A mitochondrial membrane chromatography column for immobilizing Twins Strep-Tag fusion protein, preparation method and application thereof

Abstract

The present invention provides a mitochondrial membrane chromatography column, preparation method and application of an immobilized TwinsStrep-Tag fusion protein, and belongs to the technical field of mitochondrial membrane chromatography column preparation. The method first bonds a Strep-Tactin protein containing cysteine ​​to silica gel, takes the TwinsStrep-tag fusion protein receptor on the mitochondrial membrane and specifically binds to the above-mentioned Strep-Tactin protein containing cysteine ​​to form a stable affinity system, fixes the Twins Strep-tag fusion protein on the silica gel surface, obtains a mitochondrial membrane chromatography stationary phase, and adopts a wet column packing method to prepare a mitochondrial membrane chromatography column of an immobilized TwinsStrep-Tag fusion protein. The mitochondrial membrane chromatography column prepared by the method of the present invention can target specific proteins on the mitochondrial membrane, and screen small molecule compounds that interact with the target protein from complex components with high throughput.

Description

Technical Field

[0001] The present invention belongs to the technical field of mitochondrial membrane chromatography column preparation, and specifically relates to an affinity-type mitochondrial membrane chromatography column based on immobilized Twins Strep-Tag fusion protein of Strep-Tactin (containing cysteine), and a preparation method and application thereof. Background Art

[0002] Compared with cell membrane chromatography, mitochondrial membrane chromatography can further target subcellular organelles, reducing the impact of other subcellular organelle membranes on experimental results, making drug screening more specific and mechanistic research clearer. It can quickly screen out mitochondrial regulatory molecules from complex components. However, existing research on mitochondrial membrane chromatography is relatively small and has certain problems, mainly reflected in the following aspects: First, the preparation process is complicated, tedious, and time-consuming, requiring complex equipment to extract mitochondria with high purity; second, the specificity is poor. There are many protein targets on the inner and outer mitochondrial membranes, which cannot indicate the specific mitochondrial protein targeted by the screened drug, and the mechanism is unclear, which also brings new problems to the pharmacological research of the active ingredients screened later; third, chemical bonding may destroy the protein structure; fourth, only the binding ability of mitochondrial membrane receptors with some known positive drugs and the performance of membrane proteins have been studied, and the binding ability is poor. Adsorption cannot maintain the stability of membrane proteins, and it cannot be used for high-throughput screening of drugs targeting mitochondrial membrane proteins.

[0003] The specific affinity between the Strep-Tactin recombinant protein and the Twins Strep-Tag is one of the strongest non-covalent interactions in nature. The Twins Strep-Tag is a protein fusion tag composed of 18 amino acids. Its small molecular weight barely affects the native protein structure. Because the specific binding between the Twins Strep-Tag and Strep-Tactin is reversible, it is commonly used for purification of recombinant proteins. The present invention uses the Twins Strep-Tag to link the mitochondrial membrane to the aminopropyl stationary phase, maximizing the protein's biological activity and addressing the weak physical adsorption binding between the mitochondrial membrane and the stationary matrix.

[0004] Therefore, since there are currently no reports on the transmembrane protein receptor immobilization technology of Twins Strep-Tag and Strep-Tactin recombinant proteins, in order to provide an efficient and stable mitochondrial membrane chromatography stationary phase, a method for preparing an affinity mitochondrial membrane chromatography column based on immobilized Twins Strep-Tag fusion protein with Strep-Tactin (containing cysteine) was constructed. Summary of the Invention

[0005] In response to the problems existing in the above-mentioned prior art, the purpose of the present invention is to design and provide an affinity-type mitochondrial membrane chromatography column based on immobilized Twins Strep-Tag fusion protein of Strep-Tactin (containing cysteine), as well as its preparation method and application. The mitochondrial membrane chromatography column has good stability and strong specificity, and the preparation method is simple to operate and easy to implement.

