Preparation method and application of chemokine receptor CXCR4 homotetramer
By co-expressing CXCR4, CXCL12 and beta-arrestin1 in insect cells, stable homotetramer assembly and purification of CXCR4 is solved, and the problem of difficulty in stably preparing CXCR4 quaternary assembly in the prior art is solved, and the efficiency of related applications is improved.
Patent Information
- Application Number
- CN202510058329.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-14
- Publication Date
- 2025-04-01
AI Technical Summary
The prior art is difficult to stabilize the preparation and mass production of regular four-level assemblies of the chemokine receptor CXCR4, which limits the research and development of conformational antibodies, biochemical detection reagent test strips and related medical technologies of related assemblies.
Stable homotetramer assembly and purification of CXCR4 was achieved by co-expressing CXCR4, CXCL12 and beta-arrestin1 in insect cells, and under the action of the agonist CXCL12 and the inhibitor beta-arrestin1.
The stable preparation of CXCR4 homotetramers was achieved, and its application efficiency was improved in the preparation of immune antibody, biochemical detection reagent test strips and targeted drug experimental screening.
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Figure CN119736339A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and specifically relates to a preparation method and application of a chemokine receptor CXCR4 homotetramer. Background Art
[0002] Chemokine receptor CXC-4 (CXCR4), also known as fusin or CD184, is an important chemokine receptor and known drug target. CXCR4 is essential for regulating a variety of cellular processes, including lymphocyte trafficking, hematopoiesis, embryonic development, angiogenesis, viral infection and inflammation. CXCR4 has always been a popular target for drug development, and representative drugs targeting CXCR4 include Plerixafor. Unlike other CXC-type chemokine receptors, CXCR4 has only one endogenous ligand, which is the CXC-motif chemokine 12 (CXCL12), also known as stromal cell-derived factor 1 (SDF-1), which is ubiquitously expressed in various tissues and cells. Other CXC-type chemokine receptors can cross-recognize multiple CXC-motif chemokines.
[0003] Like other CXC-type chemokines, CXCR4 couples to downstream Gi proteins after activation, and the activated state is in the form of monomers. The crystal structure shows that the inhibitory state of CXCR4 binding to antagonists is in the form of homo- or hetero-dimers. In recent years, more studies have shown that although the chemokine receptors coupled to G proteins after activation are monomers, chemokine receptors can be detected on the cell surface to assemble into homo- or hetero-aggregates, presenting a quaternary assembly structure of proteins. Studies have shown that the quaternary structure assembly of chemokines is crucial for the regulation of receptor function, providing new ideas for the development of new drugs targeting chemokines. However, the regulatory mechanism of chemokine receptor assembly has been unclear, and the chemokine receptors recombinantly expressed and purified by conventional protein engineering techniques are in the form of monomers, because chemokine receptors cannot spontaneously complete assembly. The activated state of chemokine receptors is in the form of monomers, and the recombinantly expressed and purified chemokine receptors are in the form of monomers, which may be related to the self-activation activity of chemokine receptors. After the addition of antagonists, the purified chemokine receptors are in the form of homo- or hetero-dimers. Although experiments have clearly detected regular assembly forms of chemokine receptors such as tetramers on the cell surface, due to their extremely small content and the unclear regulatory mechanism of chemokine receptor assembly, there is no stable and effective technical method to prepare regular assemblies of chemokine receptors in large quantities, which limits the development of conformational antibodies, biochemical detection reagents and test strips, and related medical technologies that recognize related assemblies.
[0004] We found for the first time in our study that under intracellular conditions, CXCR4 can stably form homotetramers under the simultaneous action of agonist CXCL12 and inhibitory protein beta-arrestin1, and can be stably separated and purified. Based on this, after condition exploration and optimization, the present invention provides a technical solution for preparing CXCR4 homotetramers, which provides a basis for the stable preparation of CXCR4 homotetramers, and the application of CXCR4 homotetramers prepared based on the method in the preparation of immune antibodies, preparation of biochemical detection reagents and test strips, and screening of targeted drug experiments. Summary of the invention
[0005] This project provides a method for preparing CXCR4 homotetramers through recombinant co-expression and separation and purification, and provides an application method for protein preparation for related applications of CXCR4 homotetramers.
