A modified GPR35 and its application

Abstract

This disclosure provides a modified GPR35 and its uses. Specifically, this disclosure provides a modified GPR35, wherein the modified GPR35 comprises one or more of the following sequences: (a1) an amino acid sequence as shown in SEQ ID NO: 32; (a2) an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 32. The modified GPR35 provided by this disclosure fuses and modifies protein sequences that are not originally suitable for studying inactive states, making it possible to study the structure of GPR35 using cryo-electron microscopy. It can be used for GPR35 structure determination, GPR35 activity analysis, screening of nucleic acid-encoded small molecule libraries, computer-aided drug design and screening, etc.

Description

Technical Field

[0001] This disclosure belongs to the field of biotechnology, and specifically, this disclosure relates to a modified GPR35 and its uses. Background Technology

[0002] G protein-coupled receptors (GPCRs) are the largest class of cell membrane receptors in the human body. 1 The human body has over 800 G protein-coupled receptor (GPL) members, including approximately 370 non-olfactory GPLs and over 400 olfactory receptors. Different GPLs participate in mediating a range of important biological functions, from chemosensory recognition to endocrine molecule-related regulation. Statistics show that approximately 35% of FDA-approved clinical drugs target about 135 different GPLs. In ongoing clinical trials, over 20% of the drugs being tested target GPLs. 2 Nevertheless, approximately 50% of non-olfactory G protein-coupled receptors (GPCRs) may be potential therapeutic targets, yet no related new drugs have undergone clinical trials, requiring further exploration and research. This demonstrates the value and potential of the GPCR family in the field of new drug development. 3 .

[0003] Through a series of technological innovations, including the rapid deployment of cryo-sample preparation technology, the development of direct electron detectors, and the improvement of 3D reconstruction software, cryo-electron microscopy single-particle microscopy has been widely applied to the study of protein and biomolecular structures at near-atomic resolution. 4 In 2017, the Nobel Prize in Chemistry was awarded to three scientists for their outstanding contributions to the development of cryo-electron microscopy, marking a new era in the field of structural biology. Cryo-electron microscopy has surpassed traditional X-ray crystallography and nuclear magnetic resonance techniques, becoming the most cutting-edge and popular structural research method in structural biology and new drug development. 5 .

[0004] G protein-coupled receptors are proteins with a classic seven-transmembrane structure and are highly dynamic in structure. 6 Some classic methods for studying the structure of G protein-coupled receptors include thermostability mutations in the receptor, and fusing T4 lysozyme into the N segment or the third disordered region of the receptor. 7 b562 fusion protein (BRIL) 8Fusion proteins are used to develop antibodies or nanobodies targeting the intracellular or extracellular side of GPCRs, forming GPCR-G protein or arrestin protein complexes. Introducing fusion proteins into the third transmembrane helix within the cell can effectively improve the structural stability of GPCRs and increase the molecular weight of the receptor. In the era of traditional crystallography, fusion proteins could increase the likelihood of receptor-receptor crystal stacking. With the development of cryo-electron microscopy, the value of using fusion proteins for structural analysis has been diminished, partly because GPCRs and G proteins or downstream signaling protein complexes are more conducive to explaining the biological function of the receptor. However, the challenge for new drug development companies is that, for some important GPCRs, antagonists targeting the inactive state of the receptor have greater clinical therapeutic value. How to leverage the advantages of cryo-electron microscopy to rapidly and efficiently obtain the structure of the receptor in its inactive state and utilize structure-based drug screening / optimization strategies for new drug development is a challenge facing this field.

[0005] The BRIL used for fusion with G protein-coupled receptors is a heat-resistant apocytochrome b562 mutant protein (also known as cytochrome b562RI) (Protein Data Bank ID: PDB 1M6T) with the amino acid sequence of apocytochrome b562 modified by inserting the amino acid residues M7W, H102I, and R106L. As one of the most classic fusion proteins in the G protein-coupled receptor family, BRIL fusion proteins have been widely used to date in the structural analysis of more than 20 G protein-coupled receptors. 9 The classic quadruple helix structure of this protein is tightly packed together, allowing the fifth and sixth transmembrane helices of the G protein-coupled receptor to form a relatively stable conformation after fusion. Traditional BRIL proteins have too small a molecular weight to be suitable for direct cryo-electron microscopy structural analysis. To facilitate structural analysis using BRIL fusion proteins, researchers have also developed antibody fragments targeting BRIL proteins and applied them to the structural analysis of multiple G protein-coupled receptors. 10 Because this antibody fragment has poor stability and is prone to aggregation after expression, developing nanobodies with better stability that can bind to the BRIL protein would be helpful for the analysis of GPCR-BRIL fusion proteins using cryo-electron microscopy.

[0006] GPR35 (G protein-coupled receptor 35) is a member of the human GPCR family, and it was first reported to have been cloned in 1998. 11Sequence analysis showed that GPR35 has the closest homology with GPR55, lysophosphatidylcholine receptors LPAR4, LPAR5 and LPAR6, and niacin receptor HM74. 12 Tissue distribution studies showed that GPR35 was abundant in the small intestine and pancreas, followed by significant expression in the colon, spleen, and immune cells (monocytes, neutrophils, T cells, and dendritic cells). 13 Studies have shown that endogenous agonists of GPR35 may include kynurenic acid. 14 And 2-oleoyl-LPA 15 GPR35 may be linked to a range of pathological conditions, including inflammation, asthma, hypertension, and diabetes. Therefore, GPR35 is an important potential therapeutic target. 16 Developing novel agonist and antagonist ligands targeting GPR35 will help decipher its true physiological significance and provide insights for the clinical development of therapeutic drugs for related diseases. However, as mentioned earlier, clinical drug development requires targeted antagonists and inactive structures for AI-assisted structure-based drug development. Meanwhile, the wild-type GPR35 protein is highly flexible and unstable, making it unsuitable for structural studies. More stable GPR35 is needed for various applications, including screening DNA-encoded small molecule libraries targeting protein targets, generating specific antibodies for animal immunization, and screening synthetic nanobody libraries for developing extracellular targeting biopharmaceuticals.

[0007] Therefore, further development is needed for BRIL proteins that are suitable for fusion with target proteins (especially G protein-coupled receptors) and used for structural analysis. At the same time, a modified GPR35 with better stability and more suitable for structural analysis needs to be developed. Summary of the Invention

[0008] The problem the invention aims to solve

[0009] To address the aforementioned problems in the existing technology, this disclosure presents a modified GPR35, formed by fusing the modified mBRIL provided in this disclosure with GPR35. The resulting fusion protein can further form a complex with anti-BRIL nanobodies. The modified GPR35 provided in this disclosure can be used for the structural analysis of GPR35, and for the development of targeted small molecule and biomolecular drugs.

[0010] Solution for solving the problem

[0011] A first aspect of this disclosure provides a modified GPR35, wherein the modified GPR35 comprises one or more of the following sequences:

[0012] (a1) The amino acid sequence as shown in SEQ ID NO: 32;

[0013] (a2) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 32, and retaining the function of the amino acid sequence shown in SEQ ID NO: 32;

[0014] (a3) 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: 32, and which retains the function of the amino acid sequence shown in SEQ ID NO: 32; or,

[0015] (a4) An amino acid sequence encoded by a nucleotide sequence, said nucleotide sequence hybridizing with a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 32 under stringent conditions, and said amino acid sequence retaining the function of the amino acid sequence shown in SEQ ID NO: 32, said stringent conditions being moderately stringent, moderately-highly stringent, highly stringent, or very highly stringent.

[0016] In some embodiments, the N-terminus of the modified GPR35 further includes a β2-adrenergic receptor or a variant thereof, as shown in SEQ ID NO: 33.

[0017] In some specific implementations, the modified GPR35 comprises one or more of the following sequences:

[0018] (b1) The amino acid sequence as shown in SEQ ID NO: 34;

[0019] (b2) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 34, and retaining the function of the amino acid sequence shown in SEQ ID NO: 34;

[0020] (b3) 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: 34, and which retains the function of the amino acid sequence shown in SEQ ID NO: 34; or,

[0021] (b4) An amino acid sequence encoded by a nucleotide sequence hybridizing with a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 34 under stringent conditions, and the amino acid sequence retains the function of the amino acid sequence shown in SEQ ID NO: 34, wherein the stringent conditions are moderately stringent, moderately-highly stringent, highly stringent, or very highly stringent.

[0022] In some embodiments, the modified GPR35 further includes a tag, a protease cleavage site, a signal peptide, a peptide linker, or any combination thereof.

[0023] In some specific implementations, the modified GPR35 includes a tag at its N-terminus and / or C-terminus; and / or,

[0024] The modified GPR35 contains a signal peptide at its N-terminus; and / or

[0025] The protease cleavage site is located between two adjacent elements.

[0026] In some more specific embodiments, the modified GPR35 comprises one or more of the following sequences:

[0027] (c1) The amino acid sequence as shown in SEQ ID NO: 42;

[0028] (c2) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 42, and retaining the function of the amino acid sequence shown in SEQ ID NO: 42;

[0029] (c3) 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: 42, and which retains the function of the amino acid sequence shown in SEQ ID NO: 42; or,

[0030] (c4) An amino acid sequence encoded by a nucleotide sequence, said nucleotide sequence hybridizing with a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 42 under stringent conditions, and said amino acid sequence retaining the function of the amino acid sequence shown in SEQ ID NO: 42, said stringent conditions being moderately stringent, moderately-highly stringent, highly stringent, or very highly stringent.

