Reversible affinity purification system and method based on subject-object interaction and application

Abstract

The invention discloses a reversible affinity purification system and method based on subject-object interaction and application, the system comprises: a functionalized solid phase carrier, the surface of which is covalently modified with a subject molecule; a bait molecule, wherein a guest molecule is connected to the bait molecule; wherein the guest molecule and the host molecule can generate specific and reversible host-guest interaction, so that the bait molecule is fixed on the functionalized solid-phase carrier; and moreover, the combination of the guest molecules and the host molecules can be reversibly dissociated by adding free competitive guest molecules, so that elution of the bait molecules or the compound thereof from the functionalized solid-phase carrier is realized. The method has the dynamic reversible regulation characteristic, high-activity target protein can be obtained through mild competitive elution, interference of endogenous biotin can be effectively avoided, and the functionalized solid-phase carrier can be repeatedly utilized and has higher environmental suitability for complex biological samples.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a reversible affinity purification system, method, and application based on host-guest interaction. Background Technology

[0002] In life science research and drug development, clarifying the interactions between biomolecules and accurately isolating target proteins are core prerequisites for elucidating the mechanisms of life activities and discovering potential drug targets. Currently, pull-down technology is the mainstream method for achieving these goals. Its core principle is to capture interacting target proteins using labeled decoy molecules, followed by separation, purification, and identification. With its direct and efficient advantages, this technology has been widely applied to the screening and verification of various biomolecular interactions, including protein-small molecule compounds, protein-protein interactions, and protein-nucleic acid interactions. It is a key technological support for elucidating signaling pathways and functional complex compositions in basic research, as well as for discovering target proteins and verifying drug-target interactions in drug development.

[0003] Existing technologies largely rely on the biotin-streptavidin system. The binding of streptavidin to biotin is a highly optimized molecular "lock-and-key" mechanism, depending on various intermolecular forces such as hydrogen bonds, hydrophobic interactions, and van der Waals forces, forming an extremely stable complex. The core principle of this system is to utilize the ultra-high affinity non-covalent interaction between biotin and streptavidin (binding constant Kd≈10). -14 This system (M) achieves the fixation of the decoy and the capture and separation of the target protein. Its core implementation steps include: 1. Labeling the decoy with biotin using a chemical cross-linking method; 2. Incubating the biotin-labeled decoy molecules with a streptavidin-modified solid support (such as magnetic beads or agarose gel), allowing the decoy molecules to be fixed to the support surface through biotin-streptavidin interaction; 3. Adding a sample containing the potential target protein (such as cell lysate), and incubating to allow the decoy molecules to form a complex with the target protein; 4. Removing impurities through multiple washes, followed by elution of the target protein under strong denaturation or extreme pH conditions, ultimately obtaining the purified target protein for subsequent identification. This system has the advantages of high binding specificity, strong stability, and mature technology, and is currently the mainstream technology for studying biomolecular interactions.

[0004] However, despite the widespread application of this system, significant technical bottlenecks still exist in practice: The elution conditions are harsh, and the target protein is easily inactivated: the binding of biotin to streptavidin is almost irreversible, which means that the subsequent elution of the captured target protein complex must be carried out under harsh conditions such as strong denaturants, extreme pH, or high temperature. These conditions can easily cause denaturation, aggregation, or inactivation of the target protein, which seriously affects its potential for subsequent functional studies such as structural analysis and enzyme activity assays.

[0005] Endogenous interference leads to false positives: Many biological samples (such as cells, tissues, and serum) naturally contain endogenous biotinylated proteins. These endogenous biotinylate proteins can nonspecifically bind to the streptavidin solid-phase carrier in the system, generating high background noise and false positive signals, which seriously interferes with the accuracy and reliability of experimental results.

[0006] The carriers are not reusable and are costly: Due to the irreversible binding of biotin-streptavidin, solid-phase carriers (such as magnetic beads and agarose beads) become ineffective after one capture-elution cycle, making them impossible to regenerate and reuse, which increases experimental costs.

[0007] Limited system compatibility: The activity of streptavidin is greatly affected by environmental conditions such as pH and ionic strength. In biological samples with complex composition (such as cell lysates and tissue homogenates), its binding efficiency and stability may decrease, which limits the application of this system under more extensive or more severe physiological conditions.

