An affinity biochromatographic column for in situ synthesis of membrane proteins, its preparation method and application

Abstract

This invention relates to the field of biochromatography, specifically to an in-situ synthetic membrane protein affinity biochromatographic column, its preparation method, and its applications. The method employs cell-free protein expression and covalent immobilization affinity chromatography techniques to construct a novel in-situ synthetic membrane protein biochromatographic column. This invention enables, for the first time, the unidirectional insertion of a single membrane protein with a natural quaternary structure onto a stationary phase bound to liposomes. This invention eliminates the need for cell culture or recombinant protein preparation; it allows for the rapid synthesis of single, high-purity membrane proteins with stable and controllable content; and the membrane protein maintains its natural transmembrane structure and consistent orientation on the stationary phase. Flexible cDNA construction provides possibilities for drug binding site analysis. This invention can be applied to the screening of lead compounds targeting membrane proteins and the determination of equilibrium dissociation constants, providing a practical method for the rapid preparation of membrane protein immobilized affinity biochromatography or other biological solid-phase materials.

Description

Technical Field

[0001] This invention relates to the field of biochromatography, specifically, to a novel in-situ synthesis membrane protein affinity chromatography method that employs cell-free protein expression and covalent immobilization affinity chromatography. Background Technology

[0002] Membrane proteins are responsible for the exchange of external substances and information, and perform multiple functions such as intercellular contact, surface recognition, signal transduction, enzyme activity, and transport, making them the most promising group of potential drug targets. It is estimated that 60% of drug targets are membrane proteins. However, due to the low abundance and complex structure of membrane proteins, drug discovery based on membrane proteins is relatively difficult. To date, only 1% of proteins with three-dimensional structures have been successfully resolved. In vitro purification and mimicking native conformations are bottlenecks in drug screening and ligand interaction analysis targeting membrane proteins.

[0003] The lipid bilayer environment is essential for membrane proteins to maintain their native conformation and physiological activity. Membrane protein biochromatographic techniques, such as cell membrane chromatography (CMC) and cell membrane affinity chromatography (CMAC), are widely used in membrane protein research. CMC, which directly extracts cell membranes and immobilizes them on silica gel as a chromatographic stationary phase, is an effective method for screening active components of membrane receptors. Combining stationary phase modification techniques and protein overexpression strategies with CMC can achieve specific screening for specific proteins. CMAC, which directly obtains target membrane proteins from cell membranes or subsequently reconstitutes them into immobilized artificial membranes and immobilizes them on a stationary phase, has been widely used in multiple stages of drug discovery. In recent years, CMAC columns containing target proteins have been extended to characterize the type and process of binding between candidate lead drugs and target proteins. However, CMC or CMAC still has some limitations: 1. Directly obtaining cell membranes as a source of membrane proteins results in low protein specificity and abundance, which is not conducive to accurate screening of membrane protein affinity components and interaction analysis; 2. During the process of fixing cell membranes or reconstructed protein-lipid structures onto the immobilization phase carrier, the orientation of membrane proteins is random, and the exposure of active binding sites is uncontrollable; 3. Preparing a sufficient number of recombinant membrane proteins is an expensive, tedious, and time-consuming task.

[0004] Cell-free expression (CFE) technology provides an ideal pathway for the expression of membrane proteins. Its principle is to use the DNA or mRNA of the target protein as a template, and in the presence of cell-extracted amino acids, RNA polymerase, and energy substances, achieve in vitro protein expression. CFE overcomes the physiological limitations of living cells, significantly improving the efficiency and yield of protein synthesis while reducing protein misfolding and aggregation. CFE is particularly suitable for the synthesis of membrane proteins. By providing an artificial biomimetic membrane in the expression system, membrane proteins can be directly expressed, co-translated, and inserted into the phospholipid membrane during synthesis. More importantly, the vector insertion of the protein into the biomimetic membrane during synthesis can be observed. CFE has been used for high-throughput protein expression, protein drug screening, and synthesis. For example, protein in situ arrays can rapidly and efficiently synthesize proteins using CFE and apply them to the study of protein-protein interaction networks on chips. CFE is a practical method for obtaining a sufficient number of oriented, consistent functional membrane proteins. However, to date, the preparation of membrane protein biochromatographic stationary phases on silica gel using CFE has not yet been achieved. Summary of the Invention