[0031] A second aspect of this disclosure provides a polynucleotide encoding the modified GPR35 described in the first aspect of this disclosure.

[0032] A third aspect of this disclosure provides an expression vector comprising the polynucleotides described in the second aspect of this disclosure.

[0033] A fourth aspect of this disclosure provides a host cell comprising the expression vector described in the third aspect of this disclosure.

[0034] The fifth aspect of this disclosure provides the use of the modified GPR35 described in the first aspect of this disclosure, the polynucleotide described in the second aspect of this disclosure, the expression vector described in the third aspect of this disclosure, or the host cell described in the fourth aspect of this disclosure in the context of GPR35 structural analysis, fluorescent molecular labeling, fusion of phosphorylated peptides or signaling proteins, GPR35 activity analysis, screening of nucleic acid-encoded small molecule libraries, computer-aided drug design, and drug screening.

[0035] The effects of the invention

[0036] This invention innovatively combines molecular structure prediction and molecular design to construct a modified GPR35. By fusing a modified BRIL with GPR35, a protein sequence that was originally unsuitable for studying inactive state structures is fused and modified, making it possible to study the structure of GPR35 using cryo-electron microscopy. Attached Figure Description

[0037] Figure 1 PCR amplification and verification of the four mutants in the vector; lanes 1 to 4 represent mBRIL-H1 ​​to mBRIL-H4, respectively; M represents the standard marker.

[0038] Figure 2 Results of nickel affinity purification of mBRIL-H1 ​​to mBRIL-H4 proteins; among which, Figure 2 A in the text is mBRIL-H1. Figure 2 B in the text stands for mBRIL-H2. Figure 2 C in the text is mBRIL-H3. Figure 2 In this table, D stands for mBRIL-H4; M: protein standard marker; C: whole cell sample; S: supernatant after disruption; P: precipitate after disruption; FT: nickel affinity flow-through sample; W1-W5: eluted samples of other proteins; E1: eluted sample of target protein; B: residual nickel column material.

[0039] Figure 3 Results of size exclusion chromatography purification of mBRIL-H1 ​​to mBRIL-H4 proteins. Figure 3 A and B in the equation are mBRIL-H1. Figure 3 C and D in the text are mBRIL-H2. Figure 3 E and F in the text are mBRIL-H3. Figure 3G and H in the text refer to mBRIL-H4; S: sample before loading; other sample labels are the collection tube numbers.

[0040] Figure 4 The molecular size exclusion chromatography purification results of the anti-mBRIL nanobody, among which, Figure 4 A in the figure represents a size exclusion chromatography purification diagram. Figure 4 B in Figure 4 SDS-PAGE purity was performed on collection tube A in the sample collection tubes, and B5 to C1 were the sample collection tube numbers, respectively.

[0041] Figure 5 Tests were conducted on the complexes of mBRIL-H1 ​​to mBRIL-H4 with anti-mBRIL nanobodies; among which... Figure 5 A in the text is mBRIL-H1. Figure 5 B in the text stands for mBRIL-H2. Figure 5 C in the text is mBRIL-H3. Figure 5 D in the text is mBRIL-H4.

[0042] Figure 6 Dual-tag purification steps for modified GPR35. D: Whole cells; P: Membrane precipitate; S: Membrane supernatant; FT1: Nickel affinity flow-through; W1-W4: Nickel affinity washing of contaminating proteins; E1: Nickel affinity elution; FT2: Flag affinity flow-through; W5: Flag affinity washing of contaminating proteins; E2-E4: Flag affinity elution; F: Flag column stock; M: Marker / 5μL; The boxes indicate samples collected for concentration and further purification.

[0043] Figure 7 Size exclusion chromatography purification results of modified GPR35; Figure 7 In the diagram, A represents size exclusion chromatography purification, indicating that the protein is in a uniform aggregated state. Figure 7 B in Figure 7 The A collection tube was used for SDS-PAGE purity identification. pre is the sample before loading. A5 to B15 are the sample collection tube numbers, and M is the marker. The box contains the sample collected for concentration and further experimental evaluation.

[0044] Figure 8 The affinity of modified GPR35 for anti-mBRIL nanobodies was determined by ELISA.

[0045] Figure 9 Size exclusion chromatography was used to evaluate the modified GPR35-anti-mBRIL nanobody complex. Figure 9 In the diagram, A represents the size exclusion chromatography purification chromatogram; Figure 9 B in the text is Figure 9In the diagram, A represents the SDS-PAGE identification results, R represents the modified GPR35 alone, mix represents the modified GPR35 and anti-mBRIL nanobody complex, A5 to B12 are the sample collection tube numbers, and M is the marker. The co-migration of modified GPR35 and anti-mBRIL nanobody indicates that they form a complex. The solid box indicates the GPR35-mBRIL complex, and the dashed box indicates excess nanobody.

[0046] Figure 10 Affinity determination of modified GPR35 with antagonist ML-145. Prism software fitting results showed that the affinity of GPR35 with antagonist ML-145 was approximately 24 nM.

[0047] Figure 11 Negative staining results of the modified GPR35-anti-mBRIL nanobody complex. Figure 11 In the image, A represents a magnification of 57,000 times. Figure 11 B in the image represents a magnification of 92,000 times.

[0048] Figure 12 Frozen photographs and 2D structural features of the modified GPR35-anti-mBRIL nanobody complex. Figure 12 A in the image: Cryogenic grid photograph; Figure 12 B in the image: Frozen photograph of the sample; Figure 12 C in the figure represents the preliminary two-dimensional classification result.

[0049] Figure 13 A schematic diagram of the construction process for the modified GPR35. Detailed Implementation

[0050] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0051] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0052] In this specification, the terms "substantially" or "truly" are used to indicate that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, and more preferably 1%.

[0053] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0054] In this specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both the scenario in which the event occurs and the scenario in which the event does not occur.

[0055] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0056] According to this disclosure, the terms “polypeptide,” “protein,” and “peptide” are used interchangeably herein to refer to a polymeric form of amino acids of any length, including encoded and non-coding amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a similar peptide backbone.

[0057] According to this disclosure, the terms "nucleic acid molecule," "polynucleotide," "polynucleotide," and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides 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.

[0058] According to this disclosure, the terms "G protein-coupled receptor" or "GPCR" or "GPR" refer to transmembrane receptors capable of transmitting signals from the extracellular space to the intracellular space via G protein pathways and / or inhibitory protein pathways. Hundreds of such receptors are known in the art; see, for example, Fredriksson et al., Mol. Pharmacol. 63: 1256-1272, 2003, and Vassilatis, DK, 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 having seven regions between 22 and 24 hydrophobic amino acids, said regions forming seven α-helices, each helix spanning the cell membrane. Each span is identified by numbering, i.e., transmembrane-1 (TM1), transmembrane-2 (TM2), etc., and in the invention may also be referred to as the first transmembrane helix, the second transmembrane helix, etc. Transmembrane helices are also linked through amino acid regions between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the extracellular or "extracellular" side of the cell membrane, referred to as "extracellular" regions 1, 2, and 3 (EC1, EC2, and EC3), respectively. Transmembrane helices are also linked through amino acid regions between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the intracellular or "intracellular" side of the cell membrane, referred to as "intracellular" regions 1, 2, and 3 (IC1, IC2, and IC3), respectively. The receptor's carboxyl ("C") terminus is located in the intracellular space, and its amino ("N") terminus is located in the extracellular space. Any of these regions can be conveniently identified by analyzing the primary amino acid sequence of the GPCR.

[0059] According to this disclosure, the term "ligand" or "receptor ligand" refers to a molecule that specifically binds to a GPCR, either intracellularly or extracellularly. Unless for limiting purposes, a ligand can be a protein, (poly)peptide, lipid, small molecule, protein scaffold, antibody, antibody fragment, nucleic acid, or carbohydrate. Ligands can be synthetic or naturally occurring. The term "ligand" includes "natural ligand," which is an endogenous, naturally occurring ligand of a natural GPCR. In most cases, a ligand is a "regulator" that increases or decreases the intracellular response upon contact (e.g., binding) with a cell-expressed GPCR. Examples of ligands as regulators include agonists, partial agonists, inverse agonists, and antagonists. An "agonist" refers to a ligand that increases the signaling activity of a receptor by binding to it. A full agonist can maximally stimulate the receptor; a partial agonist cannot induce full activity even at saturation concentrations. Partial agonists can also function as "blockers" by preventing the binding of stronger agonists. An "antagonist" refers to a ligand that binds to a receptor without stimulating any activity. "Antagonists" are also known as "blockers" because of their ability to prevent the binding of other ligands and thus block agonist-induced activity. Furthermore, "inverse agonists" refer to antagonists that, in addition to blocking the agonist effect, reduce the receptor's basal or constitutive activity to levels below those of unbound receptors.

[0060] According to this disclosure, the three-letter and single-letter codes for amino acids used are as described in J. biol. chem, 243, p3558 (1968).

[0061] According to this disclosure, the term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, 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.

[0062] According to this disclosure, an amino acid "addition" refers to the addition of an amino acid to the C-terminus or N-terminus of an amino acid sequence. According to this disclosure, an amino acid "deletion" refers to the deletion of one, two, or three or more amino acids from an amino acid sequence. According to this disclosure, an amino acid "insertion" refers to the insertion of an amino acid residue at an appropriate position in an amino acid sequence; the inserted amino acid residues may be all or partly adjacent to each other, or none of the inserted amino acids may be adjacent to each other.

