A cell membrane chromatographic column based on biotin ligase directional covalent fixation and its preparation method and application

Abstract

The application discloses a kind of cell membrane chromatographic column based on biotin ligase directional covalent fixation and its preparation method and application, belong to chromatography science and technology field. By bonding 2-imino biotin to silica gel, then using biotin ligase specific recognition with Avi-Tag cell membrane fragments, and stable covalent bonding occurs with 2-imino biotin silica gel, so that cell membrane is fixed on the surface of silica gel to obtain Avi-Tag bridged cell membrane chromatographic stationary phase, finally using wet column packing, obtain cell membrane chromatographic column based on biotin ligase directional covalent fixation. The cell membrane chromatographic column can prolong the service life of cell membrane chromatographic column, improve the stability of cell membrane chromatographic column during use, and the short peptide tag (Avi-Tag) used can improve the non-specific adsorption and poor specificity of the cell membrane chromatographic column prepared based on protein tag technology to a certain extent, providing technical support for the wide application of cell membrane chromatographic column.

Description

Technical Field

[0001] This invention belongs to the field of affinity chromatography science and technology, specifically relating to a biotin ligase-based directional covalently immobilized cell membrane chromatography column, its preparation method, and its application. Background Technology

[0002] Cell membrane chromatography involves immobilizing cell membranes onto a carrier to create a stationary phase. Chromatographic techniques are then used to study the interactions between drugs and receptors in the mobile phase. It is also a technique that integrates chromatographic separation and activity recognition, and it has unique advantages in screening active ingredients in complex systems.

[0003] Traditional cell membrane chromatography mainly employs two methods of cell membrane immobilization: physical adsorption and chemical bonding. The drawbacks of traditional cell membrane chromatography columns prepared using silica gel adsorption are: due to the weak interaction forces (hydrophobic interactions, van der Waals forces, etc.) between the cell membrane and silica gel, the cell membrane gradually detaches from the silica gel surface as the mobile phase is continuously pumped into the chromatographic system, resulting in reduced column efficiency and a short lifespan. Furthermore, the complex types of cell surface receptors lead to unclear targets and low specificity in drug screening. With the development of biotechnology, the construction and application of receptor-specific overexpression cells have effectively solved the problem of low specificity. Subsequent developments in chemical bonding targeting the C-terminus, N-terminus, and side-chain active groups of target receptors have also improved the cell membrane detachment problem to some extent. However, the intense immobilization conditions affect receptor activity, even causing membrane receptors to lose their recognition activity, thus hindering their widespread application.

[0004] In recent years, the combination of affinity tag immobilization technology and cell membrane chromatography has enabled the mild and orderly immobilization of target receptors on cell membranes, and the cell membranes prepared by this method exhibit high stability. Affinity tags are divided into protein tags and short peptide tags. Currently, Snap-Tag and Halo-Tag protein tags have been successfully introduced into the preparation of cell membrane chromatography stationary phases. However, the large spatial structure of protein tags makes the tags themselves, as secondary receptors, exhibit non-specific adsorption of small molecule compounds and significantly affect the structure of the target receptor protein. Therefore, it is crucial to seek another type of affinity tag, namely short peptide tags, and successfully introduce them into the preparation of cell membrane chromatography stationary phases. Protein tag immobilization is achieved through its specific reaction with small molecule ligands. The separation of fusion proteins can be achieved by simply immobilizing small molecule ligands. However, the immobilization of short peptide tags usually relies on macromolecular antibodies or ligand proteins to achieve chemical binding. Typically, when using short peptide tags as fusion tags, it is necessary to immobilize macromolecular ligands, which also faces problems such as ligand inactivation, increased non-specific adsorption, and high cost. To avoid the introduction of protein tags and macromolecular ligands, it is urgent to explore small molecule conjugates of short peptide tags and develop a method that reduces non-specific adsorption, does not affect the activity of the target protein, and can improve or maintain the stability and specificity of cell membrane chromatography columns. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a biotin ligase-based directional covalently immobilized cell membrane chromatography column, its preparation method and application, which reduces non-specific adsorption while maintaining the high stability, high specificity and column life of the chemically bonded cell membrane chromatography column without affecting the activity of the target protein.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] This invention discloses a biotin ligase-based directional covalently immobilized cell membrane chromatography column, which is filled with a biotin ligase-based directional covalently immobilized cell membrane chromatography stationary phase. The biotin ligase-based directional covalently immobilized cell membrane chromatography stationary phase is prepared by mixing 2-iminobiotin-modified silica gel with a high-expression cell membrane solution of Avi-Tag fused to the C-terminus / N-terminus of the target receptor protein, under the catalysis of a biotin ligase reaction.

