Natural-state protein immobilization method

Abstract

The invention provides a natural protein immobilization method. The method comprises the following steps: step 1, reacting a reactant with target natural-state protein; step 2, preparation of a reactant modification interface; and step 3, fully mixing the natural-state protein 5-HTT with the obtained reactant modification interface, stirring at room temperature for 6 hours, and washing with PBS for 3 times to complete high-activity immobilization of the natural-state 5-HTT. Through the proximity effect of ligand-functional protein affinity interaction, a nucleophilic amino acid residue at a specific site of the native protein and a ligand electrophilic group are induced to be subjected to a covalent reaction, the ligand is released through synchronous self-cleavage, and a ligand binding domain is exposed, so that high-activity one-step capture of the native protein is realized; the technical problems of low immobilization efficiency, serious protein activity loss and low specificity of low-abundance natural state functional protein in a complex system are successfully solved.

Description

Technical Field

[0001] The invention belongs to the technical field of a natural protein immobilization method, and in particular relates to a natural protein immobilization method. Background Art

[0002] The existing methods for immobilizing natural functional proteins include the following: (1) physical adsorption method, (2) random covalent immobilization method, (3) immobilization method based on affinity peptide tag, (4) site-specific covalent immobilization method based on click chemistry, (5) site-specific covalent immobilization method based on enzyme tag autocatalysis, and (6) site-specific covalent immobilization method based on small tags of non-natural amino acids. The above methods still have shortcomings, specifically:

[0003] (1) Physical adsorption method

[0004] This method is the earliest and more mature protein immobilization method developed in the literature, and has the advantages of being simple, easy to operate, and having a large protein loading capacity. However, due to its essence of weak intermolecular interactions, the immobilized protein coating is often easy to lose, and there are shortcomings such as inconsistent protein orientation and conformation.

[0005] (2) Random covalent immobilization method

[0006] This type of method utilizes the amino, carboxyl and thiol groups in the protein structure to undergo covalent reactions with active groups modified on the surface of solid materials to prepare immobilized proteins, effectively solving the problem of easy loss of immobilized proteins by physical adsorption. However, due to the low reaction selectivity of this method, there are often shortcomings such as serious loss of biological activity and function of the immobilized proteins.

[0007] (3) Immobilization method based on affinity peptide tag

[0008] This type of method uses the affinity between histidine tags, streptavidin tags and glutathione transferase tags and their specific molecules to prepare immobilized proteins. Due to its certain reaction selectivity, it can solve the problem of serious protein activity loss in physical adsorption and random covalent immobilization methods, but due to the affinity (Kd≈10 -6 -10 -9 M) belongs to the category of weak forces, and the easy loss of protein coating is still a problem that is difficult to solve by this method.

[0009] (4) Site-specific covalent immobilization method based on click chemistry

[0010] This type of method generally uses chemical reactions such as Staudinger, Diels-Alder and thiol-ene addition to achieve directional fixation of proteins. It has the characteristics of mild reaction conditions and high efficiency. Although this method can solve the problems of easy loss of protein coating and low uniformity, it requires high-purity protein to achieve immobilization.

[0011] (5) Site-specific covalent immobilization method based on enzyme tag autocatalysis

[0012] This type of method uses active enzymes such as halogenated alkane dehalogenase, O6-alkylguanine-DNA alkyltransferase and epidermal growth factor tyrosine kinase active domain as protein tags, recombines them into the inactive carbon terminus of the target protein, and utilizes the highly selective bioorthogonal reaction between the enzyme tag and the substrate small molecule modified carrier to achieve one-step covalent immobilization of recombinant functional proteins in complex systems; however, due to the large size of the tag molecule (19-37 kDa), the original activity and function of the target protein are changed, and parameters such as the ligand recognition specificity and separation ability of the immobilized protein chromatographic stationary phase are affected to a certain extent.

