Modified G protein-coupled receptors and their use

Abstract

This application relates to the field of protein engineering, and more specifically to a modified G protein-coupled receptor, a complex comprising the modified G protein-coupled receptor, and the use thereof. This application provides a modified G protein-coupled receptor comprising, in order from the N-terminus to the C-terminus, the N-terminus, a first transmembrane domain, a first intracellular loop, a second transmembrane domain, a first extracellular loop, a third transmembrane domain, a second intracellular loop, a fourth transmembrane domain, a second extracellular loop, a fifth transmembrane domain, a third intracellular loop, a sixth transmembrane domain, a third extracellular loop, a seventh transmembrane domain, and a C-terminus, wherein at least a portion of the first intracellular loop, the second intracellular loop and / or the third intracellular loop is replaced with an optional first linker, a first portion of a fluorescent protein, and an optional second linker, wherein the first linker and the second linker independently comprise one or more amino acids.

Description

Cross-reference to Related Applications

[0001] This application cites Chinese Patent Application No. 2023107655060, titled "Methods for Modifying G Protein-Coupled Receptors and Their Use", filed on June 27, 2023, the entire content of which is incorporated herein by reference.

Technical Field

[0002] This application belongs to the technical field of protein engineering, and specifically relates to modified G protein-coupled receptors, complexes containing the modified G protein-coupled receptors, and their uses.

Background Art

[0003] Most drug molecules exert their effects in the body by binding to corresponding target proteins, thereby altering physiological processes in the human body and achieving the goal of treating diseases by activating or inhibiting the function of the target proteins. G protein-coupled receptors (GPCRs) are target proteins in the human body that have significant drug development value. Currently, about 40% of commercially available drugs target G protein-coupled receptors, and many more drugs targeting these proteins are under development. There are numerous GPCRs in the human body, with over 800 discovered. These GPCRs share several commonalities in their spatial structure. Specifically, each has seven transmembrane helices, with the loop structure between the N-terminus and three helices located extracellularly and the loop structure between the C-terminus and another three helices located intracellularly. These proteins reside on the cell membrane and function as receptors for signaling molecules, transmitting signals into the cell. Foreign signaling molecules bind to the extracellular portion of the GPCR, causing a conformational change in the receptor's transmembrane helices. As a result, the intracellular portion of the receptor binds to G proteins, triggering intracellular physiological effects. GPCRs are involved in various physiological processes and play a crucial role in maintaining normal life activities in the human body. Drug molecules can bind to GPCRs and regulate intracellular physiological processes by exerting agonist or inhibitory effects on them. Therefore, theoretically, GPCRs can be widely used in drug discovery and screening.

[0004] However, many GPCRs cannot exist stably without a ligand, limiting their application in drug discovery and screening. In recent years, the rapid development of DNA-coding compound library screening and mass spectrometry techniques has enabled the identification of compounds that bind to specific target proteins from a mixture of compounds. As a result, target proteins used for screening now have high demands, including excellent stability, binding properties similar to natural wild-type protein ligands, and especially the absence of ligand-binding sites. In the discovery of therapeutic antibody drugs, purified target proteins with the above characteristics offer a clear advantage over cells overexpressing the target protein, whether using hybridoma technology, single B-cell technology, or various display technologies. This significantly accelerates the research and development process and avoids the influence of other background proteins. On the other hand, using these stable, pure proteins in combination with biophysical techniques such as surface plasmon resonance (SPR) to accurately measure the affinity between target GPCRs and compounds or antibodies is highly advantageous for drug optimization and improvement.

[0005] On the other hand, analyzing the complex structures of drug molecules or potential drug molecules with their target GPCR proteins is of crucial importance for studying their mechanisms of action and for further drug development. Currently, cryo-electron microscopy is widely used for GPCR structural analysis, and numerous GPCR structures have been obtained. However, the majority of these structures are complex structures formed when a GPCR binds to an agonist and then to a G protein. Such complexes can meet the requirements of cryo-electron microscopy for the size of the target protein molecule. On the other hand, when a GPCR is in a non-agonist state, bound to an antagonist, reverse agonist, or negative regulator, it does not form a stable complex with a G protein. Since the molecular weight of individual GPCR proteins is mostly in the range of 30-60 kDa, it is difficult to analyze their structure using cryo-electron microscopy.

[0006] Therefore, in this field, there is an urgent need to improve the stability of G protein-coupled receptors in the ligand-free state and to analyze the structure of G protein-coupled receptors in the non-agonist state using cryo-electron microscopy. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] To solve the above problems, this application provides a modified G protein-coupled receptor, a complex containing the modified G protein-coupled receptor, and the use of the same. [Means for solving the problem]

[0008] In a first aspect, the present application provides a modified G protein-coupled receptor comprising, from the N-terminus to the C-terminus, the N-terminus, a first transmembrane domain, a first intracellular loop, a second transmembrane domain, a first extracellular loop, a third transmembrane domain, a second intracellular loop, a fourth transmembrane domain, a second extracellular loop, a fifth transmembrane domain, a third intracellular loop, a sixth transmembrane domain, a third extracellular loop, a seventh transmembrane domain, and a C-terminus, wherein at least a portion of the first intracellular loop, the second intracellular loop and / or the third intracellular loop is replaced with an optional first linker, a first portion of a fluorescent protein, and an optional second linker, the first linker and the second linker independently comprising one or more amino acids.

[0009] In a second aspect, the present application provides a complex comprising a modified G protein-coupled receptor as described in the first aspect of the present application and a second portion of a fluorescent protein, wherein the first portion of the fluorescent protein and the second portion of the fluorescent protein are bound to each other.

[0010] In a third aspect, the present application provides the use of a modified G protein-coupled receptor or a complex described in a second aspect of the present application in ligand affinity measurement, drug discovery, or screening.

[0011] In a fourth aspect, the present invention provides the use of the complex described in the second aspect of the present invention in three-dimensional structural analysis using cryo-electron microscopy. [Effects of the Invention]

[0012] The modified G protein-coupled receptor and the corresponding complex described herein exhibit relatively good stability in the ligand-free state, their ligand-binding sites are unoccupied, and they possess ligand-binding activity similar to that of the wild-type G protein-coupled receptor, thereby making them suitable for drug discovery and screening. Furthermore, the complex described herein can be used for three-dimensional structural analysis using cryo-electron microscopy, providing further guidance for drug development and optimization. [Brief explanation of the drawing]

[0013] [Figure 1] This is a schematic diagram of GPCR protein engineering modification.

[0014] [Figure 2] This figure shows the identification of the GFP-clamp protein after purification. Figure 2A shows the results of molecular sieve chromatography, and Figure 2B shows the identification results by SDS gel electrophoresis.

[0015] [Figure 3] The results of screening when at least a portion of the third intracellular loop of the A2A adenosine receptor is replaced with any first linker, the first portion of the fluorescent protein (the 10th-11th β-sheets of GFP, simply called GFP10-11), and any second linker, with the replaced positions indicated.

[0016] [Figure 4]Screening results when at least a part of the third intracellular loop of the A2A adenosine receptor is replaced with any first linker, GFP10-11, and any second linker, and the amino acid sequence information of any first linker and any second linker is shown.

[0017] [Figure 5] Map of the pFastBac4R insect cell expression vector modified based on the pFastBac Dual vector.

[0018] [Figure 6] Diagram showing the purification and identification of the A2A adenosine receptor modified protein complex and the structural analysis results of the complex with the small molecule inhibitor ZM241385. Here, Figure 6A shows the results of molecular sieve chromatography, Figure 6B shows the identification results by SDS gel electrophoresis, Figure 6C shows the two-dimensional particle classification results of cryo-electron microscopy data, Figure 6D shows the FSC curve reconstructed three-dimensionally from cryo-electron microscopy data, Figure 6E shows the overall density of the protein complex reconstructed three-dimensionally by cryo-electron microscopy, and Figure 6F shows the density of the ZM241385 molecule reconstructed three-dimensionally by cryo-electron microscopy.

[0019] [Figure 7] Map of the mammalian cell expression vector pBacMam4R.

[0020] [Figure 8] Diagram showing the purification and identification of the CNR1 cannabinoid receptor modified protein complex and the structural analysis results of the complex with the small molecule inhibitor taranabant. Here, Figure 8A shows the results of molecular sieve chromatography, and Figure 8B shows the identification results by SDS gel electrophoresis.

[0021] [Figure 9]Figure 9A shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of the clamp protein (GFP-clamp-Cys) with the 186-Cys mutation after purification. Figure 9B shows the results of molecular sieve chromatography and SDS gel electrophoresis.

[0022] [Figure 10] Figure 10 shows the purification and identification of a CNR1 cannabinoid receptor modified protein complex crosslinked via disulfide bonds, and the structural analysis results of the complex with the inhibitor taranabant. Here, Figure 10A shows the results of molecular sieve chromatography, Figure 10B shows the identification results by SDS gel electrophoresis, Figure 10C shows the FSC curve reconstructed in 3D from cryo-electron microscopy data, Figure 10D shows the overall density of the protein complex reconstructed in 3D by cryo-electron microscopy, and Figure 10E shows the density of taranabant molecules.

[0023] [Figure 11] Figure 11 shows the results of purification and identification of the NK1R neurokinin receptor modified protein complex crosslinked via disulfide bonds, and the structural analysis of the complex with the inhibitor aprepitant. Here, Figure 11A shows the results of molecular sieve chromatography, Figure 11B shows the identification results by SDS gel electrophoresis, Figure 11C shows the FSC curve reconstructed in 3D from cryo-electron microscopy data, Figure 11D shows the overall density of the protein complex reconstructed in 3D by cryo-electron microscopy, and Figure 11E shows the density of aprepitant molecules.

[0024] [Figure 12]Figure 12 shows the purification and identification of the A2A adenosine receptor modified protein complex crosslinked via disulfide bonds, and the structural analysis results of the complex with the adenosine molecule, which is the agonist. Here, Figure 12A shows the results of molecular sieve chromatography, Figure 12B shows the identification results by SDS gel electrophoresis, Figure 12C shows the FSC curve reconstructed in 3D from cryo-electron microscopy data, Figure 12D shows the overall density of the protein complex reconstructed in 3D by cryo-electron microscopy, and Figure 12E shows the density of the adenosine molecule.

[0025] [Figure 13] This figure shows the purification, identification, and structural analysis results of a CCR8 chemokine receptor modified protein complex crosslinked via disulfide bonds. Here, Figure 13A shows the results of molecular sieve chromatography, Figure 13B shows the identification results by SDS gel electrophoresis, Figure 13C shows the FSC curve reconstructed in three dimensions from cryo-electron microscopy data, and Figure 13D shows the overall density of the protein complex reconstructed in three dimensions by cryo-electron microscopy.

[0026] [Figure 14] This figure shows the purification, identification, and structural analysis results of the GPRC5D receptor modified protein complex crosslinked via disulfide bonds. Here, Figure 14A shows the results of molecular sieve chromatography, Figure 14B shows the identification results by SDS gel electrophoresis, Figure 14C shows the FSC curve reconstructed in 3D from cryo-electron microscopy data, and Figure 14D shows the overall density of the protein complex reconstructed in 3D by cryo-electron microscopy.

[0027] [Figure 15] This figure shows the identification results of the GPR52-Cys-GFP10-11 protein after purification by molecular sieve chromatography and SDS gel electrophoresis. Figure 15A shows the results of molecular sieve chromatography, and Figure 15B shows the identification results by SDS gel electrophoresis.

[0028] [Figure 16] This figure shows the identification results of the purified MC4R-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 16A shows the results of molecular sieve chromatography, and Figure 16B shows the identification results by SDS gel electrophoresis.

[0029] [Figure 17] Figure 17A shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of the purified GNRHR-Cys-GFP10-11 protein. Figure 17B shows the results of molecular sieve chromatography and SDS gel electrophoresis identification.

[0030] [Figure 18] This figure shows the identification results of the purified PTGDR2-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 18A shows the results of molecular sieve chromatography, and Figure 18B shows the identification results by SDS gel electrophoresis.

[0031] [Figure 19] Figure 19A shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of the purified HTR2C-Cys-GFP10-11 protein. Figure 19B shows the results of molecular sieve chromatography and SDS gel electrophoresis identification.

[0032] [Figure 20] Figure 20 shows the identification results of the ADRA2B-Cys-GFP10-11 protein after purification by molecular sieve chromatography and SDS gel electrophoresis. Figure 20A shows the results of molecular sieve chromatography, and Figure 20B shows the identification results by SDS gel electrophoresis.

[0033] [Figure 21] This figure shows the identification results of the ADRB1-Cys-GFP10-11 protein after purification by molecular sieve chromatography and SDS gel electrophoresis. Figure 21A shows the results of molecular sieve chromatography, and Figure 21B shows the identification results by SDS gel electrophoresis.

[0034] [Figure 22] Figure 22A shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of the purified ADRB2-Cys-GFP10-11 protein. Figure 22B shows the results of molecular sieve chromatography and SDS gel electrophoresis identification.

[0035] [Figure 23] This figure shows the identification results of the purified C5AR1-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 23A shows the results of molecular sieve chromatography, and Figure 23B shows the identification results by SDS gel electrophoresis.

[0036] [Figure 24] Figure 24 shows the identification results of the purified CCR2-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 24A shows the results of molecular sieve chromatography, and Figure 24B shows the identification results by SDS gel electrophoresis.

[0037] [Figure 25] This figure shows the identification results of the purified CCR5-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 25A shows the results of molecular sieve chromatography, and Figure 25B shows the identification results by SDS gel electrophoresis.

[0038] [Figure 26]This figure shows the identification results of the purified CCR6-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 26A shows the results of molecular sieve chromatography, and Figure 26B shows the identification results by SDS gel electrophoresis.

[0039] [Figure 27] Figure 27A shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of the purified CCR7-Cys-GFP10-11 protein. Figure 27B shows the results of molecular sieve chromatography and SDS gel electrophoresis identification.