[0006] The present invention first bonds 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester to aminopropyl silica gel as a linker for bonding Strep-Tactin (containing cysteine) to the silica gel. Then, the Twins Strep-Tag fusion receptor on the mitochondrial membrane can specifically bind to the substrate by affinity, thereby indirectly fixing the fusion protein on the silica gel surface to prepare a mitochondrial membrane stationary phase. The column is then loaded into a chromatographic column by a wet method to form a mitochondrial membrane chromatographic column based on immobilized Strep-Tactin fusion protein. The mitochondrial membrane chromatographic column of the immobilized Strep-Tactin fusion protein has high stability and strong specificity. The problem of poor specificity of existing mitochondrial membrane chromatographic columns is solved.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] On the one hand, the present invention provides a mitochondrial membrane chromatography stationary phase for immobilizing Twins Strep-Tag fusion protein, wherein the mitochondrial membrane chromatography stationary phase is obtained by bonding a Strep-Tactin protein containing cysteine ​​to silica gel, and taking the Twins Strep-tag fusion protein receptor on the mitochondrial membrane to specifically bind to the above-mentioned Strep-Tactin protein containing cysteine, so that the Twins Strep-tag fusion protein is fixed on the silica gel surface to obtain a mitochondrial membrane chromatography stationary phase.

[0009] In the mitochondrial membrane chromatography stationary phase for immobilizing Twins Strep-Tag fusion protein, the silica gel is aminopropyl silica gel; the Strep-Tactin protein containing cysteine ​​is chemically bonded to the silica gel through 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester.

[0010] In a second aspect, the present invention provides a mitochondrial membrane chromatography column immobilized with Twins Strep-Tag fusion protein, wherein the mitochondrial membrane chromatography column is prepared by wet column packing of the mitochondrial membrane chromatography stationary phase based on the immobilized fusion protein of Strep-Tactin.

[0011] In a third aspect, the present invention provides a method for preparing a mitochondrial membrane chromatography stationary phase for immobilizing Twins Strep-Tag fusion protein as described in any one of the preceding claims, comprising the following steps:

[0012] (1) Weighing aminopropyl silica gel and activating it; weighing 4-dimethylaminopyridine and 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester and dissolving them in a dry N,N-dimethylformamide solution to obtain a solution;

[0013] (2) Add the activated aminopropyl silica gel to the solution, stir the reaction thoroughly at room temperature in the dark, collect the precipitate after centrifugation, wash and dry to obtain a silica gel stationary phase modified with 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester;

[0014] (3) Weigh the Strep-Tactin containing cysteine, wash it with potassium phosphate buffer, and dissolve it in potassium phosphate buffer to prepare a suspension. Add the above-mentioned 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester modified silica gel stationary phase, stir in the dark, and react fully to obtain the Strep-Tactin modified silica gel stationary phase containing cysteine;

[0015] (4) Cultivate Twins Strep-Tag fusion protein high-expressing cell lines. When the cell count is not less than 10 7 After 1 hour, the culture medium was removed to obtain Twins Strep-Tag fusion protein cells and mitochondria were isolated;

[0016] (5) Ultrasonic disruption of the mitochondria to prepare a mitochondrial membrane suspension, which was added to the cysteine-containing Strep-Tactin-modified silica gel stationary phase obtained in step (3) above. After uniform stirring for 0.5 to 1 h, a mitochondrial membrane stationary phase with the cysteine-containing Strep-Tactin-modified silica gel as a carrier was obtained.

[0017] The preparation method is characterized in that the activation conditions in step (1) include: drying in an oven at 80-120°C for 12-24 hours; the mass ratio of the added 4-dimethylaminopyridine, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester and the activated aminopropyl silica gel is 12:25:400-12:25:100;

[0018] The stirring reaction time in step (2) is 12 to 24 hours.

[0019] The preparation method is characterized in that the mass ratio of the cysteine-containing Strep-Tactin to the silica gel stationary phase modified with 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester in step (3) is 1:10~2:5; the light-proof stirring conditions are: temperature 4°C, time 30~40h; the pH of the potassium phosphate buffer solution is 7.0, and the concentration is 1.3mol / L.

[0020] In the preparation method, the specific step of isolating mitochondria in step (4) includes: suspending the obtained TwinsStrep-Tag fusion protein cells with a hypotonic buffer, incubating on ice, disrupting them, and isolating mitochondria by differential centrifugation in the presence of a mitochondrial separation medium.

[0021] In the preparation method, the hypotonic buffer is a mixed solution of 10 mmol / L Tris base, 10 mmol / L sodium chloride, and 2.5 mmol / L magnesium chloride; and the mitochondrial separation medium is 10 mmol / L Tris base, 250 mmol / L sucrose, and 1 mmol / L EDTA disodium salt.