[0005] The purpose of this invention is to provide a novel in-situ synthetic membrane protein affinity biochromatographic column, its preparation method, and its applications. This column combines the advantages of cell-free expression (CFE) and 3-mercaptopropyltrimethoxysilane (MPTS) modified bioaffinity chromatography, enabling in-situ synthesis and unidirectional insertion of membrane proteins onto a silica-liposome carrier. It can be applied to the screening of potential active components targeting specific membrane proteins in complex systems, as well as the characterization of the interactions between membrane proteins and their ligands.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides an in-situ synthetic membrane protein affinity biochromatographic column, wherein the membrane protein affinity biochromatographic column is used to synthesize membrane proteins in situ on a silica-liposome carrier; the preparation method is as follows: an empty stationary phase is prepared by vacuum vortex mixing of MPTS-modified silica and liposomes; then, the cDNA of the membrane protein and the silica attached to the liposomes are simultaneously added to the expression system to synthesize the membrane protein, thereby obtaining an insertable membrane protein stationary phase; the prepared stationary phase is loaded into the chromatographic column to obtain the in-situ synthetic membrane protein affinity chromatographic column.

[0008] The chromatographic column of this invention combines the advantages of CFE and MPTS-modified bioaffinity chromatography. It achieves, for the first time, in-situ synthesis and unidirectional insertion of membrane proteins into liposome-immobilized silica supports. It can be applied to the screening of active components in complex systems targeting membrane proteins and the determination of equilibrium dissociation constants, and provides a new technical means for the rapid preparation of membrane protein-immobilized affinity chromatography or other biological solid-phase materials.

[0009] The in-situ synthetic membrane protein affinity chromatography column described in this invention eliminates the need for complex cell culture and protein purification processes, enabling rapid one-step preparation of a biochromatographic stationary phase containing a single, stable amount of the target membrane protein. The in-situ synthetic membrane protein technology utilizes a tag constructed on cDNA to characterize the unidirectional insertion of membrane proteins into liposome membranes.

[0010] Furthermore, the preparation method of the in-situ synthetic membrane protein affinity chromatography column includes the following steps:

[0011] A) Dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), and NBD-dioleoylphosphatidylglycerol (NBD-DOPG) were dissolved in chloroform / methanol (1:1, v / v) at a molar ratio of 80:20:1 to a final concentration of 20 mg / mL and then vacuum rotary evaporated. The lipid membrane was suspended in phosphate buffer to obtain a lipid mixture of 20 mg / mL, which was sonicated for 10 minutes to form liposome vesicles. Then, the vesicles were extruded using a liposome extruder to form vesicles with a diameter of less than 100 nm.

[0012] B) An empty stationary phase was prepared by vortexing MPTS-modified silica gel (activated at 120°C before use) with liposomes and incubating overnight at 4°C; cDNA of membrane proteins and silica gel encapsulating liposomes were added to the expression system simultaneously, and membrane proteins were synthesized by oscillation at 37°C and 300 rpm; centrifugation and washing were performed to obtain the biological stationary phase for in-situ synthesis of membrane proteins.

[0013] C) The prepared stationary phase is loaded into the microcolumn core using a column packer to obtain the in-situ synthetic membrane protein affinity biochromatographic column.

[0014] Furthermore, the membrane protein is PDGFRβ (platelet-derived growth factor receptor-β), Genebank number: NM_002609.4.

[0015] It is applicable to most membrane proteins, such as TGFβR, EGFR, and G protein-coupled receptor (GPCR) family proteins.

[0016] Furthermore, in step B, centrifugation at 3000×g and washing three times yields an insert-type stationary phase for in-situ synthesis of membrane proteins.

[0017] 10. Further, in step C, the microcolumn (400 μm, id) is packed using an Sp-403k column packer. Even further, 5 mg of the prepared stationary phase is dissolved in 500 μL of PBS, placed in a sample vial, and magnetically stirred at a constant speed of 15 rpm; high-pressure nitrogen is used as the pressure source, and the mixture is packed into a 400 μm inner diameter capillary column at a pressure of 5 MPa.

[0018] A second aspect of the present invention provides an application of the in-situ synthetic membrane protein affinity chromatography column described above in the screening of active components in complex systems targeting membrane proteins.