[0013] (6) Site-specific covalent immobilization of small tags based on non-natural amino acids

[0014] This type of method introduces non-natural amino acids at specific sites of the target protein, and immobilizes the target protein on the surface of the solid matrix through click chemistry reaction between the non-natural amino acids and specific groups modified on the surface of the solid matrix. This type of method still has certain limitations: first, due to the limitations of genetic engineering technology, the insertion of non-natural amino acids has not been reported in the study of functional proteins in living cells and organisms; second, due to the limitations of the selectivity of click chemistry reactions, high-purity functional proteins are still required, and it is difficult to apply functional protein immobilization research in complex systems.

[0015] Based on the problems existing in the above technologies, there is an urgent need for a method to solve the low immobilization efficiency, severe loss of protein activity and low specificity of low-abundance natural functional proteins in complex systems. Summary of the invention

[0016] The purpose of the present invention is to provide a method for immobilizing natural proteins, which successfully solves the technical problems of low immobilization efficiency, severe protein activity loss and low specificity of low-abundance natural functional proteins in complex systems.

[0017] The present invention is achieved through the following technical solutions:

[0018] The present invention relates to a method for immobilizing a natural protein, comprising the following steps:

[0019] Step 1, reactants react with target native proteins; wherein the target native proteins include: soluble proteins, membrane proteins, G protein-coupled receptors, and nuclear receptors;

[0020] Step 2, preparation of reactant-modified interface;

[0021] Step 3: The native protein and the obtained reactant-modified interface were fully mixed, stirred at room temperature for 6 hours, and washed with PBS three times to complete the high-activity fixation of the native 5-HTT.

[0022] Preferably, the reactant consists of a ligand group and an active group;

[0023] The ligand groups include: small molecule compound ligands, nucleic acid aptamers, peptide and protein ligands, antibodies and other molecules that have affinity with the target natural protein;

[0024] Reactive groups include: p-toluenesulfonyl group, acyl imidazole group, dibromophenylbenzoate, N-sulfonylpyridine group, N-acyl-N-alkylsulfonamide group, phenyl ester group, sulfonyl azide and N-thioethylbenzamide group, see Figure 2 shown.

[0025] Preferably, the ligand group contains a known active ligand of the target natural protein, which binds to the target natural protein by affinity to produce a proximity effect; it can significantly increase the local effective concentration; the active group contains an electrophilic group, which covalently reacts with the nucleophilic amino acid residues near the ligand binding domain of the target natural protein, and when the covalent reaction occurs, the ligand group will be cleaved and released.

[0026] Preferably, in step 1, the preparation method of the reactant is specifically as follows: using fluvoxamine (1 part) as a raw material, dissolving it in DMF, adding 1H-imidazole-5-acetic acid hydrochloride (1.5-2 parts), HATU (1.5-2 parts), and DIEA (4-6 parts) in sequence, and heating at room temperature. 2 Stir for 4 to 6 hours, add water, CH 2 Cl 2 Extraction, drying over anhydrous sodium sulfate, and SiO 2 Column, CH 2 Cl 2 :CH 3 OH=20:1→10:1, to obtain acyl imidazole fluvoxamine, with a yield of about 81%.

[0027] Preferably, in step 2, the preparation of the reactant-modified interface is specifically as follows:

[0028] 1.0 g of aminopropyl silica gel (amino content: 200 μmol / g) was suspended in DMF, and Hydroxy-PEG3-acid (1 part), HATU (0.5-2 parts), and DIEA (1.5-6 parts) were added in sequence, mechanically stirred for 4-6 hours, filtered, washed with DMF and methanol for several times in sequence, and dried at 60°C;

[0029] Take the above dried silica gel, suspend it in acetonitrile, add N,N'-disuccinimidyl carbonate (1-4 parts) and triethylamine (1-4 parts), N 2 Stir at 40°C for 3-5 h under protection, filter, wash with acetonitrile several times, and dry at 60°C.

[0030] The above-mentioned dry silica gel was suspended in DMF, and acyl imidazole fluvoxamine (1 part) and DIEA (2-3 parts) were added. The mixture was mechanically stirred for 8-12 hours, and washed with DMF and methanol for several times in sequence. The silica gel modified with acyl imidazole fluvoxamine was obtained by drying at 60°C. The synthetic route is as follows: Figure 4 shown.