[0006] The present invention provides the following technical solutions:
[0007] The present invention provides a method for assembling CXCR4 homotetramers by insect cell co-expression, comprising the following steps: (1) Introducing the nucleotide sequences of CXCR4, CXCL12, and beta-arrestin1 into bacmid and infecting insect cells; (2) suspension culture for 96 h at 27 °C, 110 rpm, and 5% CO2; (3) removing the culture medium by centrifugation and harvesting the cells, wherein a large number of CXC4 homotetramers are distributed in the cell membrane; Preferably, in step (1), the affinity tag sequence is connected to the protease recognition sequence and then connected to the N-terminus of the CXCR4 protein sequence; Preferably, the insect cell in step (1) is an sf9 insect cell; Preferably, the MOI of the infection ratio in step (1) is 2, 0.5, or 1; Preferably, the centrifugal speed in step (3) is 1000 g, the centrifugal time is 15 minutes, and the centrifugal temperature is 4 °C.
[0008] The present invention provides a method for purifying CXCR4 homotetramers from the cultured cells, comprising the following steps: (1) Add hypotonic buffer and stir to make the cells absorb water and swell and rupture under the action of shear force; (2) collecting cell membranes by centrifugation; (3) extracting cell membrane proteins by adding mild detergent to dissolve the cell membrane; (4) removing insoluble precipitate by centrifugation; (5) purification of the CXCR4 homotetrameric complex by a two-step chromatography method; (6) Quality testing of purified samples by gel electrophoresis, immunoblotting, and electron microscopy; (7) Prepare samples that have passed quality inspection by concentration, quick freezing with liquid nitrogen, and cryopreservation; Preferably, the hypotonic buffer in step (1) is: 20 mM HEPES pH 7.4, 50 mM NaCl, 5 mM KCl, 5 mM MgCl2, 5 mM CaCl2; Preferably, the centrifugal acceleration in step (2) is 30000 g and the centrifugation time is 30 minutes; Preferably, the detergent in step (3) is 0.8% by weight volume final concentration of LMNG (Lauryl Maltose Neopentyl Glycol) and 0.2% by weight volume final concentration of CHS (Cholesteryl HemiSuccinate); Preferably, the centrifugal acceleration in step (4) is 30000 g and the centrifugation time is 40 minutes; Preferably, the two-step chromatography in step (5) is FLAG tag affinity chromatography and gel exclusion chromatography respectively; Preferably, the method of steps (1-5) is carried out at a low temperature of 4 °C; Preferably, the gel electrophoresis in step (6) is denaturing polyacrylamide gel electrophoresis SDS-PAGE, and the immunoblotting is Western-Blotting; Preferably, the concentration in step (7) is concentrated by ultrafiltration membrane under centrifugation, the ultrafiltration membrane cutoff size is 100 kDa, and the centrifugal acceleration is 3000 g; Preferably, the storage temperature in step (7) is -80 °C.
[0009] The present invention also provides a use of the CXCR4 homotetramer protein prepared by the technical method in screening conformational antibodies in immunized animals.
[0010] The present invention also provides a use of the CXCR4 homotetramer protein prepared by the technical method in screening drugs targeting the CXCR4 homotetramer.
