Membrane-like system containing FGFR (fibroblast growth factor receptor), phospholipid bilayer and MSP (membrane skeleton protein) and preparation method of membrane-like system

Abstract

The invention provides a membrane-like system containing FGFR (Fibroblast Growth Factor Receptor), a phospholipid bilayer and membrane skeleton protein and a preparation method of the membrane-like system, and particularly relates to a method for assembling a nanodisk (nanodisk) by using the FGFR and application of the nanodisk containing the FGFR in the aspects of antibody screening and drug screening.

Description

[0001] This application is a divisional application of the application filed on May 31, 2017, with application number 201710402086.4 and invention title "Membrane-like system containing FGFR, phospholipid bilayer and membrane skeletal protein MSP and preparation method thereof". Technical Field

[0002] This invention belongs to the field of biotechnology, and in particular relates to a membrane-like system of FGFR containing a phospholipid bilayer and its assembly method. Background Technology

[0003] Fibroblast growth factor receptors (FGFRs) belong to the tyrosine kinase superfamily. The human FGFR family has four members: FGFR1, FGFR2, FGFR3, and FGFR4. The FGFR family plays crucial roles in the physiological and metabolic activities of organisms, including embryonic development, injured tissue repair, angiogenesis, cell proliferation, cell migration, and cell differentiation. Abnormal signaling pathways may lead to cancer. Therefore, FGFRs have become potential drug targets in oncology, and researching their signaling pathways, mechanisms of action, and mechanisms is of great significance for related disease research and drug development.

[0004] Structurally, FGFRs are generally divided into three parts: an extracellular ligand-binding region, a transmembrane region, and an intracellular kinase region. At the protein level, FGFR1, FGFR2, FGFR3, and FGFR4 share approximately 55%-72% homology, thus exhibiting high homology. The extracellular region mainly includes a hydrophobic signal peptide, 2-3 immunoglobulin-like domains (D1-D3), and a threonine-containing cassette between the D1 and D2 regions. The D2 and D3 regions are ligand-binding sites (FGF) and heparin sulfate (HPSG). The transmembrane region functions to transmit substances and signals from the extracellular region to the intracellular region. Within the intracellular region, the segment closest to the cell membrane is called the juxtamembrane region, which connects to the intracellular tyrosine kinase region. The C-terminus is the final terminus of the intracellular region. In vitro studies of FGFR are currently limited to single-domain studies, without investigating its function at the overall structural level. Therefore, constructing a membrane-like system of FGFR containing a phospholipid bilayer (including the transmembrane region, extracellular ligand-binding region, and intracellular kinase region of FGFR) in vitro, simulating the physiological conditions of natural FGFR, would be of profound significance for the accurate and realistic study of FGFR function. Furthermore, this membrane-like system would provide a valuable tool for FGFR functional studies, FGFR antibody preparation, and the screening and modification of FGFR inhibitors. Summary of the Invention

[0005] To address the aforementioned problems, the inventors conducted in-depth research and developed an artificial FGFR-like membrane system containing a phospholipid bilayer and its preparation method, and utilized the artificial FGFR-like membrane system containing a phospholipid bilayer in multiple aspects.

[0006] Specifically, the present invention provides an artificial FGFR-like membrane system containing a phospholipid bilayer, the membrane system comprising:

[0007] 1) Fibroblast growth factor receptor (FGFR).

[0008] 2) Phospholipid bilayer,

[0009] 3) Membrane scaffold protein (MSP).

[0010] MSP encapsulates phospholipids to form a phospholipid bilayer. The extracellular region of FGFR is on one side of the phospholipid bilayer, and the intracellular region of FGFR is on the other side of the phospholipid bilayer. The transmembrane region of FGFR is encapsulated by a phosphate bilayer and extends throughout the entire phospholipid bilayer.

[0011] In a preferred embodiment, the phospholipid bilayer is disc-shaped.

[0012] In a preferred embodiment, the FGFR includes FGFR1c (the amino acid sequence of which is shown in SEQ ID NO:1), FGFR1b (the amino acid sequence of which is shown in SEQ ID NO:2), FGFR2c (the amino acid sequence of which is shown in SEQ ID NO:3), FGFR2b (the amino acid sequence of which is shown in SEQ ID NO:4), FGFR3c (the amino acid sequence of which is shown in SEQ ID NO:5), FGFR3b (the amino acid sequence of which is shown in SEQ ID NO:6), and FGFR4 (the amino acid sequence of which is shown in SEQ ID NO:7), or, provided that it includes a transmembrane region, an amino acid sequence having at least 60%, preferably at least 80%, more preferably at least 95%, and more preferably at least 99% homology with any of the above.