[0063] According to this disclosure, an amino acid "substitution" refers to the replacement of an amino acid residue at a certain position in an amino acid sequence by another amino acid residue; wherein, "substitution" can be a conserved amino acid substitution.

[0064] According to this disclosure, "conservative modification," "conservative substitution," or "conservative replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), such that it can be frequently altered without changing the protein's biological activity. Those skilled in the art will appreciate that, in general, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, the substitution of structurally or functionally similar amino acids is unlikely to impair biological activity. Exemplary conserved substitutions are described in the following "Exemplary Conservative Amino Acid Substitutions."

[0065] Exemplary amino acid conservative substitution

[0066] 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; Val 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

[0067] According to this disclosure, "moderate to very high stringency conditions" includes "moderate stringency conditions," "moderate to high stringency conditions," "high stringency conditions," or "very high stringency conditions," which describe the conditions for nucleic acid hybridization and washing. For guidance on performing hybridization reactions, see Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1–6.3.6, which is incorporated herein by reference. Aqueous and non-aqueous methods are described in that literature, and either can be used. For example, specific hybridization conditions are as follows: (1) Low-toughness hybridization conditions: 6× sodium chloride / sodium citrate (SSC) at about 45°C, then at at least 50°C, washed twice in 0.2× SSC, 0.1% SDS (for low-toughness conditions, the washing temperature can be increased to 55°C); (2) Medium-toughness hybridization conditions: 6× SSC at about 45°C, then at 60°C, washed once or more in 0.2× SSC, 0.1% SDS; (3) High-toughness hybridization conditions: 6× SSC at about 45°C, then at 65°C, washed once or more in 0.2× SSC, 0.1% SDS, preferably; (4) Very high-toughness hybridization conditions: 0.5M sodium phosphate, 7% SDS at 65°C, then at 65°C, washed once or more in 0.2× SSC, 1% SDS.

[0068] According to this disclosure, "homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of 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 × 100. For example, at optimal sequence alignment, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in two sequences match or are homologous, then the two sequences are 95% homologous. Typically, comparisons are made when aligning two sequences to give the maximum percentage of homology. For example, comparisons can be performed using the BLAST algorithm, where the algorithm's parameters are selected to give the maximum match between the sequences over the entire length of each reference sequence. The following references relate to the BLAST algorithm frequently used in sequence analysis: BLAST ALGORITHMS: Altschul, SF et al., (1990) J. Mol. Biol. 215: 403-410; Gish, W. et al., (1993) Nature Genet. 3: 266-272; Madden, TL et al., (1996) Meth. Enzymol. 266: 131-141; Altschul, SF et al., (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J. et al., (1997) Genome Res. 7: 649-656. Other common BLAST algorithms, such as those provided by NCBI BLAST, are also well-known to those skilled in the art.

[0069] According to this disclosure, the term "codon optimization" refers to the configuration of a nucleotide sequence encoding a polypeptide to contain codons preferred by the host cell or organism in order to improve gene expression and translation efficiency in the host cell or organism.

[0070] According to this disclosure, the term "tag" refers to a short peptide that is fused or linked to a target protein (e.g., the modified GPR35 of this disclosure) and thereby facilitates the soluble expression, detection, and / or purification of the recombinant protein. The tag may be fused or linked to the N-terminus and / or C-terminus of the target protein (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. Examples of such tags include, but are not limited to, histidine tags, glutathione transferase (GST) tags, maltose-binding protein (MBP) tags, thioredoxin (Trx) tags, NusA tags, disulfide isomerase DsbA tags, DsbC tags, SUMO tags, msyB tags, TF tags, priming factor tags, ubiquitin tags, Myc tags, Flag tags, fluorescent protein (e.g., GFP) tags, biotin tags, and avidin tags.

[0071] According to this disclosure, the terms "signal peptide," "signal sequence," or "signal peptide sequence" refer to a short peptide that, when fused with a target protein (such as the modified GPR35 of this disclosure), promotes the secretion of the target protein expressed by the cell onto the cell membrane or extracellularly. The signal peptide is typically located at the N-terminus of the target protein, and various signal peptides are known to those skilled in the art, such as, but not limited to, erythropoietin signal sequences, human insulin signal sequences, human interleukin-2 signal sequences, albumin signal sequences, etc.

[0072] According to this 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 are described in numerous prior art documents. Those skilled in the art can use appropriate protease cleavage sites in fusion proteins and perform cleavage with corresponding proteases, depending on the specific circumstances. The use of protease cleavage sites can be advantageous, for example, as they can be used to cleave signal peptides and / or tags from fusion proteins to obtain mature proteins with desired activities.

[0073] According to this disclosure, the term "peptide linker" refers to a short peptide used to link two molecules (e.g., proteins). Typically, fusion proteins, such as target protein 1-peptide linker-target protein 2, are obtained by introducing (e.g., by PCR amplification or ligase) a polynucleotide sequence encoding the short peptide between two DNA fragments encoding the two target proteins to be linked, and then expressing the protein.

[0074] According to this disclosure, the term "vector" refers to a nucleic acid delivery vehicle in which polynucleotides can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, cosmids, etc.

[0075] According to this disclosure, the terms "cell," "cell line," and "cell culture" are used interchangeably, and all such names include progeny. Therefore, the terms "transformant" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of transfections. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in terms of DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells. Where different names are intended, this is clearly apparent from the context.

[0076] Detailed technical solution

[0077] <Modified GPR35>

[0078] In some embodiments of this disclosure, a modified GPR35 is provided, wherein the modified GPR35 comprises one or more of the following sequences:

[0079] (a1) The amino acid sequence as shown in SEQ ID NO: 32;

[0080] (a2) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 32, and retaining the function of the amino acid sequence shown in SEQ ID NO: 32;

[0081] (a3) 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: 32, and which retains the function of the amino acid sequence shown in SEQ ID NO: 32; or,

[0082] (a4) An amino acid sequence encoded by a nucleotide sequence, said nucleotide sequence hybridizing with a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 32 under stringent conditions, and said amino acid sequence retaining the function of the amino acid sequence shown in SEQ ID NO: 32, said stringent conditions being moderately stringent, moderately-highly stringent, highly stringent, or very highly stringent.

[0083] In some preferred embodiments, the modified GPR35 comprises the amino acid sequence shown in SEQ ID NO: 32. In some more preferred embodiments, the amino acid sequence of the modified GPR35 is shown in SEQ ID NO: 32.

[0084] The modified GPR35 disclosed herein, fused with the modified BRIL disclosed herein, solves the problem that the wild-type GPR35 protein is too flexible and unsuitable for structural studies, improves stability, and can be applied to AI-assisted structure-based drug development. Furthermore, it can be used for screening DNA-encoded small molecule libraries based on protein targets, animal immunization to generate specific antibodies, and screening synthetic nanobody libraries for the development of extracellular biomolecular drugs.

[0085] To further improve the stability and expression level of the modified GPR35, in some embodiments, the N-terminus of the modified GPR35 further includes a β2-adrenergic receptor or a variant thereof as shown in SEQ ID NO: 33. In some specific embodiments, the modified GPR35 comprises one or more of the following sequences:

[0086] (b1) The amino acid sequence as shown in SEQ ID NO: 34;

[0087] (b2) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 34, and retaining the function of the amino acid sequence shown in SEQ ID NO: 34;

[0088] (b3) 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: 34, and which retains the function of the amino acid sequence shown in SEQ ID NO: 34; or,

[0089] (b4) An amino acid sequence encoded by a nucleotide sequence hybridizing with a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 34 under stringent conditions, and the amino acid sequence retains the function of the amino acid sequence shown in SEQ ID NO: 34, wherein the stringent conditions are moderately stringent, moderately-highly stringent, highly stringent, or very highly stringent.

[0090] To improve the expression, purification, and subsequent processing of the modified GPR35, the modified GPR35 further includes a tag, a protease cleavage site, a signal peptide, a peptide linker, or any combination thereof.

[0091] In some specific embodiments, the modified GPR35 includes a tag at its N-terminus and / or C-terminus. In some alternative embodiments, the tag is selected from at least one of the following: histidine tag, glutathione transferase (GST) tag, maltose-binding protein (MBP) tag, thioredoxin (Trx) tag, NusA tag, disulfide isomerase DsbA tag, DsbC tag, SUMO tag, msyB tag, TF tag, priming factor tag, ubiquitin tag, Myc tag, Flag tag, fluorescent protein (e.g., GFP) tag, biotin tag, and avidin tag.

[0092] In some specific embodiments, the modified GPR35 includes a signal peptide at its N-terminus. In some alternative embodiments, the signal peptide is selected from at least one of the following: erythropoietin signal sequence, human insulin signal sequence, human interleukin-2 signal sequence, and albumin signal sequence.

[0093] In some specific embodiments, the protease cleavage site is located between two adjacent elements. Examples include, but are not limited to, the β2-adrenergic receptor sequence and the GPR35 domain, or the tag sequence.

[0094] In some more specific embodiments, the modified GPR35 comprises one or more of the following sequences:

[0095] (c1) The amino acid sequence as shown in SEQ ID NO: 42;

[0096] (c2) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 42, and retaining the function of the amino acid sequence shown in SEQ ID NO: 42;

[0097] (c3) 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: 42, and which retains the function of the amino acid sequence shown in SEQ ID NO: 42; or,

[0098] (c4) An amino acid sequence encoded by a nucleotide sequence, said nucleotide sequence hybridizing with a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 42 under stringent conditions, and said amino acid sequence retaining the function of the amino acid sequence shown in SEQ ID NO: 42, said stringent conditions being moderately stringent, moderately-highly stringent, highly stringent, or very highly stringent.