[0040] [Figure 28] Figure 28A shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of the purified CHRM2-Cys-GFP10-11 protein. Figure 28B shows the results of molecular sieve chromatography and SDS gel electrophoresis identification.

[0041] [Figure 29] This figure shows the identification results of the purified CLTR2-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 29A shows the results of molecular sieve chromatography, and Figure 29B shows the identification results by SDS gel electrophoresis.

[0042] [Figure 30] This figure shows the identification results of the purified CXCR2-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 30A shows the results of molecular sieve chromatography, and Figure 30B shows the identification results by SDS gel electrophoresis.

[0043] [Figure 31]This figure shows the identification results of the purified CXCR4-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 31A shows the results of molecular sieve chromatography, and Figure 31B shows the identification results by SDS gel electrophoresis.

[0044] [Figure 32] Figure 32A shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of the purified DRD2-Cys-GFP10-11 protein. Figure 32B shows the results of molecular sieve chromatography and SDS gel electrophoresis identification.

[0045] [Figure 33] This figure shows the identification results of the DRD3-Cys-GFP10-11 protein after purification by molecular sieve chromatography and SDS gel electrophoresis. Figure 33A shows the results of molecular sieve chromatography, and Figure 33B shows the identification results by SDS gel electrophoresis.

[0046] [Figure 34] This figure shows the identification results of the GPBAR-Cys-GFP10-11 protein after purification by molecular sieve chromatography and SDS gel electrophoresis. Figure 34A shows the results of molecular sieve chromatography, and Figure 34B shows the identification results by SDS gel electrophoresis.

[0047] [Figure 35] This figure shows the identification results of the HRH1-Cys-GFP10-11 protein after purification by molecular sieve chromatography and SDS gel electrophoresis. Figure 35A shows the results of molecular sieve chromatography, and Figure 35B shows the identification results by SDS gel electrophoresis.

[0048] [Figure 36]Figure 36 shows the identification results of the purified HTR1A-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Here, Figure 36A shows the results of molecular sieve chromatography, and Figure 36B shows the identification results by SDS gel electrophoresis.

[0049] [Figure 37] This figure shows the identification results of the HTR1B-Cys-GFP10-11 protein after purification by molecular sieve chromatography and SDS gel electrophoresis. Figure 37A shows the results of molecular sieve chromatography, and Figure 37B shows the identification results by SDS gel electrophoresis.

[0050] [Figure 38] Figure 38 shows the identification results of the HTR2B-Cys-GFP10-11 protein after purification by molecular sieve chromatography and SDS gel electrophoresis. Here, Figure 38A shows the results of molecular sieve chromatography, and Figure 38B shows the identification results by SDS gel electrophoresis.

[0051] [Figure 39] Figure 39A shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of the purified LPAR1-Cys-GFP10-11 protein. Figure 39B shows the results of molecular sieve chromatography and SDS gel electrophoresis identification.

[0052] [Figure 40] Figure 40 shows the identification results of the purified MTNR1B-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 40A shows the results of molecular sieve chromatography, and Figure 40B shows the identification results by SDS gel electrophoresis.

[0053] [Figure 41]This figure shows the identification results of the purified NPY1R-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 41A shows the results of molecular sieve chromatography, and Figure 41B shows the identification results by SDS gel electrophoresis.

[0054] [Figure 42] Figure 42A shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of the purified OPRD-Cys-GFP10-11 protein. Figure 42B shows the results of molecular sieve chromatography and SDS gel electrophoresis identification.

[0055] [Figure 43] Figure 43A shows the results of molecular sieve chromatography and SDS gel electrophoresis for the purification of the OX2R-Cys-GFP10-11 protein, where Figure 43B shows the results of molecular sieve chromatography and SDS gel electrophoresis, respectively.

[0056] [Figure 44] Figure 44 shows the identification results of the purified PTGDR-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Here, Figure 44A shows the results of molecular sieve chromatography, and Figure 44B shows the identification results by SDS gel electrophoresis.

[0057] [Figure 45] Figure 45A shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of the purified GPR146-Cys-GFP10-11 protein. Figure 45B shows the results of molecular sieve chromatography and SDS gel electrophoresis identification.

[0058] [Figure 46]This figure shows the identification results of the purified MCHR1-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 46A shows the results of molecular sieve chromatography, and Figure 46B shows the identification results by SDS gel electrophoresis.

[0059] [Figure 47] This figure shows the identification results of the purified TAAR1-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 47A shows the results of molecular sieve chromatography, and Figure 47B shows the identification results by SDS gel electrophoresis.

[0060] [Figure 48] Figure 48 shows the identification results of the purified AGTR1-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 48A shows the results of molecular sieve chromatography, and Figure 48B shows the identification results by SDS gel electrophoresis.

[0061] [Figure 49] Figure 49A shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of the purified FPR1-Cys-GFP10-11 protein. Figure 49B shows the results of molecular sieve chromatography and SDS gel electrophoresis identification.

[0062] [Figure 50] Figure 50 shows the identification results of the purified GALR1-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 50A shows the results of molecular sieve chromatography, and Figure 50B shows the identification results by SDS gel electrophoresis.

[0063] [Figure 51]This figure shows the identification results of the GHSR-Cys-GFP10-11 protein after purification by molecular sieve chromatography and SDS gel electrophoresis. Figure 51A shows the results of molecular sieve chromatography, and Figure 51B shows the identification results by SDS gel electrophoresis.

[0064] [Figure 52] Figure 52A shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of the purified CCKAR-Cys-GFP10-11 protein. Figure 52B shows the results of molecular sieve chromatography and SDS gel electrophoresis identification.

[0065] [Figure 53] Figure 53A shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of the purified MTLR-Cys-GFP10-11 protein. Figure 53B shows the results of molecular sieve chromatography and SDS gel electrophoresis identification.

[0066] [Figure 54] Figure 54 shows the identification results of the purified EDNRA-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Here, Figure 54A shows the results of molecular sieve chromatography, and Figure 54B shows the identification results by SDS gel electrophoresis.

[0067] [Figure 55] Figure 55A shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of the purified PRLHR-Cys-GFP10-11 protein. Figure 55B shows the results of molecular sieve chromatography and SDS gel electrophoresis identification.

[0068] [Figure 56]This figure shows the identification results of the purified NPFF1-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 56A shows the results of molecular sieve chromatography, and Figure 56B shows the identification results by SDS gel electrophoresis.

[0069] [Figure 57] This figure shows the identification results of the purified CXCR1-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 57A shows the results of molecular sieve chromatography, and Figure 57B shows the identification results by SDS gel electrophoresis.

[0070] [Figure 58] Figure 58 shows the identification results of the TRFR-Cys-GFP10-11 protein after purification by molecular sieve chromatography and SDS gel electrophoresis. Figure 58A shows the results of molecular sieve chromatography, and Figure 58B shows the identification results by SDS gel electrophoresis.

[0071] [Figure 59] Figure 59 shows the identification results of the purified CML1-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Here, Figure 59A shows the results of molecular sieve chromatography, and Figure 59B shows the identification results by SDS gel electrophoresis.

[0072] [Figure 60] This figure shows the identification results of the QRFPR-Cys-GFP10-11 protein after purification by molecular sieve chromatography and SDS gel electrophoresis. Figure 60A shows the results of molecular sieve chromatography, and Figure 60B shows the identification results by SDS gel electrophoresis.

[0073] [Figure 61]This figure shows the identification results of the purified SSTR2-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 61A shows the results of molecular sieve chromatography, and Figure 61B shows the identification results by SDS gel electrophoresis.

[0074] [Figure 62] This figure shows the identification results of the purified FFAR1-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 62A shows the results of molecular sieve chromatography, and Figure 62B shows the identification results by SDS gel electrophoresis.

[0075] [Figure 63] This figure shows the identification results of the purified PTAFR-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 63A shows the results of molecular sieve chromatography, and Figure 63B shows the identification results by SDS gel electrophoresis.

[0076] [Figure 64] Figure 64 shows the identification results of the purified P2RY1-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Here, Figure 64A shows the results of molecular sieve chromatography, and Figure 64B shows the identification results by SDS gel electrophoresis.

[0077] [Figure 65] Figure 65A shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of the HCAR2-Cys-GFP10-11 protein after purification. Figure 65B shows the results of molecular sieve chromatography and SDS gel electrophoresis identification.

[0078] [Figure 66]This figure shows the identification results of the purified SUCR1-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 66A shows the results of molecular sieve chromatography, and Figure 66B shows the identification results by SDS gel electrophoresis.

[0079] [Figure 67] This figure shows the identification results of the APJ-Cys-GFP10-11 protein after purification by molecular sieve chromatography and SDS gel electrophoresis. Figure 67A shows the results of molecular sieve chromatography, and Figure 67B shows the identification results by SDS gel electrophoresis.

[0080] [Figure 68] This figure shows the identification results of the GPR39-Cys-GFP10-11 protein after purification by molecular sieve chromatography and SDS gel electrophoresis. Figure 68A shows the results of molecular sieve chromatography, and Figure 68B shows the identification results by SDS gel electrophoresis.

[0081] [Figure 69] Figure 69 shows the identification results of the GPR75-Cys-GFP10-11 protein after purification by molecular sieve chromatography and SDS gel electrophoresis. Here, Figure 69A shows the results of molecular sieve chromatography, and Figure 69B shows the identification results by SDS gel electrophoresis.

[0082] [Figure 70] Figure 70 shows the identification results of the purified CCR1-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 70A shows the results of molecular sieve chromatography, and Figure 70B shows the identification results by SDS gel electrophoresis.

[0083] [Figure 71]This figure shows the identification results of the purified CCR4-Cys-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 71A shows the results of molecular sieve chromatography, and Figure 71B shows the identification results by SDS gel electrophoresis.

[0084] [Figure 72] Figure 72A shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of the purified PTGER4-Cys-GFP10-11 protein. Figure 72B shows the results of molecular sieve chromatography and SDS gel electrophoresis identification.

[0085] [Figure 73] This figure shows the purification, identification, and structural analysis results of a glucagon receptor GCGR modified protein complex crosslinked via disulfide bonds. Here, Figure 73A shows the results of molecular sieve chromatography, Figure 73B shows the identification results by SDS gel electrophoresis, Figure 73C shows the FSC curve reconstructed in three dimensions from cryo-electron microscopy data, and Figure 73D shows the overall density of the protein complex reconstructed in three dimensions by cryo-electron microscopy.

[0086] [Figure 74] Figure 74 shows the purification and identification of wild-type CCR8 and five different CCR8 fusion proteins, as well as molecular sieve chromatography. Here, Figure 74A shows the identification results by SDS gel electrophoresis, and Figures 74B-G show the results by molecular sieve chromatography.

[0087] [Figure 75] This figure shows the identification results of the purified CRFR1-ICL1-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 75A shows the results of molecular sieve chromatography, and Figure 75B shows the identification results by SDS gel electrophoresis.

[0088] [Figure 76] This figure shows the identification results of the purified CRFR1-ICL2-GFP10-11 protein by molecular sieve chromatography and SDS gel electrophoresis. Figure 76A shows the results of molecular sieve chromatography, and Figure 76B shows the identification results by SDS gel electrophoresis.

[0089] [Figure 77] Figure 77A shows the identification results of GFP-clamp-avi-biotin protein and GFP-clamp-Cys-avi-biotin protein by SDS gel electrophoresis. Figure 77B shows the identification results of GFP-clamp-avi-biotin protein by SDS gel electrophoresis, and Figure 77B shows the identification results of GFP-clamp-Cys-avi-biotin protein by SDS gel electrophoresis.

[0090] [Figure 78] Figure 78 shows the results of purification, identification, and affinity measurement of the MC4R-GFP10-11 modified protein complex with its ligand. Figure 78A shows the results of molecular sieve chromatography, Figure 78B shows the identification results by SDS gel electrophoresis, and Figures 78C-F show the results of affinity measurement.

[0091] [Figure 79] This figure shows the results of purification, identification, and affinity measurement of the GPR75-GFP10-11 modified protein complex with its ligand. Figure 79A shows the results of molecular sieve chromatography, Figure 79B shows the identification results by SDS gel electrophoresis, and Figure 79C shows the results of affinity measurement.

[0092] [Figure 80]Figure 80 shows the results of immunodetection of the A2A adenosine receptor modified protein complex and the CNR1 cannabinoid receptor modified protein complex. Here, Figure 80A shows the results of detecting the titer of the A2A adenosine receptor modified protein complex in the serum of A2A-immunized mice, Figure 80B shows the results of detecting the titer of the CNR1 cannabinoid receptor modified protein complex in the serum of CNR1-immunized mice, Figure 80C shows the results of ELISA detection of the supernatant of the A2A adenosine receptor modified protein complex and anti-A2A hybridoma in a 96-well plate, and Figure 80D shows the results of ELISA detection of the CNR1 cannabinoid receptor modified protein complex and anti-CNR1 hybridoma in a 96-well plate. [Modes for carrying out the invention]

[0093] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the field relating to this application. For example, *Concise Dictionary of Biomedicine and Molecular Biology*, Juo, Pei-Show, 2nd edition, 2002, CRC Press; *The Dictionary of Cell and Molecular Biology*, 5th edition, 2013, Academic Press; and *Oxford Dictionary of Biochemistry and Molecular Biology*, 2nd edition, 2006, Oxford University Press provide general dictionaries for many of the terms used herein.

[0094] As used herein, the expression "A and / or B" or "A and / or B" includes three cases: (1) A, (2) B, and (3) A and B. The expression "A, B and / or C" or "A, B and / or C" includes seven cases: (1) A, (2) B, (3) C, (4) A and B, (5) A and C, (6) B and C, and (7) A, B and C. The meaning of similar expressions can be inferred by analogy.