[0013] In a preferred embodiment, the phospholipids used to form the membrane-like system comprise all phospholipids, preferably selected from POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol)) or POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate choline) or POPE (1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphate ethanolamine) or POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate-L... -serine) or DOPG (1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphate-(1'-rac-glycerol)) or DOPC (1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphate choline) or DOPE (1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphate ethanolamine) or DOPS (1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphate-L-serine) or PI(3,4)P2 1-Heptadecanoyl-2-(5Z,8Z,11Z,14Z-arachidonicoyl)-sn-glycero-3-phosphate-(1'-muscle-inositol-3',4'-bisphosphate) or PI(4,5)P2(1-Heptadecanoyl-2-(5Z,8Z,11Z,14Z-arachidonicoyl)-sn-glycero-3-phosphate-(1'-muscle-inositol-4',5'-bisphosphate)) or PI(3,4,5)P3(1-Heptadecanoyl-2-(5Z,8Z,11Z,14Z-arachidonicoyl)-sn-glycero-3-phosphate-(1'-muscle-inositol-3',4',5'-triphosphate)). The phospholipids are most preferably selected from POPG and POPC, wherein the composition of the phospholipids is a mixture of one or more of them in different proportions.

[0014] In a preferred embodiment, the proportion is that one of the phospholipids can be 1%-100%.

[0015] In a preferred embodiment, the membrane skeletal protein MSP includes MSP1D1 (the amino acid sequence of which is shown in SEQ ID NO:8), MSP1D1DH7-10 (the amino acid sequence of which is shown in SEQ ID NO:9), MSP1D1DH5 (the amino acid sequence of which is shown in SEQ ID NO:10), or an amino acid sequence having 70% or more, preferably 80% or more, even more preferably 90% or more, more preferably 95% or more, and most preferably 98% or more homology with the MSP sequence.

[0016] In a preferred embodiment, the artificial FGFR-like membrane system containing a phospholipid bilayer is constructed through the following steps:

[0017] 1) The FGFR encoding gene was inserted into a eukaryotic expression vector, and the FGFR protein was expressed in eukaryotic cells;

[0018] 2) Mix the purified FGFR protein with MSP, phospholipids, and detergent;

[0019] 3) Remove excess detergent from step 2);

[0020] 4) Separate FGFR membrane-like systems containing phospholipid bilayers.

[0021] In a preferred embodiment, the eukaryotic expression vector may be a mammalian expression vector and / or an insect cell expression vector.

[0022] In a preferred embodiment, the ratio of FGFR protein to MSP, phospholipids and detergent is 1:1-2:50-100:100-200, with a preferred ratio of 1:1:50:100.

[0023] In a preferred embodiment, the ratio of any two components among FGFR protein, MSP, phospholipids and detergent is 0.01-100 times the original ratio, preferably 0.1-10 times, more preferably 0.5-5 times, and most preferably 0.8-2.5 times.

[0024] In a preferred embodiment, the detergent is selected from polyethylene glycol octylphenyl ether (triton) or sodium cholate, more preferably sodium cholate.

[0025] In a preferred embodiment, the phospholipid is selected from POPG, POPC, POPE, POPS, DOPG, DOPC, DOPE, DOPS, DMPC, PI(3,4)P2, PI(4,5)P2, PI(3,4,5)P3 or a combination thereof, preferably POPG.

[0026] The present invention also provides a method for preparing the artificial FGFR-like membrane system containing a phospholipid bilayer, the method comprising the following steps:

[0027] 1) Insert the FGFR encoding gene into a eukaryote and express the FGFR protein in eukaryotic cells;

[0028] 2) Mix the purified FGFR protein with MSP, phospholipids and detergent in a certain proportion;

[0029] 3) Remove the detergent from step 2); and

[0030] 4) Separate FGFR membrane-like systems containing phospholipid bilayers.

[0031] In a preferred embodiment, the eukaryotic expression vector may be a mammalian expression vector and / or an insect cell expression vector.

[0032] In a preferred embodiment, the ratio of FGFR protein to MSP, phospholipids and detergent can be selected as 1:1-2:30-100:100-200, preferably 1:1:40:80.

[0033] In the preferred embodiment, the optional ratio of any two components is 0.01-100 times the original ratio, with a preferred ratio of 0.1-10 times, a more preferred ratio of 0.5-5 times, and the most preferred ratio of 0.8-2.5 times.

[0034] In a preferred embodiment, the detergent is selected from nonylphenol polyoxyethylene ether (NP-40), polyethylene glycol octylphenyl ether (triton), or sodium cholate, more preferably sodium cholate.

