Immobilized IdeS enzyme and preparation method and enzyme digestion method thereof

Abstract

The invention provides an immobilized IdeS enzyme and a preparation method and an enzyme digestion method thereof, and solves the problems that the existing IdeS enzyme can only be used once, and the protein purity of subsequent enzyme digestion is not single, the scheme mainly comprises the following steps: coupling the IdeS enzyme with Sepharose4FF-CNBr to obtain the corresponding immobilized IdeS enzyme, and the subsequent enzyme digestion process can be summarized as follows: adding an antibody stock solution and the immobilized IDES enzyme in a ratio of 1: 10, and carrying out enzyme digestion to obtain the immobilized IdeS enzyme. Incubating in a 37 DEG C constant-temperature water bath kettle for 3 hours; after 3h, 200g is centrifuged to remove enzyme; the method comprises the following steps of: taking the supernatant to pass through a protein A column, collecting the flow-through liquid, namely F (ab) 2 after enzyme digestion, and performing one-step purification to obtain the protein with higher purity after enzyme digestion, so that very high yield and purity are obtained, and the experiment cost and precious experiment time are greatly reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme cleavage, and in particular to an immobilized IdeS enzyme and a preparation method and an enzyme cleavage method thereof. Background Art

[0002] Immobilized enzyme is a new technology developed in the 1960s. The so-called immobilized enzyme refers to an enzyme that catalyzes in a certain spatial range and can be used repeatedly and continuously. Usually, enzyme catalysis reactions are carried out in aqueous solution, while immobilized enzyme is a water-soluble enzyme treated by physical or chemical methods to make it insoluble in water but still have enzyme activity.

[0003] At present, when conducting experiments such as IdeS enzyme cleavage of IgG and Fc fusion protein in vitro, the amount of enzyme used is greatly wasted because the IdeS enzyme has the characteristics of single use and cannot be reused. At the same time, it is not easy to remove the Ides enzyme in later experiments to obtain a protein of single purity, which also increases the amount of protein loss, increases the number of experimental steps, and wastes time.

[0004] To this end, we proposed an immobilized IdeS enzyme and its preparation method and enzyme cleavage method to solve the above problems. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects of the prior art. The present invention proposes an immobilized IdeS enzyme and a preparation method and an enzyme cleavage method thereof. By coupling the IdeS enzyme with CNBr-activated Sepharose 4FastFlow to prepare an immobilized enzyme, the yield and purity of the enzyme cleavage are greatly improved. Compared with the single IdeS enzyme, the utilization rate is improved, the tedious experimental process of removing the enzyme in the later stage is eliminated, and the experimental efficiency is greatly improved.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: an immobilized IdeS enzyme, comprising: an IdeS enzyme and Sepharose 4Fast Flow obtained by amino coupling.

[0007] A method for preparing the immobilized IdeS enzyme comprises the following steps:

[0008] S1-1, weigh 2 g of Sepharose 4 Fast Flow and add 15 mL of 1 mM HCl to mix, then swell for 30 min at a swelling ratio of 1:3, centrifuge to remove the supernatant, repeatedly add 15 mL of 1 mM HCl to mix, and centrifuge to remove the supernatant to obtain the corresponding gel solution;

[0009] S1-2, take 1 mL of the above gel solution and add 0.5 mg of IdeS enzyme to it, and add coupling buffer at the same time, make the volume to 2.5 mL, and shake at room temperature for 1.5 hours;

[0010] S1-3, centrifuge to remove the supernatant, and measure OD280 0.03, OD 260 0.03, OD 320 0.006;

[0011] S1-4, add 100mM Tris-HCl with 4 times the volume of gel solution and seal, shake at room temperature for 2h, then wash and store.

[0012] Furthermore, the washing process in step S1-4 is specifically as follows:

[0013] Wash with 5 volumes of 1*PBS until neutral;

[0014] Wash with 5 volumes of pure water;

[0015] Wash with 2 volumes of 25% ETOH and save.