[0008] Therefore, providing a novel pull-down technique that can achieve gentle elution to maintain target protein activity, avoid interference from endogenous substances, and allow for the reusability of solid-phase carriers has become a pressing technical problem to be solved in this field. Summary of the Invention

[0009] To address the aforementioned technical problems, the present invention aims to provide a reversible affinity purification system, method, and application based on host-guest interactions. This invention possesses dynamic and reversible regulatory characteristics, enabling the acquisition of highly active target proteins through gentle competitive elution. It effectively avoids interference from endogenous biotin, and the functionalized solid-phase carrier is reusable, exhibiting stronger environmental adaptability to complex biological samples.

[0010] To achieve the above-mentioned technical objectives and effects, the present invention is implemented through the following technical solution: This invention provides a reversible affinity purification system based on host-guest interactions, comprising: (a) A functionalized solid support whose surface is covalently modified with host molecules; (b) A decoy molecule, wherein a guest molecule is attached to the decoy molecule; The guest molecule and the host molecule can undergo specific and reversible host-guest interactions, thereby immobilizing the decoy molecule on the functionalized solid-phase support; and the binding between the guest molecule and the host molecule can be reversibly dissociated by the addition of free competing guest molecules, thereby achieving the elution of the decoy molecule or its complex from the functionalized solid-phase support.

[0011] Furthermore, the host molecule is cucurbit[7]urea, and the guest molecule includes ferrocene, bisadamantane, bicyclo[2.2.2]octane, 1,4-diazabicyclo[2.2.2]octane or adamantane derivatives.

[0012] Furthermore, the functionalized solid support is prepared by covalently linking azide-modified cucurbit[7]urea to the surface of an alkyne-modified solid matrix via a click chemistry reaction.

[0013] Preferably, the decoy molecule is a small molecule compound, protein, polypeptide, antibody, or nucleic acid.

[0014] Preferably, the solid matrix in the functionalized solid support is a magnetic bead.

[0015] Another aspect of the present invention provides a reversible affinity purification method for biomolecules, used to separate target molecules that interact with decoy molecules. The method utilizes the reversible affinity purification system and includes the following steps: (1) Decoy molecules connected with guest molecules are fixed onto a functionalized solid support with host molecules on its surface through host-guest interactions; (2) To bring immobilized decoy molecules into contact with biological samples containing potentially interacting target molecules; (3) Separate and wash the functionalized solid support; (4) The decoy molecule-target molecule complex is eluted from the functionalized solid support by adding free competitive guest molecules, thereby obtaining the purified target molecule.

[0016] Furthermore, in step (4), the free competitive guest molecule is an adamantane-based compound, and its concentration in the eluent is 0.1 mM to 10 mM.

[0017] The present invention also provides a kit comprising the reversible affinity purification system described above.

[0018] Furthermore, the kit also contains free competing guest molecules.

[0019] This invention also provides the application of the above-mentioned reversible affinity purification system, or reversible affinity purification method, or kit in the study of biomolecular interactions.

[0020] The beneficial effects of this invention are as follows: 1. It achieves gentle and efficient elution of target proteins while fully preserving their biological activity.

[0021] This invention utilizes the high-affinity and reversible host-guest interaction between host molecules (such as cucurbita[7]urea) and guest molecules (such as ferrocene). By introducing free competitive guest molecules (such as amantadine), the target protein complex can be completely eluted under mild physiological buffer conditions (such as neutral PBS buffer). This elution process does not require denaturing agents, extreme pH or high temperature, thereby maximally preserving the native conformation, folding state and biological activity of the target protein. This allows the eluted target protein to be directly used for subsequent in-depth studies that require strict protein activity, such as crystal structure analysis, enzyme kinetics determination, and cell function verification, greatly expanding the downstream application value of Pull-Down technology.

[0022] 2. It avoids interference from endogenous biotin, significantly improving the specificity and signal-to-noise ratio of the detection.