[0008] Preferably, the biotin ligase includes BirA, BioID, TurboID, and other enzymes with biotin ligation function.

[0009] This invention also discloses a method for preparing a biotin ligase-based directional covalently immobilized cell membrane chromatography column. The method involves constructing a stable overexpression cell line fused with Avi-Tag at the C-terminus / N-terminus of a target receptor protein, mixing 2-iminobiotin-modified silica gel with a solution of cell membrane fragments from the overexpression stable cell line, and obtaining a biotin ligase-catalyzed directional covalently immobilized cell membrane chromatography stationary phase for Avi-Tag. This stationary phase is then packed into a chromatography column to obtain the biotin ligase-based directional covalently immobilized cell membrane chromatography column.

[0010] Preferably, the specific steps are as follows:

[0011] 1) Succinic anhydride was mixed with amino silica gel to obtain carboxyl-modified silica gel; carboxyl silica gel was mixed with 2-iminobiotin to obtain 2-iminobiotin-modified silica gel.

[0012] 2) Cultivate stable cell lines overexpressing the target receptor protein fused with Avi-Tag at the C-terminus / N-terminus. The cell pellet is broken up, centrifuged, and the supernatant is collected. The cell membrane pellet is centrifuged again to obtain a cell membrane pellet. The cell membrane pellet is mixed with ultrapure water to obtain a cell membrane suspension. The cell membrane suspension is then mixed with 2-iminobiotin-modified silica gel. Under the catalysis of biotin ligase, an Avi-Tag-bridged directional covalently fixed cell membrane chromatographic stationary phase is prepared.

[0013] 3) The Avi-Tag-bridged directional covalently immobilized cell membrane chromatography stationary phase was packed into a column to prepare a biotin ligase-based directional covalently immobilized cell membrane chromatography column.

[0014] More preferably, in step 1), the solvents used for the two mixing processes are dichloromethane and DMF, respectively.

[0015] Preferably, in step 1), after the first mixing, the mixture is washed with dichloromethane, and the filter cake is dried to obtain carboxyl-modified silica gel; after the second mixing, the mixture is filtered, washed with DMF, and the filter cake is dried to obtain 2-iminobiotin-modified silica gel.

[0016] Preferably, in step 2), the method for preparing the overexpression stable cell line is as follows: construct a vector plasmid containing the C-terminus / N-terminus fusion of the receptor protein Avi-Tag, and transfect it into HEK293 cells after being loaded with lentivirus. Use the resistance gene in the plasmid to screen the transfected cells to obtain the overexpression stable cell line of the target receptor protein C-terminus / N-terminus fusion Avi-Tag.

[0017] Preferably, in step 2), the cell membrane suspension is mixed with 2-iminobiotin-modified silica gel, biotin ligase, Biomix A and Biomix B, and then shaken overnight at 4°C and 100 r / min to obtain an Avi-Tag-bridged directional covalently fixed cell membrane chromatography stationary phase.

[0018] Preferably, in step 3), the column packing method is wet column packing.

