Fusion protein for expression of designable small-molecule collagen peptide and recombinant vector and recombinant expression method thereof

Abstract

The invention provides a designable fusion protein for expression of small-molecule collagen peptide as well as a recombinant vector and a recombinant expression method thereof, and belongs to the technical field of recombinant protein expression. The fusion protein for collagen peptide expression comprises the following connected fragments: a soluble tag, a purification tag, a connecting peptide, a protease digestion recognition sequence and a designed target collagen peptide, the amino acid sequence of the connecting peptide is as shown in SEQ ID NO: 1. And the recombinant fusion protein is subjected to purification, enzyme digestion treatment, re-purification, refining and separation to obtain a peptide fragment with a target molecular weight. Experiments show that the method can obtain the small molecular collagen peptide with small molecular weight, designable sequence and yield reaching the level of gram per liter on the premise of not depending on chemical synthesis and subsequent random enzymolysis, and the scheme is simple and easy to implement, stable in preparation process, high in purity and suitable for industrial large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of recombinant protein expression technology, specifically relating to a fusion protein for the design of small molecule collagen peptide expression, its recombinant vector, and a recombinant expression method. Background Technology

[0002] Collagen and its degradation products (collagen peptides) are widely used in food, health products, cosmetics, and biomedical materials. Currently, the smallest publicly available recombinant collagen peptides are mostly concentrated in the 3–5 kDa range, and there are no reproducible, scalable recombinant expression technologies reported for target peptides of smaller sizes. Traditional preparation routes mainly include chemical synthesis and decomposition. Chemical synthesis is suitable for short peptides, but the synthesis process often uses large amounts of organic solvents and strong acids, resulting in high costs and significant environmental impact for large-scale production. Decomposition involves first recombining or extracting large-molecule collagen, and then obtaining peptides through enzymatic hydrolysis or physical high-temperature and high-pressure processes. However, the resulting products have uneven length distributions, cannot be precisely designed sequences, and suffer from poor batch-to-batch consistency. Currently, there is a lack of a green, safe, and scalable method for preparing small-molecule collagen peptides that combines customizable sequences and controllable molecular weight (2-3 kDa level). Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a fusion protein for the designable expression of small molecule collagen peptides, which is obtained by tandemly linking a target small molecule collagen peptide with a soluble tag, a purification tag, a specific linker peptide, and a cleavage enzyme. Recombinant expression can not only obtain recombinant target small molecule collagen peptides, but also has a high yield, making it suitable for large-scale production.

[0004] This invention provides a fusion protein for collagen peptide expression, comprising the following linked fragments: a soluble tag, a purification tag, a linker peptide, a protease cleavage recognition sequence, and a designed target collagen peptide;

[0005] The amino acid sequence of the linker peptide is shown in SEQ ID NO:1.

[0006] Preferably, the designed target collagen peptide is formed by repeating collagen functional peptides in tandem.

[0007] The collagen functional peptides include Gly-XY tripeptide, where X and Y represent two arbitrary amino acids.

[0008] Preferably, the soluble label includes any one of the following: TRX, SUMO, MBP, and GST;

[0009] The purification tag includes a 6×His tag;

[0010] The protease includes any one of the following enzymes: TEV protease, HRV 3C protease, and thrombin.

[0011] The present invention provides a fusion gene encoding the fusion protein.

[0012] The present invention provides a recombinant vector containing the fusion gene.

[0013] The present invention provides a recombinant strain expressing the fusion protein.

[0014] This invention provides the application of the fusion gene, the recombinant vector, or the recombinant strain in the recombinant expression preparation of collagen peptides.

[0015] This invention provides a method for preparing collagen peptides through recombinant expression, comprising the following steps:

[0016] The fusion gene described in the above technical solution is recombinantly expressed in a host bacterium. The recombinant expression product is purified based on a purification tag. The purified product is subjected to enzyme digestion. After further purification and refinement, peptides of the target molecular weight are separated. The peptides are sequence identified to obtain the target collagen peptide.

[0017] Preferably, the purification method includes Ni-NTA affinity chromatography;

[0018] The binding buffer used in the Ni-NTA affinity chromatography method is 20 mM pH 7.5–8.5 Tris-HCl, 100–300 mM NaCl, and 5–20 mM imidazole.

[0019] The washing buffer used in the Ni-NTA affinity chromatography method is 20 mM pH 7.5–8.5 Tris-HCl, 100–300 mM NaCl, and 20–100 mM imidazole;

[0020] The elution buffer used in the Ni-NTA affinity chromatography method is 20 mM pH 7.5-8.5 Tris-HCl, 100-300 mM NaCl, and 100-300 mM imidazole.