[0023] Traditional systems are hampered by the non-specific binding of endogenous biotinylated proteins in the sample, resulting in high background and a high risk of false positives. The host-guest molecular pairs used in this invention (such as CB[7] / ferrocene) are artificially designed supramolecular systems that do not exist in natural biological samples. Therefore, when applied to complex biological samples such as cell lysates, serum, and tissue homogenates, non-specific binding caused by endogenous substances can be eliminated, thereby obtaining purification results with extremely low background and single bands, which greatly improves the accuracy, reliability, and reproducibility of interaction studies.

[0024] 3. It enables efficient regeneration and reuse of functionalized solid supports, reducing experimental costs.

[0025] Due to the reversibility of host-guest binding, competitive elution can not only recover the target protein, but also dissociate the guest-tagged decoy molecule from the functionalized solid-phase support. Functionalized solid-phase supports with host molecules (such as CB[7]) on their surface can be used multiple times for a new round of capture-elution cycle after simple regeneration treatment (such as removing free competitive agents).

[0026] 4. It possesses excellent chemical stability and environmental adaptability, making it suitable for a wider range of applications.

[0027] The system constructed in this invention (such as the CB[7] modified carrier) is chemically stable and unaffected by common pH and ionic strength changes in biological samples, and does not have the activity limitation problem similar to streptavidin. Its good water solubility and biocompatibility ensure that it can maintain efficient and stable binding and dissociation performance in a variety of physiological and simulated physiological environments, and has stronger adaptability and robustness to complex biological samples, thus broadening the application potential of this technology under harsh or non-standard experimental conditions.

[0028] 5. It provides a modular, customizable, and flexible platform.

[0029] This invention enables the rapid construction of specific purification systems for different research objectives by replacing different guest marker decoy molecules (small molecules, proteins, nucleic acids, etc.), forming a universal and flexible platform technology.

[0030] In summary, this invention successfully overcomes the core defects of traditional biotin-streptavidin pull-down technology. By introducing reversible host-guest interactions, it achieves elution and maintenance of target protein activity under mild conditions, effectively avoids non-specific interference from endogenous biotin, and enables the functionalized solid-phase carrier to be regenerated and reused, significantly improving the specificity, practicality, and economy of pull-down technology. Attached Figure Description

[0031] Figure 1 This is a flowchart illustrating the preparation process of functionalized solid-phase supports. Figure 2 This is a flowchart of the purification and separation method of the present invention; Figure 3 Characterization diagrams of magnetic beads (MB) and functionalized solid supports (MB@CB[7]); Figure 4 Fluorescence scans for the residual BBR binding with CB[7] by residual titration analysis; Figure 5 The image shows the WB results of the Pull-Down experiment based on biotin-streptavidin and CB[7] host-guest chemistry. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. 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 are within the scope of protection of the present invention.

[0033] This invention provides a reversible affinity purification system, method, kit, and applications based on host-guest interactions. The core of the system and method lies in utilizing the specific, high-affinity, and reversible interaction between the host molecule and the guest molecule to replace the irreversible biotin-streptavidin system in traditional pull-down techniques. This achieves gentle elution of target molecules, reuse of functionalized carriers, and effectively avoids interference from endogenous substances.

[0034] The reversible affinity purification system of the present invention mainly comprises a functionalized solid-phase support and a decoy molecule.

[0035] A functionalized solid-phase carrier is prepared by covalently modifying a host molecule on the surface of a solid matrix using chemical methods; exemplarily, the host molecule is cucurbita[7]urea. Cucurbita[7]urea has a rigid hydrophobic cavity, which can specifically encapsulate specific guest molecules and has good water solubility and biocompatibility.

[0036] The solid matrix can be any carrier commonly found in the art suitable for affinity purification, such as magnetic beads, agarose beads, or resin microspheres. In a preferred embodiment, superparamagnetic magnetic beads are used as the matrix to facilitate rapid separation and washing operations using magnetism.

[0037] Functionalization method: The functionalized solid support can be prepared by a variety of chemical reactions. In a specific example, it is prepared by click chemistry: first, the amino magnetic beads are modified with alkynylation, and then they undergo a cycloaddition reaction with azide-modified cucurbit[7]urea in the presence of a catalyst, thereby covalently and stably fixing cucurbit[7]urea on the surface of the magnetic beads.

[0038] Guest molecule-tagged decoy molecules: The decoy molecule refers to a probe used to capture the target molecule, which is chemically bonded to the guest molecule.

