A novel streptavidin filler and preparation method thereof

Abstract

The invention discloses a novel streptavidin filler and a preparation method thereof, wherein a polymer-based ball such as polystyrene / divinylbenzene, polymethacrylate, polyacrylamide, etc. is used as a matrix, the surface of which is firstly hydrophilically modified, and then an epoxy group is introduced, and then streptavidin is bonded to the activated matrix to form a streptavidin (SA) affinity filler. The SA affinity filler has good alkali resistance and has a high affinity for biotin or biotinylated samples.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical separation media, and in particular to a novel streptavidin filler and a preparation method thereof. Background Art

[0002] Affinity chromatography is a chromatographic method that separates and purifies substances through specific recognition and interaction between molecules, such as the specific interaction between enzymes and substrates, antigens and antibodies, etc. By fixing it as a ligand on the filler, the corresponding biomolecules can be adsorbed from the initial sample, and then dissociated by appropriate elution to achieve the purpose of purification. Affinity chromatography has the advantages of high selectivity, high resolution, and high binding capacity. It can often purify to a purity of >90% in one step, and can complete the separation of substances that are difficult to complete with general methods.

[0003] Streptavidin (SA) is a tetrameric protein from Streptomyces, with a size of 66 KDa. It has the ability to efficiently bind biotin, with a dissociation constant of 10 -14 , which is one of the strongest known interactions between biomolecules. The interaction between streptavidin and biotin is widely used in many aspects such as nucleic acid detection, single molecule imaging, protein interaction, clinical diagnosis, etc., and is a key research method in biotechnology.

[0004] Streptavidin is similar to avidin and can specifically bind to biotin. However, compared with avidin, streptavidin has a lower isoelectric point (isoelectric point 6.0), does not contain sugar groups, and its nonspecific binding is much lower than that of avidin, especially in immunohistochemistry and DNA molecular hybridization detection, with almost no background color development.

[0005] There are some streptavidin fillers on the market, but the biotin binding capacity is low, about 300nmol / mL, and the alkali resistance is low. Therefore, it is an urgent need in the current market to explore a streptavidin filler with high pressure resistance, alkali resistance, low nonspecific adsorption, high biotin binding capacity, simple preparation process, and rapid scale-up production. Summary of the invention

[0006] In order to solve the above technical problems, the present invention aims to provide a novel streptavidin filler and a preparation method thereof, which has good alkali resistance and high affinity for biotin or biotinylated samples.

[0007] The present invention first provides a method for preparing a novel streptavidin filler, comprising the following steps:

[0008] 1) Hydrophilic modification of the matrix: the polymer matrix is ​​dispersed in the alkaline solution 1, the epoxy reagent 1 and dextran are added, stirred and reacted, and the intermediate 1 is obtained by filtering and washing;

[0009] 2) Epoxy activation: dispersing the intermediate 1 in an alkaline solution 2, adding an epoxy reagent 2, stirring and reacting, filtering and washing to obtain an intermediate 2;

[0010] 3) Coupling streptavidin: The intermediate 2, PB buffer and streptavidin aqueous solution are mixed, the pH is adjusted, and the mixture is reacted to obtain a novel streptavidin filler.

[0011] The conditions for the hydrophilic modification of the matrix are to first disperse the polymer matrix in the alkaline solution 1 for 0.5-1 hour, then add the epoxy reagent 1 and dextran, stir at 25-70°C for 8.0-40 hours, preferably at 25-45°C for 10-24 hours, filter and wash to obtain the intermediate 1. Dextran molecules are introduced on the surface of the matrix by chemical bonding, so that the surface of the matrix is ​​rich in a large amount of -OH, showing good hydrophilicity and reducing specific adsorption.

[0012] The conditions for epoxy activation are to first disperse the intermediate 1 in the alkaline solution 2 for 0.5-2 hours, then add the epoxy reagent 2, stir at 40-105° C. for 4-24 hours, preferably at 60-75° C. for 4-6 hours, and filter and wash to obtain the intermediate 2.

[0013] Furthermore, the present invention has no limitation on the polymer matrix, and most polymers can be used. The preferred polymer matrix is ​​polystyrene / divinylbenzene, polymethacrylate, and polyacrylamide; the matrix is ​​a porous microsphere with a pore size of The particle size is 5.0-100μm.