[0099] In some preferred embodiments, the modified GPR35 comprises the amino acid sequence shown in SEQ ID NO: 42. In some more preferred embodiments, the amino acid sequence of the modified GPR35 is shown in SEQ ID NO: 42.

[0100] <Modified GPR35-related polynucleotides, expression vectors, and host cells>

[0101] In some embodiments of this disclosure, a polynucleotide is provided that encodes a modified GPR35 of this disclosure.

[0102] The polynucleotides disclosed herein can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0103] The polynucleotides encoding the modified GPR35 disclosed herein include: a coding sequence that encodes only the modified GPR35; a coding sequence for the modified GPR35 and various additional coding sequences; a coding sequence for the modified GPR35 (and optional additional coding sequences) and non-coding sequences.

[0104] The term "polynucleotide encoding modified GPR35" can include a polynucleotide encoding this modified GPR35, or it can include a polynucleotide that also includes additional coding and / or non-coding sequences.

[0105] This disclosure also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. This disclosure particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. These stringent conditions are moderately stringent, medium-high stringent, high stringent, or very high stringent conditions.

[0106] In some embodiments of this disclosure, an expression vector is provided that contains the polynucleotides of this disclosure.

[0107] In some embodiments of this disclosure, a host cell is provided that contains the expression vector of this disclosure.

[0108] <Applications of the modified GPR35>

[0109] Some embodiments of this disclosure provide the use of the modified GPR35 of this disclosure, the polynucleotide of this disclosure, the expression vector of this disclosure, or the host cell of this disclosure in GPR35 structural determination, fluorescent molecular labeling, fusion of phosphorylated peptides or signaling proteins, GPR35 activity analysis, screening of nucleic acid-encoded small molecule libraries, computer-aided drug design, and drug screening.

[0110] The present disclosure is further illustrated below with reference to the accompanying drawings and embodiments, but this is not intended to limit the present disclosure. Specific materials used in the embodiments of the present disclosure and their sources are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present disclosure. Materials of the same or similar type, model, quality, properties, or functions as the reagents and instruments described below can be used to implement the present disclosure. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, etc., used in the following embodiments are commercially available.

[0111] Example 1: Preparation of modified BRIL and anti-mBRIL nanobodies

[0112] 1. Cloning and Mutation Design of Wild-Type BRIL Sequences

[0113] 1.1 Cloning of wild-type BRIL sequences

[0114] Wild-type BRIL protein was cloned into the pET28a vector (Beijing Xianghong Biotechnology Co., Ltd.). To facilitate purification and subsequent preparation, the linker peptide GS, the tobacco mosaic virus (TEV) protease cleavage site (ENLYFQS; SEQ ID NO: 43), the corresponding genes of linker peptide GS and 8×His tag were added to the end of the wild-type BRIL protein gene. The gene synthesis steps were entrusted to Beijing Xianghong Biotechnology Co., Ltd.

[0115] The amino acid sequence of wild-type BRIL protein (SEQ ID NO: 1): MADLEDNWETLNDNLKVIEKADNAAQVKDALTKMRAAALDAQKATPPKLEDKSPDSPEMKDFRHGFDILVGQIDDALKLANEGKVKEAQAAAEQLKTTRNAYIQKYL

[0116] The amino acid sequence of the BRIL protein with added linker peptide, restriction enzyme site, linker peptide, and tag (underlined part) (SEQ ID NO: 2):

[0117] MADLEDNWETLNDNLKVIEKADNAAQVKDALTKMRAAALDAQKATPP

[0118] KLEDKSPDSPEMKDFRHGFDILVGQIDDALKLANEGKVKEAQAAAEQLK

[0119] TTRNAYIQKYLGSENLYFQSGSSHHHHHHHH

[0120] Nucleotide sequence of BRIL protein with added linker peptide, restriction enzyme site, linker peptide, and tag (SEQ ID NO: 3):

[0121] ATGGCGGATCTGGAAGATAACTGGGAAACCCTGAATGATAACCTGAA

[0122] AGTAATTGAAAAAGCGGATAACGCGGCGCAGGTGAAAGATGCGCTGA

[0123] CGAAAATGCGCGCGGCGGCCCTGGATGCGCAGAAAGCGACCCCGCCG

[0124] AAACTGGAAGATAAAAGCCCGGATAGCCCGGAAATGAAAGATTTCCG

[0125] CCATGGCTTTTGATATTCTGGTGGGCCAGATTGATGATGCGCTGAAACT

[0126] GGCCAATGAAGGCAAAGTGAAAGAAGCGCAGGCGGCCGGAACAG

[0127] CTGAAAACCACCCGTAATGCGTACATTCAGAAATATCTGGGCAGCGA

[0128] AAATCTGTACTTTCAGAGCGGCAGCCATCACCATCATCACCACCATCA

[0129] CTAA

[0130] 1.2 BRIL Mutation Design

[0131] In this embodiment, based on the epitope recognized by the 3LRH intrabody disclosed in the literature Kim, JWet al. Application of antihelix antibodies in protein structure determination. P Natl Acad Sci Usa 116,17786–17791 (2019), mutation sites were designed and mutated at different positions in the wild-type BRIL protein, so that the modified BRIL protein carries the epitope recognized by the 3LRH intrabody. The specific binding of the epitope and its effect on protein structure determination after fusion with the protein were then tested.

[0132] Specifically, based on the nucleotide sequence of the BRIL protein in step 1.1, four pairs of primers were designed to mutate the wild-type BRIL protein, forming four different BRIL mutants, namely modified BRILs (named mBRIL-H1 ​​to mBRIL-H4 in this specification). After PCR amplification, the PCR products were identified by agarose gel electrophoresis (e.g., Figure 1 As shown in the figure, the nucleotide fragments were digested with DpnI (New England Biolabs, NEB) at 37°C for 30 minutes. Subsequently, the digested nucleotide fragments were cloned into the pET28a vector (Beijing Xianghong Biotechnology Co., Ltd.), transformed into DH5a competent cells (Jinsha Biotechnology), and single-clone selection and identification were performed.

[0133] The information for the four primer pairs is as follows:

[0134]

[0135] Sequencing verification of the mutations was completed as planned. The sequences of the four BRIL mutants are as follows:

[0136] The amino acid sequence of the mBRIL-H1 ​​protein (SEQ ID NO: 12):

[0137]

[0138] The amino acid sequence of the mBRIL-H1 ​​protein with added linker peptide, restriction enzyme site, linker peptide, and tag (SEQ ID NO: 13):

[0139]

[0140] In this text, the bold part indicates the mutation location, and the underlined part indicates the linker peptide + restriction site + linker peptide + tag.

[0141] Nucleotide sequence of mBRIL-H1 ​​protein with added linker peptide, restriction enzyme site, linker peptide, and tag (SEQ ID NO: 14):

[0142] ATGGCGGATCTGAAACTGAACAAAGCGTTTCTGAGCCTGAACCTGTTT

[0143] GTAATTGAAAAAGCGGATAACGCGGCGCAGGTGAAAGATGCGCTGAC

[0144] GAAAATGCGCGCGGCGGCCCTGGATGCGCAGAAAGCGACCCCGCCGA

[0145] AACTGGAAGATAAAAGCCCGGATAGCCCGGAAATGAAAGATTTCCGC

[0146] CATGGCTTTGATATTCTGGTGGGCCAGATTGATGATGCGCTGAAACTG

[0147] GCCAATGAAGGCAAAGTGAAAGAAGCGCAGGCGGCCGGAACAGC

[0148] TGAAAACCACCCGTAATGCGTACATTCAGAAATATCTGGGCAGCGAA

[0149] AATCTGTACTTTCAGAGCGGCAGCCATCACCATCATCACCACCATCAC

[0150] TAA

[0151] The amino acid sequence of the mBRIL-H2 protein (SEQ ID NO: 15):

[0152]

[0153] The amino acid sequence of the mBRIL-H2 protein with added linker peptide, restriction enzyme site, linker peptide, and tag (SEQ ID NO: 16):

[0154]

[0155] In this text, the bold part indicates the mutation location, and the underlined part indicates the linker peptide + restriction site + linker peptide + tag.

[0156] Nucleotide sequence of mBRIL-H2 protein with added linker peptide, restriction enzyme site, linker peptide, and tag (SEQ ID NO: 17):

[0157] ATGGCGGATCTGGAAGATAACTGGGAAACCCTGAATGATAACCTGAA

[0158] AGTAATTGAAAAAGCGGATAACGCGAAACTGGTGAAAGCGTTTCTGA

[0159] GCCTGATGCGCTTTGCGGCCCTGGATGCGCAGAAAGCGACCCCGCCG

[0160] AAACTGGAAGATAAAAGCCCGGATAGCCCGGAAATGAAAGATTTCCG

[0161] CCATGGCTTTTGATATTCTGGTGGGCCAGATTGATGATGCGCTGAAACT

[0162] GGCCAATGAAGGCAAAGTGAAAGAAGCGCAGGCGGCCGGAACAG

[0163] CTGAAAACCACCCGTAATGCGTACATTCAGAAATATCTGGGCAGCGA

[0164] AAATCTGTACTTTCAGAGCGGCAGCCATCACCATCATCACCACCATCA

[0165] CTAA

[0166] The amino acid sequence of the mBRIL-H3 protein (SEQ ID NO: 18):

[0167]

[0168] The amino acid sequence of the mBRIL-H3 protein with added linker peptide, restriction enzyme site, linker peptide, and tag (SEQ ID NO: 19):

[0169]

[0170] In this text, the bold part indicates the mutation location, and the underlined part indicates the linker peptide + restriction site + linker peptide + tag.