[0039] Guest molecules: The guest molecules refer to molecules that can strongly interact with the host molecules described above. For example, the guest molecules include ferrocene, bisadamantane, bicyclo[2.2.2]octane, 1,4-diazabicyclo[2.2.2]octane, or adamantane derivatives. For example, ferrocene and its derivatives have a very high affinity for cucurbita[7]urea.

[0040] Decoy molecule: The decoy molecule can be any biomolecule whose interacting partner needs to be studied. Exemplarily, the decoy molecule can be a small molecule compound (such as a drug molecule), protein, polypeptide, antibody, or nucleic acid. It forms a "decoy-guest" conjugate by covalently linking to the guest molecule (e.g., via an amino group reacting with an NHS-activated ester).

[0041] The core working mechanism of the system is as follows: the decoy molecule, labeled with the guest molecule, is rapidly and firmly immobilized onto a functionalized solid support through host-guest interactions. When this complex is incubated with a complex biological sample, target molecules in the sample that can specifically bind to the decoy molecule are captured. Finally, by adding a free competitive guest molecule (such as adamantane) to the system, the guest molecule-decoy molecule-target molecule complex can be reversibly dissociated from the support, achieving gentle elution and recovery of the target molecule.

[0042] A method for reversible affinity purification of biomolecules using the above system exemplarily includes the following steps: Decoy immobilization: A solution of decoy molecules linked to guest molecules (such as ferrocene) is co-incubated with a functionalized solid support (such as magnetic beads) with a host molecule (such as cucurbita[7]urea) on its surface. Through host-guest interaction, the decoy molecules are specifically immobilized on the functionalized solid support. Subsequently, washing is performed to remove unbound free decoy molecules.

[0043] Target molecule capture: A functionalized carrier immobilized with bait molecules is mixed and incubated with a biological sample containing potential target molecules (e.g., pretreated cell lysate). During this process, target molecules in the sample that can interact with the bait are specifically captured onto the surface of the functionalized solid carrier. After incubation, thorough washing is performed to remove non-specifically adsorbed contaminating proteins.

[0044] Mild competitive elution: An elution buffer containing a free competitive guest molecule is added to the washed carrier-complex system. Exemplarily, the competitive guest molecule is an adamantane amine compound, such as adamantane methylamine hydrochloride. The concentration of the adamantane amine compound in the elution buffer is, for example, from 0.1 mM to 10 mM, preferably about 1 mM. The free competitive molecule competes with the guest molecule on the immobilized decoy for binding to the host molecule on the carrier, thereby gently eluting the entire decoy-target molecule complex into solution.

[0045] Target molecule collection and analysis: The functionalized solid-phase support is separated, and the eluent containing the purified target molecule is collected. This eluent can be directly used for downstream detection and analysis, such as identification and functional studies by SDS-PAGE, Western blotting, mass spectrometry, or enzyme activity assays.

[0046] Based on the above system and method, the present invention also provides an embodiment in the form of a reagent kit. The reagent kit exemplarily comprises: The aforementioned reversible affinity purification system consists of pre-fabricated functionalized solid-phase supports and decoy molecules labeled with guest molecules. These can be provided as ready-to-use suspensions or lyophilized powders.

[0047] Optionally, the kit may also contain free competing guest molecules (such as an adamantane solution) for the elution step.

[0048] Further, optionally, the kit may also include optimized binding buffer, washing buffer, elution buffer, and instructions for use.

[0049] The system, method, and kit of the present invention can be applied, by way of example, to the following biomolecular interaction research scenarios: Discovery and validation of interactions between small molecule compounds (such as drug candidates) and their target proteins.

[0050] Screening and enrichment of protein-protein interaction complexes.

[0051] Analysis of the interaction between proteins and nucleic acids (such as DNA and RNA).

[0052] To make the objectives, technical solutions, and advantages of this invention clearer, the embodiments of this invention are described in detail below with reference to a specific example. This example uses the isolation and identification of the target protein of the small molecule drug dihydroartemisinin (DHA) as an example to fully demonstrate the construction, application, and verification process of the reversible affinity purification system based on host-guest interaction.