[0031] Preferably, in step 3, the specific method for high-activity fixation of the native 5-HTT is: fully mixing the native protein with the obtained reactant-modified interface, stirring at room temperature for 4 to 8 hours, and washing with PBS three times to complete the high-activity fixation of the native 5-HTT.

[0032] Mouse hippocampal neuronal cells (HT22) were cultured to a certain density, and the cells were lysed by ultrasonic disruption, and the supernatant was collected by centrifugation at 12000 rpm to prepare cell lysate;

[0033] The cell lysate was fully mixed with the obtained acyl imidazole fluvoxamine modified silica gel, stirred at room temperature for 4 to 6 hours, and washed three times with PBS to achieve high-activity fixation of natural 5-HTT.

[0034] The principle of the invention is to induce the covalent reaction between the nucleophilic amino acid residues at specific sites of the native protein and the electrophilic groups of the ligand through the proximity effect of the ligand-functional protein affinity, and simultaneously self-cleave to release the ligand and expose the ligand binding domain, thereby achieving high-activity one-step capture of the native protein.

[0035] The present invention has the following advantages:

[0036] (1) The reaction efficiency of the present invention is high: the proximity effect exists in the specific reaction process between enzymes and substrates in vivo and is the main driving factor for the efficient conduct of many enzymatic reactions.

[0037] (2) The present invention has strong selectivity: the affinity of the ligand-receptor interaction in vivo can be as high as nM or even pM level, which can eliminate the interference of coexisting impurities and produce a proximity effect.

[0038] (3) The immobilized protein coating of the present invention has good stability: the proximity effect between the ligand and the receptor of the present invention can significantly increase its local effective concentration and promote the covalent reaction between the electrophilic group of the ligand and the nucleophilic amino acid residue of the protein.

[0039] (4) The target natural protein of the present invention has little loss of activity and function: by appropriately modifying the ligand, the inactive amino acid residues of the receptor can be precisely controlled to undergo covalent reaction and synchronously self-cleave to release the ligand, with little effect on the activity and function of the receptor.

[0040] (5) The method of the present invention can avoid the need for structural modification of the target protein in existing studies. It is not only applicable to artificial expression systems, but also can achieve efficient one-step immobilization of natural functional proteins in living cells and tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a schematic diagram of the method for immobilizing natural protein of the present invention;

[0042] Figure 2 Schematic diagram of the active group structure of the present invention;

[0043] Figure 3 is a synthetic route diagram of acyl imidazole fluvoxamine in Example 1 of the present invention;

[0044] Figure 4 is a synthetic route of silica gel modified with acyl imidazole fluvoxamine in Example 1 of the present invention;

[0045] Figure 5 This is the high-activity immobilization route map of natural 5-HTT in Example 1 of the present invention.

[0046] Figure 6 is the chromatogram of different ligands on the immobilized native 5-HTT column;

[0047] Figure 7 This is a comparison of the ligands on the immobilized Halo-5HTT, His-5HTT and native 5HTT columns. DETAILED DESCRIPTION

[0048] The present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments are only further descriptions of the present invention, but the protection scope of the present invention is not limited to the following embodiments.

[0049] Example 1

[0050] This embodiment relates to a method for immobilizing a natural protein, such as Figure 1As shown, in this example, the serotonin transporter (5-HTT) is used as the target protein, the 5-HTT ligand fluvoxamine is used as the ligand core structure, and the acyl imidazole is used as the active group core structure to establish a high-activity immobilization strategy for natural 5-HTT. The specific steps are as follows:

[0051] Step 1, Synthesis of Acylimidazole Fluvoxamine

[0052] Fluvoxamine (1 part) was used as the raw material and dissolved in DMF. 1H-imidazole-5-acetic acid hydrochloride (1.5 parts), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.5 parts) and N,N-diisopropylethylamine (5 parts) were added in sequence. 2 Stir for 5 h, add water, CH 2 Cl 2 Extraction, drying over anhydrous sodium sulfate, and SiO 2 Column, CH 2 Cl 2 :CH 3 OH=20:1→10:1, acyl imidazole fluvoxamine was obtained with a yield of about 81%. The synthetic route is shown in Figure 3 .