[0016] An enzymatic digestion method based on the immobilized IdeS enzyme comprises the following steps:

[0017] S2-1, mix the antibody stock solution with the immobilized IdeS enzyme at a mass ratio of 1:10, and incubate in a constant temperature water bath at 37°C for 3 hours;

[0018] S2-2, take 200 g of the mixed solution and centrifuge to remove the enzyme;

[0019] S2-3. Take the supernatant and pass it through the protein A column, collect the flow-through, that is, the F(ab)2 after enzyme cleavage, and purify it in one step to obtain the enzyme-cleaved protein.

[0020] Compared with the prior art, the beneficial effects of the present invention include: the method of coupling IdeS enzyme with Sepharose 4FF-CNBr can obtain an active immobilized enzyme, thus avoiding the problem that the existing Ides enzyme cannot be reused, and a protein with a single purity can be easily obtained after enzyme cleavage, thereby indirectly reducing the loss of protein, reducing the experimental steps and the requirements for experimental experiments, and the experimental yield and degree are higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The disclosure of the present invention is described with reference to the accompanying drawings. It should be understood that the drawings are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. In the accompanying drawings, the same reference numerals are used to refer to the same components. Among them:

[0022] Figure 1 The schematic diagram shows the effect of immobilized IdeS enzyme cleavage according to one embodiment of the present invention. DETAILED DESCRIPTION

[0023] It is easy to understand that according to the technical solution of the present invention, without changing the essential spirit of the present invention, a person skilled in the art can propose a variety of interchangeable structural modes and implementation modes. Therefore, the following specific implementation modes and drawings are only exemplary descriptions of the technical solution of the present invention, and should not be regarded as the whole of the present invention or as a limitation or restriction to the technical solution of the present invention.

[0024] According to one embodiment of the present invention, Figure 1 Shown.

[0025] FabRICATOR (IdeS) is a unique cysteine ​​protease that cleaves IgG at a single amino acid site below the hinge region. It can cleave IgG from a variety of sources (human, mouse, rabbit, monkey, sheep, chimeric IgG, Fc fusion protein, etc.).

[0026] Sepharose 4Fast Flow is a highly cross-linked agarose matrix. CNBr-activated Sepharose 4Fast Flow is a pre-activated affinity matrix that combines the advantages of CNBr coupling with the high fluidity and stability of Sepharose 4Fast Flow. It can couple biospecific ligands to the CNBr-activated mechanism to prepare affinity chromatography media. Proteins and other molecules containing primary amino groups can be directly coupled to the pre-activated gel.

[0027] Immobilized IdeS The IdeS enzyme is coupled to CNBr-activated Sepharose 4Fast Flow via amino groups. The resulting affinity matrix can separate specific species from complex mixtures, often in a single step with very high yield and purity.

[0028] The present invention borrows the CNBr-activated Sepharose 4Fast Flow mechanism and couples the IdeS enzyme with the CNBr-activated Sepharose 4Fast Flow to prepare an immobilized enzyme, thereby greatly improving the yield and purity of the enzyme cleavage. Compared with the single IdeS enzyme, the utilization rate is improved, the tedious experimental process of enzyme removal in the later stage is eliminated, and the experimental efficiency is greatly improved.

[0029] Specifically, for the preparation method of the immobilized IdeS enzyme obtained by the above coupling,

[0030] Experimental equipment:

[0031] 1. Decolorization shaker;

[0032] 2. Centrifuge tube.

[0033] Experimental Materials:

[0034] 1. Sepharose 4FF CNBr;

[0035] 2.IdeS enzyme;

[0036] 3. Coupling buffer: PBS, pH 7.4;

[0037] 4. Blocking buffer: 100 mM Tris-HCl solution pH 8.3;

[0038] 5. Save buffer: 25% ETOH.

[0039] The corresponding operation steps are as follows:

[0040] S1-1, weigh 2 g of Sepharose 4 Fast Flow and add 15 mL of 1 mM HCl to mix, then swell for 30 min at a swelling ratio of 1:3, centrifuge to remove the supernatant, repeatedly add 15 mL of 1 mM HCl to mix, and centrifuge to remove the supernatant to obtain the corresponding gel solution;

[0041] S1-2, take 1 mL of the above gel solution and add 0.5 mg of IdeS enzyme to it, and add coupling buffer at the same time, make the volume to 2.5 mL, and shake at room temperature for 1.5 hours;

[0042] S1-3, centrifuge to remove the supernatant, and measure OD280 0.03, OD 260 0.03, OD 320 0.006;

[0043] S1-4, add 100mM Tris-HCl with 4 times the volume of gel solution and seal, shake at room temperature for 2h, then wash and store.