[0022] The application of the mitochondrial membrane chromatography stationary phase of the immobilized Twins Strep-Tag fusion protein or the mitochondrial membrane chromatography column of the immobilized Twins Strep-Tag fusion protein in screening active pharmaceutical ingredients.

[0023] The application of the mitochondrial membrane chromatography stationary phase for immobilizing Twins Strep-Tag fusion protein or the mitochondrial membrane chromatography column for immobilizing Twins Strep-Tag fusion protein in screening regulatory molecules targeting Twins Strep-Tag fusion protein.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The affinity-type mitochondrial membrane chromatography column based on the Twins Strep-Tag fusion protein containing cysteine ​​Strep-Tactin disclosed in the present invention covalently bonds Strep-Tactin (containing cysteine) to silica gel, so that the Twins Strep-Tag fusion receptor on the mitochondrial membrane can specifically interact with the substrate to form a stable affinity system, thereby indirectly fixing the Twins Strep-Tag fusion protein on the silica gel surface and enriching the mitochondrial membrane from the mixed system on the silica gel surface.

[0026] On the one hand, the present invention can achieve the fixation of mitochondrial membrane on silica gel in a stable affinity binding manner, thereby improving the stability of the mitochondrial membrane chromatography column. On the other hand, due to the fusion of the target protein and Twins Strep-Tag, it can specifically interact with the substrate Strep-Tactin, that is, only mitochondrial membrane target receptors are present on the mitochondrial membrane chromatography column, without interference from other membrane receptors, which greatly improves its specificity. The mitochondrial membrane chromatography column of the present invention can target specific proteins on the mitochondrial membrane, and based on the specific binding between the target protein and the active effector molecule, it can high-throughput screen small molecule compounds that interact with the target protein from complex components, and is suitable for the screening of effector molecules and drug molecules for specific proteins on the mitochondrial targeting membrane. At the same time, the preparation method is simple to operate and easy to implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Flow chart for the preparation of affinity mitochondrial membrane chromatography columns based on the cysteine-containing Strep-Tactin Twins Strep-Tag fusion protein;

[0028] Figure 2 These are the infrared spectra of aminopropyl silica gel (NH2-SiO2), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester modified silica gel (SMCC-SiO2), and Strep-Tactin (containing cysteine) modified silica gel (Strep-Tactin-SiO2);

[0029] Figure 3 These are the X-ray photoelectron spectra of aminopropyl-modified silica gel (NH2-SiO2), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester-modified silica gel (SMCC-SiO2), and Strep-Tactin (containing cysteine)-modified silica gel (Strep-Tactin-SiO2). DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0031] The terms "comprise," "comprises," 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 elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, product, or apparatus.

[0032] Example 1: Preparation of a mitochondrial membrane chromatography column immobilized with Twins Strep-Tag fusion protein

[0033] The flow chart of the preparation of affinity mitochondrial membrane chromatography column based on the Twins Strep-Tag fusion protein containing cysteine ​​Strep-Tactin of the present invention is as follows: Figure 1 As shown, the preparation method of the immobilized Twins Strep-Tag fusion protein mitochondrial membrane chromatography column comprises the following steps:

[0034] 1) Activation of aminopropyl silica stationary phase

[0035] Weigh 400 mg of macroporous aminopropyl silica gel and dry it at 100° C. for 24 h to obtain activated aminopropyl silica gel for later use.

[0036] 2) Preparation of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester bonded aminopropyl silica gel stationary phase

[0037] First, 12 mg of 4-dimethylaminopyridine and 25 mg of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester were dissolved in dry N,N-dimethylformamide solution, and then 400 mg of activated aminopropyl silica gel was gradually added. The mixture was stirred in the dark at room temperature for 24 hours, centrifuged at 1000×g for 5 minutes, and the precipitate was collected and washed 5 times with dry N,N-dimethylformamide solution. It was dried at 100°C for 24 hours to obtain 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester bonded to aminopropyl silica gel stationary phase.