[0053] Example: Reversible affinity purification based on cucurbita[7]urea (CB[7]) / ferrocene host-guest system This embodiment aims to verify the feasibility of the system, method, kit, and application of the present invention, with the traditional biotin-streptavidin system as a control.

[0054] 1. Preparation of materials and reagents The main materials and reagents used are as follows: Control materials: commercially available biotin-labeled dihydroartemisinin (Biotin-DHA) and commercially available biotin-labeled protein Pull-Down kit (containing streptavidin magnetic beads and corresponding buffers).

[0055] Preparation of materials for functionalized solid-phase supports: Main material: Cucurbita[7]urea containing azide-modified cucurbita[7]-N3; Solid matrix: Aminated magnetic beads (particle size 1 μm, concentration 10 mg / mL). Modifying agents: N-hydroxysuccinimide (NHS) activated 6-heptyneic acid, copper sulfate, sodium ascorbate; Blocking agent: Bovine serum albumin (BSA); Guest molecule-tagged decoy molecule: Ferrocene (Fc) modified dihydroartemisinin (Fc-DHA): .

[0056] Competitive guest molecules: adamantane methylamine hydrochloride (AM) and 1,4-phenylenediamine (PXDA) used to verify the regeneration of the carrier.

[0057] Buffer system: NaHCO3 buffer (pH 8.3, 0.1M), PBS buffer (pH 7.4, 10 mM), MES buffer (pH 6, containing 50 mM NaCl, 100 mM), PBST wash buffer (PBS buffer containing 0.05% Tween-20), elution buffer (PBS buffer containing 1 mM amantadine hydrochloride (AM), Tris-HCl buffer (100 mM), EDTA buffer (PBS buffer containing 15 mM EDTA).

[0058] Biological sample: Whole-cell lysate of esophageal squamous cell carcinoma KYSE150.

[0059] Auxiliary reagents: a mixture of protease inhibitors (phenylmethylsulfonyl fluoride, PMSF, final concentration 1 mM; dithiothreitol, DTT, final concentration 5 mM).

[0060] Characterization and detection reagents: berberine hydrochloride (BBR, used for fluorescence characterization), SDS-PAGE electrophoresis reagents, and Western blotting (WB) related reagents (including primary antibody against ENO1, etc.).

[0061] 2. Specific steps and parameters (1) Preparation of functionalized solid supports This step specifically demonstrates the preparation of functionalized solid-phase supports with covalently modified cucurbit[7]urea on the surface via click chemical reaction, as follows: Figure 1 As shown.

[0062] ① Alkyne modification of magnetic beads: Place 100 μL of aminated magnetic beads in a centrifuge tube and wash three times with PBS buffer. After each wash, magnetically adsorb for 30 seconds to remove the supernatant. Resuspend the magnetic beads in 180 μL of PBS buffer, and then add 20 μL of NHS-activated 6-heptanynic acid solution (100 mM concentration, prepared with PBS buffer). Incubate at room temperature in the dark with shaking for 200 minutes (shaking speed 100 rpm). After incubation, add Tris-HCl buffer and incubate at room temperature with shaking for 10 minutes (shaking speed 100 rpm). Repeat this step twice to fully quench the reaction. Magneticly adsorb for 30 seconds to remove the supernatant; wash three times with PBS-T washing buffer, and finally magnetically wash three times with PBS buffer. Resuspend in 100 μL of PBS buffer to complete the alkyne modification of the magnetic beads.

[0063] ② CB[7] modified magnetic beads: Add 50 μL of CB[7]-N3 solution (5 mM concentration, prepared with PBS buffer) and 10 μL of catalyst (freshly prepared by mixing copper sulfate (100 mM) and sodium ascorbate (100 mM) in a volume ratio of 1:2) to the above alkynylated magnetic bead system. Incubate at room temperature in the dark with shaking for 4 hours (shaking speed 80 r / min) to achieve covalent fixation of CB[7] through click chemical reaction.

[0064] ③ Blocking and purification: After incubation, the magnetic beads were separated by magnetic adsorption, washed three times with EDTA buffer, and then washed three times with PBS buffer. 100 μL of PBS buffer containing 1% bovine serum albumin (BSA) was added, and the beads were blocked by shaking at room temperature for 30 minutes to block non-specific sites. Finally, the beads were washed twice with PBS buffer, resuspended in 100 μL of PBS buffer, and stored at 4°C for later use. CB[7] functionalized magnetic beads, i.e., functionalized solid-phase carriers, were obtained.