[0095] In a first embodiment, the present application provides a modified G protein-coupled receptor comprising, from the N-terminus to the C-terminus, the N-terminus, a first transmembrane domain, a first intracellular loop, a second transmembrane domain, a first extracellular loop, a third transmembrane domain, a second intracellular loop, a fourth transmembrane domain, a second extracellular loop, a fifth transmembrane domain, a third intracellular loop, a sixth transmembrane domain, a third extracellular loop, a seventh transmembrane domain, and a C-terminus, wherein at least a portion of the first intracellular loop, the second intracellular loop and / or the third intracellular loop is replaced with an optional first linker, a first portion of a fluorescent protein, and an optional second linker, the first linker and the second linker independently comprising one or more amino acids.

[0096] The modified G protein-coupled receptor of this application exhibits relatively good stability in the ligand-free state, its ligand-binding site is not occupied, and it has ligand-binding activity similar to that of the wild-type G protein-coupled receptor; therefore, it can be used for drug discovery and screening.

[0097] In this specification, "G protein-coupled receptors" are understood in their broadest sense. This is a general term referring to a large class of membrane protein receptors. A common characteristic of these receptors is that they consist of a polypeptide chain containing seven transmembrane α-helices, and that a G protein (guanylate-binding protein) binding site is present at both the C-terminus of the polypeptide chain and at the intracellular loop (third intracellular loop) connecting the 5th and 6th transmembrane helices (counting from the N-terminus of the polypeptide chain).

[0098] In this specification, unless otherwise specified or the context clearly contradicts it, the expression “G protein-coupled receptor” includes both wild-type and modified G protein-coupled receptors.

[0099] Because G protein-coupled receptors have similar combinations of overall and secondary structures, the embodiments of this application select representative members from a large number of subfamilies (e.g., A2A, CNR1, NK1R, CCR8, GPRC5D, GCGR, MC4R, GPR75, GPR52, GNRHR, PTGDR2, HTR2C, ADRA2B, ADRB1, ADRB2, C5AR1, CCR2, CCR5, CCR6, CCR7, CHRM2, CLTR2, CXCR2, CXCR4, DRD2, DRD3, GPBAR, HRH1, HTR1A, HTR1B, HTR2B, LPAR1, MTNR1B, NPY1R, OP) Experiments were conducted using selected GPCRs (RD, OX2R, PTGDR, GPR146, MCHR1, TAAR1, AGTR1, FPR1, GALR1, GHSR, CCKAR, MTLR, EDNRA, PRLHR, NPFF1, CXCR1, TRFR, CML1, QRFPR, SSTR2, FFAR1, PTAFR, P2RY1, HCAR2, SUCR1, APJ, GPR39, GPR75, PTGER4, CCR1, CCR4) and all yielded stable proteins. Therefore, it can be reasonably inferred that the modified GPCRs of this application are applicable to a wide range of GPCRs and cover a broad spectrum of GPCRs. It includes 5-HT1A, 5-HT1B, 5-HT1D, -5-HT1E, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT4, 5HT5A, 5-HT6, 5-HT7, M1, M2, M3, M4, M5, A1, A2A, A2B, A3, ADA1A, ADA1B, ADA1D, ADA2A, ADA2B, ADA2C, ADRB1, ADRB2, ADRB3, C3a, C5a, C5L2, AT1, AT2, APJ, GPBA, BB1, BB2, BB3, B1, B2, CB1, CB2, CCR1, C CR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCK1, CCK2, D1, D2, D3, D4, D5, ETA, ETB, GPER, FPR1, FPR2 / ALX, FPR3, FFA1, FFA2, FFA3, GPR42, GAL1, GAL2, GAL3, GHSR, FSH, LH, TSH, GnRH, GnRH2, H1, H2, H3, H4,HCA1、HCA2、HCA3、KISSR、BLT1、BLT2、CysLT1、CysLT2、OXE、FPR2 / ALX、LPA1、LPA2、LPA3、LPA4、LPA5、S1P1、S1P2、S1P3、S1P4、S1P5、MCH1、MCH2、MC1、MC2、MC3、MC4、MC5、MT1、MT2、MTLR、NMU1、NMU2、NPFF1、NPFF2、NPS、NPBW1、NPBW2、Y1、Y2、Y4、Y5、NTS1、NTS2、delta、kappa、mu、NOP、OX1、OX2、P2Y1、P2Y2、P2Y4、P2Y6、P2Y11、P2Y12、P2Y13、P2Y14、QRFP、PAF、PKR1、PKR2、PRRP、DP1、DP2、EP1、EP2、EP3、EP4、FP、IP1、TP、PAR1、PAR2、PAR3、PAR4、RXFP1、RXFP2、RXFP3、RXFP4、SST1、SST2、SST3、SST4、SST5、NK1、NK2、NK3、TRH1、TA1、UT、V1A、V1B、V2、OT、CCRL2、CMKLR1、GPR1、GPR3、GPR4、GPR6、GPR12、GPR15、GPR17、GPR18、GPR19、GPR20、GPR21、GPR22、GPR25、GPR26、GPR27、GPR31、GPR32、GPR33、GPR34、GPR35、GPR37、GPR37L1、GPR39、GPR42、GPR45、GPR50、GPR52、GPR55、GPR61、GPR62、GPR63、GPR65、GPR68、GPR75、GPR78、GPR79、GPR82、GPR83、GPR84、GPR85、GPR87、GPR88、GPR101、GPR119、GPR120、GPR132、GPR135、GPR139、GPR141、GPR142、GPR146、GPR148、GPR149、GPR150、GPR151、GPR152、GPR153、GPR160、GPR161、GPR162、GPR171、GPR173、GPR174、GPR176、GPR182、GPR183、LGR4、LGR5、LGR6、LPAR6、MAS1、MAS1L、MRGPRD、MRGPRE、MRGPRF、MRGPRG、MRGPRX1、MRGPRX2、MRGPRX3、MRGPRX4、OPN3、OPN5、OXGR1、P2RY8、P2RY10、SUCNR1, TAAR2, TAAR3, TAAR4, TAAR5, TAAR6, TAAR8, TAAR9, CCPB2, CCRL1, FY, CT, CALRL, CRF1, CRF2, GHRH, GIP, GLP-1, GLP-2, GCGR, SCTR, PTH1, PTH2, PAC1, VPAC1, VPAC2, BAI1, BAI2, BAI3, CD97, CELSR1, CELSR2, CELSR3, ELTD1, EMR1, EMR2, EMR3, EMR4P, GPR56, GPR64, GPR97, GPR98, GPR110, GPR111, GPR112, GPR113, GPR114, GPR115, GPR116, This includes, but is not limited to, GPR123, GPR124, GPR125, GPR126, GPR128, GPR133, GPR143, GPR144, GPR157, LPHN1, LPHN2, LPHN3, CaS, GPRC6, GABAB1, GABAB2, mGlu1, mGlu2, mGlu3, mGlu4, mGlu5, mGlu6, mGlu7, mGlu8, GPR156, GPR158, GPR179, GPRC5A, GPRC5B, GPRC5C, GPRC5D, frizzled, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, and SMO.

[0100] In some embodiments, the first and second linkers can exist independently of each other. That is, there may be only the first linker, only the second linker, neither, or both the first and second linkers.

[0101] In some embodiments, the first and second linkers may be independently selected from peptide fragments containing 1 to 50 amino acids, 1 to 40 amino acids, 1 to 30 amino acids, 1 to 20 amino acids, 1 to 10 amino acids, 1 to 9 amino acids, 1 to 8 amino acids, 1 to 7 amino acids, 1 to 6 amino acids, or 1 to 5 amino acids.

[0102] In some embodiments, the first linker and the second linker may independently be peptide fragments consisting of one amino acid, two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, nine amino acids, ten amino acids, or more amino acids.

[0103] In some embodiments, the first linker and the second linker may be the same or different.

[0104] In this specification, "fluorescent protein" includes both wild-type fluorescent proteins and modified fluorescent proteins (obtained by cleavage, elongation, insertion, deletion, substitution, etc.).

[0105] In some embodiments, the fluorescent protein includes one or more of the following: green fluorescent protein (GFP), red fluorescent protein, yellow fluorescent protein, blue fluorescent protein, cyan fluorescent variant, and enhanced green fluorescent protein.

[0106] In some embodiments, the fluorescent protein includes an amino acid sequence represented by SEQ ID NO: 81, or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid sequence represented by SEQ ID NO: 81.

[0107] In some embodiments, the first portion of the fluorescent protein comprises the 10th to 11th β-sheets, the 1st to 2nd β-sheets, the 8th to 11th β-sheets, or the 1st to 4th β-sheets of the fluorescent protein.

[0108] In some embodiments, the first portion of the fluorescent protein includes the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0109] As used herein, the term “identity” refers to the degree of similarity between a pair of sequences (nucleotides or amino acids). Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the quotient by 100 to calculate a percentage. Gaps are not considered when assessing identity. Therefore, two copies of an entirely identical sequence have 100% identity, while sequences containing deletions, additions, or substitutions may have a lower degree of identity. Those skilled in the art are familiar with computer programs that can be used to determine sequence identity, such as those employing algorithms like BLAST. BLAST nucleotide searches are performed using the NBLAST program, and BLAST protein searches are performed using the BLASTP program, where the default parameters of each program are used.

[0110] In some embodiments, the amino acid at position 34.51 in the second intracellular loop, according to the Ballesteros-Weinstein numbering system, is cysteine.

[0111] In some embodiments, the amino acid at position 34.51 in the second intracellular loop, according to the Ballesteros-Weinstein numbering system, is naturally cysteine. In some embodiments, the amino acid at position 34.51 in the second intracellular loop, according to the Ballesteros-Weinstein numbering system, is mutated to cysteine ​​(e.g., denoted as GPCR-Cys-GFP10-11).

[0112] In some embodiments, the modified G protein-coupled receptor includes an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with any one of the amino acid sequences shown in SEQ ID NOs: 3 to 71.

[0113] In a second aspect, the present application provides a complex comprising the modified G protein-coupled receptor described in the first aspect of the present application and a second portion of a fluorescent protein, wherein the first portion of the fluorescent protein and the second portion of the fluorescent protein are bound to each other.

[0114] The complex of this invention exhibits relatively good stability in the ligand-free state, its ligand-binding site is unoccupied, and it has ligand-binding activity similar to that of a wild-type G protein-coupled receptor, thereby making it suitable for drug discovery and screening. Furthermore, the complex of this invention can be used for three-dimensional structural analysis using cryo-electron microscopy, providing further guidance for drug development and optimization.

[0115] In some embodiments, there is no linking group between the first and second portions of the fluorescent protein. In other words, the first and second portions of the fluorescent protein are linked by van der Waals forces.

[0116] In some embodiments, the first and second portions of the fluorescent protein combine to form a complete fluorescent protein.

[0117] In some embodiments, the first and second portions of the fluorescent protein are bound by co-expression of the modified G protein-coupled receptor and the second portion of the fluorescent protein.

[0118] In some embodiments, co-expression is performed in a eukaryotic cell expression system.

[0119] In some embodiments, the eukaryotic cell is an insect cell or a mammalian cell. In some embodiments, the insect cell is a fall armyworm (Spodoptera frugiperda) cell Sf-9. In some embodiments, the mammalian cell is a HEK293F cell.

[0120] In some embodiments, the modified G protein-coupled receptor and the second portion of the fluorescent protein (e.g., GFP1-9 proteins) are expressed by different promoters, such as a polyhedral gene promoter and a P10 promoter, respectively.

[0121] In some embodiments, the N-terminus of the vector used for co-expression includes a signal peptide sequence and / or a maltose-binding protein fusion tag. In some embodiments, the downstream of the vector used for co-expression includes an internal ribosome entry site.

[0122] In some embodiments, the gene sequence encoding the second portion of the fluorescent protein (e.g., GFP1-9 proteins) is cloned into the internal ribosome entry site of the expression vector.

[0123] In some embodiments, the complete fluorescent protein may be a wild-type fluorescent protein or a modified fluorescent protein.

[0124] In some embodiments, the second portion of the fluorescent protein includes the 1st to 9th β-sheets, the 3rd to 11th β-sheets, the 1st to 7th β-sheets, or the 5th to 11th β-sheets of the fluorescent protein.

[0125] In some embodiments, the first portion of the fluorescent protein comprises the 10th to 11th β-sheets of the fluorescent protein, and the second portion of the fluorescent protein comprises the 1st to 9th β-sheets of the fluorescent protein. In some embodiments, the first portion of the fluorescent protein comprises the 1st to 2nd β-sheets of the fluorescent protein, and the second portion of the fluorescent protein comprises the 3rd to 11th β-sheets of the fluorescent protein. In some embodiments, the first portion of the fluorescent protein comprises the 8th to 11th β-sheets of the fluorescent protein, and the second portion of the fluorescent protein comprises the 1st to 7th β-sheets of the fluorescent protein. In some embodiments, the first portion of the fluorescent protein comprises the 1st to 4th β-sheets of the fluorescent protein, and the second portion of the fluorescent protein comprises the 5th to 11th β-sheets of the fluorescent protein.

[0126] In some embodiments, the second portion of the fluorescent protein includes the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid sequence shown in SEQ ID NO: 2.

[0127] In some embodiments, the first portion of the fluorescent protein includes an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and the second portion of the fluorescent protein includes an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid sequence shown in SEQ ID NO: 2.

[0128] In some embodiments, the first portion of the fluorescent protein includes the amino acid sequence shown in SEQ ID NO: 1, and the second portion of the fluorescent protein includes the amino acid sequence shown in SEQ ID NO: 2.

[0129] In some embodiments, the complex further comprises clamp proteins bound to the first and second portions of the fluorescent protein.

[0130] In some embodiments, there are no linking groups between the clamp protein and the first portion of the fluorescent protein, and between the clamp protein and the second portion of the fluorescent protein. In other words, the clamp protein is bound to the first and second portions of the fluorescent protein by van der Waals forces.