[0011] Beneficial effects of the present invention: (1) The technical method provided by the present invention uses insect cells for recombinant expression and bacmids for inoculation. Bacmids are capable of self-replication and assembly to achieve long-term stable production; (2) Although the assembly phenomenon of chemokine receptors can be easily detected on the cell surface by single-molecule fluorescence and other technical methods, the extraction of stable assemblies has always been a technical difficulty. The assembly of CXCR4 is strictly regulated. CXCR4 monomers will not spontaneously assemble under in vitro conditions and do not have an obvious dynamic equilibrium between monomers and aggregates (except for unstable protein denaturation and oligomerization nucleation to form precipitation). The assembly of CXCR4 is completed inside the cell, and the molecular mechanism of related regulation is at the forefront of research and has not yet been revealed. The present invention is based on the discovery that CXCR4 can form a stable homotetramer under the simultaneous action of CXCL12 and the inhibitory protein beta-arrestin1. It provides for the first time an efficient and stable technical solution for obtaining CXCR4 homotetrameric protein, which can stably produce CXCR4 homotetrameric protein products and will provide protein-related scientific research reagent products for related research and development; (3) The CXCR4 homotetramer prepared by the technical method provided by the present invention has stable and uniform properties and high purity; (4) The present invention also provides the experimentally determined near-atomic resolution molecular structure of the CXCR4 homotetramer prepared by the method, which exhibits a self-locking allosteric antagonistic inhibition state. Among them, the CXCR4 protomers are arranged according to C4 rotational symmetry, forming a disk with a diameter of about 7.12 nm, and a cavity with a diameter of about 1.56 nm in the middle. In this unique assembly and arrangement form, each CXCR4 protomer occupies the allosteric regulatory site of the adjacent protomer and acts as an "allosteric antagonist", forming a special self-inhibitory state of mutual locking. This unique self-locking inhibition structure effectively prevents the tetramer from dissociating into monomers; (5) The simple and convenient operation process of the present invention improves efficiency and reduces costs, and also lays a solid foundation for the application of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] In order to more clearly illustrate the technical solution in the specific implementation of the present invention, the following is a brief introduction to the drawings required for describing the specific implementation. Figure 1 It is a schematic diagram of the technical principle of CXCR4 homotetramer assembly; Figure 2 It is a schematic diagram of the results of gel exclusion chromatography and SDS-PAGE detection of CXCR4 homotetramer; Figure 3 It is a schematic diagram of the two-dimensional average results of the electron microscope image of the CXCR4 homotetrameric protein particle detection; Figure 4 It is a schematic diagram of the electron microscopy reconstruction density and experimentally determined molecular structure of CXCR4 homotetramer; Figure 5It is a schematic diagram of the molecular structural mechanism of the protomer arrangement and self-locking allosteric antagonism of CXCR4 homotetramer; Figure 6 This is a schematic diagram of the key amino acid sites and functional experimental analysis results of the mutual locking and allosteric antagonism of CXCR4 homotetramers; DETAILED DESCRIPTION
[0013] In order to more clearly describe the technical solution of the present invention, it is further described below in conjunction with specific embodiments, but it does not constitute a limitation on the embodiments of the present invention.
[0014] The embodiment provided by the present invention includes the following steps.
[0015] 1. Molecular construction of recombinant human CXCR4, human CXCL12, and human beta-arrestin1 The molecular construction and operation methods used in this example, unless otherwise specified, are conventional methods known to those skilled in the art, and reference books such as the Molecular Cloning Experiment Guide published by Science Press can be used for reference. The reagents and consumables used are commercially available products unless otherwise specified. (1) Based on the amino acid sequences of human CXCL12 and human beta-arrestin1, the corresponding preferred nucleotide sequences were obtained by double-stranded DNA chemical synthesis, amplified by PCR, and seamlessly linked to the polyhedrin promoter of the pFastbac1 vector by homologous recombination; (2) Based on the amino acid sequence of human CXCR4, the corresponding preferred nucleotide sequence was obtained by double-stranded DNA chemical synthesis; by designing PCR primers, a FLAG tag sequence was introduced at the N-terminus of the CXCR4 sequence, and the recognition sequence of the HRV 3C enzyme was added between the FLAG tag sequence and CXCR4; by PCR amplification and homologous recombination, it was seamlessly connected to the polyhedrin promoter of the pFastbac1 vector; (3) Transform DH5alpha competent cells, spread on LB plates containing 100 μg / mL ampicillin, and culture at 37 °C overnight; (4) Select 5 single colonies, inoculate them into 5 mL of LB liquid medium containing 50 μg / mL ampicillin, and culture at 37 °C with shaking (220 rpm) overnight; (5) Extract plasmid from overnight culture samples; (6) Use universal primers and plasmid as template for PCR, and detect PCR products by agarose gel electrophoresis; (7) For samples whose PCR product fragment size was consistent with the expected value, DNA sequencing was performed for further verification, and finally one positive plasmid each of human CXCR4, human CXCL12, and human beta-arrestin1 was screened for construction.