[0171] Nucleotide sequence of mBRIL-H3 protein with added linker peptide, restriction enzyme site, linker peptide, and tag (SEQ ID NO: 20):

[0172] ATGGCGGATCTGGAAGATAACTGGGAAACCCTGAATGATAACCTGAA

[0173] AGTAATTGAAAAAGCGGATAACGCGGCGCAGGTGAAAGATGCGCTGA

[0174] CGAAAATGCGCGCGGCGGCCCTGGATGCGCAGAAAGCGACCCCGCCG

[0175] AAACTGGAAGATAAAAGCCCGGATAGCCCGGAAATGAAAGATTTCCG

[0176] CCATGGCTTTAAACTGCTGAAAGCGTTTATTAGCCTGGCGCTGTTTTCT

[0177] GGCCAATGAAGGCAAAGTGAAAGAAGCGCAGGCGGCCGGAACAG

[0178] CTGAAAACCACCCGTAATGCGTACATTCAGAAATATCTGGGCAGCGA

[0179] AAATCTGTACTTTCAGAGCGGCAGCCATCACCATCATCACCACCATCA

[0180] CTAA

[0181] The amino acid sequence of the mBRIL-H4 protein (SEQ ID NO: 21):

[0182]

[0183] The amino acid sequence of the mBRIL-H4 protein with added linker peptide, restriction enzyme site, linker peptide, and tag (SEQ ID NO: 22):

[0184]

[0185] In this text, the bold part indicates the mutation location, and the underlined part indicates the linker peptide + restriction site + linker peptide + tag.

[0186] Add the linker peptide, restriction enzyme site, linker peptide, and tag mBRIL-H4 nucleotide sequence (SEQ ID NO: 23):

[0187] ATGGCGGATCTGGAAGATAACTGGGAAACCCTGAATGATAACCTGAA

[0188] AGTAATTGAAAAAGCGGATAACGCGGCGCAGGTGAAAGATGCGCTGA

[0189] CGAAAATGCGCGCGGCGGCCCTGGATGCGCAGAAAGCGACCCCGCCG

[0190] AAACTGGAAGATAAAAGCCCGGATAGCCCGGAAATGAAAGATTTCCG

[0191] CCATGGCTTTTGATATTCTGGTGGGCCAGATTGATGATGCGCTGAAACT

[0192] GGCCAATGAAGGCAAAGTGAAACTGGCGAAAGCGTTTGCGAGCCTGC

[0193] TGAAATTTACCCGTAATGCGTACATTCAGAAATATCTGGGCAGCGAA

[0194] AATCTGTACTTTCAGAGCGGCAGCCATCACCATCATCACCACCATCAC

[0195] TAA

[0196] 2. Expression and purification of mBRIL

[0197] 2.1 Preparation of plasmids containing mBRIL

[0198] Recombinant plasmids (pET28a vector, Beijing Xianghong Biotechnology Co., Ltd.) containing the target gene (with added linker peptide, restriction enzyme site, linker peptide, and tags mBRIL-H1 ​​to mBRIL H4) were introduced into *E. coli* DH5α competent cells (Jinsha Biotechnology) via heat shock transformation and cultured overnight (37°C) in LB medium containing 100 μg / mL kanamycin. The next day, the bacterial culture was collected, and the plasmid was extracted.

[0199] 2.2 The mBRIL plasmid was transformed into BL21(DE3) bacteria for target protein expression.

[0200] The plasmid containing the target gene extracted in step 2.1 was introduced into *E. coli* BL21(DE3) competent cells (Bomaide Biotechnology) via heat shock transformation and cultured overnight (37°C) in LB medium containing 100 μg / mL kanamycin. The next day, the cells were transferred to 1 L of LB medium for further expansion. When the OD... 600 When the expression level reached 0.8, 0.1 mM IPTG was added and the temperature was adjusted to 20°C to induce expression overnight.

[0201] 2.3mBRIL protein nickel affinity purification

[0202] After culture, the bacterial culture was collected and centrifuged at 7000 rpm for 10 minutes at 4°C, and the supernatant was removed. Cells were resuspended in 30 ml of Buffer A per 5 g of cells and homogenized by high-pressure homogenization. The lysate was collected and centrifuged at 18000 rpm for 15 minutes. The supernatant was collected for nickel column affinity incubation. Subsequently, contaminating proteins were eluted with Buffer B, 2% Buffer C, and 4% Buffer C, respectively, and the target protein was eluted with 50% Buffer C. The purity of the target protein was identified and analyzed using SDS-PAGE.

[0203] The specific components of the aforementioned buffer AC are as follows:

[0204] Buffer A: 25mM Tris-HCl pH 7.4 (RT), 150mM NaCl, 1mM PMSF, 0.0025mg / ml leupeptin;

[0205] Buffer B: 25mM Tris-HCl pH 7.4, 150mM NaCl;

[0206] Buffer C:Buffer A+1M imidazole (Imidazole)

[0207] Experimental results: such as Figure 2 As shown, mBRIL-H1 ​​to mBRIL-H4 all exhibit high levels of expression of the target protein in whole cells. The expression levels and purity of each mutant vary slightly, but the overall purity is approximately 95% or higher.

[0208] 2.4 Size Exclusion Chromatography Purification

[0209] A Superdex 200 increase 10 / 300 Column GL (Cytiva) pre-equilibrated chromatography column was used. Nickel affinity elution buffer was transferred to a 10 kDa ultrafiltration tube to concentrate the target protein. When the volume was concentrated to approximately 800 μL, size exclusion chromatography was performed for purification. The target protein sample was collected based on the UV-280 absorption peak, and SDS-PAGE gel electrophoresis was performed to determine the content and purity of the target protein.

[0210] Experimental results: such as Figure 3 As shown, size exclusion chromatography and SDS-PAGE results for mBRIL-H1 ​​to mBRIL-H4 indicate that all proteins are primarily in monomeric form. The yields of different mutants vary slightly.

[0211] 3. Expression and purification of anti-mBRIL nanobodies

[0212] 3.1 Sequence Cloning of Anti-mBRIL Nanobody

[0213] The anti-mBRIL nanobody used in this embodiment is the 3LRH intracellular antibody disclosed in the literature Kim, J Wet al. Application of antihelix antibodies in protein structure determination. P Natl Acad Sci Usa 116, 17786–17791 (2019).

[0214] The anti-mBRIL nanobody was cloned into the pET26b vector (Beijing Xianghong Biotechnology Co., Ltd.). To facilitate purification and subsequent preparation, a signal peptide was added to the N-terminus of the gene and a 6×His tag was added to the C-terminus. The gene synthesis was entrusted to Beijing Xianghong Biotechnology Co., Ltd.

[0215] Anti-mBRIL nanobody amino acid sequence (SEQ ID NO: 24): QPVLTQSPSVSAAPRQRVTISVSGSNSNIGSNTVNWIQQLPGRAPELLMYDDDLLAPGVSDRFSGSRSGTSASLTISGLQSEDEADYYAATWDDSLNGWVFGGGTKVTVLSA

[0216] The amino acid sequence of the anti-mBRIL nanobody with added signal peptide and tag (SEQ ID NO: 25): MKYLLPTAAAGLLLLAAQPAMAQPVLTQSPSVSAAPRQRVTISVSGSNSNIGSNTVNWIQQLPGRAPELLMYDDDLLAPGVSDRFSGSRSGTSASLTISGLQSEDEADYYAATWDDSLNGWVFGGGTKVTVLSAHHHHHH

[0217] The underlined portion at the N-terminus is the signal peptide, and the underlined portion at the C-terminus is the tag sequence.

[0218] The nucleotide sequence of the anti-mBRIL Nanobody with added signal peptide and tag (SEQ ID NO: 26): ATGAAATACCTGCTGCCGACCGCGGCGGCCGGTCTGCTGCTGCTGGCCGCACAGCCTGCGATGGCGCAGCCGGTGCTGACCCAAAGCCCGAGCGTGAGCGCGGCGCCGCGTCAGCGTGTTACCATTAGCGTGAGCGGTAGCAATAGCAATATTGGCTCCAATACCGTGAACTGGATTCAGCAGCTGCCTGGTCGCGCGCCGGAACTGC TGATGTACGATGATGATCTGCTGGCGCCGGGTGTATCGGATCGCTTTAGCGGCAGCCGTAGCGGCACCTCAGCCAGCCTGACCATTAGCGGCCTGCAGAGCGAAGATGAAGCCGATTATTATGCGGCGACCTGGGATGATAGTCTGAACGGCTGGGTATTTGGTGGCGGCACCAAAGTGACCGTTCTGTCGGCCCATCATCATCATCATCATTAA

[0219] 3.2 Expression and purification of anti-mBRIL nanobodies

[0220] 3.2.1 Preparation of anti-mBRIL nanobody plasmids

[0221] Recombinant plasmids containing the target gene anti-mBRIL nanobodies were introduced into *E. coli* DH5α competent cells (Jinsha Biotechnology) via heat shock transformation and cultured overnight (37°C) in LB medium containing 100 μg / mL kanamycin. The next day, the bacterial culture was collected and the plasmids were extracted.