[0131] In some embodiments, the clamp protein includes a biomarker. The biomarker may be any biomarker known to those skilled in the art, for example, any molecular pair capable of specific binding, which includes, but is not limited to, biotin / avidin (e.g., biotin / streptavidin, biotin / neutraavidin), antibody / antigen, antibody / hapten, DNA / complementary DNA or RNA, enzyme / substrate, enzyme / inhibitor, enzyme / cofactor, receptor / ligand (e.g., hormone / hormone receptor, folate / folate receptor), lectin / saccharides, Staphylococcus A protein / IgG, cation / anion, spy-tag / spy-catcher, strepto-tag (and its variants) / streptavidin (and its variants), and the like. In some embodiments, the clamp protein includes a biotin marker. In some embodiments, the clamp protein includes an avi tag. The presence of biotin tags on target GPCRs is extremely useful for screening compound drugs using purified GPCRs and for discovering therapeutic antibody drugs. This is because the target protein can be immobilized or modified through the very strong binding between biotin and streptavidin.

[0132] In some embodiments, the complex is a ternary complex formed by a modified G protein-coupled receptor, a second portion of a fluorescent protein, and a clamp protein.

[0133] In some embodiments, the molecular weight of the ternary complex is 85 kD or higher.

[0134] In some embodiments, the ternary complex can be purified by affinity chromatography and molecular sieve chromatography.

[0135] The clamp protein may be any protein that can bind to both the first and second portions of the fluorescent protein, thereby immobilizing or encapsulating the first and second portions of the fluorescent protein. For example, if the fluorescent protein is GFP, the first portion of the fluorescent protein may contain the amino acid sequence shown in SEQ ID NO: 1, the second portion of the fluorescent protein may contain the amino acid sequence shown in SEQ ID NO: 2, and the clamp protein may contain the amino acid sequence shown in SEQ ID NO: 72 (GFP-clamp).

[0136] In some embodiments, the clamp protein includes an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid sequence represented by any one of SEQ ID NOs. 72-74 and SEQ ID NO. 82.

[0137] In some embodiments, the 186th position of the clamp protein is cysteine, the position of which is shown in SEQ ID NO: 72.

[0138] In some embodiments, position 186 of the clamp protein is naturally cysteine, the position of which is referenced in SEQ ID NO: 72. In some embodiments, position 186 of the clamp protein is mutated to cysteine, the position of which is referenced in SEQ ID NO: 72, and the resulting clamp protein may contain the amino acid sequence shown in SEQ ID NO: 73 (GFP-clamp-Cys).

[0139] In some embodiments, a disulfide bond is formed between the amino acid at position 186 of the clamp protein and the amino acid at position 34.51 according to the Ballesteros-Weinstein numbering system in the second intracellular loop. In some embodiments, no linking groups other than disulfide bonds are present between the clamp protein and the first portion of the fluorescent protein, and between the clamp protein and the second portion of the fluorescent protein.

[0140] GPCRs share several commonalities in their spatial structure. Specifically, they all possess seven transmembrane helices; the loop structure between the N-terminus and three helices is located extracellularly, while the loop structure between the C-terminus and another three helices is located intracellularly. These proteins reside on the cell membrane and function as receptors for signaling molecules, transmitting signals into the cell. Foreign signaling molecules bind to the extracellular portion of the GPCR, causing a conformational change in the receptor's transmembrane helices. As a result, the intracellular portion of the receptor binds to G proteins, triggering intracellular physiological effects. GPCRs are involved in various physiological processes and play a crucial role in maintaining normal life activities in the human body. Drug molecules can bind to these receptor proteins and modulate intracellular physiological processes by exerting agonist or inhibitory effects. Analyzing the structure of complexes between these drugs or potential drugs and their target GPCR proteins provides important guidance for studying the mechanisms of action of these compounds and for further drug development.

[0141] In a third aspect, the present application provides the use of a modified G protein-coupled receptor or a complex described in a second aspect of the present application in ligand affinity measurement, drug discovery, or screening.

[0142] For use in ligand affinity measurement, drug discovery, or screening, the following conditions are not required: (1) the amino acid at position 34.51 of the second intracellular loop according to the Ballesteros-Weinstein numbering system is cysteine; (2) the amino acid at position 186 of the clamp protein is cysteine; (3) a disulfide bond is formed between position 186 of the clamp protein and the amino acid at position 34.51 of the second intracellular loop according to the Ballesteros-Weinstein numbering system. In other words, regardless of whether the above conditions are met, the modified G protein-coupled receptor described in the first aspect of this application, or the complex described in the second aspect of this application, can be used for ligand affinity measurement, drug discovery, or screening.

[0143] In a fourth aspect, the present invention provides the use of the complex described in the second aspect of the present invention in three-dimensional structural analysis using cryo-electron microscopy.

[0144] In some embodiments, the amino acid at position 34.51 according to the Ballesteros-Weinstein numbering system in the second intracellular loop of the modified G protein-coupled receptor is cysteine, the amino acid at position 186 of the clamp protein is cysteine, and a disulfide bond is formed between the amino acid at position 186 of the clamp protein and the amino acid at position 34.51 according to the Ballesteros-Weinstein numbering system in the second intracellular loop.

[0145] In some embodiments, the use of the complex described in the second aspect of this application in three-dimensional structural analysis of modified G protein-coupled receptors using cryo-electron microscopy can be provided.

[0146] In some embodiments, the use of the complex described in the second aspect of this application in three-dimensional structural analysis of the complex using cryo-electron microscopy can be provided.

[0147] In some embodiments, the use of the complex described in the second aspect of this application can be provided in the three-dimensional structural analysis of the complex formed by the compound molecule and the complex bond using cryo-electron microscopy.

[0148] In some embodiments, the compound molecule is an inhibitor or agonist of a G protein-coupled receptor.

[0149] The complex of this invention exhibits relatively good stability in the ligand-free state, its ligand-binding site is unoccupied, and it has ligand-binding activity similar to that of wild-type G protein-coupled receptors, thereby making it usable for drug discovery and screening. Furthermore, the complex of this invention can be used for three-dimensional structural analysis of modified G protein-coupled receptors, the complex itself, or complexes formed by the binding of compound molecules to the complex using cryo-electron microscopy, providing further guidance for drug development and optimization.

[0150] The modified G protein-coupled receptor provided in this application achieves a size of over 85 kD for the entire complex particle by inserting a short fusion fragment at a specific position on the GPCR and binding this fusion fragment to two other chaperone proteins, thereby bringing the chaperone proteins close enough to the GPCR to form a structure with a certain degree of rigidity. By mutating an adjacent amino acid to cysteine ​​in the GPCR and one of the chaperone proteins, a disulfide bond is formed between them, further reducing the flexibility of the entire complex. This makes it possible to analyze the three-dimensional structure of the complex formed by the GPCR and ligand compound using cryo-electron microscopy.

[0151] In one specific embodiment, the green fluorescent protein GFP is divided into two parts: GFP1-9, which includes the first to ninth sheets (i.e., β-sheets), and GFP10-11, which includes the tenth and eleventh sheets. Taking advantage of the property that these two parts can spontaneously bind to each other, the first intracellular loop between the first transmembrane helix (TM1) and the second transmembrane helix (TM2) of the GPCR, the second intracellular loop between the third transmembrane helix (TM3) and the fourth transmembrane helix (TM4), or the third intracellular loop between the fifth transmembrane helix (TM5) and the sixth transmembrane helix (TM6) is replaced with GFP10-11 by genetic engineering. In the case of the A2A adenosine receptor, GFP10-11 can be inserted between L208 (5.69a in the Ballesteros-Weinstein numbering system) and L225 (6.27a in the Ballesteros-Weinstein numbering system), and linked peptide fragments can be attached to both ends as linkers. In the case of other GPCRs, GFP10-11 can be inserted at the corresponding position of the A2A adenosine receptor (between 5.69a and 6.27 in the Ballesteros-Weinstein numbering system), or one, two, three, four, or five amino acids in the linker can be replaced with other amino acid sequences, such as the corresponding sequence of the A2A adenosine receptor. This GFP10-11 insertion fragment can be flexibly linked to the first and second helixes, the third and fourth helixes, or the fifth and sixth helixes of the GPCR, without affecting the folding of the GPCR into the correct structure, and without forming a contiguous helix with the GPCR sequence at the insertion site, thus reducing the constraints on the GPCR structure. The modified GPCR can also bind to ligand molecules. The fusion protein formed by this GPCR and GFP10-11 fragment (GPCR-GFP10-11, i.e., the modified GPCR of this application) is co-expressed with GFP1-9 fragments in cells. The GFP10-11 fragment in the GPCR-GFP10-11 fusion protein binds to GFP1-9 to form a complete fluorescent GFP molecule. In subsequent purification, a GFP-clamp protein with a specific sequence is added and bound to the GFP moiety.Modified GPCRs, such as the complex formed by the A2A adenosine receptor and the chaperone proteins GFP1-9 and GFP-clamp, exhibit strong overall rigidity and can be used for structural analysis using cryo-electron microscopy, yielding high-resolution structures of the complex formed with inhibitory compounds. Furthermore, to further increase the overall rigidity of the complex formed by the modified GPCR and the chaperone proteins GFP1-9 and GFP-clamp, a disulfide bond can be formed between the GPCR and GFP-clamp by mutating the amino acid at position 34.51 to cysteine ​​using the Ballesteros-Weinstein numbering system in the second intracellular loop of the GPCR, and by mutating the amino acid at position 186 of GFP-clamp to cysteine. This formation of a disulfide bond results in crosslinking between the GPCR and GFP-clamp, leading to the observation of a band with increased molecular weight in non-reducing SDS electrophoresis. In reduced SDS electrophoresis, the disulfide bond is reduced, and the individual bands of the two proteins remain visible, thus allowing for the identification of disulfide bond formation. The disulfide bonds designed in this way significantly reduce the overall flexibility of the complex, resulting in a highly rigid overall structure that is suitable for structural analysis by cryo-electron microscopy. A schematic diagram of the modified G protein-coupled receptor of the present invention is shown in Figure 1.

[0152] [Effects of the invention] The beneficial effects of this application are as follows:

[0153] (1) The modified G protein-coupled receptor obtained in this invention not only has a molecular weight of 85 kD or more, but the substitution fragment inserted into the GPCR (any first linker, the first portion of the fluorescent protein, and any second linker) binds simultaneously to two sites on the GPCR, and also to the two chaperone proteins of the second portion of the fluorescent protein and the clamp protein. As a result, the spatial structure rigidity of the entire complex is increased, which is advantageous for structural analysis by cryo-electron microscopy, and this beneficial effect is further enhanced by the formation of disulfide bonds.

[0154] (2) The first portion of the fluorescent protein (e.g., GFP10-11) is very short, and its insertion has little effect on GPCR expression or proper folding. Furthermore, because it does not form a continuous helix structure with the GPCR helix, it imposes fewer constraints on the GPCR's three-dimensional structure and is suitable for structural analysis of various types of GPCRs with different three-dimensional structures.

[0155] (3) The stable complex formed by the modified GPCR and the second portion of the fluorescent protein of this invention is appropriately expressed in both insect and mammalian cells.

[0156] (4) The formed complex does not easily dissociate during purification, thus eliminating the need to extensively investigate the conditions for complex formation and purification experimentally.

[0157] (5) The structure of the GPCR-agonist complex and the GPCR-agonist complex can also be analyzed.

[0158] (6) The formation of a complex with a chaperone protein (i.e., the second part of the fluorescent protein, the clamp protein) does not require the addition of a ligand. Expensive ligand compounds can be bound to the complex after sample purification or during sample preparation by freezing, significantly reducing costs.

[0159] (7) By purifying batches of protein complexes, it becomes possible to prepare various complexes with various small molecule compounds, thereby reducing time, workload, and cost.

[0160] (8) The fluorescence properties of fluorescent proteins are advantageous for screening the expression of small sample volumes using fluorescence detection size exclusion chromatography, saving costs.

[0161] (9) Time-consuming and uncertain antibody screening processes become unnecessary.

[0162] (10) It is highly versatile and can produce stable proteins for many types of GPCRs.

[0163] (11) It is possible to obtain GPCR proteins with their native three-dimensional structure without ligands, which have native ligand-binding activity similar to wild-type GPCRs and are suitable for screening compound drugs and discovering functional monoclonal antibodies.

[0164] [Examples]

[0165] Unless otherwise noted, the experimental methods used in the following examples are conventional methods. Unless otherwise noted, all reagents and drugs used in the following examples were purchased from a general biochemical reagent supplier.

[0166] Example 1: Cryo-electron microscopy analysis of the complex of A2A adenosine receptor and ZM241385

[0167] (1) GFP-clamp expression and purification

[0168] The DNA sequence encoding the GFP-clamp protein (SEQ ID NO: 72) was cloned into the E. coli expression plasmid pMALc2x, which had a maltose-binding protein fusion tag and a TEV protease cleavage site at the N-terminus and a histidine fusion tag at the C-terminus. This plasmid was used to transform Rosetta® 2(DE3) competent cells. Positive single clones were selected and cultured in small quantities in 20 mL of resistant LB medium until the cells became supersaturated. 20 mL of cells were transferred to 800 mL of ampicillin and chloramphenicol-resistant medium for amplification. When the absorbance of the bacterial suspension at 600 nm reached approximately 0.6, 0.5 mM isopropyl-β-D-thiogalactoside was added, and induction was performed overnight at 20°C. The bacterial suspension was collected in a large-capacity centrifuge flask and centrifuged at 4°C and 4,000 rpm for 30 minutes. The supernatant was discarded, and the precipitate was retained. The precipitate was resuspended in a buffer solution (500 mM NaCl, 5% glycerol, 20 mM imidazole, 50 mM Tris-HCl, pH 7.8), appropriate amounts of lysozyme, nuclease, and benzyl sulfonyl fluoride were added, and the bacteria were disrupted using an ultrasonic cell disruptor. The sonicated suspension was again centrifuged at high speed (4°C, 20000 g, 30 minutes), and the supernatant was retained. An appropriate volume of Ni-NTA resin was taken and bound to the supernatant, and the mixture was rotated at 4°C for 1.5 hours to ensure uniform mixing. After complete binding, the mixture was centrifuged at 4°C, 2,000 rpm for 2 minutes, the supernatant was discarded, and the protein-bound resin was retained. The Ni-NTA resin was transferred to a gravity column, and impurity proteins were washed with the above buffer solution for approximately 15-20 column volumes. The protein was eluted with elution buffer (500 mM NaCl, 5% glycerol, 500 mM imidazole, 50 mM Tris-HCl, pH 7.8), and the protein concentration was measured. TEV protease was added at a 1 / 10 mass ratio, and digestion was carried out overnight. The mixture was diluted with 500 mM NaCl, 5% glycerol, 50 mM Tris-HCl, pH 7.8 buffer until the imidazole concentration reached 20 mM, and then recombined with an appropriate amount of Ni-NTA resin.The resin was washed with 500 mM NaCl, 5% glycerol, 20 mM imidazole, 50 mM Tris-HCl, pH 7.8, and then the protein was eluted with 500 mM NaCl, 5% glycerol, 500 mM imidazole, 50 mM Tris-HCl, pH 7.8. The aggregation state and purity of the protein were analyzed by molecular sieve chromatography and SDS-PAGE gel (see Figure 2).