[0019] Furthermore, the membrane protein is PDGFRβ, and the active ingredient is an active compound that targets PDGFRβ to reverse liver fibrosis.

[0020] Furthermore, the application of the in-situ synthesized membrane protein affinity chromatography column in screening compounds for the treatment of liver fibrosis.

[0021] A third aspect of the present invention provides the application of the in-situ synthesized membrane protein affinity chromatography column as described above in the determination of affinity between ligands and specific regions of membrane proteins.

[0022] Compared with the prior art, the advantages of this invention are:

[0023] 1. This invention employs cell-free protein expression and covalent immobilization affinity chromatography techniques to construct a novel in-situ synthetic membrane protein biochromatographic column. The column prepared by this invention is the first to achieve unidirectional insertion of a single membrane protein with a natural quaternary structure onto a stationary phase bound to liposomes.

[0024] 2. The preparation of in-situ synthetic membrane protein affinity chromatography columns does not require cell culture or recombinant protein preparation; it can rapidly synthesize single, high-purity membrane proteins with stable and controllable content; and the membrane proteins can maintain their natural transmembrane structure and consistent orientation on the stationary phase; flexible cDNA construction provides the possibility for the analysis of drug binding sites.

[0025] 3. In-situ synthesized membrane protein affinity chromatography columns can be used for screening active ingredients in complex systems targeting membrane proteins and for determining the affinity of ligands for specific regions of membrane proteins.

[0026] 4. The method for preparing in-situ synthetic membrane protein affinity chromatography stationary phases is applicable to most membrane proteins and can be used for screening lead compounds targeting membrane proteins and determining the equilibrium dissociation constant between the protein and the active component. It can provide a practical method for the rapid preparation of membrane protein affinity biochromatography or other biological stationary phases. Attached Figure Description

[0027] Figure 1A schematic diagram of the preparation and application of an affinity biochromatographic column for the cell-free in situ synthesis of membrane protein PDGFRβ.

[0028] Figure 2 The results evaluate the effectiveness, specificity, and protein orientation of the PDGFRβ microcolumns. A represents the retention behavior of PDGF-BB and dexamethasone on the empty column; B represents the retention behavior of PDGF-BB and dexamethasone on the PDGFRβ column; and C represents the retention behavior of the Flag and His-tagged antibodies on the PDGFRβ column.

[0029] Figure 3 Results of screening for PDGFRβ-targeting affinity components from extracts of *Salvia miltiorrhiza* and *Schisandra chinensis* using the PDGFRβ-offline-UPLC / MS system. In the figures, A shows the retention behavior chromatograms of *Salvia miltiorrhiza* and *Schisandra chinensis* extracts on the PDGFRβ column; B shows the mass spectrometry results of retained and non-retained components of *Salvia miltiorrhiza* and *Schisandra chinensis* extracts on the PDGFRβ column; and C shows the retention behavior chromatograms of salvianolic acid B and gomisin D standards on the PDGFRβ column.

[0030] Figure 4 The results show the affinity analysis of the interactions between salvianolic acid B, gomisin D, and PDGFRβ. In this table, A represents the affinity determination of salvianolic acid B with PDGFRβ using frontier chromatography; B represents the affinity determination of gomisin D with PDGFRβ using frontier chromatography. DXMS represents dexamethasone, Sal B represents salvianolic acid B, and Gomisin D represents gomisin D. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to embodiments and accompanying drawings. These descriptions are for illustrative purposes only and are not intended to limit the scope of the invention.

[0032] Example 1:

[0033] Liver fibrosis is the foundation of many complex end-stage liver diseases and is also a reversible pathological process. PDGFRβ is one of the important targets for the development of drugs for liver fibrosis. The in-situ synthetic membrane protein affinity chromatography column prepared in this invention can be used to screen active compounds that target PDGFRβ to reverse liver fibrosis, providing highly specific and effective lead compounds for the treatment of liver fibrosis.