[0053] ESI-MS measured m / z [M+H] + 472.10 and [M+Na] + 449.05. 1 H NMR (600 MHz, Methanol-d 4 )δ8.26(s,1H),7.85(d,J=8.3Hz,2H),7.68(d,J=8.4Hz,2H),7.18(s,1H),4.27(t,J=5.5Hz,2H),3.65(s, 2H), 3.57(t,J=5.5Hz,2H), 3.39(t,J=6.0Hz,2H), 3.30(s,3H), 2.82(t,J=7.2Hz,2H), 1.66–1.49(m,4H).

[0054] Step 2, preparation of acyl imidazole fluvoxamine modified silica gel

[0055] (1) 1.0 g of aminopropyl silica gel (amino content: 200 μmol / g) was suspended in DMF, and Hydroxy-PEG3-acid (1 part), HATU (1 part), and DIEA (2 parts) were added in sequence, mechanically stirred for 5 h, filtered, washed with DMF and methanol in sequence for multiple times, and dried at 60° C. to obtain dry silica gel;

[0056] (2) The dried silica gel prepared in (1) was suspended in acetonitrile, and N,N'-disuccinimidyl carbonate (2 parts) and triethylamine (2 parts) were added. 2 Stir at 40°C for 3 h under protection, filter, wash with acetonitrile for several times, and dry at 60°C to obtain dry silica gel;

[0057] (3) The dried silica gel obtained in (2) was suspended in DMF, and acyl imidazole fluvoxamine (1 part) and DIEA (2.5 parts) were added. The mixture was mechanically stirred for 10 h, washed with DMF and methanol for multiple times, and dried at 60° C. to obtain acyl imidazole fluvoxamine modified silica gel. The synthetic route is as follows: Figure 4 shown.

[0058] Step 3: Highly active fixation of natural 5-HTT

[0059] Adherently culturing mouse hippocampal neuron cells (HT22) to a certain density, wherein one of 5-HTT HT22 is expressed; lysing the cells by ultrasonic disruption, centrifuging at 12000 rpm to obtain the supernatant, and preparing a cell lysate;

[0060] The cell lysate was fully mixed with the obtained acyl imidazole fluvoxamine modified silica gel, stirred at room temperature for 6 h, and washed 3 times with PBS to complete the high-activity fixation of natural 5-HTT. Figure 5 shown.

[0061] Example 2

[0062] The above-mentioned natural 5-HTT-modified silica gel was loaded into a stainless steel chromatographic column tube (4.6×30 mm) at a pressure of 200 to 400 bar using 50 mM phosphate buffer (PB, pH=7.4) as the displacement fluid to investigate the retention behavior of the 5-HTT-specific ligand on the chromatographic column (e.g. Figure 6 As shown). Taking the retention time of sodium nitrite as an indicator, the dead time of the chromatographic system was determined to be 1.0 min. Under the same chromatographic conditions, the retention times of fluvoxamine, fluoxetine and sertraline on the chromatographic column were 7.5 min, 7.2 min and 11.0 min, respectively, which were much longer than the dead time of the chromatographic system, proving that the immobilized natural 5-HTT has the activity of recognizing its ligands, and the recognition ability of different ligands is different.

[0063] Example 3

[0064] Using immobilized Halo-tagged recombinant 5-HTT and His-tagged recombinant 5-HTT columns as controls, the retention behavior of 5-HTT-specific ligands on the three columns was investigated. Figure 7As shown. The three ligands are retained on the three 5-HTT columns with different retention times, indicating that the three immobilized proteins have recognition effects on their specific ligands. The retention factor, tailing factor and half-peak width on the immobilized natural 5-HTT column are smaller, indicating that compared with the recombinant immobilized target protein containing the tag, it has less nonspecific adsorption, higher column efficiency and better activity of the immobilized protein.

[0065] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which will not affect the essence of the present invention.