[0169] (2) Optimization of the fusion protein formed by the A2A adenosine receptor and GFP10-11

[0170] The amino acid sequence of the human A2A adenosine receptor was obtained from GenBank (accession number NM_000675.6). The first amino acid of the N-terminus and the sequence from position 318 onwards at the C-terminus were removed. The third intracellular loop was located between L208 and L225. The DNA sequence encoding the GFP10-11 fragment (SEQ ID NO: 1) was inserted between these two amino acids using overlap PCR, replacing the original third intracellular loop sequence. To optimize the linked peptide to maintain the stability of the fusion protein and reduce flexibility, a series of position and linker length combinations were created, and proteins with various combinations were identified and analyzed by fluorescence size exclusion chromatography (FSEC).

[0171] First, the linkage site between the A2A adenosine receptor and GFP10-11 was optimized. GFP10-11 is linked between leucine (L), the 208th amino acid in the TM5 transmembrane domain of the A2A adenosine receptor, and arginine (R), the 220th amino acid in the TM6 transmembrane domain of the A2A adenosine receptor. Each end is positioned with a five-amino acid linked peptide fragment (i.e., a linker). The left side is glycine-serine-glycine-glycine-glycine (GSGGG) (i.e., the first linker), and the right side is glycine-glycine-serine-glycine-glycine (GGSGG) (i.e., the second linker). This combination is denoted as L208_GSGGG_GFP10-11_GGSGG_R220(a). Similarly, variants at other linking positions are L208_GSGGG_GFP10-11_GGSGG_A221(b);L208_GSGGG_GFP10-11_GGSGG_R222(c);L208_GSGGG_GFP10-11_GGSGG_T224(d);and L208_GSGGG_GFP10-11_GGSGG_L225(e). Figure 3 shows the screening results when at least a portion of the third intracellular loop of the A2A adenosine receptor is replaced with any first linker, the first site of the fluorescent protein (the 10th to 11th β-sheets of GFP, denoted as GFP10-11 in units), and any second linker, with the substitution sites indicated. Based on the screening results, GFP10-11 was ligated between leucine L, the 208th amino acid in the TM5 transmembrane domain of the A2A adenosine receptor, and leucine L, the 225th amino acid in the TM6 transmembrane domain of the A2A adenosine receptor.

[0172] Next, the linker between the A2A adenosine receptor and GFP10-11 was optimized to have different lengths from both sides of GFP10-11: L208_GSGG_GFP10-11_GGSG_L225;L208_GGG_GFP10-11_GGG_L225;L208_GG_GFP10-11_GG_L225;L208_GSGGG_GFP10-11_GGSGG_L225;L208_G_GFP10- The sequence 11_G_L225;L208_GGG_GFP10-11_GG_L225;L208_GG_GFP10-11_GGG_L225;L208_GG_GFP10-11_G_L225;L208_G_GFP10-11_GG_L225;L208_G_GFP10-11_L225;L208_GFP10-11_G_L225;L208_GFP10-11_L225 was designed to be linked together.

[0173] The above-mentioned different A2A adenosine receptor modification sequences were cloned into the mammalian cell expression vector pBacMam4R, which contains a gnar peptide sequence, a maltose-binding protein fusion tag, and a TEV protease cleavage site at the pN terminus. The vector also includes an internal ribosome entry site. By cloning the gene sequence encoding the GFP1-9 fragments (SEQ ID NO: 2) after the internal ribosome entry site, the A2A-GFP10-11 fusion protein and the GFP1-9 fragments are simultaneously expressed in cells, spontaneously binding to form a complex. The constructed vector DNA was transiently transfected into 293T cells using Lipofectamine 2000 transfection reagent. After 40 hours, cells were collected, weighed, and sonicated. The cell membranes were lysed with 1% lauryl maltose neopentyl glycol (LMNG) and 0.1% cholesterol hemisuccinate (CHS), and 50 μg of GFP-clamp protein per 100 mg of cells was added. After incubation for 1.5 hours, the supernatant was rapidly centrifuged at 17000g, and the supernatant was analyzed by fluorescence detection size exclusion chromatography (SEC). Fluorescence from the chromatography column eluate was detected using 488 nm excitation light and 510 nm synchrotron radiation (see Figure 4). The position and shape of the fluorescence peaks were used to determine characteristics such as whether the protein was correctly folded and its polymerization state. Ultimately, the L208_G_GFP10-11_L225 sequence was selected for expression and structural analysis. The amino acid sequence of the modified A2A adenosine receptor is Sequence ID No. 3.

[0174] [Table 1]

[0175] (3) Cryo-electron microscopy analysis of the complex of the A2A adenosine receptor and ZM241385

[0176] In the modified pFastbacDual plasmid, a maltose-binding protein sequence with a hemagglutinin signal sequence at its N-terminus was added downstream of the polyhedrin gene promoter (see Figure 5). The nucleotide sequence encoding the A2A adenosine receptor fused with the GFP10-11 fragments determined in (2) (SEQ ID NO: 3) was cloned to the C-terminus of the maltose-binding protein, and the nucleotide sequence encoding the GFP1-9 fragments was cloned downstream of the P10 gene promoter. Recombinant baculoviruses were generated using the standard Bac-to-Bac baculovirus expression system procedure. Specifically, competent DH10Bac-competent cells were transformed with the above plasmids, and dishes containing LB agar medium with kanamycin, gentamicin, tetracycline, IPTG, and Bluo-gal were placed in a 37°C incubator and cultured for 48 hours. After blue-white screening, white positive single colonies were picked and cultured overnight in 5 mL of LB liquid medium. The following day, bacterial cells were collected by centrifugation, and recombinant baculovirus DNA was extracted. The purified recombinant baculovirus DNA was transfected into Spodoptera frugiperda (Sf-9) cells using Cellfectin transfection reagent. After 4-5 days, P0 generation recombinant baculovirus was obtained. High-titer baculovirus was obtained by infecting Sf-9 cells with the P0 generation virus and amplifying it.

[0177] High titer baculovirus in good growth condition with a cell density of 2 × 10 6The cells were infected with Sf-9 cells at a concentration of / mL and cultured with shaking at 125 rpm for 48 hours in a 27°C constant temperature incubator, and the cells were collected by centrifugation. The cells were resuspended in buffer solution (500 mM NaCl, 5% glycerol, 50 mM Tris-HCl pH 7.8) and lysed using an ultrasonic cell disruptor. 1% final concentration of lauryl maltose neopentyl glycol (LMNG) and 0.1% cholesterol hemisuccinate (CHS) were added, and the cells were centrifuged at 4°C for 2 hours to lyse the cell membranes. The cells were centrifuged at 20,000 g for 30 minutes at 4°C. The supernatant was mixed with an appropriate volume of amylose resin and centrifuged at 4°C for 1.5 hours to ensure homogeneity. After complete binding, the cells were centrifuged at 2,000 rpm for 2 minutes at 4°C, the supernatant was discarded, and the protein-bound resin was retained. The amylose resin was transferred to a gravity column, and impurity proteins were washed with a washing buffer (500mM NaCl, 5% glycerol, 50mM Tris-HCl pH 7.8, 0.01% LMNG, 0.001% CHS). The target protein was eluted with an elution buffer (500mM NaCl, 5% glycerol, 20mM maltose, 50mM Tris-HCl pH 7.8, 0.01% LMNG, 0.001% CHS), and the target protein was collected and its concentration measured. Subsequently, the protein was concentrated to 500 μL using a 10K centrifugal filter. The concentrated protein was separated using a Superdex 200 Increase 10 / 300 GL molecular sieve chromatography column with the buffer (500mM NaCl, 5% glycerol, 50mM Tris-HCl pH 7.8, 0.01% LMNG, 0.001% CHS) as the mobile phase. Proteins located in the peak tip region of the UV absorption peak corresponding to the target protein complex were collected.The collected proteins were combined with an appropriate excess of GFP-clamp protein to form a complex. This complex was then separated again using a Superdex 200 Increase 10 / 300 GL molecular sieve chromatography column (see Figure 6A) with buffer (150mM NaCl, 20mM HEPES pH 7.4, 0.005% LMNG, 0.0005% CHS) as the mobile phase. The proteins in the peak tip region of the UV absorption peak corresponding to the target protein complex were collected, and the proteins were concentrated to 10-15 mg / mL using a centrifugal filter. The components of the obtained samples were analyzed and identified by SDS-PAGE gel electrophoresis (see Figure 6B). The results of molecular sieve chromatography and SDS gel electrophoresis showed that the proteins were well-characterized, highly pure, and contained the expected components. The purified proteins formed a non-concentrated UV absorption peak in molecular sieve chromatography, indicating good stability.

[0178] ZM241385 was dissolved in DMSO at a concentration of 100 mM, diluted 100-fold with water to a concentration of 1 mM, and added to the purified and concentrated protein sample at a volume of 10%, resulting in a final concentration of 100 μM of ZM241385. The Quantifoil Cu 300 1.2 / 1.3 electron microscope screen was hydrophilized using a PELCO easiGlow glow discharge meter. Using a Vitrobot cryosampler, 3 μL of protein sample was added to the hydrophilized screen, and blotting was performed after 4 seconds under conditions of intensity 1-6 and time 3-5 seconds. Subsequently, the sample was rapidly immersed in liquid ethane pre-cooled with liquid nitrogen to freeze, and then transferred to liquid nitrogen for storage. The prepared frozen samples were loaded into a Titan Krios cryo-electron microscope equipped with a Gatan K2 direct electron detection camera, and image data of protein particles in the sample pores was collected at an accelerating voltage of 300 kV. Data collection was performed with a pixel size of 0.55 Å, 40 frames per image, and a total electron dose of 60 e. - / Å 2 The test was performed in super-resolution mode, which has a specific parameter of / s.

[0179] The collected data was corrected for electron beam displacement using MotionCor2. The contrast transfer function parameters of each image were estimated using CryoSPARC software. Next, particles were selected and local images of protein particles were extracted. After multiple iterations of 2D classification and removal of error particles (see Figure 6C), the initial 3D density model was subjected to Ab Initio reconstruction. Heterogeneous refinement was performed on these different reconstruction models to select the type with the correct shape and best quality. The above 2D classification and 3D heterogeneous refinement can be repeated multiple times to obtain the highest quality protein particle images and density model. Finally, high-quality 3D density was obtained through homogenous refinement and non-uniform refinement (see Figure 6DD) (see Figure 6E). Based on the three-dimensional density obtained from cryo-electron microscopy data, a structural model of the protein complex was constructed using Coot software, and its molecular structure model was constructed based on the density of small molecule compounds (see F in Figure 6). Subsequently, the structure was modified using Phenix software to obtain a high-quality structural model.

[0180] Example 2: Modification, expression, and purification of human cannabinoid type I receptor (CNR1) according to the method described in Example 1.

[0181] The sequence of human cannabinoid type I receptor (CNR1) was obtained from GenBank (accession number NM_001160226.3). The N-terminal and C-terminal regions were removed from this sequence, stabilizing mutations were introduced based on the existing structure, and the GFP10-11 fragment (underlined) was fused to the third intracellular loop. The linker is indicated by a wavy line. The amino acid sequence of the modified CNR1 receptor-GFP10-11 fusion protein is sequence number 4.