[0034] The preparation of an in-situ synthetic PDGFR β affinity chromatography column includes the following steps:

[0035] 1) Preparation of liposomes and plasmids

[0036] In a 25 mL flask, DOPC, DOPE, and NBD-DOPG were dissolved in chloroform / methanol (1:1, v / v) at a molar ratio of 80:20:1 to a final concentration of 20 mg / mL. The solvent was evaporated by vacuum distillation. The lipid membrane was then suspended in phosphate buffer to obtain a lipid mixture of 20 mg / mL. This mixture was sonicated at 400 W for 10 minutes to form liposome vesicles, which were then extruded 3-4 times using a liposome extruder to form liposomes with a diameter of less than 100 nm.

[0037] Construct the PDGFRβ plasmid. Clone PDGFRβ into the pET28 plasmid vector, name it pET28-PDGFRβ, add a Flag tag to the N-terminus, and add a fusion fluorescent "cherry" and 6His tag to the C-terminus.

[0038] 2) Preparation of in-situ synthesized PDGFRβ affinity stationary phase

[0039] An empty stationary phase was prepared by vortexing MPTS-modified silica gel (activated at 120℃ before use) with liposomes and incubating overnight at 4℃. The constructed pET28-PDGFRβ plasmid and silica-liposomes were simultaneously added to the expression system, and PDGFRβ was synthesized under shaking conditions of 37℃ and 300 rpm. The PDGFRβ insert stationary phase was obtained by centrifugation at 3000×g and washing three times.

[0040] 3) Preparation of PDGFR β-affinity biochromatographic column for in-situ synthesis

[0041] The microcolumn (400 μm, id) was packed using an Sp-403k packer. 5 mg of the prepared stationary phase was dissolved in 500 μL of PBS and placed in a sample vial, then magnetically stirred at a constant speed of 15 rpm. High-pressure nitrogen was used as the pressure source, and the microcolumn was packed at a pressure of 5 MPa into a 400 μm inner diameter capillary column.

[0042] 4) Validation of the effectiveness of the in-situ synthesis PDGFR β affinity column

[0043] This invention selects PDGF-BB, the extracellular ligand of PDGFRβ, as a positive control drug and dexamethasone as a negative control drug to verify the effectiveness and selectivity of in-situ synthesized PDGFRβ affinity biochromatographic columns.

[0044] The PDGFRβ microcolumn was mounted on an Agilent 1200 series micropump and equipped with a UV detector. The mobile phase was 1 mM PBS (pH 7.4), the flow rate was 10 μL / min, and the column temperature was 37 °C. The detection wavelength was 210–280 nm. The injection volume was 0.1 μL. Chromatographic data were collected using ChemStation B.04.03 and imported into Origin 8.0 to generate chromatograms.

[0045] Test results as follows Figure 2 As shown: A represents the retention behavior of PDGF-BB and dexamethasone on an empty column; B represents the retention behavior of PDGF-BB and dexamethasone on a PDGFRβ column; C represents the retention behavior of Flag and 6His-tagged antibodies on a PDGFRβ column.

[0046] The above results indicate that PDGF-BB exhibits strong retention behavior on the PDGFRβ column, reaching a peak at 35.4 min, while no retention was observed on the negative control column, and dexamethasone showed no retention on either. Furthermore, the Flag-tagged antibody was retained on the PDGFRβ column, while the 6His-tagged antibody was not. This demonstrates that the expressed PDGFRβ not only possesses ligand recognition and binding activity but also exhibits an N-terminal outward unidirectional insertion into the lipid bilayer, ensuring that the prepared PDGFRβ affinity biochromatographic column has good screening activity.

[0047] Example 2:

[0048] To date, most developed PDGFRβ inhibitors are kinase inhibitors with poor specificity. Drugs targeting the ligand-binding domain of the PDGFRβ extracellular region hold promise for higher specificity and lower toxicity. Danshen (Salvia miltiorrhiza) and Schisandra chinensis are traditional Chinese medicines with certain anti-liver fibrosis effects. The in-situ synthetic membrane protein affinity chromatography column prepared in this invention can be used to screen potential active ingredients targeting the PDGFRβ extracellular ligand-binding domain from extracts of Danshen and Schisandra chinensis. The preparation method of the in-situ synthetic PDGFRβ affinity chromatography stationary phase is the same as in Example 1.

[0049] This invention selects extracts of Salvia miltiorrhiza and Schisandra chinensis for in-situ synthesis of PDGFRβ affinity chromatography column for screening potential anti-liver fibrosis active ingredients targeting PDGFRβ.