[0044] The washing process is as follows:

[0045] Wash with 5 volumes of 1*PBS until neutral;

[0046] Wash with 5 volumes of pure water;

[0047] Wash with 2 volumes of 25% ETOH and save.

[0048] Similarly, when conducting experiments such as IdeS enzyme cleavage of IgG and Fc fusion proteins in vitro, the amount of enzyme used is greatly wasted due to the fact that the IdeS enzyme is a single-use enzyme and cannot be reused. At the same time, it is not easy to remove the Ides enzyme to obtain a protein of single purity in later experiments, which also increases the amount of protein loss, increases the number of experimental steps, and wastes time.

[0049] The method of coupling IdeS enzyme with Sepharose 4FF-CNBr to immobilize the enzyme greatly solves the above problems. While making the IdeS enzyme reusable, it also reduces the experimental steps, saves time and cost, and further obtains a very high yield and purity, greatly reducing the experimental cost and precious experimental time. The enzyme cleavage effect is as follows: Figure 1 shown.

[0050] The specific experimental process is as follows:

[0051] S2-1, antibody stock solution and immobilized IDES enzyme were added at a ratio of 1:10 and incubated in a constant temperature water bath at 37 degrees Celsius for 3 hours;

[0052] S2-2, 3 h later, centrifugation at 200 g was performed to remove the enzyme;

[0053] S2-3. Take the supernatant and pass it through the protein A column to collect the flow-through, i.e. the F(ab)2 after enzyme cleavage. A protein with higher purity after enzyme cleavage can be obtained by one-step purification.

[0054] The technical scope of the present invention is not limited to the contents in the above description. Those skilled in the art can make various deformations and modifications to the above embodiments without departing from the technical idea of ​​the present invention, and these deformations and modifications should all fall within the protection scope of the present invention.

Claims

1. An immobilized IdeS enzyme, characterized in that: include: It is obtained by amino coupling of IdeS enzyme and Sepharose 4Fast Flow.

2. A method for preparing the immobilized IdeS enzyme according to claim 1, characterized in that: The steps include: S1-1, weigh 2 g of Sepharose 4 Fast Flow and add 15 mL of 1 mM HCl to mix, then swell for 30 min at a swelling ratio of 1:3, centrifuge to remove the supernatant, repeatedly add 15 mL of 1 mM HCl to mix, and centrifuge to remove the supernatant to obtain the corresponding gel solution; S1-2, take 1 mL of the above gel solution and add 0.5 mg of IdeS enzyme to it, and add coupling buffer at the same time, make the volume to 2.5 mL, and shake at room temperature for 1.5 hours; S1-3, centrifuge to remove the supernatant, and measure OD280 0.03, OD 260 0.03, OD 320 0.006; S1-4. Take 100 mM Tris-HCl with a volume of 4 times the gel solution and add it to block. Shake it at room temperature for 2 hours, then wash and store it.

3. The preparation method according to claim 2, characterized in that: The washing process in step S1-4 is specifically as follows: Wash with 5 volumes of 1*PBS until neutral; Wash with 5 volumes of pure water; Wash with 2 volumes of 25% ETOH and save.

4. A method for enzymatic cleavage of immobilized IdeS enzyme according to claim 1, characterized in that: The steps include: S2-1, mix the antibody stock solution with the immobilized IdeS enzyme at a mass ratio of 1:10, and incubate in a constant temperature water bath at 37°C for 3 hours; S2-2, take 200 g of the mixed solution and centrifuge to remove the enzyme; S2-3. Take the supernatant and pass it through the protein A column, collect the flow-through, that is, the F(ab)2 after enzyme cleavage, and purify it in one step to obtain the enzyme-cleaved protein.

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