[0065] (2) Decoy targeting fixation ① Incubation and binding: Take 50 μL of the CB[7] functionalized magnetic beads prepared above, add 50 μL of Fc-DHA solution (5 mM), and then add 100 μL of PBS buffer. Incubate at room temperature with shaking for 1 hour (shaking speed 50 r / min). The decoy molecules marked by guest molecules are fixed through the host-guest interaction of CB[7]-Fc.

[0066] ② Washing and purification: The magnetic beads were separated by magnetic adsorption and washed three times with PBST washing buffer. After each wash, the adsorption and separation was carried out for 30 seconds to remove unbound free decoy molecules and obtain functionalized magnetic beads with fixed decoy molecules.

[0067] (3) Capture and enrichment of target proteins ① Sample processing: Take 1 mL of lysate of esophageal cancer KYSE150 cells containing potential target proteins, add a mixture of protease inhibitors (to make the final concentration of PMSF 1 mM and DTT 5 mM), centrifuge at 12000 r / min for 10 minutes at 4℃, and take the supernatant for later use.

[0068] ② Incubation and capture: Add the supernatant of the treated cell lysate to the magnetic bead system immobilized with the bait molecules, and incubate at 4°C with shaking for 2 hours (shaking speed 50 r / min) to allow the bait molecules to form a complex with the target protein.

[0069] ③ Removal of contaminating proteins: Magnetic beads are separated by magnetic adsorption and washed 5 times with PBST washing buffer. After each wash, the beads are separated by adsorption for 30 seconds to completely remove unbound contaminating proteins.

[0070] (4) Gentle elution and collection of target proteins ① Reversible elution: Add 100 μL of elution buffer (containing 1 mM AM) to the washed magnetic bead system and incubate at room temperature with shaking for 10 minutes (shaking speed 80 r / min). The decoy molecule-target molecule complex is reversibly dissociated by the competitive binding of free competitive guest molecule AM ​​to CB[7] on the surface of the magnetic beads.

[0071] ② Collection and Detection: Magnetic beads are separated by magnetic adsorption, and the supernatant (containing the eluted target protein) is collected. This eluent can be directly used for SDS-PAGE electrophoresis to detect the purity of the target protein, and Western blotting to identify the types of target proteins.

[0072] ③ Magnetic bead regeneration: To restore the binding capacity of the functionalized solid-phase support for reuse, the magnetic beads after collecting the supernatant can be thoroughly eluted with a high concentration of competing guest molecules (e.g., 1,4-phenylenediamine (PXDA)) solution. After regeneration and washing, the CB[7] functionalized magnetic beads can be reused in subsequent affinity purification cycles.

[0073] The flowcharts of the above purification system and purification separation method are as follows: Figure 2 As shown.

[0074] 3. System characterization and effect verification experiments and data (1) Characterization of CB[7] functionalized magnetic beads Scanning electron microscopy (SEM), dynamic light scattering (DLS), zeta potential analysis, and fluorescence scanning analysis of CB[7] and berberine (BBR) confirmed the successful preparation of CB[7] functionalized magnetic beads.

[0075] ① Morphology and size characterization of CB[7] functionalized magnetic beads The SEM, DLS, and zeta potential analyses revealed changes in the morphology and size of unmodified and CB[7] modified magnetic beads. Figure 3 As shown, the specific observations and results are as follows: First, it can be observed that the morphology of the CB[7] modified magnetic beads (MB@CB[7]) is basically similar to that of the unmodified magnetic beads (MB), and the surface of MB@CB[7] is slightly shiny; by DLS, it was found that the diameter of MB is about 1033nm, while the size of MB@CB[7] is slightly larger by 1083nm; zeta potential analysis showed that MB was -7.72 mV, and the potential of MB@CB[7] was more negative at -13.29 mV.