[0050] PDGFRβ microcolumns were loaded onto an Agilent 1200 series micropump and equipped with a UV detector. The mobile phase was 1 mM PBS (pH 7.4), the flow rate was 10 μL / min, and the column temperature was 37 °C. The detection wavelength was 210–280 nm. The injection volume was 0.1 μL. Chromatographic data were collected using ChemStation B.04.03 and imported into Origin 8.0 to generate chromatograms.

[0051] Every 0.5 min, the components of Salvia miltiorrhiza and Schisandra chinensis were collected into 96-well plates, separately collecting the non-retained and retained components. After drying the samples with nitrogen, they were redissolved in 20 μL of methanol and analyzed using an Agilent 1290 UPLC-QTOF / MS. The chromatographic column was an XBridge™ C18 (100 × 2.1 mm id, 2.5 μm, Waters, Ireland), the mobile phase was solvent A (0.1% formic acid) and solvent B (acetonitrile), and the flow rate was 0.8 mL·min. -1 Linear gradient elution is employed.

[0052] Test results as follows Figure 3 As shown: A represents the chromatographic results of the extracts of Salvia miltiorrhiza and Schisandra chinensis on a PDGFRβ column; B represents the mass spectrometry results of components with and without retention on a PDGFRβ column; C represents the chromatographic results of the retention behavior of salvianolic acid B and gomisin D standards on a PDGFRβ column.

[0053] The above results indicate that the in-situ synthesized PDGFRβ affinity biochromatographic column was successfully applied to screen potential active ingredients targeting the extracellular domain of PDGFRβ in extracts of Salvia miltiorrhiza and Schisandra chinensis. The identified affinity components were salvianolic acid B and gomisin D.

[0054] Example 3

[0055] Frontier chromatography is primarily used to study the interactions between proteins and ligands. This method involves injecting solutions of the analyte compound at different concentration gradients as the mobile phase into a biochromatographic column until a breakthrough curve appears. The binding time between the compound and the protein is characterized by an increase in the time to reach the breakthrough curve as the concentration decreases. If there is no specific binding interaction between the compound and the protein, the breakthrough time remains constant regardless of the ligand concentration. The in-situ synthetic membrane protein affinity biochromatographic column prepared in this invention can be used to determine the affinity of potential active ingredients for PDGFRβ. The preparation method of the in-situ synthetic PDGFRβ affinity chromatographic stationary phase and the chromatographic analysis method are the same as in Examples 1 and 2.

[0056] In this invention, salvianolic acid B and gomisin D were selected, and the affinity of the compounds for the PDGFRβ phase was determined using an in-situ synthesized PDGFRβ affinity biochromatographic column.

[0057] Tanshinone B and Gomicin D were dissolved in DMSO to a concentration of 50 mM as a stock solution, and then diluted with PBS to a gradient concentration of 100, 50, 25, 10, 5, and 1 μM to obtain the chromatographic mobile phase. The flow rate in micro mode was 10 μL / min. The column was first equilibrated with 1 mM PBS, and then a mobile phase of a certain concentration of the compound was passed through the column to form a stable breakthrough curve. Between two different concentrations, the system was washed with PBS until the UV absorption returned to the baseline. Simultaneously, a column filled with an empty stationary phase was used as a control column to eliminate non-specific interactions and obtain the dead time t0. When the non-specific interactions were negligible compared to the specific interactions, the total amount of captured compounds (defined as q) and the ligand concentration (defined as [L]) could be expressed as an equation to calculate K. D value.

[0058] Test results as follows Figure 4 As shown: A represents the frontal chromatographic affinity determination result of salvianolic acid B on a PDGFRβ column; B represents the frontal chromatographic affinity determination result of gomisin D on a PDGFRβ column.

[0059] The above results indicate that salvianolic acid B and gomisin D exhibit a typical single-point interaction mode with PDGFRβ, K D The values ​​were 13.44 μM and 7.39 μM, respectively. This further demonstrates that the in-situ synthesized PDGFRβ affinity biochromatographic column can provide biologically active, orientation-consistent PDGFRβ for analyzing the affinity between compounds and proteins.