[0038] 3) Preparation of Strep-Tactin (containing cysteine) bonded aminopropyl silica stationary phase

[0039] Weigh 50 mg of aminopropyl silica gel modified with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and wash three times with pH 7.0 potassium phosphate buffer. Add 5 mg of Strep-Tactin (containing cysteine) dissolved in 1.3 mol / L potassium phosphate buffer at pH 7.0, and stir in the dark at 4°C for 40 h to obtain a Strep-Tactin-modified aminopropyl silica gel stationary phase.

[0040] Infrared spectroscopy and X-ray photoelectron spectroscopy were used to characterize the aminopropyl chromatographic stationary phase, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester modified silica gel stationary phase, and Strep-Tactin modified silica gel stationary phase. Figure 2 The infrared spectra of aminopropyl silica gel (NH2-SiO2), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester modified silica gel (SMCC-SiO2), and Strep-Tactin (containing cysteine) modified silica gel (Strep-Tactin-SiO2). Among them, the infrared spectrum of SMCC-SiO2 is at 1760 cm -1 The carbonyl vibration absorption peak appeared, indicating that 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester has been successfully bonded to the aminopropyl silica gel chromatographic stationary phase. -1 The methyl groups that appear correspond to the Strep-Tactin protein.

[0041] In order to further verify the prepared Strep-Tactin bonded silica gel stationary phase, X-ray photoelectron spectroscopy was used to verify it. The X-ray photoelectron spectrum peak can reflect the proportion of the elements contained. Figure 3 The following are X-ray photoelectron spectra of aminopropyl silica gel (NH2-SiO2), silica gel modified with succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC-SiO2), and silica gel modified with Strep-Tactin (containing cysteine) (Strep-Tactin-SiO2). In the XPS spectra, the nitrogen content increases from NH2-SiO2 to SMCC-SiO2 and then to Strep-Tactin-SiO2, indicating that succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate and Strep-Tactin are bonded to the silica gel. The appearance of the characteristic S signal of Strep-Tactin-SiO2 further confirms that Strep-Tactin is bonded to the silica gel.

[0042] 4) Preparation of mitochondrial membrane chromatography stationary phase

[0043] Construction of lentivirus containing the Twins Strep-Tag-CPT1A gene: HEK293 cells were infected with lentiviral plasmid particles containing the Twins Strep-Tag-CPT1A gene, and the viral fluid was collected. Briefly, HEK293T cells were cultured routinely and seeded into 6 cm culture dishes to ensure that the cell density reached 80% the next day. At this point, add 2 μg each of △8.9, VSVG, and pLV[Exp]-Puro-EF1A>Twins Strep-Tag-hCPT1A:T2A:EGFP plasmids to a sterile 1.5ml EP tube. Add 36 μL of 1 mg / mL PEI solution and 500 μL of Opti-MEM® medium. Mix thoroughly and let stand for 15 minutes. Add the mixture dropwise to the monolayer cell culture dish containing 3 mL of complete medium. Gently shake to mix thoroughly and incubate in a 37°C incubator with 5% CO2. 6-8 hours after transfection, replace the medium, add 3 mL of complete medium, and continue incubating the cells in the incubator. Collect the supernatants at 48 and 72 hours, centrifuge at 1500 rpm for 5 minutes, and collect the virus-containing supernatant.

[0044] Construction of recombinant cells expressing Twins Strep-Tag-CPT1A: HEK293 cells were infected with the virus-containing supernatant prepared as described above, and recombinant cells expressing Twins Strep-Tag-CPT1A were stably obtained by Puromycin selection. Briefly, HEK293 cells were seeded into 6-well plates (ensuring a cell density of approximately 30%-40% the next day before infection). After the cells adhered, the medium was aspirated and 1.5 mL of complete medium, 1.5 mL of the virus-containing supernatant prepared as described above, and 3 μL of 10 μg / μl polybrene were added. Fresh culture medium was replaced 24 hours after infection. Green fluorescence expression was observed under an inverted fluorescence microscope 48-96 hours after infection to estimate the efficiency of lentiviral infection of HEK293 cells. When the infection efficiency reached 60%-80%, 1:5000 Puromycin (10 mg / ml) was added for drug screening. At the same time, a HEK293 cell control dish was set up and 1:5000 Puromycin (10 mg / ml) was added as a control for whether Puromycin is effective. Puromycin-containing culture medium was replaced every other day to replace the culture medium containing a large number of dead cells until the resistant colony could be identified. The positive Twins Strep-Tag-CPT1A-HEK293 cells with the Puromycin resistance gene were cultured, subcultured, and frozen for subsequent experiments after successful identification.