[0035] In this invention, MSP is an amphiphilic protein modified from Apolipoprotein A-Ⅰ (ApoA-Ⅰ) protein, which encapsulates phospholipids to form a nanoscale disc-shaped phospholipid bilayer. Furthermore, FGFR is migrated into the nanodisc, wherein the extracellular region of FGFR is on one side of the phospholipid bilayer of the nanodisc, the intracellular region of FGFR is on the other side of the phospholipid bilayer of the nanodisc, and the transmembrane region of FGFR is encapsulated by a phosphate bilayer and extends through the entire phospholipid bilayer.

[0036] It should be noted that those skilled in the art know that the fibroblast growth factor receptor tyrosine (FGFR) family includes seven members: FGFR1c, FGFR1b, FGFR2c, FGFR2b, FGFR3c, FGFR3b, and FGFR4. They have high homology and are all single-transmembrane proteins, so they share commonalities in the assembly methods of nanodisks.

[0037] Therefore, the "FGFR protein" mentioned in this invention refers to the mature FGFR protein that naturally exists in humans. Typically, there are seven FGFR proteins, namely FGFR1c, FGFR1b, FGFR2c, FGFR2b, FGFR3c, FGFR3b, and FGFR4, whose amino acid sequences are shown in SEQ ID NO: 1-7, or, provided that the transmembrane region of the FGFR is included, have an amino acid sequence that has more than 60%, preferably more than 80%, more preferably more than 95%, and more preferably more than 99% homology with SEQ ID NO: 1-7.

[0038] In a preferred embodiment, the eukaryotic expression vector is pFastBac. TM 1, pFastBacTM -Gus, pFastBac TM HT, pFastBac TM HT-CAT, pFastBac TM Dual or pFastBac TM Dual-Gus / CAT uses a blue-white screening method to select expression vectors containing the target fragment, namely the FGFR encoding gene.

[0039] In a preferred embodiment, the eukaryotic cells are insect cells, preferably Sf9 or Sf21 cells.

[0040] In a preferred embodiment, FGFR protein is obtained by lysing cells with a lysis buffer containing Triton X-100 and purifying the protein using a heparin chromatography column. The detergent is known and commonly used by those skilled in the art.

[0041] In a preferred embodiment, the excess detergent described in step 3) is removed with biobeads.

[0042] In a preferred embodiment, the separation in step 4) is performed using a molecular sieve. Molecular sieves used in this invention are known to those skilled in the art, such as FPLC molecular sieves (Superdex 200 10 / 300 GL, GE).

[0043] In a preferred embodiment, the phospholipid is one of POPG, POPC, POPE, POPS, DOPG, DOPC, DOPE, DOPS, PIP2, PIP3, or CLP, or a mixture of two or more of these, wherein the mixing ratio can be arbitrary.

[0044] In a preferred embodiment, the detergent added is sodium cholate.

[0045] In a preferred embodiment, the ratio of MSP: phospholipid: sodium cholate: FGFR1c is 1:50:100:1.

[0046] The present invention also provides the use of the artificial FGFR membrane-like system containing a phospholipid bilayer for preparing antibodies, screening drugs, or developing FGFR activity detection kits, wherein the drug is preferably an FGFR inhibitor.

[0047] The main advantage of the nanodiscs containing FGFR prepared by this invention is that it can dissolve transmembrane proteins such as FGFR that are not easily dissolved under conditions without detergent, and that FGFR exists in an environment close to the natural phospholipid bilayer. The resulting nanodiscs are uniform in size and their dimensions are controllable. Attached Figure Description

[0048] Figure 1 FGFR1c nucleic acid electrophoresis image

[0049] Figure 2 FGFR1c protein SDS-PAGE gel electrophoresis image

[0050] Figure 3 .MSP protein SDS-PAGE gel electrophoresis image

[0051] Figure 4 Size exclusion chromatography of FGFR1c nanodiscs

[0052] Figure 5 SDS-PAGE gel electrophoresis image of FGFR1c nanodiscs

[0053] Figure 6 Transmission electron microscopy image of FGFR1c nanodiscs

[0054] Figure 7 Western blot diagram of phosphorylation of FGFR1c nanodiscs and inhibitory efficacy. Detailed Implementation