[0019] This invention also discloses the application of the above-mentioned biotin ligase-based directional covalently immobilized cell membrane chromatography column in ligand-receptor interaction studies and screening and identification of potential active components, wherein the screening and identification of potential active components refers to the screening and identification of potential active components acting on specific receptors in complex systems.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention provides a biotin ligase-based directional covalently immobilized cell membrane chromatography column. 2-Iminobiotin is covalently bonded to silica gel, and then reacted with a highly expressed cell membrane containing Avi-Tag fused to the C-terminus / N-terminus of a target receptor protein. Under the action of biotin ligase, Avi-Tag specifically covalently binds to 2-iminobiotin, thereby directionally and covalently immobilizing the target receptor on the modified silica gel surface. Compared to the low lifespan and specificity of traditional physical adsorption immobilization of cell membranes, and the problem of non-specific adsorption in protein tag immobilization, the stationary phase in this cell membrane chromatography column has the following three characteristics: 1) At the cell membrane level, no other macromolecular tags are introduced besides the target receptor; at the silica gel carrier level, no macromolecular reactants are introduced; 2) A mild enzymatic reaction is used to maximize the retention of target activity; 3) Under enzymatic catalysis, short peptide tag-target receptor fusion protein cell membrane fragments are covalently bound to the small-molecule modified silica gel carrier, effectively preventing membrane detachment and improving the stability of the stationary phase. Because the cell membrane immobilization method uses a covalently bonded short peptide tag Avi, it exhibits lower non-specific adsorption compared to protein tag-based cell membrane chromatographic columns. Furthermore, the specific reaction between Avi-Tag and 2-iminobiotin further screens the immobilized cell membrane, ensuring that only cell membrane fragments fused with Avi-Tag are immobilized, thus enhancing the column's specificity. This directional covalently immobilized cell membrane chromatographic column can be applied to ligand-receptor interaction studies and the screening and identification of potential active components acting on specific receptors in complex systems, providing technical support for the further widespread application of cell membrane chromatographic columns.

[0022] This invention provides a method for preparing a biotin ligase-based directional covalently immobilized cell membrane chromatography column. The preparation conditions are mild and the operation is simple, which can lay a good foundation for large-scale industrial production of cell membrane chromatography columns. The Avi-Tag cell membrane chromatography column prepared by this method has high stability and long service life, and has low non-specific adsorption compared with protein tag technology for immobilized cell membranes.

[0023] Furthermore, carboxyl-modified silica gel was prepared by mixing succinic anhydride with amino silica gel. The resulting carboxyl-modified silica gel has a smaller particle size than commercially available silica gel, which better meets the requirements for preparing stationary phases for cell membrane chromatography. Attached Figure Description

[0024] Figure 1 This is a flowchart illustrating the preparation of the biotin ligase-based directed covalently immobilized cell membrane chromatography column of the present invention.

[0025] Figure 2 The mass spectrometry results are for verifying the chemical reactivity of 2-iminobiotin with Avi-Tag in this invention; where (a) and (b) are the mass spectra of N-terminal acetylated Avi-Tag (Ac-Avi-Tag, Acetylation-GLNDIFEAQKIEWHE) and 2-iminobiotinylated Ac-Avi-Tag in positive ion mode, respectively.

[0026] Figure 3 Infrared spectra of the amino silica gel (SiO2-NH2), 2-iminobiotin-modified silica gel (SiO2-IB), and biotin ligase-based directional covalently immobilized cell membrane chromatography stationary phase (SiO2-CMSP) of the present invention.

[0027] Figure 4 X-ray photoelectron spectra of the amino silica gel (SiO2-NH2), 2-iminobiotin-modified silica gel (SiO2-IB), and biotin ligase-based directional covalently immobilized cell membrane chromatography stationary phase (SiO2-CMSP) of the present invention are shown below; wherein, (a) is the full spectrum of XPS scan; (b) is the fine spectrum of P2p; (c) is the fine spectrum of Si2p; and (d) is the fine spectrum of N1s.

