing, samples were taken for Western blot detection to determine the expression of the target protein.

We performed Western blot parallel experiments on wild-type GPR75 and modified GPR75. The experimental results showed that the wild-type GPR75 had no obvious expression bands, but the expression of target bands was observed in the modified GPR75 ( FIG. 5 ). It indicated that the expression level of GPR75 was improved after transformation. Since the wild-type GPR75 had no expression detected, a large amount of expression and purification of the truncated GPR75 and the modified GPR75 were further compared.

4. Mass expression and purification of protein

The P2-generation recombinant baculovirus was used to transfect 1 L of sf9 insect cells at a density of 4×10 ⁶ /mL at a ratio of 1:50, and the cells were cultured for 48 hours at 27°C and 110 rpm.

After the cell culture is over, centrifuge at 4°C and 4000rpm for 20 minutes to collect the cells, resuspend the cell pellet with 100mL Buffer A, stir at 4°C for 10 minutes to fully lyse the cells, and centrifuge the lysed cells at 4°C and 15000rpm for 10 minutes Discard the supernatant, resuspend the pellet with 25mL Buffer B and homogenize with a homogenizer, dissolve the homogenate at 4°C for 90 minutes. After dissolving the membrane, centrifuge at 4°C and 37,000rpm for 15 minutes, incubate the supernatant with nickel affinity chromatography filler for 1 hour, then use Buffer C and Buffer D to elute the impurity protein, and use Buffer E to elute the target protein. Load the eluate containing the target protein onto the Flag affinity chromatography column, use Buffer F to elute the impurity protein, and Buffer G to elute the target protein, and then use a 50KDa ultrafiltration tube to concentrate the target protein to a volume of At about 500 μL, perform gel filtration chromatography, the gel column model used is Superdex 200 Increase 10/300GL (cytiva), the buffer is Buffer H, and the protein samples at the UV-280 ultraviolet absorption peak are collected for SDS-PAGE gel The content and purity of the target protein were detected by electrophoresis.

Wherein, the above-mentioned buffer solution (Buffer) A-F specific composition is as follows:

Buffer A: 20mM Tris, pH 7.5, 2mg/mL iodoacetamide (Iodoacetamide);

Buffer B: 20mM Tris, pH 7.5, 1mg/mL iodoacetamide, 750mM NaCl, 0.5% LMNG, 0.03% CHS, 0.2% sodium cholate, 1/1000 protease inhibitor;

Buffer C: 20mM Tris, pH 7.5, 150mM NaCl, 0.05% LMNG, 0.003% CHS, 0.02% sodium cholate, 1/1000 protease inhibitor, 20mM imidazole (Imidazole);

Buffer D: 20mM Tris, pH 7.5, 150mM NaCl, 0.05% LMNG, 0.003% CHS, 0.02% sodium cholate, 1/1000 protease inhibitor, 30mM imidazole;

Buffer E: 20mM Tris, pH 7.5, 150mM NaCl, 0.05% LMNG, 0.003% CHS, 0.02% sodium cholate, 1/1000 protease inhibitor, 250mM imidazole;

Buffer F: 20mM Tris, pH 7.5, 150mM NaCl, 0.05% LMNG, 0.003% CHS, 0.02% sodium cholate, 1/1000 protease inhibitor;

Buffer G: 20mM Tris, pH 7.5, 150mM NaCl, 0.05% LMNG, 0.003% CHS, 0.02% sodium cholate, 1/1000 protease inhibitor, 0.13mg/mL Flag peptide;

Buffer H: 20mM Tris, pH 7.5, 150mM NaCl, 0.00075% LMNG, 0.0001% CHS, 0.00025% GDN, 1/1000 protease inhibitor, 100μM

Tris(2-carboxyethyl)phosphine (TCEP).