[0055] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0056] The main reagents, consumables, and instruments used in the experiment were as follows: Primers used for cloning were all synthesized by Shanghai Sangon Biotech. For polymerase chain reaction (PCR), DNA polymerase (primer star), restriction endonucleases, ligases, and Dpn I were purchased from Takara Bio Inc.; the DNA marker was purchased from Thermo Fisher Scientific Inc.; and the conventional DNA product recovery kit and agarose gel extraction kit were purchased from Tiangen Biotech Co., Ltd. E. coli DH5α strain, E. coli DH10 strain, E. coli BL21 strain, pET22b-SUMO-FGF21 plasmid, and SUMO protease were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0057] SOC culture medium, Tris, imidazole, and antibiotics were purchased from Shanghai Sangon Biotech; other salts were purchased from Sinopharm; and concentration tubes were purchased from Millipore. The FPLC instrument used was the ÄKTA system from GE. The heparin affinity chromatography column, Ni-sepharose chromatography column, and size exclusion chromatography column (Hiload 16 / 60 Superdex 75 pg) were all purchased from GE. Insect cell culture medium (Sf-900TM II SFM) was purchased from Gibco. The BCA kit was purchased from Thermo, and the FGFR tyrosine phosphorylation antibody was purchased from R&D.

[0058] Example 1. Cloning and positive screening of FGFR1c

[0059] Using the synthesized FGFR1c as a template, PCR amplification was performed. The sequence of the FGFR1c is shown in SEQ ID NO: 1. The sequences of the two primers used are as follows:

[0060] P1:5'-CCCGGATCCATGTGGAGCTGGAAGTGCCTCCTC-3' (SEQ ID NO: 12),

[0061] P2: 5'-CGCAAGCTTCAAGCGGGCTTTTGAGTCCGCCATTG-3' (SEQ ID NO: 13).

[0062] They contain restriction endonuclease sites of BamHI and HindIII at their two ends, respectively. PCR reaction conditions were: 94℃, 5 min; 60℃, 30 s; 72℃, 3 min, for 30 cycles at 72℃, 5 min each. Detection was performed by agarose gel electrophoresis. Figure 1 As shown. PCR products were purified using a kit and then sent to a biotechnology company for sequencing. pFastBac TM 1. The vector (purchased from Invitrogen) and the PCR-amplified FGFR1c DNA fragment were double-digested with restriction endonucleases BamHI and HindIII to obtain pFastBac with sticky ends produced by BamHI and HindIII digestion, respectively. TM 1. The vector and FGFR1c(22-765) DNA fragments were separated and purified by agarose gel electrophoresis, and the fragments containing pFastBac were cleaved under UV light. TM1. The target fragment of the vector and FGFR1c was recovered using a DNA mini-recovery kit. The vector and target gene content was estimated using agarose gel electrophoresis. The target gene fragment and vector were mixed at a molar ratio of approximately 3:1, and ligated at 16°C for 2 hours using a TaKaRa ligation kit. The ligation product was transformed into DH5α competent cells. Transformed strains were selected for colony PCR detection and sent to a biotechnology company for sequencing.

[0063] Take 1 ng pFastBac that has been correctly sequenced TM 1- The FGFR1c recombinant plasmid was added to 100 μL of DH10Bac competent cells, incubated on ice for 30 min, heat-shocked at 42℃ for 45 s, incubated on ice for 2 min, and then 900 μL of SOC medium (2% peptone, 0.5% yeast extract, 0.05% NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM glucose) was added and the mixture was shaken at 37℃ for 4 h. The cells were then diluted to 10⁻⁶ of their original concentration using SOC medium. -1 10 -2 10 -3 100 μL of the diluted solution was plated onto LB agar plates containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 100 μg / mL Bluo-gal, and 40 μg / mL IPTG, respectively, and incubated at 37°C for 48 h. Ten white monoclonal antibodies from the plates were inoculated into LB agar plates containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 100 μg / mL Bluo-gal, and 40 μg / mL IPTG, and incubated overnight at 37°C. Viral rod particles were extracted using the isopropanol precipitation method, and the extraction steps are as follows:

[0064] 1. Collect 3 mL of overnight cultured bacteria by centrifugation, resuspend the bacteria in 300 μL of Solution 1 (50 mM glucose, 25 mM tris-cl, 10 mM EDTA, 100 μg / mL RNase), gently shake and mix well.

[0065] 2. Add 300 μL of solution 2 (0.2 M NaOH, 1% SDS) to the well-mixed bacterial solution, mix gently, and incubate for 3-5 minutes until the solution becomes clear and semi-transparent.

[0066] 3. Slowly add 300 μL of solution 3 (3M potassium acetate, pH 5.5), mix gently, and a white precipitate will form.

[0067] 4. Centrifuge at 14000 rpm for 20 min at 4℃.

[0068] 5. Gently transfer the supernatant to a solution containing 0.9 g / L isopropanol, mix well, and incubate on ice for 20 minutes.

[0069] 6. Centrifuge at 14000 rpm for 10 min at 4℃.

[0070] 7. Remove the supernatant and wash three times with 0.5 mL of 70% ethanol solution.