[0045] Culture the Twins Strep-Tag fusion protein high-expressing cell line, and when the cell count is not less than 10 7 After the cells are separated, they are centrifuged at 1000g for 5 minutes at 4°C after being blown off. The culture medium is removed and the precipitate is taken. 10mmol / L PBS buffer is added to resuspend the mixture and centrifuged at 1000g for 5 minutes at 4°C. The culture medium remaining on the cell surface is aspirated and repeated three times to separate the cultured cells. The cells are suspended in 2mL of hypotonic buffer solution and incubated on ice for 10 minutes. After the cells swell, the plasma cell suspension is transferred to a Dounce glass homogenizer and the pestle is pulverized up and down 40 times to break the cells and release the organelles. Then, 2.5× isotonic buffer is added to make the solution 1× isotonic solution and mixed thoroughly to neutralize the hypotonic buffer solution. The homogenized sample was centrifuged at 1300g for 10 min at 4°C, and the supernatant was centrifuged at 17000g for 15 min at 4°C. The resulting precipitate was resuspended in 1× isotonic buffer and centrifuged at 10000g for 10 min at 4°C. The resulting precipitate was the mitochondria. The collected mitochondrial precipitate was resuspended in 10mmol / L PBS and ultrasonically disrupted to obtain a mitochondrial membrane suspension. The mitochondrial membrane suspension was then added to 50mg of Strep-Tactin-modified silica stationary phase, placed on a magnetic stirrer and stirred at 4°C for 1h. The mitochondrial membrane was fixed on the surface of the Strep-Tactin-modified silica stationary phase by utilizing the high specific recognition and stable affinity of Twins Strep-Tag and its specific binding substrate Strep-Tactin to obtain a mitochondrial chromatographic stationary phase.

[0046] 5) Establishment of mitochondrial membrane chromatography column

[0047] The obtained mitochondrial membrane chromatography stationary phase of immobilized Twins Strep-Tag fusion protein was wet-packed into a 10 mm (L) × 2.0 mm (ID) column core using an RPL-ZD10 column packing machine to obtain a mitochondrial membrane chromatography column based on immobilized Twins Strep-Tag fusion protein.

[0048] Example 2: Verification of the effect of mitochondrial membrane chromatography column based on immobilized Twins Strep-Tag fusion protein

[0049] Intra- and inter-column reproducibility were primarily measured using the retention time of the CPT1A agonist baicalin on a mitochondrial membrane chromatography column. Column lifetime was measured using the half-life of the baicalin retention time decay curve. A CPT1A-Strep Tactin-Tag affinity mitochondrial membrane chromatography column was prepared and placed in a liquid chromatograph. After full equilibration, 10 μL of a 10 mmol / L baicalin sample was injected five times, and the retention time for each injection was recorded. Inter-column variability was measured by simultaneously preparing three CPT1A-Strep Tactin-Tag affinity mitochondrial membrane chromatography columns using the same method. After full equilibration under the same chromatographic conditions, 10 μL of a 10 mmol / L baicalin sample was injected, and the retention time for each column was recorded. Column lifetime experiments were performed using a single CPT1A-Strep Tactin-Tag affinity mitochondrial membrane chromatography column. Samples were injected daily and recorded until the retention time decayed. The results showed that the intra-column reproducibility for five retention time runs was ≤3% RSD, and the inter-column RSD was ≤4%. The half-life of the activity retained on a single column was 7 days. These results demonstrate that the CPT1A-Strep Tactin-Tag affinity mitochondrial membrane chromatography column has excellent reproducibility, meets analytical requirements, and maintains biological activity for a long time.