[0016] 2. Preparation of bacmid (1) The three selected plasmids were transformed into DH10bac competent bacteria respectively, spread on plates containing 0.05 mg / mL kanamycin, 0.01 mg / mL tetracycline, 0.007 mg / mL gentamicin, 0.1 mg / mL X-gal and 0.04 mg / mL IPTG, and cultured at 37 °C overnight; (2) Select 5 white colonies from each of the three plates and inoculate them into 5 mL of LB liquid medium containing 0.05 mg / mL kanamycin, 0.01 mg / mL tetracycline, and 0.007 mg / mL gentamicin. Incubate at 37 °C with shaking (220 rpm) overnight. (3) The bacmid genome was extracted using the classic alkaline lysis method. The extracted bacmid genome was used as a template and PCR was performed using universal primers M13F and M13R. The DNA fragment size was verified by agarose gel electrophoresis and further verified by DNA sequencing. (4) Use the validated Bacmid to transfect Sf9 insect cells using the classic liposome method, harvest the bacmid, and use the classic cell infection method to determine the MOI (Multiplicity of Infection) value of the bacmid;
[0017] 3. Recombinant expression of CXCR4 and homotetramer assembly (technical principle see Figure 1 ) (1) Sf9 insect cells were cultured in a constant temperature suspension culture at 27 °C and a rotation speed of 110 rpm. (2) The cell density was measured using a hemocytometer. 6 Each milliliter, three bacmids, CXCR4, CXCL12, and beta-arrestin1, were simultaneously inoculated, with MOIs of 2, 0.5, and 1, respectively, and the culture volume was 1 L; (3) Cultivate at 27 °C, 110 rpm, 5% CO2 for 96 h; (4) Centrifuge at 4 °C for 15 min at 1000 g. Quickly freeze the cell pellet in liquid nitrogen and store at -80 °C.
[0018] 4. Purification of CXCR4 homotetramers (1) Resuspend the collected cell pellet in pre-cooled hypotonic buffer at 4 °C, consisting of: 20 mM HEPES pH 7.4, 50 mM NaCl, 5 mM KCl, 5 mM MgCl2, 5 mM CaCl2; gently stir for 10 minutes; (2) Low-temperature high-speed centrifugation for 30 min at a centrifugal acceleration of 30,000 g and a temperature of 4 °C, remove the supernatant and collect the cell membrane precipitate; (3) Resuspend the cell membrane pellet in pre-cooled buffer at 4 °C, the composition is: 20 mM HEPES pH7.4, 100 mM NaCl, 5 mM KCl, 5 mM MgCl2, 5 mM CaCl2; grind with a grinder to disperse the cell membrane into small fragments; (4) Add detergent to dissolve and extract membrane proteins from cell membrane fragments. The detergent composition and final mass volume concentration are: 0.8% LMNG (Lauryl Maltose Neopentyl Glycol, Anatrace) and 0.2% CHS (Cholesteryl Hemi Succinate, Anatrace). Place on a rotating device at 4 °C and mix at low speed for 2 hours to fully dissolve the membrane proteins. (5) Low-temperature high-speed centrifugation for 40 min at a centrifugal acceleration of 30,000 g and a temperature of 4 °C to remove insoluble precipitates and collect the supernatant; (6) The collected supernatant was incubated with FLAG medium at 4 °C and subjected to FLAG gravity column chromatography to affinity adsorb CXCR4 homotetramers with FLAG tags; (7) Use 10 column volumes of pre-cooled buffer at 4 °C, composed of 20 mM HEPES pH 7.4, 100 mM NaCl, 0.01% (mass volume concentration) LMNG, and 0.002% (mass volume concentration) CHS, to wash the column and elute the impurities; (8) Add 1 column volume of pre-cooled buffer at 4 °C, composed of 20 mM HEPES pH 7.4, 100 mM NaCl, 0.001% (mass volume concentration) LMNG, and 0.0002% (mass volume concentration) CHS; add HRV 3C protease with FLAG tag and incubate at 4 °C overnight; (9) Collect the CXCR4 homotetramer elution solution after the FLAG tag is removed, and use an ultrafiltration tube (cutoff specification: 100 kDa) to concentrate it at a gravity acceleration of 3000 g to a final volume of 1 mL; (10) Gel exclusion chromatography was performed at 4 °C. The column specifications were Superose 6 Increase10 / 300GL column (GE Healthcare). The mobile phase was 20 mM HEPES pH 7.4, 100 mM NaCl, 0.00075% (mass volume concentration) LMNG, and 0.0002% (mass volume concentration) CHS. The chromatographic results are shown in Figure 2. Figure 2 As shown in the left figure, the elution position of CXCR4 homotetramer is marked with an asterisk; (11) The chromatographically purified CXCR4 homotetramer solution was collected and concentrated by ultrafiltration at 3000 g using an ultrafiltration tube (cutoff specification: 100 kDa) to a final concentration of 10 mg / mL. The aliquots were quickly frozen in liquid nitrogen and then stored at -80 °C.