[0071] 8. Let stand at room temperature for 20-30 minutes to completely remove the ethanol, then add 40uL of deionized water to dissolve the extracted rod particles.

[0072] Viral rod particles were detected using PCR. The two primer sequences used in this PCR method are as follows:

[0073] P1:5'-gCGGATCCATGGCTGTGCTGGTCACAGCCACACTCTGCACC-3' (SEQ ID NO: 14),

[0074] P2: 5'-CcCAAGCTTttaCTCCTGGTTGGAGGTCAAGGCCACGATGCG-3' (SEQ ID NO: 15).

[0075] The PCR reaction conditions were: 94℃ for 3 min; 94℃ for 45 s; 55℃ for 45 s; 72℃ for 3 min, with 30 cycles from step 2 to step 4, each cycle lasting 5 min at 72℃. PCR products were detected by agarose gel electrophoresis. Results are as follows: Figure 1 As shown, the target band identified by PCR was at 2200 bp, consistent with the theoretical value.

[0076] Example 2. FGFR1c virus packaging

[0077] First, 2×10 6 One sf9 cell was placed in a T25 culture flask and 5 ml of antibody-free insect cell culture medium was added. The mixture was incubated for at least 30 minutes. Transfection was then performed using liposome transfection reagent. 12 μL of the transfection reagent and 2 μg of rod particles (isolated in Example 1) were placed separately into 100 μL of serum-free and antibiotic-free culture medium and incubated for 30 minutes. Then, the two media containing 2 μg of rod particles were added to a medium containing 12 μL of the transfection reagent and incubated for another 30 minutes. The mixture was then added dropwise into a pre-prepared T25 culture flask. After 4 days of culture, the supernatant virus was collected; this was the first-generation virus P1. 1 mL of P1 was added to a medium containing 1 × 10⁻⁶ cells. 7In a T75 culture flask containing 15-20 ml of insect cell culture medium, culture for 4 days. Collect the supernatant virus, which is the second-generation virus P2. Add 1 mL of P2 to a culture medium containing 1 × 10⁻⁶ cells. 7 Incubate cells in a T75 culture flask (15-20 ml of culture medium) for 4 days. Collect the supernatant, which is the P3 generation virus, and it can be used for protein expression.

[0078] Example 3. Expression of FGFR1c protein

[0079] Add 500 ml of SOC culture medium to a sterile 2 L Erlenmeyer flask, and then add sf9 cells to achieve a cell concentration of 0.5 × 10⁻⁶. 6 Cells / mL, cultured for approximately 20 hours until the cell concentration reaches 1.0 × 10⁻⁶. 6 When the cell count is 1 / mL, add 5 mL of FGFR1c P3 generation virus to each bottle, continue culturing for 36-48 h, collect the cells, and analyze their expression by SDS-PAGE gel electrophoresis.

[0080] Example 4. Purification of FGFR protein

[0081] The collected sF9 cells were added to lysis buffer at a certain ratio. 1g of cells (wet weight) was added to 10ml of cell lysis buffer (20mM Tris, 100mM NaCl, 1mM DTT, 0.2% Triton, 0.2mM NaVO3, pH 7.2) and lysed for 1h. Then, the cells were centrifuged at 50,000 rpm for 1h. The supernatant was collected and the protein was purified by heparin affinity chromatography. The heparin affinity chromatography column was equilibrated with equilibration buffer (20 mM Tris, 100 mM NaCl, 1 mM DTT, 0.2% Triton, 0.2 mM NaVO3). The supernatant was incubated with heparin for 1 h for purification. Impurities were eluted with elution buffer (20 mM Tris, 300 mM NaCl, 1 mM DTT, 0.2% Triton, 0.2 mM NaVO3). Finally, FGFR1c protein was eluted with buffer (20 mM Tris, 1 M NaCl, 1 mM DTT, 0.2% Triton, 0.2 mM NaVO3), and identified by SDS-PAGE gel electrophoresis. Figure 2 As shown, the molecular weight of the purified FGFR band in gel electrophoresis is around 90,000, consistent with the theoretical molecular weight.

[0082] Example 5. Construction of MSP1D1 DH5 vector

[0083] GenScript optimized the expression codons of E. coli based on the amino acid sequence of MSP1D1 DH5 and synthesized cDNA (sequence shown as SEQ ID NO: 11), and the deduced amino acid sequence is shown as (SEQ ID NO: 10).

[0084] The primers used include:

[0085] Forward primer: 5'-CGCCATATGGGTCATCATCATCATCATCATCACGATTATGATATTC-3' (SEQ ID NO: 16),

[0086] Reverse primer:

[0087] 5'-CCGGAATCCTTACTGGGTATTCAGCTTTTTAGT-3' (SEQ ID NO: 17). The MSP1D1 DH5 gene was amplified using primers.