[0222] 3.2.2 The anti-mBRIL nanobody plasmid was transformed into BL21(DE3) bacteria for target protein expression.

[0223] Plasmids containing the target gene were introduced into *E. coli* BL21(DE3) competent cells (Bomaide Biotechnology) via heat shock transformation and cultured overnight (37°C) in LB medium containing 100 μg / mL kanamycin. The next day, the cells were transferred to 1 L of LB medium for further expansion. When OD... 600 When the expression level reached 0.8, 0.1 mM IPTG was added and the temperature was adjusted to 20°C to induce expression overnight.

[0224] 3.3.3 Purification of anti-mBRIL nanobodies

[0225] The expressed *E. coli* culture was centrifuged at 7000 rpm for 10 minutes at 4°C to remove the culture medium. 30 ml of Buffer A was added per 5 g of cells for resuspending, and the cells were homogenized by high-pressure homogenization. Impurities were eluted with Buffer BH, and the target protein was eluted with Buffer I. The target protein was concentrated using a 10 kDa ultrafiltration tube to a volume of approximately 800 μL, and then purified by size exclusion chromatography using a Superdex 200 increase 10 / 300 Column GL (Cytiva) gel column and Buffer B. Protein samples from the UV-280 absorption peak were collected for SDS-PAGE gel electrophoresis to determine the content and purity of the target protein.

[0226] The specific components of the aforementioned buffer DI are as follows:

[0227] Buffer D: 25mM Tris-HCl pH7.4, 500mM NaCl, 20mM imidazole;

[0228] Buffer E: 25mM Tris-HCl pH7.4, 150mM NaCl, 20mM imidazole;

[0229] Buffer F: 25mM Tris-HCl pH7.4, 500mM NaCl, 40mM imidazole;

[0230] Buffer G: 25mM Tris-HCl pH7.4, 150mM NaCl, 40mM imidazole;

[0231] Buffer H: 25mM Tris-HCl pH7.4, 500mM NaCl, 60mM imidazole;

[0232] Buffer I: 25mM Tris-HCl pH7.4, 150mM NaCl, 300mM imidazole.

[0233] The results are as follows Figure 4 As shown, the anti-mBRIL nanobody has a high expression level, and after purification, a large amount of target protein with high purity is obtained.

[0234] 4. Assay for the complex of mBRIL and anti-mBRIL nanobodies

[0235] mBRIL-H1, mBRIL-H2, mBRIL-H3, and mBRIL-H4 were incubated with anti-mBRIL nanobody at a molar ratio of 1:1 at 4°C for 1 hour. A Buffer B pre-equilibrated chromatography column (Superdex 200 increase 5 / 150GL (Cytiva)) was used. Individual BRIL mutants were injected as control samples, while the BRIL mutant-anti-mBRIL nanobody complex was injected as the experimental group. After the experiment, the peak positions and shapes of the control and experimental groups were analyzed to evaluate the complex formation effect of the BRIL mutant and anti-mBRIL nanobody.

[0236] The results are as follows Figure 5 As shown, among the four BRIL mutants, mBRIL-H1 ​​and mBRIL-H4 can form good complexes with anti-BRIL nanobodies.

[0237] Example 2: Expression and preparation of modified GPR35

[0238] 1. Expression and preparation of modified GPR35

[0239] To further verify the application value of the modified mBRIL in fusion GPCR protein for stabilizing receptor structure and assisting in structural analysis, this embodiment modifies the fusion protein of the BRIL mutant of the GPR35 gene.

[0240] 1.1 Sequence Modification of GPR35

[0241] The human GPR35 protein sequence (UNIPORT: Q9HC97) was retrieved from the UNIPORT database. The GPR35 sequence was then fused with the mBRIL-H4 mutant, and a signal peptide and Flag / Strep / 6×His purification tags were added.

[0242] Wild-type GPR35 protein sequence (SEQ ID NO: 27):

[0243] MNGTYNTCGSSDLTWPPPAIKLGFYAYLGVLLVLGLLLNSLALWVFCCR

[0244] MQQWTETRIYMTNLAVADLCLLCTLPFVLHSLRDTSDTPLCQLSQGIYLT

[0245] NRYMSISLVTAIAVDRYVAVRHPLRARGLRSPRQAAAVCAVLWVLVIGS

[0246] LVARWLLGIQEGGFFCFRSTRHNFNSMAFPLLGFYLPLAVVVFCSLKVVTA

[0247] LAQRPPTDVGQAEATRKAARMVWANLLVFVVCFLPLHVGLTVRLAVG

[0248] WNACALLETIRRALYITSKLSDANCCLDAICYYYMAKEFQEASALAVAPS

[0249] AKAHKSQDSLCVTLA

[0250] In the above-mentioned wild-type GPR35 amino acid sequence, the single underlined part represents the amino acid sequence retained in the modified GPR35.

[0251] 1.1.1 Extending the length of the mBRIL protein helix

[0252] The mBRIL-H4 protein obtained in Example 1 was modified by removing the first methionine (M) at the N-terminus, and then the sequences ARRQL (SEQ ID NO: 28) and ERARS (SEQ ID NO: 29) were added to its N-terminus and C-terminus, respectively, to extend the helix length of the mBRIL protein and avoid conflict between BRIL and detergent microclusters.

[0253] The amino acid sequence of the resulting extended helix length mBRIL-H4 protein is as follows (SEQ ID NO: 30): ARRQLADLEDNWETLNDNLKVIEKADNAAQVKDALTKMRAAALDAQKATPPKLEDKSPDSPEMKDFRHGFDILVGQIDDALKLANEGKVKLAKAFASLLKFTRNAYIQKYLERARS

[0254] Alternatively, an extended helix length mBRIL-H1 ​​protein can be used, the amino acid sequence of which is shown below (SEQ ID NO: 31):

[0255] ARRQLADLKLNKAFLSLNLFVIEKADNAAQVKDALTKMRAAALDAQKATPPKLEDKSPDSPEMKDFRHGFDILVGQIDDALKLANEGKVKEAQAAAEQLKTTRNAYIQKYLERARS

[0256] 1.1.2 Ligation of mBRIL-H4 protein with extended helix length to GPR35 protein

[0257] The amino acids 201-207 of the wild-type GPR35 protein were deleted and replaced with the amino acid sequence of the extended helix length mBRIL-H4 protein. The resulting sequence (SEQ ID NO: 32) is as follows:

[0258]

[0259] In this sequence, a single underscore represents the amino acid sequence from GPR35, while a double underscore represents the amino acid sequence of the mBRIL-H4 protein with an extended helix length.

[0260] 1.1.3 N-terminal fusion with β2-adrenergic receptor sequence

[0261] In this embodiment, to further improve the stability and expression level of the receptor, a 24-amino acid sequence of the human β2-adrenergic receptor (UNIPORT: P07550) was fused to the N-terminus.

[0262] The amino acid sequence of the β2-adrenergic receptor used in the modified GPR35 in this embodiment is (SEQ ID NO: 33):

[0263] MGQPGNGSAFLLAPNRSHAPDHDV

[0264] In this embodiment, the sequence of the modified GPR35 with the addition of a β2-adrenergic receptor sequence is as follows (SEQ ID NO: 34):

[0265]

[0266] In this sequence, a single underline represents the amino acid sequence from GPR35, a double underline represents the amino acid sequence of the mBRIL-H4 protein with an extended helix length, and a dotted underline represents the amino acid sequence of the β2-adrenergic receptor.

[0267] 1.1.4 Addition of signal peptide, restriction enzyme site, and tag sequence

[0268] In this embodiment, Flag tag sequences, Strep tag sequences, and 6×His tag sequences were designed at the N-terminus and C-terminus of the modified GPR35, respectively, to facilitate the purification of heterologously expressed modified GPR35. A signal sequence was further added to the N-terminus. Finally, this embodiment designed multiple restriction enzyme sites (3C / TEV) and a Sortase A fusion sequence for the protein sequence, which facilitates subsequent reverse purification, matrix immobilization, and site-specific fluorescent labeling of the purified protein as needed. The modified sequence can be conveniently applied to nucleic acid-encoded small molecule drug libraries and drug binding experiments.

[0269] Specifically, in this embodiment, the modified GPR35 from the N end to the C end is as follows:

[0270] Erythropoietin signal sequence (MKTIIALSYIFCLVFA; SEQ ID NO: 35);

[0271] Flag tag sequence (DYKDDDDA; SEQ ID NO: 36);

[0272] β2-adrenergic receptor sequence (MGQPGNGSAFLLAPNRSHAPDHDV; SEQ ID NO: 33);

[0273] TEV protease cleavage site (ENLYFQG; SEQ ID NO: 37; which is another form of the TEV protease cleavage site shown in SEQ ID NO: 43);

[0274] A modified GPR35 sequence (SEQ ID NO: 32) was linked by an extended helix length mBRIL-H4 protein sequence;

[0275] Sortase A fusion sequence (LPETG; SEQ ID NO: 38); it is a "USB-like interface" and the linker site of the Sortase A enzyme.

[0276] Strep tag sequence (SAWSHPQFEK; SEQ ID NO: 39);

[0277] HRV 3C protease cleavage site (LEVLFQGP; SEQ ID NO: 40);

[0278] Connecting peptides (GS);

[0279] 6×His tag sequence (HHHHHH; SEQ ID NO: 41).