[0021] Preferably, the digestion buffer used for the enzyme digestion treatment is 20 mM pH 7.5–8.5 Tris-HCl and 100–300 mM NaCl;

[0022] During the enzymatic digestion treatment, the mass ratio of the purified product to the protease is 20–100:1.

[0023] The enzyme digestion treatment time is 4 to 24 hours.

[0024] This invention provides a fusion protein for collagen peptide expression. The invention designs a target collagen peptide segment linked to a soluble tag, a purification tag, a linker peptide, and a protease digestion recognition sequence. The soluble tag makes the recombinantly expressed fusion protein readily soluble in water, facilitating subsequent extraction. The purification tag facilitates the separation and purification of the fusion protein from other proteins. The protease digestion recognition sequence is used to separate the target collagen peptide from the fusion protein. The amino acid sequence of the linker peptide, as shown in SEQ ID NO:1, regulates the three-dimensional conformation of the fusion protein, effectively increasing the yield of the target collagen peptide, thereby achieving the goal of recombinantly expressing small molecule collagen peptides as needed.

[0025] This invention also provides a method for preparing collagen peptides through recombinant expression. The fusion gene described in the above-mentioned technical solution is recombinantly expressed in a host bacterium. The resulting recombinant expression product is purified using a purification tag. The purified product is then subjected to enzymatic digestion. After further purification and refinement, peptides of the target molecular weight are separated, and the peptides are sequence-identified to obtain the target collagen peptide. Experiments show that this method can obtain small-molecule collagen peptides with low molecular weight, designable sequences, and yields reaching the gram-per-liter level without relying on chemical synthesis and subsequent random enzymatic digestion. Furthermore, the method is simple, easy to implement, has a stable preparation process, and produces high purity, making it suitable for large-scale industrial production. Attached Figure Description

[0026] Figure 1 This is a chromatogram of the recombinant vector containing the fusion gene constructed in Example 1 of the present invention;

[0027] Figure 2 Electrophoretic images of different elution fractions were obtained using Tricine-SDS-PAGE.

[0028] Figure 3 The results of Ni-NTA affinity chromatography were obtained after TEV cleavage of the recombinant expression product.

[0029] Figure 4 For gel filtration elution detection results;

[0030] Figure 5 Results of Tricine-SDS-PAGE analysis for different collected peak fractions;

[0031] Figure 6 The results are from MALDI-TOF-MS.

[0032] Figure 7 The results show the small molecule collagen peptide (type III) (colpep3) recombinantly expressed using different linker peptides. Detailed Implementation

[0033] This invention provides a fusion protein for collagen peptide expression, comprising the following linked fragments: a soluble tag, a purification tag, a linker peptide, a protease cleavage recognition sequence, and a designed target collagen peptide; the amino acid sequence of the linker peptide is shown in SEQ ID NO:1.

[0034] In this invention, the designed target collagen peptide is preferably formed by tandem repeating collagen functional peptides. This invention does not impose any particular limitation on the preparation method of the collagen functional peptide; any collagen functional peptide of interest in the art can be used. In the embodiments of this invention, the collagen functional peptide is represented by the Gly-XY tripeptide, where X and Y represent two arbitrary amino acids. The tandem copy number of the repeating unit is preferably 7–10, and can be 8–9. In the embodiments of this invention, the amino acid sequence of the designed target collagen peptide (colpep3) is as shown in SEQ ID NO:2.

[0035] In this invention, the soluble tag preferably includes any one of the following: TRX, SUMO, MBP, and GST. In this embodiment, the soluble expression of the fusion protein is illustrated using a TRX tag as an example. The soluble tag guides the soluble expression of the fusion protein, improves its solubility in water, and thus facilitates the efficient extraction of the fusion protein.

[0036] In this invention, the purification tag preferably includes a 6×His tag. In this embodiment, a purification method for the fusion protein is described using a 6×His tag as an example. The amino acid sequence of the 6×His tag is as shown in SEQ ID NO:11 (HHHHHH). The purification tag provides a target site for specific binding during the purification process, thereby achieving the purpose of removing contaminating proteins.

[0037] In this invention, the protease preferably includes any one of the following enzymes: TEV protease, HRV 3C protease, and thrombin. In this embodiment, TEV protease is used as a representative example to illustrate the enzymatic cleavage and isolation of the target collagen peptide from the fusion protein. The cleavage recognition sequence of the TEV protease is ENLYFQ↓G (SEQ ID NO:12) or an equivalent site, where "↓" represents the cleavage site.

[0038] The present invention provides a fusion gene encoding the fusion protein.