[0088] The PCR product of MSP1D1 DH5 and the PET28a plasmid were digested with restriction endonucleases NdeI and EcoRI, respectively. They were then separated and purified by agarose gel electrophoresis. Regions containing the pET28a vector and the MSP1D1 DH5 target fragment were cut under UV light. The target fragment was recovered using a DNA mini-recovery kit (purchased from TIANGEN) and extracted according to the kit's instructions. The vector and target gene content were determined by agarose gel electrophoresis. The target gene fragment and vector were mixed at a molar ratio of approximately 3:1 and ligated at 16°C for 2 hours using a TaKaRa ligation kit. The ligation product was transformed into DH5α competent cells. Transformed strains were selected for colony PCR detection and sent to a biotechnology company for sequencing. Plasmids were extracted from the correct clones, and the extracted pET28a-MSP1D1 DH5 plasmid was transformed into Escherichia coli BL21(DE3) strain (Novagen) to obtain positive single clones with kanamycin resistance. These clones were then stored as expression strains in an ultra-low temperature freezer at -80 °C.

[0089] Example 6. MSP1D1 DH5 protein expression

[0090] The specific steps of the experiment are as follows:

[0091] (1) Take out the MSP1D1 DH5 bacterial culture stored at -80℃ and place it on ice. Take out 200uL and inoculate it into a test tube containing 5ml LB medium. Add kanamycin to a final concentration of 50mg and incubate overnight at 37℃.

[0092] (2) Transfer to 500 ml LB medium at a ratio of 1:100 (5 ml), and add kanamycin to a final concentration of 50 mg;

[0093] (3) Incubate at 37℃ until OD 600 When the concentration reaches between 0.8 and 1.0, add 250 μL of IPTG (final concentration 0.5 M), induce expression at 25°C for 3.5-4 h, and collect the bacterial cells by centrifugation.

[0094] (4) Store the centrifuged precipitate at -80℃. SDS-PAGE detection is required for samples before and after induction.

[0095] Example 7. Purification of MSP1 D1 DH5 protein

[0096] The pET28a-MSP1D1 DH5 vector was obtained from our laboratory. The coding sequence for MSP is SEQ ID NO: 11. A 6*His tag is inserted at the N-terminus of the MSP to facilitate the purification of the target protein. For the MSP series proteins, we used a two-step chromatography method: Ni-NTA affinity column chromatography and FPLC molecular sieve chromatography. The specific experimental steps are as follows:

[0097] (1) Ultrasonic disruption: Take out the bacterial solution stored at -80℃ and thaw it in a room temperature water bath, shaking it several times during the process. After it is completely thawed, pour it into a small glass beaker, place it in an ice bath, and set the ultrasonic disruption instrument parameters to 3 seconds of operation, 4 seconds of rest, and 5 minutes of duration, repeating this process three times. Then, centrifuge the cell disruption solution at 14000 rpm for 30 minutes (46# rotor). Take out the supernatant and store it at 4℃. Before centrifuging after ultrasonic disruption, take 20 μL of the sample and centrifuge it at 13000 rpm for 1 minute. Add 4 μL of 6* SDS loading buffer to the supernatant to prepare the disrupted supernatant electrophoresis sample, and add 20 μL of 2* SDS loading buffer to the precipitate to prepare the disrupted precipitate electrophoresis sample. Store both samples at -20℃.

[0098] (2) Ni-NTA affinity column purification: Before use, the Ni-NTA affinity column needs to be regenerated and equilibrated with buffer. The treatment method is as follows: wash with 0.5M NaOH, 100mM EDTA, 100mM Ni2SO4 and 20% ethanol for 2 column volumes respectively. Wash with water for 5 column volumes before and after each step to remove the impurities that were previously attached to the column. Finally, equilibrate with suspension buffer for 5 column volumes. Mix the supernatant after sonication and centrifugation with an appropriate amount of nickel packing material and bind at 4℃ for more than 2 hours. After binding, slowly load the sample and collect the elution liquid. After loading the sample, wash with equilibration buffer for 2 column volumes and collect the impurities that were not attached to the column. Then, elute with gradient buffer with continuously increasing imidazole concentration and collect each protein peak. Take 20ul from each elution peak receiving tube for SDS-PAGE detection, and store the rest at 4℃.

[0099] (3) Concentration: Based on the SDS-PAGE detection results, the elution peak receiving tube was concentrated using an ultrafiltration concentration tube (10kD cutoff), centrifuged at 3000rpm and 4℃ until it was concentrated to about 5ml.