[0019] 5. Quality detection of CXCR4 homotetramers (1) The prepared CXCR4 homotetramer protein band was detected by SDS-PAGE gel electrophoresis, such as Figure 2 As shown in the picture on the right; (2) Electron microscopy was used to detect CXCR4 homotetramer samples and collect data; the two-dimensional average morphology of CXCR4 homotetramer protein particles was as follows: Figure 3 As shown, there are various 2D projection orientations, in which the CXCR4 protomer exhibits a C4 rotationally symmetric arrangement and clear structural details can be observed; (3) Further analysis and processing of electron microscopy data can obtain the near-atomic resolution molecular structure of the CXCR4 homotetramer and establish an atomic coordinate model, such as Figure 4 As shown in Figure 1, the characteristics of the CXCR4 homotetramer include four-fold rotational symmetry, a nearly disc-shaped structure with a diameter of 7.12 nm and a height of 6.42 nm, and a circular channel with a diameter of 1.56 nm in the middle of the disc; the molecular structure shows that the CXCR4 homotetramer formed by the unique arrangement of CXCR4 protomers is a self-locking allosteric antagonistic inhibitory state, such as Figure 5 As shown; (4) Further functional experimental analysis explained the key amino acid sites and molecular mechanisms of mutual locking and allosteric antagonism of CXCR4 homotetramers, such as Figure 6 As shown, a sufficient basis is provided for the application of the CXCR4 homotetramer protein prepared by the technical method of the present invention in conformational antibody immunoscreening and drug screening targeting CXCR4 homotetramer.
Claims
1. A method for recombinantly constructing a chemokine receptor CXCR4 homotetramer, characterized in that: Co-expression of chemokine receptor CXCR4, chemokine CXCL12 and inhibitory protein beta-arrestin1 was used to promote the formation of CXCR4 homotetramers.
2. A method for preparing a chemokine receptor CXCR4 homotetramer, characterized in that: The steps include: a. constructing the nucleic acid sequence encoding CXCR4, CXCL12, and beta-arrestin1 into a vector, and recombining and preparing a bacmid; b. Co-expression via insect cells; c. Collect the culture and extract the membrane protein using detergent; d. Purifying CXCR4 homotetramers by chromatography; e. Perform quality testing on purified samples; Preferably, in step a, a protease recognition sequence is connected to the N-terminus of the CXCR4 protein sequence, and then an affinity tag sequence is connected; Preferably, the insect cells in step b are Sf9 cells, which are cultured in suspension on a shaker at a temperature of 27° C. and a rotation speed of 110 rpm; Preferably, in step b, the MOI values of the inoculation amounts of CXCR4, CXCL12, and beta-arrestin1 are 2, 0.5, and 1, respectively; Preferably, in step b, when the recombinant protein is expressed, the CO2 concentration is increased from the air concentration to 5%; Preferably, the expression time of the recombinant protein in step b is 96 hours; Preferably, the extract in step c comprises: 20 mM HEPES pH 7.4, 100 mM NaCl, 5 mM KCl, 5 mMMgCl2, 5 mM CaCl2, 0.8% (mass volume concentration) LMNG (Lauryl Maltose Neopentyl Glycol, Anatrace) and 0.2% (mass volume concentration) CHS (Cholesteryl HemiSuccinate, Anatrace); Preferably, step d adopts a two-step chromatography method, first performing affinity chromatography and then performing gel exclusion chromatography; Preferably, in step e, SDS-PAGE gel electrophoresis and immunoblotting are used to detect the purity of the sample, and electron microscopy is used to detect the properties of the protein particles in the sample.
3. The preparation method according to claim 1-2, characterized in that: The prepared CXCR4 homotetramer is a self-locked inhibitory state of CXCR4.
4. Application of the method for preparing recombinant CXCR4 homotetramers as described in claims 1-2 in industrial production.
5. The method for preparing recombinant CXCR4 homotetramers as described in claims 1-2, and its commercial application in preparing recombinant CXCR4 homotetramer proteins to prepare conformational antibodies that recognize CXCR4 homotetramers.
6. The method for preparing recombinant CXCR4 homotetramers as described in claims 1-2, and its commercial application in preparing recombinant CXCR4 homotetramer proteins to screen drugs targeting CXCR4 homotetramers.