[0014] Preferably, the pore size of the matrix is Particle size is 80 μm; optimized to pore size Particle size 60μm and then optimized to pore size Particle size: 50μm.

[0015] Furthermore, the alkaline solution 1 and the alkaline solution 2 are inorganic bases, including sodium hydroxide, potassium hydroxide, etc. The concentration of the alkaline solution 1 is 0.5-2.0M, and the concentration of the alkaline solution 2 is 0.5-6.0M. Preferably, the concentration of the alkaline solution 2 is 0.5-4.0M.

[0016] The alkaline solution 1 and the alkaline solution 2 may use the same inorganic alkaline solution or different alkaline solutions.

[0017] Furthermore, the epoxy reagent 1 and the epoxy reagent 2 are selected from epichlorohydrin, ethylene glycol glycidyl ether, 1,4-butanediol diglycidyl ether, 1,2-cyclohexanediol diglycidyl ether or pentaerythritol glycidyl ether, and the epoxy reagent 1 and the epoxy reagent 2 can use the same compound or different compounds.

[0018] Furthermore, the molecular weight of the dextran is 2000-200000, for example, 2000, 6000, 20000, 40000, 200000, etc.

[0019] Furthermore, the mass ratio of the matrix to the dextran is 1.0:0.02-0.4, and the mass ratio of the matrix to the epoxy reagent 1 is 1.0:1.0-1.5.

[0020] Furthermore, the mass ratio of the matrix to the epoxy reagent 2 is 1.0:2.0-2.5.

[0021] Furthermore, the pH range is 5.0-12.0, and the preferred pH range is 6.5-9.0. When coupling streptavidin, the reaction environment can be either acidic or alkaline, but the coupling effect will be better under alkaline conditions; the reagent used to adjust the pH is an inorganic base such as sodium hydroxide, potassium hydroxide, sodium carbonate, etc.

[0022] Furthermore, when coupling with streptavidin, the reaction is carried out at 25-45° C. for 16-24 hours, preferably at 25-40° C. for 16-18 hours.

[0023] Furthermore, before adding the coupled streptavidin, the intermediate 2 is first dispersed in water for 0.5-2 hours. Generally, it is not necessary to disperse it first, but the effect will be better if it is dispersed first and then coupled.

[0024] In addition, the present invention also provides a novel streptavidin filler prepared by the above scheme.

[0025] Beneficial effects:

[0026] The novel streptavidin filler of the present invention has a high affinity for biotin or biotinylated substances, and the maximum biotin binding amount can reach 377 nmol / mL. Since the macroporous base ball of the polymer matrix is ​​used and the surface is treated with hydrophilicity, it has good alkali resistance. After soaking in 0.1M NaOH solution for 48 hours, the biotin binding amount decreases by less than 0.8%. In addition, the filler also has good pressure resistance, high mass transfer efficiency and excellent hydrophilicity, and non-specific adsorption is extremely low; in addition, the process of the present invention is simple and easy to scale up for production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the spectrum of biotin purified by the filler prepared in Example 1. DETAILED DESCRIPTION

[0028] The present invention will be further described in detail below in conjunction with specific embodiments. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0030] Example 1

[0031] S1. Hydrophilic modification of matrix

[0032] Accurately weigh 10 g of polymethacrylate base ball on a balance and add it into a 500 mL three-necked flask, add 200 mL of 0.5 mol / L NaOH solution into the reaction flask and stir for 1.0 hour, add 10 g of epichlorohydrin and 4.0 g of dextran 2000, stir at 25 °C for 10.0 hours, filter with a Buchner funnel after the reaction, wash with 200 mL of ethanol, and then wash with ultrapure water until the pH test paper shows neutral, drain and set aside to obtain intermediate 1.

[0033] S2, Epoxy Activation

[0034] The intermediate 1 was transferred to a 500 mL three-necked flask, and 200 mL of 1.5 mol / L sodium hydroxide aqueous solution was added. The mixture was stirred for 1.0 hour, and then 20 g of ethylene glycol glycidyl ether was added. The mixture was stirred at 25°C for 4 hours. After the reaction was completed, it was filtered with a Buchner funnel and washed with ultrapure water until neutral to obtain the intermediate 2.