[0039] In this invention, the fusion gene can be codon-optimized according to the type of subsequent expression host. In an embodiment of this invention, when the fusion protein is TrX-His6-Linker-tev-colpep3, the nucleotide sequence of the fusion gene is shown in SEQ ID NO:13(ATGAGCGATAAAATTATTCATCTGAC).

[0040] The present invention provides a recombinant vector containing the fusion gene.

[0041] In this invention, the backbone vector of the recombinant vector can be selected according to the type of host bacteria, including prokaryotic expression vectors or eukaryotic expression vectors. The prokaryotic expression vector preferably includes pET-28a(+). In this embodiment of the invention, to illustrate the recombinant expression method of the target collagen peptide, the recombinant vector is constructed by artificial synthesis, or it can be completed through homologous recombination.

[0042] The present invention provides a recombinant strain expressing the fusion protein.

[0043] In this invention, the host bacteria of the recombinant strain preferably includes a prokaryotic expression system or a eukaryotic expression system. The prokaryotic expression system is preferably *Escherichia coli* strain KRX or BL21(DE3). This invention does not impose any particular limitation on the preparation method of the recombinant strain; any transformation method well-known in the art can be used.

[0044] This invention provides the application of the fusion gene, the recombinant vector, or the recombinant strain in the recombinant expression preparation of collagen peptides.

[0045] This invention provides a method for preparing collagen peptides through recombinant expression, comprising the following steps:

[0046] The fusion gene described in the above technical solution is recombinantly expressed in a host bacterium. The recombinant expression product is purified based on a purification tag. The purified product is subjected to enzyme digestion. After further purification and refinement, peptides of the target molecular weight are separated. The peptides are sequence identified to obtain the target collagen peptide.

[0047] In this invention, the recombinant expression is preferably cultured in LB or TB medium, and at OD... 600 Induction culture begins when the temperature is 0.5–0.8, and can be 0.6–0.7. For the induction culture, L-rhamnose or IPTG is preferably added to the culture system. The working concentration of L-rhamnose is preferably 0.05%–0.2%, and can be 0.1%–0.15%; the working concentration of IPTG is preferably 0.1–1 mM, and can be 0.2–0.8 mM, or can be 0.4–0.6 mM. The induction culture temperature is preferably 16–37℃, and can be 20–32℃, or can be 25–28℃. The induction culture time is preferably 1–5 h, and can be 2–3 h.

[0048] In this invention, after recombinant expression, the recombinant expression product is preferably isolated from the bacterial cells. The separation method preferably involves sonication in a lysis buffer, followed by centrifugation to collect the lysis products. The lysis buffer is preferably 20 mM Tris-HCl pH 8.0, 200 mM NaCl, 10 mM imidazole, 10 μg / mL DNase, and 1 mg / mL lysozyme. The sonication power is preferably 150–250 W, and can be 200 W. The total sonication time is preferably 6–8 min, and can be 7 min. The sonication on / off sequence is preferably 1 s on / 1 s off, and the maximum controlled temperature is preferably 7°C. The centrifugal force is preferably 30000–45000 g, and can be 40000 g. The centrifugation temperature is preferably 0–8°C, and can be 4°C. The centrifugation time is preferably 20–40 min, and can be 30 min.

[0049] In this invention, the purification method preferably includes Ni-NTA affinity chromatography. The binding buffer used in the Ni-NTA affinity chromatography is 20 mM pH 7.5–8.5 Tris-HCl, 100–300 mM NaCl, and 5–20 mM imidazole; the washing buffer used in the Ni-NTA affinity chromatography is 20 mM pH 7.5–8.5 Tris-HCl, 100–300 mM NaCl, and 20–100 mM imidazole; the elution buffer used in the Ni-NTA affinity chromatography is 20 mM pH 7.5–8.5 Tris-HCl, 100–300 mM NaCl, and 100–300 mM imidazole. The Ni-NTA affinity chromatography column used in the purification process is a 2 ml column purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0050] In this invention, the digestion buffer used for the enzymatic digestion treatment is 20 mM pH 7.5–8.5 Tris-HCl and 100–300 mM NaCl, or 20 mM pH 7.8–8.2 Tris-HCl and 150–250 mM NaCl; or 20 mM pH 8.0 Tris-HCl and 200 mM NaCl. During the enzymatic digestion treatment, the mass ratio of the purified product to the protease is preferably 20–100:1; it can be 30–80:1; or it can be 50:1. The enzyme used for the digestion treatment is preferably TEV protease. The digestion treatment time is preferably 4–24 h, or 6–20 h, 8–18 h, 10–15 h, or 12 h.