[0182] [Table 2]

[0183] The nucleotide sequence encoding the CNR1 receptor-GFP10-11 fusion protein was cloned into the mammalian cell expression vector pBacMam4R (see Figure 7), which had a signal peptide sequence and a maltose-binding protein fusion tag appended to its N-terminus. The internal ribosome entry site (IRES) downstream of this sequence can co-express GFP1-9 fragments. The cloned plasmid was amplified, and a large amount of plasmid DNA was extracted using an endotoxin-free plasmid bulk extraction kit. This DNA was transfected into HEK293F cells grown well in PEI-40K. 24 hours after transfection, 5 mM sodium butyrate was added. After another 24 hours, cell fluorescence was observed, and cells were collected by centrifugation at 2,000 rpm for 2 minutes. The cells were resuspended in buffer solution (500 mM NaCl, 5% glycerol, 50 mM Tris-HCl, pH 7.8) and lysed using an ultrasonic cell disruptor. The cell membrane was lysed by adding 1% lauryl maltose neopentyl glycol (LMNG) and 0.1% cholesterol hemisuccinate (CHS) and rotating the column at 4°C for 2 hours. The column was then centrifuged at 20,000 g for 30 minutes at 4°C. The supernatant was mixed with an appropriate volume of amylose resin and rotated at 4°C for 1.5 hours to ensure thorough mixing. After complete binding, the column was centrifuged at 2,000 rpm at 4°C for 2 minutes, the supernatant was discarded, and the resin containing the bound proteins was retained. The amylose resin was transferred to a gravity column and washed with a washing buffer (500 mM NaCl, 5% glycerol, 50 mM Tris-HCl pH 7.8, 0.01% LMNG, 0.001% CHS) to remove impurity proteins. The target protein was eluted with elution buffer (500 mM NaCl, 5% glycerol, 20 mM maltose, 50 mM Tris-HCl pH 7.8, 0.01% LMNG, 0.001% CHS), collected, and its concentration was measured. The protein was then concentrated to 500 μL using a 10K centrifugal filter.The concentrated proteins were separated using a Superdex 200 Increase 10 / 300 GL molecular sieve chromatography column with buffer (500 mM NaCl, 5% glycerol, 50 mM Tris-HCl pH 7.8, 0.01% LMNG, 0.001% CHS) as the mobile phase. Proteins in the peak tip region of the UV absorption peak corresponding to the target protein complex were collected. An appropriate excess of GFP-clamp protein was added to the collected proteins to form a complex, and the proteins were separated again using a Superdex 200 Increase 10 / 300 GL molecular sieve chromatography column with buffer (150 mM NaCl, 20 mM HEPES pH 7.4, 0.005% LMNG, 0.0005% CHS) as the mobile phase. Proteins in the peak tip region of the UV absorption peak corresponding to the target protein complex were collected and concentrated to 10-15 mg / mL using a centrifugal filter. The components of the obtained samples were analyzed and identified by SDS-PAGE gel electrophoresis.

[0184] Molecular sieve chromatography and SDS gel electrophoresis results showed that the protein was well-characterized (see Figures 8A and B), highly pure, and contained the expected components. The purified protein formed a non-concentrated UV absorption peak in molecular sieve chromatography, indicating good stability.

[0185] Example 3: Expression and purification of GFP-clamp cysteine ​​mutant and GFP-clamp-Cys mutant proteins

[0186] The glutamic acid at position 186 of GFP-clamp was mutated to cysteine ​​(outlined area), but the N-terminal maltose-binding protein and TEV protease cleavage site, as well as the C-terminal histidine tag and other parts, remained unchanged. The amino acid sequence of GFP-clamp-Cys is sequence number 73.

[0187] [Table 3]

[0188] A plasmid containing the nucleotide sequence encoding the above-mentioned GFP-clamp-Cys was used to transform Rosetta® 2(DE3) competent cells. Positive single clones were selected and cultured in small quantities in 20 mL of resistant LB medium until the cells became supersaturated. 20 mL of cells were transferred to 800 mL of ampicillin and chloramphenicol-resistant medium for amplification. When the absorbance of the bacterial suspension at 600 nm reached approximately 0.6, 0.5 mM isopropyl-β-D-thiogalactoside was added, and induction was carried out overnight at 20°C. The bacterial suspension was collected in a large centrifuge flask and centrifuged at 4°C and 4,000 rpm for 30 minutes. The supernatant was discarded, and the precipitate was retained. The precipitate was resuspended in a buffer containing 500 mM NaCl, 5% glycerol, 20 mM imidazole, 2 mM tris(2-carbonylethyl) phosphate (TCEP), 50 mM Tris-HCl, pH 7.8. Appropriate amounts of lysozyme, nuclease, and benzyl sulfonyl fluoride were added, and the bacteria were disrupted using an ultrasonic cell disruptor. The sonicated suspension was again centrifuged at high speed (4°C, 20000g, 30 minutes), and the supernatant was retained. An appropriate volume of Ni-NTA resin was taken and bound to the supernatant, and mixed uniformly by swishing at 4°C for 1.5 hours. After complete binding, the mixture was centrifuged at 4°C, 2000 rpm for 2 minutes, the supernatant was discarded, and the resin with bound proteins was retained. The Ni-NTA resin was transferred to a gravity column, and impurity proteins were washed with the above buffer for approximately 15-20 column volumes. The proteins were eluted with elution buffer (500mM NaCl, 5% glycerol, 500mM imidazole, 50mM Tris-HCl, 2mM TCEP, pH 7.8), and the protein concentration was measured. TEV protease was added at a 1 / 10 mass ratio, and digestion was carried out overnight. The imidazole solution was diluted with a buffer containing 500 mM NaCl, 5% glycerol, 50 mM Tris-HCl, 2 mM TCEP, and pH 7.8 until the imidazole concentration reached 20 mM, and then recombined with an appropriate amount of Ni-NTA resin.The resin was washed with a buffer containing 500 mM NaCl, 5% glycerol, 20 mM imidazole, 2 mM TCEP, 50 mM Tris-HCl, pH 7.8, and then the protein was eluted with 500 mM NaCl, 5% glycerol, 500 mM imidazole, 50 mM Tris-HCl, 2 mM TCEP, pH 7.8. The protein buffer was replaced with 500 mM NaCl, 5% glycerol, 50 mM Tris-HCl, pH 7.8 by desalting column chromatography. The identification results of the purified GFP-clamp-Cys protein by molecular sieve chromatography and SDS gel electrophoresis are shown in Figure 9.

[0189] Example 4: Structural analysis of the complex of a cannabinoid type I receptor (CNR1) cysteine ​​mutant and the inhibitor talanabant.

[0190] Based on the fusion protein sequence of CNR1 and GFP10-11 in Example 2, the leucine at position 222 of the second intracellular loop (34.51 in the Ballesteros-Weinstein numbering system) was mutated to cysteine ​​(outlined portion). The amino acid sequence of the modified CNR1-Cys_GFP10-11 is sequence number 5.

[0191] [Table 4]

[0192] The nucleotide sequence encoding Sequence ID No. 5 was cloned into the mammalian cell expression vector pBacMam4R, which had a signal peptide sequence and a maltose-binding protein fusion tag appended to its N-terminus. The internal ribosome entry site (IRES) downstream of this sequence can co-express GFP1-9 fragments. The cloned plasmid was amplified, and a large amount of plasmid DNA was extracted using an endotoxin-free plasmid bulk extraction kit. This DNA was transfected into HEK293F cells grown well using PEI-40K. 24 hours after transfection, 5 mM sodium butyrate was added. After another 24 hours, cell fluorescence was observed, and cells were collected by centrifugation at 2,000 rpm for 2 minutes. The cells were resuspended in buffer solution (500 mM NaCl, 5% glycerol, 50 mM Tris-HCl, pH 7.8), purified GFP-clamp-Cys protein was added, and the cells were disrupted using an ultrasonic cell disruptor. The cell membrane was lysed by adding 1% lauryl maltose neopentyl glycol (LMNG) and 0.1% cholesterol hemisuccinate (CHS) and rotating the column at 4°C for 2 hours. The column was centrifuged at 20,000 g for 30 minutes at 4°C. The supernatant was mixed with an appropriate volume of Ni-NTA resin and rotated at 4°C for 1.5 hours to ensure homogeneity. After complete binding, the column was centrifuged at 2000 rpm at 4°C for 2 minutes, the supernatant was discarded, and the resin with the bound proteins was retained. The resin was transferred to a gravity column and washed with a washing buffer (500 mM NaCl, 5% glycerol, 20 mM imidazole, 50 mM Tris-HCl, pH 7.8, 0.01% LMNG, 0.001% CHS) to remove impurity proteins. The target protein was eluted with elution buffer (500 mM NaCl, 5% glycerol, 500 mM imidazole, 50 mM Tris-HCl, pH 7.8, 0.01% LMNG, 0.001% CHS).The protein eluted from the Ni-NTA resin was bound to an appropriate volume of amylose resin and mixed uniformly by rotation at 4°C for 1.5 hours. Then, the mixture was centrifuged at 4°C and 2,000 rpm for 2 minutes, the supernatant was discarded, and the amylose resin was transferred to a gravity column. Impurity proteins were washed with washing buffer (500 mM NaCl, 5% glycerol, 50 mM Tris-HCl, pH 7.8, 0.01% LMNG, 0.001% CHS). Next, the target protein was eluted with elution buffer (500 mM NaCl, 5% glycerol, 20 mM maltose, 50 mM Tris-HCl, pH 7.8, 0.01% LMNG, 0.001% CHS). 1 mg of GFP-clamp-Cys was added, and the protein was concentrated to 500 μL using a 10K centrifugal filter. Further purification was performed using a Superdex 200 Increase 10 / 300 GL molecular sieve chromatography column (see Figure 10A) with buffer (150mM NaCl, 20mM HEPES pH 7.4, 0.005% LMNG, 0.0005% CHS) as the mobile phase. Proteins in the peak tip region of the UV absorption peak corresponding to the target protein complex were collected. The samples were mixed with reducing SDS loading buffer and non-reducing SDS loading buffer, respectively, and processed at 37°C for 30 minutes. The bands of the obtained sample components were analyzed by SDS gel electrophoresis (see Figure 10B; Sample 1 is the sample processed under reducing conditions, and Sample 2 is the sample processed under non-reducing conditions; in subsequent examples, even in SDS gel electrophoresis of different GPCR samples, Sample 1 is the sample processed under reducing conditions and Sample 2 is the sample processed under non-reducing conditions). Identification results by molecular sieve chromatography and SDS gel electrophoresis showed that the protein had good properties, high purity, and contained the expected components. The purified protein formed a non-concentrated UV absorption peak in molecular sieve chromatography, indicating good stability.

[0193] A 200 mM stock solution was prepared by dissolving the CNR1 receptor antagonist taranabant in DMSO. This stock solution was diluted 8-fold with DMSO and then 100-fold with water to obtain a 250 μM taranabant solution. This solution was added at 10% volume to the above-mentioned CNR1 protein sample purified by molecular sieve chromatography, and the protein was concentrated to 10-15 mg / mL using a 10K centrifugal filter. The Quantifoil Cu 300 1.2 / 1.3 electron microscope screen was hydrophilized using a PELCO easiGlow glow discharge meter. Using a Vitrobot cryosampler, 4 μL of the protein sample was added to the hydrophilized screen, and blotting was performed after 4 seconds under conditions of intensity 1-6 and time 3-5 seconds. Subsequently, the sample was rapidly immersed in liquid ethane pre-cooled with liquid nitrogen to freeze, and then transferred to liquid nitrogen for storage. The prepared frozen samples were loaded into a Titan Krios cryo-electron microscope equipped with a Gatan K2 direct electron detection camera, and image data of protein particles within the sample pores was collected at an acceleration voltage of 300 kV. Data acquisition was performed with a pixel size of 0.55 Å, 40 frames per image, and a total electron dose of 60 e. - / Å 2 The test was performed in / s super-resolution mode.

[0194] The collected data was corrected for electron beam displacement using MotionCor2. The contrast transfer function parameters of each image were estimated using CryoSPARC software. Next, particles were selected, and local images of protein particles were extracted. After multiple iterations of 2D classification and removal of error particles, the initial 3D density model was subjected to ab initio reconstruction. Heterogeneous refinement was performed on these different reconstruction models to select the type with the correct shape and best quality. The above 2D classification and 3D heterogeneous refinement can be repeated multiple times to obtain the highest quality protein particle images and density models. Finally, high-quality 3D density was obtained through homogenous refinement and non-uniform refinement (see Figure 10C) (see Figure 10D). Based on the 3D density obtained from the cryo-electron microscopy data, a structural model of the protein complex was constructed using Coot software, and its molecular structure model was constructed based on the density of small molecule compounds (see Figure 10E). Subsequently, we used Phenix software to modify the structure and obtain a high-quality structural model.

[0195] Example 5: Structural analysis of the complex of neurokinin receptor NK1R and inhibitor aprepitant

[0196] The amino acid sequence of NK1R was obtained from GenBank (accession number NM_001058.4). The C-terminal region was removed from this sequence, and stabilization mutations were performed based on the existing structure, and the GFP10-11 fragment (underlined) was fused to the third intracellular loop. The linker is indicated by a wavy line. In the second intracellular loop, leucine at position 138 (34.51 in the Ballesteros-Weinstein numbering system) was mutated to cysteine ​​(boxed). The amino acid sequence of the modified NK1R-Cys_GFP10-11 fusion protein is sequence number 6.

[0197] [Table 5]

[0198] The purification and identification methods for the modified NK1R-Cys_GFP10-11 fusion protein are shown in Example 4. The results of molecular sieve chromatography of the modified NK1R-Cys_GFP10-11 fusion protein are shown in Figure 11A, and the identification results by SDS gel electrophoresis are shown in Figure 11B. Molecular sieve chromatography and SDS gel electrophoresis showed that the protein was well-characterized, highly pure, and contained the expected components. The purified protein formed a non-concentrated UV absorption peak in molecular sieve chromatography, indicating good stability.

[0199] A stock solution of 187 mM was prepared by dissolving the NK1R receptor antagonist aprepitant in DMSO. This stock solution was diluted 16-fold with DMSO and then 100-fold with water to obtain a 116 μM taranabant solution. This was added to an NK1R protein sample purified by molecular sieve chromatography at a volume of 10%, and the protein was concentrated to 10-15 mg / ml using a centrifuge filter-10K. For specific methods of structural analysis, refer to Example 4. After homogeneity correction and heterogeneity correction (see Figure 11C), a high-quality 3D density was obtained (see Figure 11D). Based on the 3D density obtained from cryo-electron microscopy data, a structural model of the protein complex was constructed using Coot software, and its molecular structure model was further constructed based on the density of the aprepitant molecule (see Figure 11E). Subsequently, structural correction was performed using Phenix software to obtain a high-quality structural model.

[0200] Example 6: Structural analysis of the complex between the A2A adenosine receptor and the agonist adenosine.

[0201] In Example 1, the GFP10-11 fragment was fused with a cysteine ​​(outlined portion) at position 110 of the second intracellular loop of the A2A adenosine receptor sequence. The amino acid sequence of the modified A2A-Cys_GFP10-11 fusion protein is sequence number 7.