Experimental results: As shown in Figure 6, in the gel filtration chromatography experiment, when the elution volume of Buffer H reached 11.7mL, the modified GPR75 began to show UV-280 absorption peak, and the maximum absorption value was 228.2mAu. The liquid elution volume was 12.6 mL. As shown in Figure 6, compared with the truncated GPR75 (without BRIL fusion and without deletion of the N-terminal random sequence of wild-type GPR75), the expression level of the modified GPR75 was significantly increased. The engineered GPR75 also has a higher proportion of monomer peaks. The modified GPR75 protein has a molecular weight of 58.03KDa, as shown in Figure 7A and Figure 7B. After purification by affinity chromatography and gel filtration chromatography, the protein obtained by SDS-PAGE gel electrophoresis analysis shows that its purity is about 90 %. Cryo-electron microscopy data show that the aggregation state of the sample is good, which can be used for further structural analysis as shown in Figure 7A and Figure 7B.

test case

Test example 1, activity identification of modified GPR75 protein

In order to verify whether the modified GPR75 protein prepared and purified in the example has activity, this test example utilizes G protein-coupled receptors to activate the downstream Gq protein and promote the hydrolysis efficiency of Gq protein to GTP to be measured ²¹ . The experimental steps were carried out according to the instructions of the GTPase-Glo ^TM Assay (Promega) kit. The principle of this method is that when 10 μM GTP molecules are added to the experimental system, since the Gq protein has GTP hydrolysis activity, the GTP molecules in the system will be gradually consumed. The modified GPR75 protein without ligand binding has a background level of activation activity, which will accelerate the consumption of GTP in the system.

The specific steps are as follows: First, the buffer conditions used in the experiment were configured: 20 mM Tris pH 7.5, 100 mM NaCl, 0.01% MNG, 100 μM TCEP, 5 mM MgCl ₂ . In the experiment, the purified control buffer, Gq protein, modified GPR75 protein, modified GPR75 protein-Gq protein complex, and modified GPR75 protein-Gq protein-20-HETE ligand complex were mixed with 10 μM GTP , wherein the concentrations of the modified GPR75 protein and Gq protein were both 3 μM, and the concentration of 20-HETE was 10 μM. The above samples were incubated at room temperature for 2 hours. Then configure the Glo reaction solution: dilute the Glo reagent in the kit 500 times into double distilled water, and add 10 μM ADP molecules. Take 20 μL of the Glo reaction solution and add it to the 5 samples in the previous step at a volume ratio of 1:1, and incubate for 30 minutes. Subsequently, 40 μL of Glo detection solution was added to the previous step at a volume ratio of 1:1. Finally, the samples were dispensed into 384-well plates and read with an Ensight microplate reader (perkinelmer).

Compared with the GTP hydrolysis activity of the Gq protein itself, the hydrolysis activity of the Gq protein was enhanced after the modified GPR75 protein was added ( FIG. 8A ). This shows that the engineered GPR75 protein in the state of no ligand binding has a certain level of background activity. In this test case, it was found that the ligand 20-HETE of the GPR75 protein reported in the literature showed the effect of inhibiting the activation of the modified GPR75 protein. In order to further study the effect of 20-HETE, the IC ₅₀ of 20-HETE was determined in this test example ( FIG. 8B ), and the result showed that the IC ₅₀ value of 20-HETE was about 2nM. This shows that the modified GPR75 has a certain activity. It can be seen that the modified GPR75 provided by the present disclosure can bind to the ligand of wild-type GPR75, such as 20-HETE, and can be applied to GPR75 structure analysis, GPR75 activity analysis, and related nucleic acid coding Small molecule library screening, computer-aided drug design and drug screening, etc. And as demonstrated in the examples, compared with wild-type and truncated GPR75, the modified GPR75 provided by the present disclosure has a higher expression level, which is suitable for subsequent applications such as structural analysis.

Test example 2. The modified GPR75 protein is used for structural analysis

In order to further use the modified GPR75 protein for structural analysis, this test example incubated the modified GPR75 protein prepared and purified in the examples with the fab fragment of anti-BRIL, and obtained the purification of the complex through molecular sieves (Fig. 9A). The purified complex was verified in further SDS-PAGE experiments (Fig. 9B). And, as shown in Figure 9C, 2D sorting of single particles by cryo-EM revealed the signature of receptors and complexes with anti-BRIL fab fragments. Test examples 1 and 2 show that in the modified GPR75, the GPR75 protein partially retains the ligand-binding site of the wild-type GPR75, and the BRIL fusion protein can also be recognized by its antibody, which can be used as a marker in the analysis of the electron microscope structure. Subsequently, in this test example, the obtained stable complex was frozen for sample preparation, electron microscope data collection and structural analysis. The single-particle two-dimensional classification data showed that the modified 75 protein-anti-BRIL fab fragment formed a relatively stable complex, which laid a solid foundation for further structural analysis of the modified GPR75 protein.