[0280] The modified GPR35 protein amino acid sequence (SEQ ID NO: 42):

[0281]

[0282] In this sequence, a single underline represents the amino acid sequence from GPR35, a double underline represents the amino acid sequence of the mBRIL-H4 protein with an extended helix length, and a dotted underline represents the amino acid sequence of the β2-adrenergic receptor.

[0283] 1.2 Cloning, Virus Preparation and Expression of Modified GPR35

[0284] 1.2.1 The sequence-modified GPR35 gene was synthesized by Beijing Xianghong Biotechnology Co., Ltd., and then ligated into the pFastbac-1 plasmid.

[0285] 1.2.2 The recombinant pFastbac plasmid containing the target gene (Beijing Xianghong Biotechnology Co., Ltd.) was introduced into *E. coli* DH10Bac competent cells (BioBomei Biotechnology) via heat shock transformation. The cells were cultured at 37°C for 48 hours in LB solid medium containing 50 μg / mL kanamycin (BioBomei), 7 μg / mL gentamicin (BioBomei), 10 μg / mL tetracycline (BioBomei), 200 μg / mL X-gal (inalco), and 40 μg / mL IPTG (inalco). Uniform white spots were then transferred to 3 mL of LB liquid medium containing the three antibiotics (50 μg / mL kanamycin, 7 μg / mL gentamicin, and 10 μg / mL tetracycline) and cultured overnight at 37°C and 200 rpm until the bacterial culture OD... 600 When the value is approximately 0.6, the recombinant baculovirus plasmid is extracted.

[0286] 1.2.3 Take 1 mL of Insect Medium (Graces), add 15 μL of FuGENE transfection reagent (Promega) and 5 μg of recombinant baculovirus plasmid, and incubate at room temperature for 15 minutes. Resuspend the plasmid in this mixture at 10⁻¹² × 10⁻¹⁰. 6 SF9 insect cells (Expression Systems) were cultured at 27°C and 200 rpm for 4 hours, followed by the addition of 5 mL of ESF921 insect cell culture medium (Expression Systems) and continued culturing at 27°C and 200 rpm for another 48 hours. After 48 hours of culture, the SF9 cells were transferred to 100 mL Erlenmeyer flasks and cultured at 27°C and 110 rpm until a cell density of 2-4 × 10⁻⁴ cells / mL was reached. 6 When the concentration is 1 / mL, the supernatant obtained by centrifugation at 2500 rpm for 10 minutes at room temperature is the P1 generation recombinant baculovirus.

[0287] 1.2.4 Transfect 100 mL of cells with P1 generation recombinant baculovirus at a ratio of 1:10000 to a density of 1.5 × 10⁻⁶ cells. 6 / mL sf9 insect cells were cultured at 27℃ and 110rpm. When the cell density reached about 6×106 / mL and the cell volume was enlarged and relatively uniform, the cells were centrifuged at 2500rpm for 10 minutes at room temperature. The supernatant was filtered through a 0.22μm syringe filter to obtain the P2 generation recombinant baculovirus.

[0288] 1.2.5 Small-scale protein purification experiment

[0289] P2 generation recombinant baculovirus was transfected into 20 mL at ratios of 1:50, 1:100, and 1:200, at a density of 4 × 10⁻⁶. 6 / mL sf9 insect cells were cultured at 27℃ and 110 rpm for 48 hours. After culture, samples were taken for Western blot analysis to determine the expression of the target protein. Since a 1:50 ratio yielded the highest expression level of the target protein, this ratio was subsequently selected for large-scale expression preparation.

[0290] 1.2.6 Protein purification and large-scale expression

[0291] P2 generation recombinant baculovirus was transfected at a ratio of 1:50 to a density of 4 × 10⁻⁶ cells / L. 6 / mL sf9 insect cells were cultured at 27°C and 110 rpm for 48 hours to induce viral infection. After cell culture, the cells were collected by centrifugation at 4000 rpm for 20 minutes at 4°C and then frozen for later use.

[0292] 1.3 Purification of Modified GPR35

[0293] 1.3.1 Nickel affinity purification and Flag affinity tag purification of modified GPR35

[0294] Resuspend 20g of dry cells in 100ml of Buffer J and incubate at 4°C for 15min. Centrifuge at 15000rpm for 10min and collect the cell pellet. Resuspend 20g of initial cells in 50ml of Buffer K and manually homogenize 30 times using a glass homogenizer. Incubate at 4°C with stirring for 1.5h, then centrifuge at 41000rpm for 1h at 4°C. After centrifugation, collect the supernatant and co-incubate with 1ml of Ni-NTA column stock (Cytiva). After 1h, transfer the liquid to a gravity column and elute with Buffer L for contaminating proteins. Subsequently, elute with 20mM imidazole + Buffer L and 30mM imidazole + Buffer L, respectively. Finally, elute the target protein with Buffer L containing 250mM imidazole.

[0295] After binding nickel affinity elution buffer to 1 ml Flag medium (Tiandi Renhe Company) for 30 minutes, impurities were eluted with Buffer L, followed by elution of the target protein with Buffer M.

[0296] The specific components of the aforementioned buffer JM are as follows:

[0297] Buffer J (20 mM tris, 0.2% iodoacetamide, 10 U / ml benzoxonase, 0.0025% leupetin);

[0298] Buffer K (20mM Tris-HCl pH 7.5, 150mM NaCl, 0.5% LMNG, 0.03% CHS, 0.2% sodium cholate, 0.0025mg / mL Leupeptin, 0.1% iodoacetamide);

[0299] Buffer L (20mM Tris-HCl pH 7.5, 150mM NaCl, 0.05% LMNG, 0.003% CHS, 0.02% sodium cholate, 0.0025mg / mL Leupeptin);

[0300] Buffer M (20mM Tris-HCl pH 7.5, 150mM NaCl, 0.05% LMNG, 0.003% CHS, 0.02% sodium cholate, 0.0025mg / mL Leupeptin, 0.3mg / mL Flag).

[0301] The results are as follows Figure 6 As shown, the modified GPR35 can be purified using dual tags with high purity.

[0302] 1.3.2 Molecular sieve purification of modified GPR35

[0303] The eluted liquid was added to a 50 kDa ultrafiltration tube (Millipore) and the target protein was concentrated and enriched. When the sample was concentrated to about 100 μL, it was injected into a Superdex 200 increase 5 / 150 (Cytiva) gel column pre-equilibrated with Buffer N. The target protein was collected according to the UV-280 absorption peak indication, and then the sample was analyzed by SDS-PAGE gel electrophoresis, concentrated, and frozen.

[0304] The specific components of the aforementioned buffer (Buffer N) are as follows:

[0305] Buffer N (20mM Tris-HCl pH 7.5, 150mM NaCl, 0.00075% LMNG, 0.0001% CHS, 0.0025% GDN).

[0306] The results are as follows Figure 7 As shown, the modified GPR35 exhibits a monopolymer state with high purity.

[0307] 1.4 ELISA detection of the affinity between modified GPR35 and anti-mBRIL nanobody

[0308] 1.4.1 Coating of anti-mBRIL nanobodies

[0309] The high-concentration mBRIL nanobody was diluted with CBS diluent (pH 9.6) to a concentration of 5 μg / mL. The antibody was coated overnight in 96-well plates at 4°C with a coating volume of 100 μL / well. The next day, the coating solution and antibody solution were discarded, and the plates were washed three times with 250 μL of PBST solution and patted dry. 200 μL / well of blocking buffer (2% (m / v) skim milk powder + PBS) was added, and the plates were gently mixed and blocked at room temperature for 2 hours. Subsequently, the blocking buffer was discarded, and the plates were washed three times with 250 μL of PBST and patted dry.

[0310] 1.4.2 Dilution of Modified GPR35 Antigen

[0311] The modified GPR35 antigen was diluted 2-fold starting from a high concentration (50 μg / mL), with eight dilutions in the following buffer: (2% (m / v) skim milk powder + PBS + 0.05% Tween 20 + 0.002% LMNG + 0.00012% CHS). Subsequently, 100 μL of the diluted antigen was transferred to a plate coated with mBRIL nanobodies and incubated gently at room temperature for 1 hour. The plate was then washed three times with (PBST + 0.002% LMNG + 0.00012% CHS) and patted dry.

[0312] 1.4.3 Secondary Antibody Treatment

[0313] Add 100 μL / well of diluted HRP-labeled secondary antibody (0.8 μg / mL) (PBST + 0.002% LMNG + 0.00012% CHS) to the sample plate. Also add anti-FLAG-tagged mouse monoclonal antibody (Anti-DYKDDDDK (Binds to...)) at a concentration of 100 μL / well. Incubate with Tagepitope (Antibody, Mouse Monoclonal) at room temperature with gentle shaking for 1 hour. Then wash four times with (PBST + 0.002% LMNG + 0.00012% CHS) and pat dry.

[0314] 1.4.4 Color development and data analysis.

[0315] Add 100 μL of TMB single-component chromogenic solution (Solepro) to each well, and terminate the reaction with 50 μL of stop solution (Solepro) per well. Read the values ​​at 450 nm using a microplate reader. Experimental data were fitted with a GraphPad Prism5 to obtain a curve, and the EC50 value was calculated.

[0316] The results are as follows Figure 8 As shown, the modified GPR35 specifically binds to the anti-BRIL nanobody with an affinity of approximately 0.47 μM.