[0028] Figure 5Field emission scanning electron microscope (FESEM) images of the amino silica gel (SiO2-NH2), 2-iminobiotin-modified silica gel (SiO2-IB), and biotin ligase-based directional covalently immobilized cell membrane chromatography stationary phase (SiO2-CMSP) of the present invention; wherein, (a), (b), and (c) have a scale bar of 5 μm, representing amino silica gel, 2-iminobiotin-modified silica gel, and MrgprX2-Avi-Tag cell membrane chromatography stationary phase, respectively; (d), (e), and (f) have a scale bar of 2 μm, representing amino silica gel, 2-iminobiotin-modified silica gel, and MrgprX2-Avi-Tag cell membrane chromatography stationary phase, respectively;

[0029] Figure 6 Transmission electron microscopy (TEM) images of the amino silica gel (SiO2-NH2), 2-iminobiotin-modified silica gel (SiO2-IB), and biotin ligase-based directional covalently immobilized cell membrane chromatography stationary phase (SiO2-CMSP) of the present invention; wherein, (a), (b), and (c) have a scale bar of 2 μm, representing amino silica gel, 2-iminobiotin-modified silica gel, and MrgprX2-Avi-Tag cell membrane chromatography stationary phase, respectively; (d), (e), and (f) have a scale bar of 50 nm, representing amino silica gel, 2-iminobiotin-modified amino silica gel, and MrgprX2-Avi-Tag cell membrane chromatography stationary phase, respectively.

[0030] Figure 7 Immunofluorescence electron microscope images of the 2-iminobiotin-modified silica gel and the biotin ligase-based directional covalently fixed cell membrane chromatography stationary phase of the present invention are shown below. Among them, (a) and (b) are bright field images of the MrgprX2-Avi-Tag cell membrane chromatography stationary phase and the 2-iminobiotin-modified silica gel, respectively, with a magnification of 20x; (c) and (d) are green fluorescence images of the MrgprX2-Avi-Tag cell membrane chromatography stationary phase and the 2-iminobiotin-modified silica gel, respectively, with a magnification of 20x. Detailed Implementation

[0031] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0032] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0033] The present invention will now be described in further detail with reference to the accompanying drawings:

[0034] The present invention provides a biotin ligase-based directional covalently immobilized cell membrane chromatography column, wherein the directional covalently immobilized cell membrane chromatography column is filled with a biotin ligase-based directional covalently immobilized cell membrane chromatography stationary phase;

[0035] The biotin ligase-based directional covalently immobilized cell membrane chromatography stationary phase is prepared by mixing 2-iminobiotin-modified silica gel with a high-expression cell membrane solution of Avi-Tag fused to the C-terminus / N-terminus of the target receptor protein, and reacting under the catalysis of biotin ligase; the biotin ligase includes, but is not limited to, BirA, BioID, TurboID and other enzymes with biotin ligation function.

[0036] This invention provides a method for preparing a biotin ligase-based directional covalently immobilized cell membrane chromatography column, such as... Figure 1 As shown, firstly, amino silica gel is modified to obtain carboxylated silica gel. 2-Iminobiotin is then mixed with carboxylated silica gel to prepare 2-iminobiotin-modified silica gel. Simultaneously, overexpressing cell lines fused with Avi-Tag at the C-terminus or N-terminus of the target receptor protein are cultured to prepare a cell membrane suspension. The cell membrane suspension is then mixed with 2-iminobiotin-modified silica gel. Avi-Tag can form covalent bonds with 2-iminobiotin through a specific reaction under the action of biotin ligase, thus directionally and covalently immobilizing the target receptor on the silica gel surface, obtaining a directionally covalently immobilized cell membrane chromatography stationary phase of Avi-Tag. Finally, the directionally covalently immobilized cell membrane chromatography stationary phase of Avi-Tag is wet-packed onto a column to obtain a directionally covalently immobilized cell membrane chromatography column based on biotin ligase.

[0037] The target receptor protein is MrgprX2 as an example; the constructed cells are HEK293 cells overexpressed and stably transfected with Avi-Tag fused to the C-terminus of MrgprX2 (referred to as MrgprX2-Avi-Tag cells), and the biotin ligase is BirA as an example. The specific operation steps are as follows:

[0038] 1) Biotinylation verification of 2-iminobiotin and Avi-Tag

[0039] Using a micropipette, 500 μL of 0.1 mg / mL N-terminally acetylated Avi-Tag (purchased from Nanjing Genscript Biotech Co., Ltd.), 67 μL of Biomix A (0.5 M bicine buffer, pH 8.3, purchased from AVIDITY, LLC), 67 μL of homemade Biomix B (100 mM ATP (purchased from Shanghai Ron Chemical Technology Co., Ltd.), 100 mM magnesium acetate (MgOAc, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.), 500 mM 2-iminobiotin (purchased from Shanghai Mairui Biochemical Technology Co., Ltd.)), 29.3 μL of water, and 6.7 μL of BirA (purchased from AVIDITY, LLC) were respectively transferred to 1.5 mL centrifuge tubes and incubated at 30 °C for 35 minutes. After the reaction was completed and the solution was desalted, the product was analyzed by HPLC-MS. Figure 2 result, Figure 2 In the middle (a) and (b), the mass spectra of N-terminal acetylated Avi-Tag (Ac-Avi-Tag) and 2-iminobiotinylated Ac-Avi-Tag are respectively in positive ion mode. The results show that 2-iminobiotin binds to acetyl-terminated Avi-Tag, indicating that biotin ligases represented by BirA can specifically catalyze the ligation of 2-iminobiotin to lysine residues of Avi-Tag.

[0040] 2) Preparation of 2-Iminobiotin-modified silica gel

[0041] Weigh 500 mg of succinic anhydride into a 25 mL round-bottom flask, add 10 mL of dichloromethane to dissolve it, then add 500 μL of triethylamine, stir, and add 1 g of amino silica gel (Model: Innoval, 5 μm). After stirring magnetically at room temperature for 48 hours with 3.5% carbon loading, the mixture was filtered, washed six times with dichloromethane, and the filter cake was collected in a petri dish and dried at 37°C for 12 hours to obtain carboxyl-modified silica gel (SiO2-COOH).

[0042] Weigh 383.1 mg EDCI and 57.5 mg NHS into a 25 mL round-bottom flask, add 10 mL DMF to dissolve, then add 200 μL triethylamine, stir, and add 100 mg carboxyl-modified silica gel. After stirring magnetically at room temperature for 3 h, centrifuge at 2300 r / min for 5 min to remove the supernatant and retain the precipitate. Weigh 8 mg 2-iminobiotin, add 1 mL DMF to dissolve, and then transfer the solution to the round-bottom flask containing the precipitate. After stirring at room temperature for 4 h, wash six times with DMF, collect the precipitate in a petri dish, and dry at 37 °C for 12 h to obtain 2-iminobiotin-modified silica gel (SiO2-IB).

[0043] 3) Preparation of Avi-Tag-bridged directional covalently immobilized cell membrane chromatography stationary phase

[0044] A plasmid containing the C-terminus of the MrgprX2 receptor protein fused with Avi-Tag was constructed using genetic engineering technology. After being loaded with lentivirus, the plasmid was transfected into HEK293 cells. The transfected cells were screened using the resistance gene in the plasmid to obtain stably transfected HEK293 cells overexpressing the MrgprX2 receptor protein C-terminus fused with Avi-Tag. The cells were constructed by Jimon Biotechnology (Shanghai) Co., Ltd.

[0045] Cells in the logarithmic growth phase (at least 2 × 10⁻⁶) were collected by digestion with 0.25% trypsin. 7 Cell membranes were centrifuged at 1000×g for 10 min at 4℃, and the culture medium was discarded. The cells were washed twice with physiological saline, then resuspended in hypotonic buffer, and sonicated for 30 min before homogenization. The suspension was centrifuged at 1000×g for 10 min, and the supernatant was collected. The supernatant was then centrifuged at 12000×g for 20 min at 4℃, and the supernatant was discarded. 5 mL of hypotonic buffer was added to resuspend the precipitate, and the mixture was centrifuged again at 12000×g. The cell membrane precipitate was collected and resuspended in 398.75 μL of pre-cooled ultrapure water to obtain a cell membrane suspension for later use. This entire process was performed on ice.