The descriptions presented above of the exemplary embodiments are only intended to illustrate the technical solutions of the present disclosure and are not intended to be exhaustive nor to limit the present disclosure to the precise forms described. Obviously, many modifications and variations are possible to those skilled in the art based on the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the present disclosure and its practical application, so that others skilled in the art can easily understand, implement and use the various exemplary embodiments of the present disclosure and various alternatives thereof and modified form. It is intended that the scope of protection of the present disclosure be defined by the claims appended hereto and their equivalents.

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Claims (10)

1. A modified GPR75, characterized in that the modified GPR75 comprises:

   a first domain comprising an amino acid sequence derived from a β2 adrenoceptor; and,

   The second structural domain, the second structural domain is the random sequence between the fifth transmembrane helix and the sixth transmembrane helix and the random sequence at the N-terminal and C-terminal in the wild-type GPR75, and is derived from BRIL The amino acid sequence of the fusion protein connects the domain between the fifth transmembrane helix and the sixth transmembrane helix.
2. The modified GPR75 according to claim 1, wherein the first structural domain comprises the amino acid sequence shown in SEQ ID NO: 4 or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 4 source amino acid sequence; and/or

   The second domain comprises the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO: 3.
3. The modified GPR75 according to claim 1 or 2, wherein the modified GPR75 comprises one or more of the following sequences:

   (i) amino acid sequence as shown in SEQ ID NO:5;

   (ii) at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO:5 The amino acid sequence, and it retains the function of binding the specific ligand of the amino acid sequence shown in SEQ ID NO:5;

   (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO: 5, and it retains the combination of the amino acid sequence shown in SEQ ID NO: 5 function of the specific ligand; or,

   (iv) an amino acid sequence encoded by a nucleotide sequence that hybridizes to a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 5 under stringent conditions, and the amino acid sequence remains in The amino acid sequence shown in SEQ ID NO:5 binds to the function of a specific ligand, and the stringent conditions are medium stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.
4. The modified GPR75 according to claim 1 or 2, wherein the modified GPR75 further comprises a label, a protease cleavage site, a signal peptide, a peptide linker or any combination thereof.
5. The modified GPR75 according to claim 4, wherein the modified GPR75 comprises a tag at its N-terminus and/or C-terminus; and/or,

   The engineered GPR75 comprises a signal peptide at its N-terminus; and/or,

   The protease cleavage site is located between two adjacent elements; the elements are selected from the group consisting of a first domain, a second domain, a tag, a signal peptide and a peptide linker.
6. The modified GPR75 according to claim 4, wherein the modified GPR75 comprises one or more of the following sequences:

   (i) amino acid sequence as shown in SEQ ID NO:13;

   (ii) at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 13 The amino acid sequence, and it retains the function of binding the specific ligand of the amino acid sequence shown in SEQ ID NO:13;

   (iii) an amino acid sequence in which 1 or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO: 13, and which retains the combination of the amino acid sequence shown in SEQ ID NO: 13 function of the specific ligand; or,

   (iv) an amino acid sequence encoded by a nucleotide sequence that hybridizes to a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 13 under stringent conditions, and the amino acid sequence is retained by The amino acid sequence shown in SEQ ID No: 13 binds to the function of a specific ligand, and the stringent conditions are medium stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.
7. A polynucleotide encoding the modified GPR75 according to any one of claims 1 to 6.
8. An expression vector comprising the polynucleotide according to claim 7.
9. A host cell comprising the expression vector of claim 8.
10. The modified GPR75 according to any one of claims 1 to 6, the polynucleotide according to claim 7, the expression vector according to claim 8 or the host cell according to claim 9 are used in GPR75 structure analysis, fluorescent molecular labeling, fusion of phosphorylated polypeptides or signaling proteins, GPR75 activity analysis, screening of nucleic acid-encoded small molecule libraries, computer-aided drug design and drug screening.

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