[0100] (4) Further purification of FPLC molecular sieve: Assemble the molecular sieve (HiLoad 16 / 60 Superdex75, GE) and equilibrate it with at least one column volume of the filtered FPLC equilibration buffer; centrifuge the concentrate obtained in the previous step at 13000 rpm for 10 min at 4℃, and transfer the supernatant to another new EP tube; take 5 μL from this tube as the SDS-PAGE electrophoresis sample; load the remaining processed concentrate onto the molecular sieve (be careful not to aspirate air bubbles or precipitates); control the injection rate and pay attention to the column pressure; collect the protein solution of each peak, and take 10 μL from each tube for SDS-PAGE electrophoresis detection, and store the rest at 4℃.

[0101] (5) Secondary concentration: Based on the SDS-PAGE electrophoresis results, the receiving tubes of each molecular sieve peak were concentrated using an ultrafiltration concentration tube (10kD cutoff), centrifuged at 3000rpm at 4℃ until concentrated to about 1.5ml, and stored at 4℃.

[0102] (6) Concentration determination: Take 5 μL of the final protein sample and dilute it to 200 μL with molecular sieve buffer. Measure the absorbance at A280 using a UV spectrophotometer. The protein concentration can be obtained by calculation.

[0103] Example 8. Preparation of phospholipid mother liquor

[0104] (1) The phospholipids purchased in this experiment are all powders, while phospholipid stock solutions are commonly used in assembly. Therefore, all phospholipids used must first be dissolved in sodium cholate solution. The preparation process of the stock solution is as follows:

[0105] (2) Weigh the required phospholipid powder, dissolve it in chloroform solution, and slowly blow it dry with nitrogen gas to spread it evenly on the bottom of the container to form a transparent or white film. In this example, 30 mg of POPC is weighed.

[0106] (3) Seal the container with sealing film and leave a few pores, then put it into a freeze dryer and freeze dry it for 3-4 hours to remove residual chloroform.

[0107] (4) Add an appropriate amount of pre-prepared sodium cholate solution (usually the concentration of sodium cholate is twice the concentration of phospholipid) to the lyophilized phospholipid film, and then mix at low speed on a rotary shaker overnight to obtain phospholipid stock solution. In this example, 1 mL of 80 mM sodium cholate solution (20 mM Tris, 100 mM NaCl, pH 7.2) is added.

[0108] (5) The mixed phospholipid mother liquor can be stored at -80℃ for a long time.

[0109] Example 9. Assembly of FGFR nanodisks

[0110] (1) The purified MSP protein and the phospholipid mother liquor prepared in Example 8 were mixed in a certain ratio, where the molar ratio of MSP DH5:phospholipid:sodium cholate:FGFR1c was 1:50:100:0.2; and incubated at 4°C for 2 hours.

[0111] (2) After incubation, add an appropriate amount of hydrophobic adsorbent Bio-Beads SM-2 (Bio-Rad, Hercules, CA) to remove sodium cholate molecules; the amount of hydrophobic adsorbent Bio-Beads is proportional to the sodium cholate content in the assembly solution, usually 0.5-0.8g of bio-Beads can adsorb and remove the sodium cholate molecules. The process is mixed in a rotary shaker at room temperature for 4-8h.

[0112] (3) After mixing, aspirate the supernatant, centrifuge at 13000 rpm for 10 min at 4℃, and further purify by FPLC molecular sieve (Superdex 200 10 / 300 GL, GE); Figure 3 As shown. Peak 1 was analyzed by SDS-PAG gel electrophoresis, then concentrated using an ultrafiltration concentrator (30kD cutoff), and centrifuged at 3000 rpm at 4℃. SDS-PAGE gel electrophoresis was then performed for analysis, as shown below. Figure 4 As shown, FGFR1c and MSP both appear in peak 1.

[0113] Example 10. Electron microscopy characterization of FGFR nanodisks

[0114] The purified sample was stained with phosphotungstic acid, and 10 μL was titrated onto a 400-mesh copper grid for characterization. The nanodiscs were clearly visible in the electron microscope images. Figure 5 As shown, the nanodisks were successfully assembled.