[0076] ② Quantitative loading by fluorescence scanning: The loading of CB[7] was calculated by residual titration. Since berberine hydrochloride (BBR) has almost no fluorescence emission in aqueous solution, but its emission increases by about 500 times after being encapsulated with CB[7] in water through host-guest reaction, BBR is used as a fluorescent indicator to confirm the presence of CB[7] on magnetic beads.

[0077] The experiment was divided into three groups: Group A (CB[7] magnetic beads), Group B (unmodified amino magnetic beads), and Group C (standard control group with only BBR). 0.25 mg of magnetic beads were added to the experimental and control groups along with 100 μL of BBR (50 μM) solution, while the standard control group only received 100 μL of BBR (50 μM) solution. After sufficient reaction in the dark, 50 μL of the supernatant from each group was aspirated and 50 μL of CB[7] (200 μM) was added. After sufficient reaction, the fluorescence intensity was measured. Figure 4 As shown.

[0078] In the experiment, a known amount of BBR specifically binds to CB[7] immobilized on the magnetic beads, and the amount of BBR remaining in the supernatant after the reaction is inversely proportional to the loading of CB[7] on the magnetic beads. By setting up a control group B (unmodified magnetic beads), the non-specific adsorption background of BBR on the magnetic bead matrix was eliminated; group C provided a baseline for the initial total amount of BBR. By measuring the difference in fluorescence intensity of the remaining BBR in the supernatants of the three groups (group B value - group A value), the number of CB[7] molecules covalently bound per unit mass of magnetic beads, i.e., the loading (7.20 nmol / mg), can be accurately calculated. This data directly proves the successful and efficient immobilization of CB[7] on the surface of the support.

[0079] (2) Western Blot (WB) verification of compound Pull-Down experiment To verify the feasibility of the subject-guest interaction pull-down scheme of the present invention, a drug-target protein pair with known interactions was selected for a control experiment. Dihydroartemisinin (DHA) is an effective anticancer drug for esophageal squamous cell carcinoma KYSE150. Through DHA pull-down proteomics, it was demonstrated that ENO1 protein is one of the binding target proteins of DHA. This experiment was conducted by comparing the effects of commercially available biotin-labeled DHA (Biotin-DHA) with the traditional streptavidin system, and the ferrocene (Fc)-labeled DHA (Fc-DHA) and CB[7] magnetic bead system of the present invention on enriching ENO1 protein in KYSE150 cell lysate.

[0080] Experimental grouping and operating procedures: Control group (Biotin-Streptavidin technology): Strictly follow the instructions for the commercial Pull-Down kit.

[0081] ① Take 50 μL of streptavidin magnetic beads, wash them 3 times with washing buffer, and separate them by magnetic adsorption; ② Add 50 μL of commercially available biotin-labeled dihydroartemisinin (Biotin-DHA) solution and incubate at room temperature with shaking for 1 hour (50 r / min) to fix the bait; ③ Add KYSE150 cell lysis buffer and incubate at 4°C with shaking for 2 hours (50 r / min) to capture target proteins; ④ Elute the target protein using the strong denaturing buffer provided with the kit, following the kit instructions; ⑤ Collect the eluent and subject it to high-temperature denaturation treatment.

[0082] ⑥ Perform SDS-PAGE and Western Blot (WB) to verify the protein bands. This experiment was repeated twice in parallel.

[0083] Experimental group (FC-CB[7] subject-object technology): using the system and method described in this invention.

[0084] ① Take 50 μL of the prepared CB[7] functionalized magnetic beads, wash them 3 times with washing buffer, and separate them by magnetic adsorption for later use; ② Fc-labeled DHA (Fc-DHA) and CB[7] functionalized magnetic beads were incubated at room temperature for 1 hour (50 r / min) to achieve targeted fixation of the decoy; ③ Add KYSE150 cell lysis buffer and incubate at 4°C with shaking for 2 hours (50 r / min) to capture target proteins; ④ Perform gentle elution: Add PBS buffer containing 1 mmol / L amantadine hydrochloride (AM) and incubate at room temperature with shaking for 30 minutes; ⑤ Collect the eluent and subject it to high-temperature denaturation treatment; ⑥ Perform SDS-PAGE and Western Blot (WB) to verify the protein bands.