[0202] [Table 6]

[0203] For the purification and identification of the modified A2A-Cys_GFP10-11 fusion protein, refer to Example 4. The results of molecular sieve chromatography of the modified A2A-Cys_GFP10-11 fusion protein are shown in Figure 12A, and the identification results by SDS gel electrophoresis are shown in Figure 12B. Molecular sieve chromatography and SDS gel electrophoresis showed that the protein was well-characterized, highly pure, and contained the expected components. The purified protein formed a non-concentrated UV absorption peak in molecular sieve chromatography, indicating good stability.

[0204] The A2A adenosine receptor complex, purified by molecular sieve chromatography, was concentrated to 10-15 mg / ml using a centrifugal filter (molecular weight cutoff 10 kDa). Adenosine was dissolved in pure water to prepare a 10 mM stock solution. This was added to the concentrated protein solution by volume at 10%, resulting in a final concentration of 1 mM. For specific methods of structural analysis, refer to Example 4. After homogeneity correction and heterogeneity correction (see Figure 12C), high-quality three-dimensional density was obtained (see Figure 12D). Based on the three-dimensional density obtained from cryo-electron microscopy data, a structural model of the protein complex was constructed using Coot software, and its molecular structure model was constructed based on the density of adenosine molecules (see Figure 12E). Subsequently, structural correction was performed using Phenix software to obtain a high-quality structural model.

[0205] Example 7: Structural analysis of the chemokine receptor CCR8

[0206] The chemokine receptor CCR8 sequence is an amino acid sequence obtained from GenBank (accession number NM_005201.4). The first amino acid was removed from this sequence, and the GFP10-11 fragment (underlined) was fused to the third intracellular loop. The linker is indicated by a wavy line. V139 (34.51 in the Ballesteros-Weinstein numbering system) in the second intracellular loop was mutated to cysteine ​​(boxed). The amino acid sequence of the modified fusion protein is sequence number 8.

[0207] [Table 7]

[0208] For the purification and identification methods of the modified fusion protein, refer to Example 4. The results of molecular sieve chromatography of the modified fusion protein are shown in Figure 13A, and the identification results by SDS gel electrophoresis are shown in Figure 13B. Molecular sieve chromatography and SDS gel electrophoresis showed that the protein had good properties, high purity, and contained the expected components. The purified protein formed a non-concentrated UV absorption peak in molecular sieve chromatography, indicating good stability.

[0209] The CCR8 protein complex, purified by molecular sieve chromatography, was concentrated to 6 mg / ml using a centrifugal filter (molecular weight cutoff 10 kDa). For specific structural analysis methods, refer to Example 4. High-quality 3D density was obtained after homogeneity and heterogeneity correction (see Figure 13C) (see Figure 13D). Based on the 3D density obtained from cryo-electron microscopy data, a structural model of the protein complex was constructed using Coot software, and then structural correction was performed using Phenix software to obtain a high-quality structural model.

[0210] Therefore, the modified chemokine receptor CCR8 according to the present application has good stability without adding a ligand. To further demonstrate the superiority of the modified GPCR according to the present application, for comparison, an unmodified wild-type CCR8 protein and a modified CCR8 protein fused with several other fragments commonly used in the GPCR field in the third intracellular loop were constructed. The amino acid sequences of these proteins are as follows, where the fusion fragment sequences are underlined.

[0211] Wild-type CCR8 (CCR8wt, Sequence ID 75)

[0212]

Table 8

[0213] CCR8-T4L (T4 lysozyme, SEQ ID NO: 76)

[0214]

Table 9

[0215] CCR8-Rubredoxin (SEQ ID NO: 77)

[0216]

Table 10

[0217] CCR8-BRIL (Cyrochromium b562 RIL, SEQ ID NO: 78)

[0218]

Table 11

[0219] CCR8-Flavodoxin (SEQ ID NO: 79)

[0220]

Table 12

[0221] CCR8-PGS (Pyrococcus glycogen synthase, SEQ ID NO: 80)

[0222]

Table 13

[0223] These proteins have an MBP tag at the N-terminus and a His tag at the C-terminus. The expression and purification methods were the same as in Example 4, except that GFP-clamp was not added during purification. The results (see Figure 74) showed that the obtained proteins exhibited broad peaks on a molecular sieve chromatography column. This indicates that these native or modified CCR8 proteins cannot form a native three-dimensional structure, cannot aggregate, and cannot exist stably in the ligand-free state.

[0224] Example 8: Structural analysis of the (GPCR C family) orphan receptor GPRC5D

[0225] The orphan receptor GPRC5D sequence is the amino acid sequence obtained from GenBank (accession number NM_018654.2). The first amino acid was removed from this sequence, and the GFP10-11 fragment (underlined) was fused to the third intracellular loop. The linker is indicated by a wavy line. The 121st amino acid in the second intracellular loop is cysteine (boxed), and no mutation was observed. The amino acid sequence of the modified fusion protein is SEQ ID NO: 9.

[0226]

Table 14

[0227] For the purification and identification of the modified fusion protein, refer to Example 4. The results of molecular sieve chromatography of the modified fusion protein are shown in Figure 14A. The protein sample was taken from the second UV absorption peak indicated by the arrow. The identification results by SDS gel electrophoresis are shown in Figure 14B. Molecular sieve chromatography and SDS gel electrophoresis showed that the protein was well-characterized, high-purity, and contained the expected components. The purified protein formed a non-concentrated UV absorption peak in molecular sieve chromatography, indicating good stability.

[0228] The CCR8 protein complex, purified by molecular sieve chromatography, was concentrated to 6 mg / ml using a centrifugal filter (molecular weight cutoff 10 kDa). For specific structural analysis methods, refer to Example 4. High-quality 3D density was obtained after homogeneity and heterogeneity correction (see Figure 14C) (see Figure 14D). Based on the 3D density obtained from cryo-electron microscopy data, a structural model of the protein complex was constructed using Coot software, and then structural correction was performed using Phenix software to obtain a high-quality structural model.

[0229] Example 9: Structural analysis of the glucagon receptor GCGR (GPCR B1 family)

[0230] The glucagon receptor GCGR sequence is an amino acid sequence obtained from GenBank (accession number NM_000160.5). The N-terminal signal peptide sequence was removed from this sequence, and the GFP10-11 fragment (underlined) was fused to the third intracellular loop. The linker is indicated by a wavy line. T234 (34.51 in the Ballesteros-Weinstein numbering system) in the second intracellular loop was mutated to cysteine ​​(boxed). The amino acid sequence of the modified fusion protein is sequence number 10.

[0231] [Table 15]

[0232] For the method for purifying and identifying the modified fusion protein, refer to Example 4. The results of molecular sieve chromatography of the modified fusion protein are shown in A of FIG. 73, and the identification results by SDS gel electrophoresis are shown in B of FIG. 73. From molecular sieve chromatography and SDS gel electrophoresis, it was shown that the protein had good characteristics, high purity, and contained the expected components. The purified protein formed a non-concentrated UV absorption peak in molecular sieve chromatography, indicating good stability.

[0233] The GCGR protein complex purified by molecular sieve chromatography was concentrated to 8 mg / ml using a centrifugal filter (molecular weight cut-off 10 kDa). For the specific method of structural analysis, refer to Example 4. After homogeneous correction and inhomogeneous correction (refer to C of FIG. 73), high-quality three-dimensional density was obtained (refer to D of FIG. 73). Based on the three-dimensional density obtained from cryo-electron microscopy data, a structural model of the protein complex was constructed using Coot software, and then structural refinement was performed using Phenix software to obtain a high-quality structural model.

[0234] Example 10: Other forms of the modified GPCR of the present application

[0235] The CRFR receptor sequence is the amino acid sequence obtained from GenBank (accession number NP_001138618). The N-terminal signal peptide sequence was removed from the sequence, and the GFP10-11 fragment (underlined) was fused to the first intracellular loop. The amino acid sequence of the modified fusion protein is SEQ ID NO: 11.

[0236]

Table 16

[0237] For the purification and identification of the modified fusion protein, refer to Example 4. The results of molecular sieve chromatography of the modified fusion protein are shown in Figure 75A, and the identification results by SDS gel electrophoresis are shown in Figure 75B. Molecular sieve chromatography and SDS gel electrophoresis showed that the protein had good properties, high purity, and contained the expected components. The purified protein formed a non-concentrated UV absorption peak in molecular sieve chromatography, indicating good stability.

[0238] The N-terminal signal peptide sequence of the CRFR receptor was removed, and the GFP10-11 fragment (underlined) was fused to the second intracellular loop. The amino acid sequence of the modified fusion protein is sequence number 12.

[0239] [Table 17]

[0240] For the purification and identification of the modified fusion protein, refer to Example 4. The results of molecular sieve chromatography of the modified fusion protein are shown in Figure 76A, and the identification results by SDS gel electrophoresis are shown in Figure 76B. Molecular sieve chromatography and SDS gel electrophoresis showed that the protein had good properties, high purity, and contained the expected components. The purified protein formed a non-concentrated UV absorption peak in molecular sieve chromatography, indicating good stability.

[0241] The results showed that GFP10-11 fragment fusion in the first or second intracellular loop of a GPCR allows for the formation of stable complexes with GFP1-9 and GFP-clamp proteins.

[0242] Example 11: Preparation of biotinylated GFP-clamp

[0243] By utilizing the property of a modified GPCR-GFP10-11 fusion protein to form a stable complex with GFP1-9 and GFP-clamp, we attached an Avi tag to GFP-clamp and co-expressed it with the biotin ligase BirA, thereby achieving biotinylation of GFP-clamp-avi in ​​cells. When GPCR-GFP10-11 was purified using the purified GFP-clamp-avi-biotin, the biotin tag was attached to the resulting complex. This complex can be used for screening compound drugs and antibody drugs, or for affinity measurements using biophysical methods.

[0244] The amino acid sequence of GFP-clamp-avi is as follows (SEQ ID NO: 74), with an Avi tag (wavy line) at its C-terminus.

[0245] [Table 18]

[0246] GFP-clamp-avi and biotin ligase BirA were co-expressed in Rosetta™ 2(DE3) competent cells and purified in the same manner as in Example 1(1) to obtain biotinylated GFP-clamp-avi-biotin. The high degree of biotinylation of the protein was confirmed by binding experiments with streptavidin magnetic beads, and almost all of the protein bound to the streptavidin magnetic beads. The SDS gel electrophoresis results of the GFP-clamp-avi-biotin protein are shown in Figure 77A. SDS gel electrophoresis showed that the protein had good properties, high purity, and contained the expected components. The purified protein formed a non-concentrated UV absorption peak in molecular sieve chromatography, indicating good stability.

[0247] The glutamic acid at position 186 of GFP-clamp-Avi has been mutated to cysteine ​​(outlined area). The N-terminal maltose-binding protein and TEV protease cleavage site, as well as the C-terminal histidine tag and other parts, remain unchanged. The amino acid sequence of GFP-clamp-Cys-Avi is sequence number 82.

[0248] [Table 19]

[0249] GFP-clamp-Cys-avi and biotin ligase BirA were co-expressed in Rosetta™ 2(DE3) competent cells and purified in the same manner as in Example 3 to obtain biotinylated GFP-clamp-avi-biotin. The high degree of biotinylation of the protein was confirmed by binding experiments with streptavidin magnetic beads, and almost all of the protein bound to the streptavidin magnetic beads. The SDS gel electrophoresis results of the GFP-clamp-Cys-avi-biotin protein are shown in Figure 77B. The SDS gel electrophoresis results showed that the protein had good properties, high purity, and contained the expected components. The purified protein showed a non-concentrated UV absorption peak in molecular sieve chromatography, indicating good stability.

[0250] Example 12: Binding characteristics of the modified MC4R according to the present invention and its ligand.

[0251] The GFP10-11 fragment (underlined) was fused to the third intracellular loop of melanocortin receptor 4 (MC4R). The linker is indicated by a wavy line. The modified sequence is sequence number 13.

[0252] [Table 20]

[0253] The purification and identification methods for the modified MC4R fusion protein are described in Example 4, except that GFP-clamp-avi-biotin was added instead of GFP-clamp during purification. The results of molecular sieve chromatography of the modified fusion protein are shown in Figure 78A, and the identification results by SDS gel electrophoresis are shown in Figure 78B. Molecular sieve chromatography and SDS gel electrophoresis showed that the protein had good characteristics, high purity, and contained the expected components. The purified protein formed a non-concentrated UV absorption peak in molecular sieve chromatography, indicating good stability.

[0254] Using purified MC4R, affinity for four known ligands was measured by surface plasmon resonance (SPR). The instrument used was a Biacore 8K with an SPR sensor chip SAD200M. The protein fixation buffer and running buffer were both 20 mM HEPES, 150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM EDTA, 0.005% LMNG, 0.0005% CHS, pH 7.4. The affinity measurement results are shown in Figure 78 (C-F).

[0255] The results indicate that the properties of the modified MC4R protein according to this application are stable, and that the affinity measurements with several known compounds are close to those reported. This suggests that the ligand-binding site of the modified GPCR has a structure consistent with that of the native protein and can be used for screening compound libraries.

[0256] Example 13: Binding characteristics of the modified GPR75 and its ligand according to the present application

[0257] The GFP10-11 fragment (underlined) was fused to the third intracellular loop of the obesity-related receptor GPR75 (1-395). The modified sequence is sequence number 14.

[0258] [Table 21]

[0259] The purification and identification methods for the modified GPR75 fusion protein are described in Example 4, except that GFP-clamp-avi-biotin was added instead of GFP-clamp during purification. The results of molecular sieve chromatography of the modified fusion protein are shown in Figure 79A, and the identification results by SDS gel electrophoresis are shown in Figure 79B. Molecular sieve chromatography and SDS gel electrophoresis showed that the protein had good characteristics, high purity, and contained the expected components. The purified protein formed a non-concentrated UV absorption peak in molecular sieve chromatography, indicating good stability.