[0046] According to the instructions (BirA Biotin-Protein Ligase Kit, purchased from AVIDITY, LLC), 10 mg of 2-iminobiotin-modified silica gel prepared in step 2), 1.25 μL of BirA enzyme (1 mg / mL, purchased from AVIDITY, LLC), 50 μL of BiomixA (0.5 M bicine buffer, pH 8.3, purchased from AVIDITY, LLC), and 50 μL of self-made BiomixB (100 mM ATP (purchased from Shanghai Ron Chemical Technology Co., Ltd.), 100 mM magnesium acetate (MgOAc, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.)) were added sequentially. The mixture was shaken overnight at 4°C, and after washing three times with physiological saline, the MrgprX2-Avi-Tag cell membrane chromatography stationary phase (CMSP) was obtained.

[0047] 4) Characterization of Avi-Tag-bridged directional covalently immobilized cell membrane chromatography stationary phase

[0048] To verify the synthesized stationary phase, we used infrared spectroscopy to analyze the changes in each functional group. For example... Figure 3 As shown, 1698cm -1 The absorption peak at 1640 cm⁻¹ corresponds to the C=N bond in 2-iminobiotin. -1 The absorption peak at 2943 cm⁻¹ corresponds to the C=O double bond in the amide bond, indicating that IB (i.e., 2-iminobiotin) was successfully bonded; -1 The absorption peak at this point is a characteristic absorption peak of CH3 at the terminal of lipids and CH3 in proteins of the cell membrane, indicating that cell membrane bonding is successful.

[0049] X-ray photoelectron spectroscopy (XPS) was used to characterize amino silica gel, 2-iminobiotin-modified silica gel, and MrgprX2-Avi-Tag cell membrane chromatography stationary phase. Figure 4 In the middle (a), XPS scan full spectrum of amino silica gel (SiO2-NH2), 2-iminobiotin modified silica gel (SiO2-IB), and MrgprX2-Avi-Tag cell membrane chromatography stationary phase (CMSP) is shown. Figure 4 (b) shows the fine spectral scan of P2p; Figure 4 (c) is a fine spectral scan of Si2p; Figure 4Image (d) shows the fine spectrum scan of N1s. In the fine spectrum scan of N1s, a gradual increase in the characteristic signal of nitrogen (N1s) was observed at 399.7 eV. This is due to the condensation reaction between the carboxyl group of carboxylated silica gel and the amino group of 2-iminobiotin, resulting in the bonding of 2-iminobiotin to the silica gel. This indicates that 2-iminobiotin was successfully bonded to the carboxylated silica gel support. In the fine spectrum scans of Si2p and P2p, the characteristic signal of silicon (Si) decreased significantly, while the characteristic signal of phosphorus (P2p) was detected at 134 eV. This indicates that the cell membrane was successfully immobilized on the support surface.

[0050] To further verify the above results, the surface morphology of the three materials was characterized using field emission scanning electron microscopy (SEM), and the results are as follows: Figure 5 After modification with 2-iminobiotin, the surface of the amino silica gel stationary phase did not change significantly because the modification of small molecules has little effect on the surface morphology. However, after reacting with the cell membrane solution, the surface of the stationary phase underwent significant changes. Since the amino silica gel used is porous silica, the surface pores were significantly covered after bonding with the cell membrane, and the surface of the stationary phase appeared smooth. This is consistent with the results of characterization of 2-iminobiotin-modified silica gel and MrgprX2-Avi-Tag cell membrane chromatography stationary phase using transmission electron microscopy (TEM). Figure 6 The presence of a membrane on the surface of the MrgprX2-Avi-Tag cell membrane chromatography stationary phase is more clearly visible in the image, which also indicates the successful preparation of the Avi-Tag directional covalently fixed cell membrane chromatography stationary phase.

[0051] Finally, we used immunofluorescence to characterize the cell membrane chromatographic stationary phase. For example... Figure 7 As shown, Figure 7 In the middle, (a) and (b) are bright-field fluorescence electron microscope images of the MrgprX2-Avi-Tag cell membrane chromatography stationary phase and 2-iminobiotin-modified silica gel, respectively. Figure 7 (c) and (d) are fluorescence images obtained by fluorescence electron microscopy of the MrgprX2-Avi-Tag cell membrane chromatography stationary phase and the 2-iminobiotin-modified silica gel, respectively, at a magnification of 20x. As can be seen from the images, the MrgprX2-Avi-Tag cell membrane chromatography stationary phase fluoresces upon excitation, while the 2-iminobiotin-modified silica gel shows almost no fluorescence. This indicates the presence of MrgprX2 on the surface of the MrgprX2-Avi-Tag cell membrane chromatography stationary phase. This result also demonstrates the successful preparation of the Avi-Tag-bridged, directionally covalently fixed cell membrane chromatography stationary phase.