[0317] 1.5 Preparation of a complex of modified GPR35 and anti-mBRIL nanobody

[0318] The purified modified GPR35 and anti-mBRIL nanobody were mixed at a molar ratio of 1:1.2 and pre-incubated on ice for 1 hour. After pretreatment, the mixture was injected into a Superdex 200 increase 5 / 150 (Cytiva) gel column pre-equilibrated with Buffer N. Target proteins were collected based on the UV-280 absorption peak indication, and the complex samples of modified GPR35 and anti-mBRIL nanobody were subsequently analyzed by SDS-PAGE gel electrophoresis.

[0319] The results are as follows Figure 9 As shown, the modified GPR35 can form a complex with the anti-BRIL nanobody on the molecular sieve.

[0320] Example 3: Activity verification of modified GPR35: Affinity experiment with antagonist molecule ML-145

[0321] 1. Fixation of GPR35 samples modified by surface plasmon resonance (SPR)

[0322] The CM5 chip (Cytiva) was installed according to the Biacore T200 (Cytiva) standard operating procedure, and pretreated with SPR buffer (PBS-P+ (pH 7.4), 0.00075% LMNG, 0.00025% GDN, 0.0001% CHS). After baseline stabilization, EDC and NHS (Cytiva) were injected through the injection port and allowed to interact with the chip for 7 min to complete chip surface activation. For channel 3, the modified GPR35 prepared in Example 2 was diluted to 50 μg / ml with sodium acetate at pH 5.0 and injected through the injection port for fixation; after the response reached 8855.5 RU, it was blocked with ethanolamine for 7 min. Channel 1, as the reference channel, was not fixed with ligands and was directly blocked with ethanolamine after activation.

[0323] 2. Concentration titration experiment of SPR-modified GPR35 and antagonist ML-145

[0324] Replace the running buffer with SPR buffer pretreatment containing 5% DMSO. First, dilute the 10mM ML-145 (MCE, HY-107536) stock solution to 500μM using 1.05×PBS-P+ buffer. Then, ML-145 is diluted 2-fold starting from 400nM, resulting in multiple concentrations. Repeat concentrations and a 0 concentration are set. The diluent is SPR buffer containing 5% DMSO. A multi-cycle program is set with a 3-1 flow path, a flow rate of 30μL / min, a binding time of 120s, a natural dissociation time of 120s, and solvent correction is applied.

[0325] The results are as follows Figure 10 As shown, the modified GPR35 specifically binds to ML-145. Data correction and single-binding-specific binding equation fitting were performed on the SPR data using Prism software (GraphPad). The results showed that the affinity of ML-145 for the modified GPR35 was approximately 24 nM. This is consistent with the reported affinity of approximately 20 nM between ML-145 and wild-type GPR35. 16 The similarity indicates that the modified GPR35 has antagonist binding activity.

[0326] Example 4: Negative staining evaluation and frozen data analysis of modified GPR35 and anti-mBRIL nanobody complex.

[0327] 1. Negative Staining Assessment

[0328] Dilute the sample to a suitable concentration; perform hydrophilic treatment on the ultrathin carbon membrane used for negative staining, followed by glow discharge; drop the diluted sample onto the grid and let it stand for 1 minute, then absorb the sample with filter paper, wash the sample three times with ultrapure water, and stain it three times with uranium acetate solution. After the last staining, let it stand for 45 seconds, absorb the excess stain with filter paper, and allow it to dry naturally to form a gradient; place the prepared grid into the negative staining sample storage box and mark its position; observe the prepared sample using a 120kV Talos L120C TEM scanning / transmission electron microscope (purchased from Thermo Fisher Scientific).

[0329] The results are as follows Figure 11 As shown, negative staining studies indicate that the modified GPR35-anti-mBRIL nanobody complex exhibits good overall homogeneity, with slight aggregation, which does not affect structural studies.

[0330] 2. Frozen sample preparation

[0331] Because the prepared modified GPR35-anti-mBRIL nanobody complex exhibited good homogeneity, it was frozen for sample preparation, and the modified protein was used for single-particle structure studies. The sample preparation process is as follows:

[0332] Turn on the Vitrobot cryo-electron microscope sample preparation system (purchased from Thermo Fisher Scientific), and set the temperature and humidity to 8°C and 100%, respectively; dilute the sample to the required concentration for freezing;

[0333] The foam cup was filled with liquid nitrogen, and the copper ring in the middle was filled with liquid ethane; gold carrier mesh (Au quantifoil R1.2 / 1.3, purchased from Quantifoil GmbH, Germany) and GraFuture were used. TM - The GO carrier mesh (http: / / shuimubio.uunn.cn / technology / 1) was subjected to hydrophilic treatment and then glow discharge; the Vitrobot cryo-electron microscopy sample preparation system (purchased from Thermo Fisher Scientific) was set up to prepare frozen samples with a loading volume of 4 μL; the prepared carrier mesh was observed and data was collected using a Falcon 4 camera on a 300 kV Krios G4 cryo-electron microscope (purchased from Thermo Fisher Scientific).

[0334] 3. Analysis of cryogenic data from the modified GPR35

[0335] The original TIFF format data was compressed, aligned, and dose-weighted using the drift correction software (MotionCor2), and finally the multiple frames were combined into a single image with a pixel size of [missing information]. Single-frame photos were obtained. The pre-processed photos were then imported into cryoSPARC for further data analysis. The cryoSPARC data analysis process is as follows:

[0336] The parameters of the contrast transfer function (CTF) were determined using the Patch CTF estimation (multi) program; the particle size of the protein particles was determined using the Manualpicker; particles were automatically picked up using the Use circular blob method using the Blob picker; particles were extracted from micrographs; and finally, suitable particles were selected through three rounds of 2D classification.

[0337] The results are as follows Figure 12 As shown, GPR35 modified by mBRIL fusion and combined with the anti-mBRIL nanobody scheme showed clear transmembrane secondary structure features in the 2D classification results. The sample was further studied at high resolution using single-particle cryo-electron microscopy.

[0338] References

[0339] 1.Hilger,D.,Masureel,M.&Kobilka,B.K.Structure and dynamics of GPCRsignaling complexes.Nature Structural&;Molecular Biology 25,4–12(2018).

[0340] 2.Hauser,A.S.,Attwood,M.M.,Rask-Andersen,M., H.B.&Gloriam,D.E.Trends in GPCR drug discovery:new agents,targets and indications.NatureReviews Drug Discovery 16,829–842(2017).

[0341] 3.Kumari,P.,Ghosh,E.&Shukla,A.K.Emerging Approaches to GPCR LigandScreening for Drug Discovery.Trends Mol Med 21,687–701(2015).

[0342] 4.Callaway,E.Revolutionary cryo-EM is taking over structuralbiology.Nature578,201–201(2020).

[0343] 5.Renaud,J.-P.et al.Cryo-EM in drug discovery:achievements,limitations andprospects.Nat Rev Drug Discov 17,471–492(2018).

[0344] 6.Latorraca,N.R.,Venkatakrishnan,A.J.&Dror,R.O.GPCR Dynamics:Structures in Motion.Chemical Reviews 117,139–155(2017).

[0345] 7.Zou,Y.,Weis,W.I.&Kobilka,B.K.N-Terminal T4 LysozymeFusionFacilitates Crystallization of a G Protein Coupled Receptor.Plos One 7,e46039(2012).

[0346] 8.Chun,E.et al.Fusion Partner Toolchest for the StabilizationandCrystallization of G Protein-Coupled Receptors.Structure 20,967–976(2012).9.Kooistra,A.J.et al.GPCRdb in 2021:integrating GPCR sequence,structureandfunction.Nucleic Acids Res 49,gkaa1080-(2020).

[0347] 10.Miyagi,H.et al.The discovery of a new antibody for BRIL-fusedGPCRstructure determination.Sci Rep-uk 10,11669(2020).

[0348] 11.O’Dowd,B.F.et al.Discovery of Three Novel G-Protein-CoupledReceptorGenes.Genomics 47,310–313(1998).

[0349] 12.Fredriksson,R., M.C.,Lundin,L.-G.& H.B.TheG-protein-coupled receptors in the human genome form five mainfamilies.Phylogenetic analysis,paralogon groups,and fingerprints.MolecularPharmacology 63,1256–1272(2003).

[0350] 13.Taniguchi,Y.,Tonai-Kachi,H.&Shinjo,K.Zaprinast,a wellt,a wellwelguanosine monophosphate-specific phosphodiesterase inhibitor,is anagonistfor GPR35.Febs Lett 580,5003–5008(2006).

[0351] 14.Wang,J.et al.Kynurenic Acid as a Ligand for Orphan G Protein-coupledReceptor GPR35*.J Biol Chem 281,22021–22028(2006).

[0352] 15.Southern,C.et al.Screeningβ-Arrestin Recruitment for theIdentification ofNatural Ligands for Orphan G-Protein–Coupled Receptors.JBiomol Screen 18,599–609(2012).

[0353] 16.MacKenzie,A.E.,Lappin,J.E.,Taylor,D.L.,Nicklin,S.A.&Milligan,G.GPR35 as a Novel Therapeutic Target.Front Endocrinol 2,68(2011).

Claims

1. A modified GPR35, wherein, The amino acid sequence of the modified GPR35 is shown in SEQ ID NO: 34 or SEQ ID NO:

42.

2. A polynucleotide encoding the modified GPR35 of claim 1.

3. An expression vector comprising the polynucleotide of claim 2.

4. A host cell comprising the expression vector of claim 3.

5. Use of the modified GPR35 of claim 1, the polynucleotide of claim 2, the expression vector of claim 3, or the host cell of claim 4 in the structural determination of GPR35.

Images