Claims

1. A method for immobilizing a natural protein, characterized in that: The following steps are involved: Step 1, reactants react with target native proteins; wherein the target native proteins include: soluble proteins, membrane proteins, G protein-coupled receptors, and nuclear receptors; Step 2, preparation of reactant-modified interface; Step 3, high-activity fixation of natural 5-HTT.

2. The method for immobilizing a natural protein according to claim 1, characterized in that: The reactant consists of a ligand group and an active group; The ligand groups include: small molecule compound ligands, nucleic acid aptamers, peptide and protein ligands, antibodies and molecules that have affinity with the target natural protein; Reactive groups include: p-toluenesulfonyl group, acyl imidazole group, dibromophenylbenzoate, N-sulfonylpyridine group, N-acyl-N-alkylsulfonamide group, phenyl ester group, sulfonyl azide and N-thioethylbenzamide group.

3. The method for immobilizing a natural protein according to claim 2, wherein: The ligand group contains a known active ligand of the target natural protein, which binds to the target natural protein by affinity to produce a proximity effect; the active group contains an electrophilic group, which covalently reacts with the nucleophilic amino acid residues near the ligand binding domain of the target natural protein. After the covalent reaction occurs, the ligand group will be cleaved and released.

4. The method for immobilizing a natural protein according to claim 1, wherein: In step 1, the preparation method of the reactant is specifically as follows: using fluvoxamine as a raw material, dissolving it in DMF, adding 1H-imidazole-5-acetic acid hydrochloride, HATU, and DIEA in sequence, stirring for 4 to 6 hours under N2 protection at room temperature, adding water, extracting with CH2Cl2, drying with anhydrous sodium sulfate, passing through a SiO2 column, CH2Cl2:CH3OH=20:1→10:1, and obtaining acyl imidazole fluvoxamine.

5. The method for immobilizing a natural protein according to claim 4, characterized in that: The amount of fluvoxamine used is 1 part; the amount of 1H-imidazole-5-acetic acid hydrochloride used is 1.5-2 parts; the amount of HATU used is 1.5-2 parts; and the amount of DIEA used is 4-6 parts.

6. The method for immobilizing a natural protein according to claim 1, wherein: In step 2, the preparation of the reactant-modified interface is specifically as follows: Suspend aminopropyl silica gel in DMF, add Hydroxy-PEG3-acid, HATU, and DIEA in sequence, stir mechanically for 4 to 6 hours, filter, wash with DMF and methanol in sequence for multiple times, and dry at 60°C to obtain dry silica gel; Take dry silica gel, suspend it in acetonitrile, add N,N'-disuccinimidyl carbonate and triethylamine, stir at 40℃ for 2-3h under N2 protection, filter, wash with acetonitrile several times, and dry at 60℃; The above-mentioned dry silica gel was suspended in DMF, acyl imidazole fluvoxamine and DIEA were added, mechanically stirred for 10 hours, washed with DMF and methanol for multiple times, and dried at 60° C. to obtain acyl imidazole fluvoxamine modified silica gel.

7. The method for immobilizing a natural protein according to claim 6, wherein: The dosage of the aminopropyl silica gel is 1.0 g, wherein the amino content is 200 μmol / g; the dosage of the Hydroxy-PEG3-acid is 1 part, the dosage of the HATU is 0.5 to 2 parts, the dosage of the DIEA is 1.5 to 6 parts, the dosage of the N,N'-disuccinimidyl carbonate is 1 to 4 parts; the dosage of the triethylamine is 1 to 4 parts; and the dosage of the acyl imidazole fluvoxamine is 1 part.

8. The method for immobilizing a natural protein according to claim 1, wherein: In step 3, the specific method for high-activity fixation of the native 5-HTT is: fully mix the native protein with the obtained reactant-modified interface, stir at room temperature for 4 to 8 hours, and wash 3 times with PBS to complete the high-activity fixation of the native 5-HTT.

9. The method for immobilizing a natural protein according to claim 8, characterized in that: The native protein 5-HTT is an expressed protein in mouse hippocampal neuronal cells.

Images