[0052] 5) Establishment of a biotin ligase-based directional covalently immobilized cell membrane chromatography column and cell membrane chromatography system

[0053] The MrgprX2-Avi-Tag cell membrane chromatography stationary phase obtained in step 3) was wet-packed into a 5.0 mm × 1.0 mm (ID) column core using a column packer. The column core was then inserted into the column sleeve, and the nut was tightened to obtain the cell membrane chromatography column. This column was then connected to a liquid chromatograph to establish a cell membrane chromatography system based on biotin ligase-directed covalent fixation.

[0054] 6) Systematic validation of a biotinylate-based directed covalently immobilized cell membrane chromatography model

[0055] Using the retention time of the publicly reported MrgprX2 receptor agonist sinomenine hydrochloride as the evaluation index, we compared the intra-column and inter-column differences between Snap-Tag-based directional covalently immobilized MrgprX2 cell membrane chromatography columns and biotin ligase-based directional covalently immobilized MrgprX2 cell membrane chromatography columns.

[0056] After equilibration of both cell membrane chromatography columns, 10 μL of 0.05 mg / mL sinomenine hydrochloride was injected six times consecutively into each column. The mobile phase was aqueous solution, and the flow rate was 0.2 mL / min. The retention times of sinomenine hydrochloride were recorded, and the relative standard deviations (RSD, %) of the six retention times on the two columns were compared. Following the same method, Snap-Tag-based and biotin ligase-based directional covalently immobilized MrgprX2 cell membrane chromatography columns were prepared. After equilibration, 10 μL of 0.05 mg / mL sinomenine hydrochloride was injected into each column, and the retention times were recorded. The results are shown in Table 1. Table 1: Repeatability and activity studies of Snap-Tag-based and biotin ligase-based directional covalently immobilized MrgprX2 cell membrane chromatography columns.

[0057]

[0058] *: All data are expressed as RSD (%) of retention time of sinomenine hydrochloride on CMC columns; 1: Six consecutive injections of sinomenine hydrochloride on the same column (n=6); 2: Sinomenine hydrochloride on three different CMC columns.

[0059] Table 1 shows that both the Snap-Tag-based and biotin ligase-based directional covalently immobilized cell membrane chromatography columns exhibit good intra-column and inter-column variability, making them suitable for screening and analyzing active ingredients in complex systems. Furthermore, the biotin ligase-based MrgprX2 cell membrane chromatography column showed a lower RSD value for positive drugs in the inter-column variability assessment, indicating better reproducibility than the Snap-Tag-based column. Next, the activity time of the two cell membrane chromatography columns was investigated. For both the biotin ligase-based and Snap-Tag-based MrgprX2 cell membrane chromatography columns, the activity time was recorded after a 6-day continuous monitoring period, with 10 μL of 0.05 mg / mL sinomenine hydrochloride injected daily. Three days later, sinomenine hydrochloride was still significantly retained on both the Snap-Tag-based and biotin-ligase-based MrgprX2 cell membrane chromatography columns, but the retention time RSD value within three days was smaller on the biotin-ligase-based MrgprX2 cell membrane chromatography column; after six days, sinomenine hydrochloride was still well retained on the biotin-ligase-based MrgprX2 cell membrane chromatography column.

[0060] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A biotin ligase-based directional covalently immobilized cell membrane chromatographic column, characterized in that, The directional covalently immobilized cell membrane chromatography column is filled with a biotin ligase-based directional covalently immobilized cell membrane chromatography stationary phase; the biotin ligase-based directional covalently immobilized cell membrane chromatography stationary phase is prepared by mixing 2-iminobiotin-modified silica gel with a high-expression cell membrane solution of Avi-Tag fused to the C-terminus / N-terminus of the target receptor protein, and then reacting the mixture under the catalysis of biotin ligase.