[0260] The affinity of purified GPR75 to the CCL5 ligand was measured by surface plasmon resonance (SPR). The instrument used was a Biacore 8K with a Series S Sensor SA Chip: SA-2429. The protein fixation buffer and running buffer consisted of 20 mM HEPES, 150 mM NaCl, 0.005% LMNG, 0.0005% CHS, and pH 7.4. The affinity measurement results are shown in Figure 79C.

[0261] The results indicate that the properties of the modified GPR75 protein according to this application are stable, and that the affinity measurement results with CCL5 are close to those reported. This demonstrates that the ligand-binding site of the modified GPCR has a structure consistent with that of the native protein and can be used for screening compound libraries.

[0262] Example 14: Use of the modified A2A adenosine receptor and cannabinoid type I receptor (CNR1) according to the present application in antibody discovery.

[0263] The modified sequences of the A2A receptor and the CNR1 receptor are the same as those in Examples 1 and 2, respectively. These two modified receptors were co-expressed with GFP1-9, GFP-clamp-avi-biotin was added, and the mixtures were purified according to the same method as in Example 1 to obtain biotin-tagged complexes.

[0264] ELISA detection of antibody titers in serum from BALB / c mice immunized with virus-like particles (VLPs) of A2A and CNR1 receptors: Streptavidin (0.1 M carbonate buffer, pH 9.6, 2.5 μg / well) was added to 96-well microplates, coated, and incubated overnight at 4°C. Next, 350 μL of 40 mg / ml bovine serum albumin (BSA) was added to each well for blocking. The wells were washed once with PBSL solution (10 mM PBS, pH 7.4, 0.01% LMNG, 0.001% CHS). 1 μg of purified biotin-containing GPCR protein complex was added to each well and incubated at room temperature for 1 hour. The wells were washed once with PBSL solution. Each well was blocked with PBSL containing 40 mg / ml bovine serum albumin (BSA). Mouse serum was diluted in PBS at different dilutions, added to each well, incubated at room temperature for 1 hour, and washed four times with PBSL solution. Horseradish peroxidase-conjugated goat anti-mouse antibody was added, incubated at room temperature for 1 hour, and washed four times with PBSL solution. 100 μL of TMB substrate was added to each well and incubated in the dark at room temperature for 10-20 minutes to allow color development. After the color development had stabilized, 50 μL of stop solution was added to each well, and the absorbance at 450 nm wavelength was read for each well using a microplate reader. The detection results for serum titers of A2A and CNR1 immunized mice are shown in Figures 80A and 80B, respectively.

[0265] BALB / c mice immunized with virus-like particles (VLPs) of target proteins A2A and CNR1 were euthanized, immersed in 75% ethanol solution for 5 minutes, removed, their abdomens wiped, their limbs fixed, the abdominal skin, muscles, and peritoneum incised, and the spleen removed and temporarily stored in PBS containing bivalent antibodies. The mouse spleens were washed with serum-free RPMI-1640 medium and placed on a 70 μm cell sieve. They were ground using a grinding rod, with RPMI-1640 medium continuously added during grinding. The spleen cells that passed through the sieve were collected in a centrifuge tube, centrifuged at 300 g for 10 minutes, the supernatant was discarded, and the cells were resuspended in RPMI-1640 medium and washed twice. The cells were counted. Simultaneously, well-developed mouse myeloma SP2 / 0 cells were resuspended and collected from the culture dish, centrifuged at 300g for 5 minutes, and then resuspended in serum-free RPMI-1640 medium for counting.

[0266] Resuspended 1-2 × 10 8One mouse splenocyte was mixed with SP2 / 0 cells in a 10:1 ratio, centrifuged at 300g for 10 minutes, and the supernatant was removed. The centrifuge tube was placed in a 37°C water bath. Using a pipette, 1 ml of 50% PEG1450 solution preheated to 37°C was drawn in, and the pipette tip was placed in the cell precipitate. The PEG solution was slowly added while gently stirring to ensure that the cell-PEG solution was completely mixed. The addition of the PEG solution was completed within 1 minute. Gentle stirring was continued for another minute. Then, 1 ml of serum-free RPMI-1640 medium was drawn in and slowly added to the cell-PEG mixture in the same manner, while gently stirring. While stirring, 10 ml of serum-free RPMI-1640 medium was slowly added. The mixture was centrifuged at 300g for 10 minutes, and the cell precipitate was collected. The cells were resuspended in 1 ml of RPMI-1640 medium containing 10% FBS, and the supernatant was removed. The cells were resuspended in 120 ml of HAT semi-solid selective medium (RPMI-1640 medium containing 10% FBS, 1x HAT selective reagent, 1x hybridoma feeder additive factor, 1x penicillin / streptomycin, 1.68% methylcellulose), mixed uniformly, and then dispensed into eight 100 mm cell culture dishes. They were cultured statically at 37°C in a 5% CO2 incubator. After 10-14 days, the hybridoma cells that had successfully fused grew into visible cell aggregates.

[0267] 200 μl of RPMI-1640 medium (containing 10% FBS, 0.5-fold diluted hybridoma feeder additive, and 1-fold diluted penicillin / streptomycin) was added to each well of a 96-well cell culture plate. Single cell aggregates were sequentially transferred from the semi-solid medium to each well of the 96-well plate and grown until they covered most of the bottom of the well. During growth, hybridoma cells secreted antibodies into the culture supernatant.

[0268] Following the same steps as the serum titer detection described above, ELISA detection was performed to detect target-specific antibodies in each well.

[0269] Streptavidin (0.1 M carbonate buffer, pH 9.6, 2.5 μg / well) was added to a 96-well ELISA plate, coated, and coated overnight at 4°C. 350 μL of 40 mg / ml bovine serum albumin BSA was added to each well and blocked. The wells were washed once with PBSL solution (10 mM PBS, pH 7.4, 0.01% LMNG, 0.001% CHS). 1 μg of purified biotin-containing GPCR protein complex was added to each well and incubated at room temperature for 1 hour. The wells were washed once with PBSL solution. Each well was blocked with PBSL containing 40 mg / ml bovine serum albumin BSA. Cultured hybridoma cell supernatant was collected from each well of a 96-well cell culture plate and transferred to the corresponding wells of a 96-well ELISA plate. The cells were incubated at room temperature for 1 hour, and the wells were washed four times with PBSL solution. Horseradish peroxidase-conjugated goat anti-mouse antibody was added and incubated at room temperature for 1 hour, after which the wells were washed four times with PBSL solution. 100 μL of TMB substrate was added to each well and incubated at room temperature in the dark for 10-20 minutes to allow color development. After the color development had stabilized, 50 μL of stop solution was added to each well, and the absorbance at 450 nm wavelength was read for each well using a microplate reader. The ELISA detection results for one 96-well plate of anti-A2 hybridoma and one 96-well plate of anti-CNR1 hybridoma are shown in Figures 80C and 80D, respectively.

[0270] The results demonstrate that the modified GPCRs described in this application can be effectively used for antibody discovery. A key challenge in the discovery of therapeutic GPCR antibodies using mouse immunization and hybridoma technology is that the generated and identified antibodies must be able to recognize GPCRs with their native three-dimensional structure on the surface of human cells. First, mice were immunized with GPCRs with their native three-dimensional structure to stimulate the mouse immune system and produce antibodies that recognize GPCRs with their native three-dimensional structure. Subsequently, hybridoma cells were created and identified using GPCRs with their native three-dimensional structure. Considering that many antibodies can bind to target proteins but are unsuitable as therapeutic antibodies due to factors such as affinity and stability, as many hybridoma cell lines that secrete specific antibodies as possible are obtained during the hybridoma creation and screening stages. For multitransmembrane proteins like GPCRs, the extracellular region is very small, so the proportion of B cells that produce GPCR protein antibodies in spleen cells after immunization is usually very low (as can be seen from the proportion of positive cells in Figure 80). Therefore, in many cases, it is necessary to create a large number (at least several thousand) of hybridoma cells and screen them. ELISA is characterized by its high sensitivity and high throughput, making it suitable for rapidly identifying cells that secrete antibodies that recognize GPCRs with their native three-dimensional structure. However, high-purity protein is required to identify specific antibodies and avoid screening hybridoma cells that secrete other nonspecific antibodies. Furthermore, the amount of purified protein required to identify a large number of hybridoma cells is relatively large. The present invention provides a simple way to obtain high-purity GPCR protein, which, when used in ELISA, eliminates interference from other background proteins and allows for rapid screening of hybridoma cells that secrete antibodies specific to the target GPCR (see Figure 80). In particular, the present invention generates GPCR proteins with their native extracellular structure and screens for antibodies that recognize the native structure of GPCRs, which is extremely important for therapeutic antibodies.

[0271] Example 15: Another modified G protein-coupled receptor according to the present application

[0272] The amino acid sequences of the following GPCRs were modified in the same manner as those of CNR1 and NK1R described above. 1. At least a portion of the third intracellular loop of these GPCRs was replaced with a GFP10-11 fragment (SEQ ID NO: 1) with linkers added to both sides. 2. In the second intracellular loop, the amino acid at position 34.51 according to the Ballesteros-Weinstein numbering system was mutated to cysteine. The amino acid sequences of the modified GPCRs are as follows: [Table 22] JPEG2026521948000024.jpg243163JPEG2026521948000025.jpg244163JPEG2026521948000026.jpg244164JPEG2026521948000027.jpg243164 JPEG2026521948000028.jpg243164JPEG2026521948000029.jpg244164JPEG2026521948000030.jpg244164JPEG2026521948000031.jpg243163 JPEG2026521948000032.jpg244163JPEG2026521948000033.jpg243164JPEG2026521948000034.jpg236163JPEG2026521948000035.jpg243163 JPEG2026521948000036.jpg242164JPEG2026521948000037.jpg242163JPEG2026521948000038.jpg242163JPEG2026521948000039.jpg129164

[0273] The nucleotide sequences encoding the modified GPCR and GFP10-11 fusion protein described above were cloned into the mammalian cell expression vector pBacMam4R, and transfected into HEK293F cells using the same steps as in the above example, resulting in co-expression of the GPCR, GFP10-11 fusion protein, and GFP1-9 fragments. After obtaining the cells, the GFP-clamp-Cys protein obtained in Example 3 was added, and the cells were purified using the same steps as in Example 7. The purified proteins were analyzed by molecular sieve chromatography and SDS gel electrophoresis, and the results are shown in Figures 15-74. The results showed that these modified GPCRs had good characteristics, high purity, and contained the expected components. The purified proteins formed a non-concentrated UV absorption peak in molecular sieve chromatography, indicating good stability.

Claims

1. A modified G protein-coupled receptor, the modified G protein-coupled receptor comprising, in order from the N-terminus to the C-terminus, the N-terminus, a first transmembrane domain, a first intracellular loop, a second transmembrane domain, a first extracellular loop, a third transmembrane domain, a second intracellular loop, a fourth transmembrane domain, a second extracellular loop, a fifth transmembrane domain, a third intracellular loop, a sixth transmembrane domain, a third extracellular loop, a seventh transmembrane domain, and a C-terminus, wherein at least a portion of the first intracellular loop, the second intracellular loop and / or the third intracellular loop is replaced with an optional first linker, a first portion of a fluorescent protein, and an optional second linker, and the first linker and the second linker independently comprise one or more amino acids.

2. The modified G protein-coupled receptor according to claim 1, wherein the fluorescent protein comprises one or more of the following: green fluorescent protein, red fluorescent protein, yellow fluorescent protein, blue fluorescent protein, cyan fluorescent variant, and enhanced green fluorescent protein.

3. The modified G protein-coupled receptor according to claim 1 or 2, wherein the first portion of the fluorescent protein comprises the 10th to 11th β-sheets, the 1st to 2nd β-sheets, the 8th to 11th β-sheets, or the 1st to 4th β-sheets of the fluorescent protein.

4. The modified G protein-coupled receptor according to any one of claims 1 to 3, wherein the first portion of the fluorescent protein includes an amino acid sequence that has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5% sequence identity with the amino acid sequence that has at least 99%.

5. A modified G protein-coupled receptor according to any one of claims 1 to 4, wherein in the second intracellular loop, the amino acid at position 34.51 according to the Ballesteros-Weinstein numbering system is cysteine.

6. The modified G protein-coupled receptor according to any one of claims 1 to 5, wherein the modified G protein-coupled receptor includes an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 3 to 71.

7. A complex comprising a modified G protein-coupled receptor according to any one of claims 1 to 6 and a second portion of a fluorescent protein, wherein the first portion of the fluorescent protein and the second portion of the fluorescent protein are bound to each other.

8. The complex according to claim 7, wherein the second portion of the fluorescent protein comprises the 1st to 9th β-sheets, the 3rd to 11th β-sheets, the 1st to 7th β-sheets, or the 5th to 11th β-sheets of the fluorescent protein.

9. The complex according to claim 7 or 8, wherein the second portion of the fluorescent protein includes the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid sequence shown in SEQ ID NO:

2.

10. The complex according to any one of claims 7 to 9, further comprising a clamp protein, wherein the clamp protein is bound to a first portion and a second portion of the fluorescent protein.

11. The complex according to claim 10, wherein the clamp protein includes an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5% sequence identity with the amino acid sequence represented by any one of SEQ ID NOs. 72-74 or SEQ ID NO.

82.

12. The complex according to claim 11, wherein the 186th position of the clamp protein is cysteine, and the position is as shown in SEQ ID NO:

72.

13. The complex according to claim 11 or 12, wherein a disulfide bond is formed between the amino acid at position 186 of the clamp protein and the amino acid at position 34.51 according to the Ballesteros-Weinstein numbering system in the second intracellular loop.

14. Use of a modified G protein-coupled receptor according to any one of claims 1 to 6 or a complex according to any one of claims 7 to 13 in ligand affinity measurement, drug discovery, or screening.

15. Use of the complex according to any one of claims 7 to 13 in three-dimensional structural analysis using cryo-electron microscopy.

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