[0115] Example 11. Validation of phosphorylation of FGFR nanodisc

[0116] For the activity verification experiment, the nanodiscs containing FGFR1c from Example 9 (dissolved in 20 mM Tris-HCl, 100 mM NaCl, pH 7.2) were aliquoted into four EP tubes, and dovirtinib was added to each tube to achieve final concentrations of 1000 nM, 500 nM, 250 nM, and 0 nM. Then, 10 mM MgCl2 and 20 μM ATP were added to each tube, and the mixture was incubated at 37°C for 15 min. Two volumes of loading buffer (20 mM Tris-HCl pH 8.0, 100 mM DTT, 2% SDS, 20% glycerol, and 0.016% Coomassie Brilliant Blue) were added, and the mixture was heated at 95°C for 10 min. Western blot analysis was performed after the reaction, and the results were verified using an FGFR tyrosine phosphorylation antibody (R&D). Figure 7 As shown, the FGFR assembled into the nanodiscs can autophosphorylate in vitro, indicating that the assembled FGFR has biological activity. Figure 7 The results also demonstrated that the assembled FGFR could be inhibited by the FGFR inhibitor dovirtinib. In conclusion, FGFR-containing nanodiscs can be used for drug screening, and further, they can be used for the preparation of FGFR antibodies and the development of FGFR activity assay kits.

Claims

1. An artificial membrane-like system comprising FGFR, a phospholipid bilayer, and a membrane skeletal protein MSP, said membrane-like system comprising: 1) Fibroblast growth factor receptor (FGFR). 2) Phospholipid bilayer, 3) Membrane skeletal proteins (MSPs). in, The MSP encapsulates phospholipids to form a phospholipid bilayer. The extracellular region of the FGFR is on one side of the phospholipid bilayer, and the intracellular region of the FGFR is on the other side of the phospholipid bilayer. The transmembrane region of the FGFR is encapsulated by a phosphate bilayer and extends throughout the entire phospholipid bilayer.

2. The artificial membrane-like system containing FGFR, phospholipid bilayer, and membrane skeletal protein MSP according to claim 1, the preparation process includes the following steps: 1) Insert the FGFR target gene into an expression vector and express the FGFR protein in cells; 2) Mix the purified FGFR protein with MSP, phospholipids, and detergent; 3) Remove excess detergent from step 2); and 4) Separate FGFR membrane-like systems containing phospholipid bilayers.

3. A method for preparing an artificial membrane-like system containing FGFR, a phospholipid bilayer, and a membrane skeletal protein MSP, the method comprising the following steps: 1) Insert the FGFR target gene into an expression vector and express the FGFR protein in cells; 2) Mix the purified FGFR protein with MSP protein, phospholipids, and detergent; 3) Remove excess detergent from step 2); and 4) Separate FGFR membrane-like systems containing phospholipid bilayers.

4. The artificial membrane-like system containing FGFR, phospholipid bilayer and membrane skeletal protein MSP according to claim 1 or 2, or the method according to claim 3, wherein the phospholipid is selected from POPG, POPC, POPE, POPS, DOPG, DOPC, DOPE, DOPS, DMPC, PI(3,4)P2, PI(4,5)P2, PI(3,4,5)P3 or any combination thereof, preferably POPG.

5. The artificial membrane-like system containing FGFR, phospholipid bilayer and membrane skeletal protein MSP according to claim 1, wherein the FGFR includes FGFR1c, FGFR1b, FGFR2c, FGFR2b, FGFR3c, FGFR3b, FGFR4 or mutants thereof, wherein the mutants retain the specific biological activity and function of FGFR.

6. The artificial membrane-like system containing FGFR, a phospholipid bilayer, and a membrane skeletal protein MSP according to claim 2, or the method according to claim 3, wherein the expression vector is a eukaryotic expression vector, preferably selected from pFastBac TM 1, pFastBac TM -Gus, pFastBac TM HT, pFastBac TM HT-CAT, pFastBac TM Dual or pFastBac TM Dual-Gus / CAT, wherein the cells are eukaryotic cells, preferably insect cells, and more preferably Sf9 or Sf21 cells.

7. The artificial membrane-like system containing FGFR, phospholipid bilayer and membrane skeletal protein MSP according to claim 2, or the method according to claim 3, wherein the ratio of FGFR protein to MSP protein, phospholipid and detergent is 1:1-2:50-100:100-200, preferably 1:1:50:

100.

8. The artificial membrane-like system containing FGFR, phospholipid bilayer and membrane skeletal protein MSP according to claim 2, or the method according to claim 3, wherein the separation in step 4) is performed using a molecular sieve.

9. The artificial membrane-like system containing FGFR, phospholipid bilayer and membrane skeletal protein MSP according to claim 2, or the method according to claim 3, wherein the detergent is selected from nonylphenol polyoxyethylene ether (NP-40), polyethylene glycol octylphenyl ether, or sodium cholate, preferably sodium cholate.

10. The use of the artificial membrane-like system containing FGFR, a phospholipid bilayer and a membrane skeletal protein MSP as described in claim 1 or 2 for the preparation of antibodies, drug screening or development of FGFR activity detection kits, wherein the drug is preferably an FGFR inhibitor.

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