2. The biotin ligase-based directional covalently immobilized cell membrane chromatography column as described in claim 1, characterized in that, The biotin ligases include BirA, BioID, TurboID, and other enzymes with biotin-ligating functions.

3. A method for preparing a biotin-ligase-based directional covalently immobilized cell membrane chromatography column, characterized in that, A stable cell line overexpressing Avi-Tag was constructed by fusing the C-terminus / N-terminus of the target receptor protein. 2-Iminobiotin-modified silica gel was mixed with a solution of cell membrane fragments from the stable cell line. Under the catalysis of biotin ligase, a directional covalently immobilized cell membrane chromatography stationary phase of Avi-Tag was obtained. This stationary phase was then packed into a chromatographic column to prepare a biotin ligase-based directional covalently immobilized cell membrane chromatography column.

4. The method for preparing a biotin ligase-based directional covalently immobilized cell membrane chromatography column as described in claim 3, characterized in that, The specific steps are as follows: 1) Succinic anhydride was mixed with amino silica gel to obtain carboxyl-modified silica gel; carboxyl silica gel was mixed with 2-iminobiotin to obtain 2-iminobiotin-modified silica gel. 2) Cultivate stable cell lines overexpressing the target receptor protein fused with Avi-Tag at the C-terminus / N-terminus. The cell pellet is broken up, centrifuged, and the supernatant is collected. The cell membrane pellet is centrifuged again to obtain a cell membrane pellet. The cell membrane pellet is mixed with ultrapure water to obtain a cell membrane suspension. The cell membrane suspension is then mixed with 2-iminobiotin-modified silica gel. Under the catalysis of biotin ligase, an Avi-Tag-bridged directional covalently fixed cell membrane chromatographic stationary phase is prepared. 3) The Avi-Tag-bridged directional covalently immobilized cell membrane chromatography stationary phase was packed into a column to prepare a biotin ligase-based directional covalently immobilized cell membrane chromatography column.

5. The method for preparing a biotin ligase-based directional covalently immobilized cell membrane chromatography column as described in claim 4, characterized in that, In step 1), the solvents used for the two mixing processes are dichloromethane and DMF, respectively.

6. The method for preparing a biotin-ligase-based directional covalently immobilized cell membrane chromatography column as described in claim 4, characterized in that, In step 1), after the first mixing, the mixture is washed with dichloromethane and the filter cake is dried to obtain carboxyl-modified silica gel; after the second mixing, the mixture is filtered, washed with DMF, and the filter cake is dried to obtain 2-iminobiotin-modified silica gel.

7. The method for preparing a biotin-ligase-based directional covalently immobilized cell membrane chromatography column as described in claim 4, characterized in that, In step 2), the method for preparing the overexpression stable cell line is as follows: construct a vector plasmid containing the C-terminus / N-terminus fusion of the receptor protein Avi-Tag, and transfect it into HEK293 cells after being loaded with lentivirus. Use the resistance gene in the plasmid to screen the transfected cells to obtain the overexpression stable cell line of the target receptor protein C-terminus / N-terminus fusion Avi-Tag.

8. The method for preparing a biotin ligase-based directional covalently immobilized cell membrane chromatography column as described in claim 4, characterized in that, In step 2), the cell membrane suspension is mixed with 2-iminobiotin-modified silica gel, biotin ligase, Biomix A and Biomix B, and then shaken overnight at 4°C and 100 r / min to obtain an Avi-Tag-bridged directional covalently fixed cell membrane chromatography stationary phase.

9. The method for preparing a biotin ligase-based directional covalently immobilized cell membrane chromatography column as described in claim 4, characterized in that, In step 3), the column loading method is wet column loading.

10. The application of a biotin ligase-based directional covalently immobilized cell membrane chromatography column as described in claim 1 or 2 in ligand-receptor interaction studies and screening and identification of potential active components, characterized in that... The screening and identification of potential active components refers to the screening and identification of potential active components that act on specific receptors in complex systems.

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