[0050] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A mitochondrial membrane chromatography stationary phase for immobilizing Twins Strep-Tag fusion protein, characterized in that: The mitochondrial membrane chromatography stationary phase is obtained by bonding the Strep-Tactin protein containing cysteine ​​to silica gel, taking the Twins Strep-tag fusion protein receptor on the mitochondrial membrane to specifically bind to the Strep-Tactin protein containing cysteine ​​to form a stable affinity system, so that the Twins Strep-tag fusion protein is fixed on the silica gel surface to obtain the mitochondrial membrane chromatography stationary phase; The silica gel is aminopropyl silica gel; the Strep-Tactin protein containing cysteine ​​is chemically bonded to the silica gel through 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester; The preparation method of mitochondrial membrane chromatography stationary phase comprises the following steps: (1) Weighing aminopropyl silica gel and activating it; weighing 4-dimethylaminopyridine and 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester and dissolving them in a dry N,N-dimethylformamide solution to obtain a solution; (2) Add the activated aminopropyl silica gel to the solution, stir the reaction thoroughly at room temperature in the dark, collect the precipitate after centrifugation, wash and dry to obtain a silica gel stationary phase modified with 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester; (3) Weigh the Strep-Tactin containing cysteine, wash it with potassium phosphate buffer, and dissolve it in potassium phosphate buffer to prepare a suspension. Add the above-mentioned 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester modified silica gel stationary phase, stir in the dark, and react fully to obtain the Strep-Tactin modified silica gel stationary phase containing cysteine; (4) Cultivate Twins Strep-Tag fusion protein high-expressing cell lines. When the cell count is not less than 10 7 After 1 hour, the culture medium was removed to obtain Twins Strep-Tag fusion protein cells and mitochondria were isolated; (5) Ultrasonic disruption of the mitochondria to prepare a mitochondrial membrane suspension, which was added to the cysteine-containing Strep-Tactin-modified silica gel stationary phase obtained in step (3) above. After uniform stirring for 0.5 to 1 h, a mitochondrial membrane stationary phase with the cysteine-containing Strep-Tactin-modified silica gel as a carrier was obtained.

2. The mitochondrial membrane chromatography stationary phase for immobilizing Twins Strep-Tag fusion protein according to claim 1, characterized in that: The activation conditions in step (1) include: drying in an oven at 80-120° C. for 12-24 hours; the mass ratio of 4-dimethylaminopyridine, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester, and activated aminopropyl silica gel is 12:25:400-12:25:100; The stirring reaction time in step (2) is 12 to 24 hours.

3. The mitochondrial membrane chromatography stationary phase for immobilizing Twins Strep-Tag fusion protein according to claim 1, characterized in that: The mass ratio of the cysteine-containing Strep-Tactin to the silica gel stationary phase modified with 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester in step (3) is 1:10 to 2:5; the light-proof stirring conditions are: temperature 4°C, time 30 to 40 hours; the pH of the potassium phosphate buffer solution is 7.0, and the concentration is 1.3 mol / L.

4. The mitochondrial membrane chromatography stationary phase for immobilizing Twins Strep-Tag fusion protein according to claim 1, characterized in that: The specific steps of isolating mitochondria in step (4) include: suspending the obtained Twins Strep-Tag fusion protein cells with hypotonic buffer, incubating on ice, disrupting them, and isolating mitochondria by differential centrifugation in the presence of mitochondrial separation medium.

5. The mitochondrial membrane chromatography stationary phase for immobilizing Twins Strep-Tag fusion protein according to claim 4, characterized in that: The hypotonic buffer solution is a mixed solution of 10 mmol / L Tris base, 10 mmol / L sodium chloride, and 2.5 mmol / L magnesium chloride; the mitochondrial separation medium is 10 mmol / L Tris base, 250 mmol / L sucrose, and 1 mmol / L EDTA disodium salt.

6. A mitochondrial membrane chromatography column immobilized with Twins Strep-Tag fusion protein, characterized in that: The mitochondrial membrane chromatography column is prepared by packing the mitochondrial membrane chromatography stationary phase of the immobilized Twins Strep-Tag fusion protein according to claim 1 using a wet column method.

7. Use of the mitochondrial membrane chromatography stationary phase immobilized with Twins Strep-Tag fusion protein according to claim 1, or the mitochondrial membrane chromatography column immobilized with Twins Strep-Tag fusion protein according to claim 6, in screening active pharmaceutical ingredients.

8. Use of the mitochondrial membrane chromatography stationary phase immobilized with Twins Strep-Tag fusion protein according to claim 1, or the mitochondrial membrane chromatography column immobilized with Twins Strep-Tag fusion protein according to claim 6, in screening effector molecules targeting mitochondria.

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