[0035] S3, coupling ligand

[0036] The intermediate 2 was added to a 250 mL three-necked flask, 50 mL of 20 mM PB buffer, 4.0 mL of 2.0 mg / mL streptavidin (SA) aqueous solution, the pH was adjusted to 8.0, and the mixture was reacted at 40 °C for 20 h. After the reaction, the mixture was filtered with a Buchner funnel, and the filler was washed with 500 mL of ethanol and 500 mL of ultrapure water in turn. Finally, the filler was stored in 20% ethanol and refrigerated at 2-8 °C.

[0037] Example 2

[0038] The difference from Example 1 is that, in the hydrophilic modification of the S1 matrix, dextran 2000 is replaced with dextran 4000, and the rest of the process is the same as Example 1.

[0039] Example 3

[0040] The difference from Example 1 is that, in the hydrophilic modification of the S1 matrix, dextran 2000 is replaced with dextran 6000, and the rest of the process is the same as Example 1.

[0041] Example 4

[0042] The difference from Example 1 is that in the S2 epoxy activation, ethylene glycol glycidyl ether is replaced with 1,4-butanediol diglycidyl ether, and the rest of the process is the same as Example 1.

[0043] Comparison example

[0044] Compared with Example 1, the difference is that SA is not added in the S3 coupling ligand step, and the rest of the process is the same as Example 1. Adsorption and elution test of SA affinity filler

[0045] Chromatography column: The SA affinity filler prepared in Example 1 was filled into a 4.6*50 mm chromatography column;

[0046] Instrument: Protein purification system SCG;

[0047] Flow rate: 0.34 mL / min;

[0048] Detector: UV 280nm;

[0049] Mobile phase: A: 20 mM NaH2PO4 + 150 mM NaCl, pH 7.4;

[0050] B: 8 M guanidine hydrochloride, pH 1.5;

[0051] Sample volume: 5 μL (3.4 mg / mL biotin);

[0052] Column temperature: room temperature;

[0053] method:

[0054] Equilibration: 100% A, 12 min;

[0055] Elution: 100% B, 16 min;

[0056] Table 1 below shows the test results of Examples 1-4 and the control example.

[0057] Table 1

[0058]

[0059]

[0060] Table 2 below shows the results of the natron test of Example 1 and Example 4.

[0061] (NaOH conditions: immerse the filler in 0.1M NaOH solution for 48 hours)

[0062] Table 2

[0063]

[0064] The above are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a novel streptavidin filler, characterized in that: The following steps are involved: 1) Hydrophilic modification of the matrix: the polymer matrix is ​​dispersed in the alkaline solution 1, the epoxy reagent 1 and dextran are added, stirred and reacted, and the intermediate 1 is obtained by filtering and washing; 2) Epoxy activation: dispersing the intermediate 1 in an alkaline solution 2, adding an epoxy reagent 2, stirring and reacting, filtering and washing to obtain an intermediate 2; 3) Coupling with streptavidin: first disperse the intermediate 2 in water for 0.5-2 hours, then mix with PB buffer and streptavidin aqueous solution, adjust the pH to 6.5-9.0, react, and obtain a novel streptavidin filler; The polymer matrix is ​​selected from polystyrene / divinylbenzene, polymethacrylate, and polyacrylamide; the matrix is ​​porous microspheres with a pore size of 500-10000Å and a particle size of 5.0-100μm.

2. The preparation method according to claim 1, characterized in that: The alkaline solution 1 and the alkaline solution 2 are inorganic bases, including sodium hydroxide and potassium hydroxide. The concentration of the alkaline solution 1 is 0.5-2.0M, and the concentration of the alkaline solution 2 is 0.5-6.0M.

3. The preparation method according to claim 1, characterized in that: The epoxy reagent 1 and the epoxy reagent 2 are selected from epichlorohydrin, ethylene glycol glycidyl ether, 1,4-butanediol diglycidyl ether, 1,2-cyclohexanediol diglycidyl ether or pentaerythritol glycidyl ether.

4. The preparation method according to claim 1, characterized in that: The molecular weight of the dextran is 2000-200000.

5. The preparation method according to claim 1, characterized in that: The mass ratio of the matrix to the dextran is 1.0:0.02-0.4, and the mass ratio of the matrix to the epoxy reagent 1 is 1.0:1.0-1.

5.

6. The preparation method according to claim 1, characterized in that: The mass ratio of the matrix to the epoxy reagent 2 is 1.0:2.0-2.

5.

7. A novel streptavidin filler, characterized in that: Prepared by the method according to any one of claims 1 to 6.

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