[0085] In addition, to verify the reproducibility of the carrier, the magnetic beads were subsequently eluted and regenerated using a high-concentration 1,4-phenylenediamine (PXDA) solution.

[0086] Experimental results ( Figure 5 ): WB results as follows Figure 5 As shown, the lanes from left to right are: A: streptavidin magnetic beads + biotin; B: streptavidin magnetic beads + biotin-DHA; C: CB[7] functionalized magnetic beads + Fc; D: CB[7] functionalized magnetic beads + Fc-DHA.

[0087] Results analysis: Compared with commercially available biotin-labeled dihydroartemisinin and its matching kit (control group, lane B), the ENO1 protein bands enriched by the DHA-Fc-CB[7] magnetic bead system constructed in this invention (experimental group, lane D) were consistent in position, more uniform in band, and had a clearer background, indicating that its separation effect was better and its specificity was stronger. This result directly verifies the feasibility of the host-guest interaction Pull-Down scheme described in this invention. At the same time, effective elution of the target protein complex can be achieved simply by adding a free competitive guest (AM), and the CB[7] functionalized magnetic beads can be regenerated by the competitive agent, which has the advantage of repeated use.

[0088] The target protein capture efficiency of the system and method of this invention is comparable to that of the traditional biotin-streptavidin technology, which can reliably meet the basic requirements for screening small molecule-protein interactions. Moreover, the mild competitive elution method adopted in this invention can achieve the dissociation of the complex under physiologically compatible conditions, thereby maximizing the preservation of the native conformation and biological activity of the target protein, providing a key guarantee for its subsequent functional verification and in-depth research.

[0089] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0090] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A reversible affinity purification system based on host-guest interactions, characterized in that, include: (a) A functionalized solid support whose surface is covalently modified with host molecules; (b) A decoy molecule, wherein a guest molecule is attached to the decoy molecule; The guest molecule and the host molecule can undergo specific and reversible host-guest interactions, thereby immobilizing the decoy molecule on the functionalized solid-phase support; and the binding between the guest molecule and the host molecule can be reversibly dissociated by the addition of free competing guest molecules, thereby achieving the elution of the decoy molecule or its complex from the functionalized solid-phase support.

2. The reversible affinity purification system based on host-guest interaction according to claim 1, characterized in that, The host molecule is cucurbit[7]urea, and the guest molecule includes ferrocene, bisadamantane, bicyclo[2.2.2]octane, 1,4-diazabicyclo[2.2.2]octane or adamantane derivatives.

3. The reversible affinity purification system based on host-guest interaction according to claim 1 or 2, characterized in that, The functionalized solid support was prepared by covalently linking azide-modified cucurbit[7]urea to the surface of an alkynyl-modified solid matrix via a click chemistry reaction.

4. The reversible affinity purification system based on host-guest interaction according to claim 1, characterized in that, The decoy molecule is a small molecule compound, protein, polypeptide, antibody, or nucleic acid.

5. The reversible affinity purification system based on host-guest interaction according to claim 1, characterized in that, The solid matrix in the functionalized solid support is magnetic beads.

6. A reversible affinity purification method for biomolecules, used to separate target molecules that interact with decoy molecules, characterized in that, The method uses the reversible affinity purification system according to any one of claims 1 to 5 and includes the following steps: (1) Decoy molecules connected with guest molecules are fixed onto a functionalized solid support with host molecules on its surface through host-guest interactions; (2) To bring immobilized decoy molecules into contact with biological samples containing potentially interacting target molecules; (3) Separate and wash the functionalized solid support; (4) The decoy molecule-target molecule complex is eluted from the functionalized solid support by adding free competitive guest molecules, thereby obtaining the purified target molecule.

7. The reversible affinity purification method according to claim 6, characterized in that, In step (4), the free competitive guest molecule is an adamantane compound, and its concentration in the eluent is 0.1 mM to 10 mM.

8. A reagent kit, characterized in that, The reversible affinity purification system comprising any one of claims 1 to 5.

9. The reagent kit according to claim 8, characterized in that, It also includes free competing object molecules.

10. The application of the reversible affinity purification system of any one of claims 1 to 5, or the reversible affinity purification method of any one of claims 6 to 7, or the kit of any one of claims 8 to 9 in the study of biomolecular interactions.

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