[0060] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. An in-situ synthetic membrane protein affinity biochromatographic column, characterized in that, The membrane protein affinity biochromatographic column is used to synthesize membrane proteins in situ on a silica-liposome carrier. The preparation method is as follows: Silica gel modified with 3-mercaptopropyltrimethoxysilane (MPTS) is mixed with liposomes under vacuum vortex to prepare an empty stationary phase. Then, the cDNA of the membrane protein and the silica gel with attached liposomes are simultaneously added to a cell-free expression system to synthesize the membrane protein, resulting in an insertable membrane protein stationary phase. The prepared stationary phase is then loaded into the chromatographic column to obtain the in situ synthesized membrane protein affinity biochromatographic column.

2. The in-situ synthetic membrane protein affinity biochromatographic column according to claim 1, characterized in that, The method for preparing the in-situ synthetic membrane protein affinity biochromatographic column includes the following steps: A) Dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, and NBD-dioleoylphosphatidylglycerol were dissolved in chloroform / methanol at a molar ratio of 80:20:1 to a final concentration of 20 mg / mL and then vacuum-evaporated. The lipid membrane was suspended in phosphate buffer to obtain a lipid mixture of 20 mg / mL. The mixture was sonicated for 10 minutes to form liposome vesicles, and then extruded using a liposome extruder to form vesicles with a diameter of less than 100 nm. B) An empty stationary phase was prepared by vortexing MPTS-modified silica gel with liposomes and incubating overnight at 4°C; cDNA of membrane proteins and silica gel encapsulating liposomes were simultaneously added to the cell-free expression system, and membrane proteins were synthesized by oscillation at 37°C and 300 rpm; the biological stationary phase for in-situ synthesis of membrane proteins was obtained by centrifugation and washing. C) The prepared stationary phase is loaded into the microcolumn core using a column packer to obtain the in-situ synthetic membrane protein affinity biochromatographic column.

3. The in-situ synthetic membrane protein affinity biochromatographic column according to claim 1, characterized in that, The membrane protein in question is PDGFRβ.

4. The in-situ synthetic membrane protein affinity biochromatographic column according to claim 2, characterized in that, In step B), centrifugation at 3000×g and washing three times yielded an insert-type stationary phase for in situ synthesis of membrane proteins.

5. The in-situ synthetic membrane protein affinity biochromatographic column according to claim 2, characterized in that, Step C) The 400 μm id microcolumn was filled using an Sp-403k column packer.

6. The in-situ synthetic membrane protein affinity biochromatographic column according to claim 5, characterized in that, The prepared stationary phase was dissolved in 500 μL of phosphate buffered PBS and placed in a sample vial. The solution was then magnetically stirred at a constant speed of 15 rpm. High-pressure nitrogen was used as the pressure source and the solution was packed into a capillary column with an inner diameter of 400 μm at a pressure of 5 MPa.

7. A method for preparing an in-situ synthetic membrane protein affinity biochromatographic column as described in claim 1, characterized in that, Includes the following steps: A) Dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, and NBD-dioleoylphosphatidylglycerol were dissolved in chloroform / methanol at a molar ratio of 80:20:1 to a final concentration of 20 mg / mL and then vacuum-evaporated. The lipid membrane was suspended in phosphate buffer to obtain a lipid mixture of 20 mg / mL. The mixture was sonicated for 10 minutes to form liposome vesicles, and then extruded using a liposome extruder to form vesicles with a diameter of less than 100 nm. B) An empty stationary phase was prepared by vortexing MPTS-modified silica gel with liposomes and incubating overnight at 4°C; cDNA of membrane proteins and silica gel encapsulating liposomes were added to the expression system simultaneously, and membrane proteins were synthesized by shaking at 37°C and 300 rpm; the biological stationary phase for in-situ synthesis of membrane proteins was obtained by centrifugation and washing. C) The prepared stationary phase is loaded into the microcolumn core using a column packer to obtain the in-situ synthetic membrane protein affinity biochromatographic column.

8. The application of an in-situ synthetic membrane protein affinity biochromatographic column as described in any one of claims 1-6 in the screening of active components in complex systems targeting membrane proteins.

9. The application of an in-situ synthesized membrane protein affinity biochromatographic column as described in any one of claims 1-6 in the determination of affinity between ligands and specific regions of membrane proteins.

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