[0051] In this invention, the re-purification is the same as the purification method described above, and will not be repeated here. The purification preferably includes gel filtration (e.g., Superdex 75) or reversed-phase HPLC. During gel filtration, the gel filtration buffer is preferably 50 mM pH 6.0–7.0 phosphate and 150–300 mM NaCl; it can also be 50 mM pH 6.0–7.0 phosphate and 180–220 mM NaCl; or it can be 50 mM pH 6.50 phosphate and 200 mM NaCl.

[0052] In this invention, the sequence identification is preferably performed using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry.

[0053] The method provided by this invention enables the preparation of collagen peptides with controllable and smaller molecular weights, stably preparing collagen peptides with a molecular weight of approximately 3 kDa or less. These collagen peptides can be designed on demand, enabling targeted development of functionalization or specific biological activities. The entire process is green, safe, and mild, conducted in an aqueous system without the need for strong acids or alkalis. It also features high yield and scalability, with gram-level yields achievable through 1L fermentation. Furthermore, it offers advantages such as high purity and thorough analytical validation, as verified by Ni-NTA affinity chromatography, gel filtration, and MALDI-TOF-MS, demonstrating high purity and accurate molecular weight.

[0054] Terminology Explanation:

[0055] TRX: Thioredoxin, a thioredoxin fusion tag that improves protein solubility.

[0056] Ni-NTA: Nickel ion-nitrotriacetic acid affinity chromatography medium used for the purification of His-tagged proteins.

[0057] Tricine-SDS-PAGE: An electrophoretic method suitable for analyzing low molecular weight peptides.

[0058] The following detailed description, in conjunction with embodiments, illustrates a fusion protein for designing small molecule collagen peptide expression, its recombinant vector, and its recombinant expression method provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0059] Example 1

[0060] A fusion protein for the designable expression of small molecule collagen peptides

[0061] Fusion protein framework design

[0062] Construct a fusion protein framework using the following tandem gene elements: The amino acid sequence is as shown in SEQ ID NO:3

[0063]

[0064] The colpep3 is a small molecule collagen peptide (type III), which is a Gly-XY repeating unit tandemly, resulting in a target collagen peptide with a length of 30 amino acids (GEPGGKGERGAPGEKGEGGPPGVAGPPGGS, SEQ ID NO:2).

[0065] Linker is a flexible linker peptide with the following amino acid sequence: SSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDK (SEQ ID NO:1).

[0066] The fusion gene sequence corresponding to the fusion protein framework is as follows: (SEQ ID NO:13).

[0067] Example 2

[0068] A method for recombinant expression and purification of small molecule collagen peptides (type III)

[0069] 1. Construction of recombinant vectors

[0070] The fusion gene sequence designed in Example 1 was codon-optimized in *E. coli* and cloned into the pET-28a(+) sequence between TTTGTTTAACTTTAAGAAGGAGATATACC (SEQ ID NO:4) and CTCGAGCACCACCACCACCACCACTGA (SEQ ID NO:5). This was synthesized by Genscript Biotech Co., Ltd. The chromatogram of the synthesized recombinant vector is shown below. Figure 1 .

[0071] 2. Recombinant Expression Methods

[0072] The recombinant vector was transformed into competent cells of the host bacterium E. coli KRX, and after incubation at 37°C in TB medium, when OD... 600 When the concentration was 0.6, 1 mM L-rhamnose was added to the final concentration, and expression was induced at 16°C for 3 h. The cells were then cultured in selection medium containing 100 μg / mL Ampicillin. After cell collection, the cells were resuspended in lysis buffer (20 mM Tris-HCl pH 8.0, 200 mM NaCl, 10 mM imidazole, 10 μg / mL DNase, 1 mg / mL lysozyme) and lysed under sonication (200 W power, 7 min total time, 1 s on / 1 s off, maximum temperature 7°C). The cells were then centrifuged at 40000 g at 4°C for 30 min, and the protein fraction was collected. Fermentation induced in 1 L of culture medium yielded approximately 2 g of crude fusion protein.

[0073] 3. Purification by Ni-NTA affinity chromatography column

[0074] The crude fusion protein was purified by Ni-NTA affinity chromatography. Adsorption was performed for 30 min, followed by washing with 50 mM imidazole in binding buffer containing 20 mM imidazole, and elution with 20 mM, 50 mM, 100 mM, 150 mM, and 250 mM imidazole solutions to obtain the TRX-His6 fusion protein. Tricine-SDS-PAGE was used to analyze each elution fraction. Results are shown below. Figure 2 .

[0075] 4. TEV digestion and re-purification

[0076] TEV enzyme and the target collagen peptide were mixed at a mass ratio of 1:50 and digested for 20 h in dialysis buffer (20 mM Tris-HCl pH 8.0, 200 mM NaCl, MWCO 500 Da) at 4 °C. The digestion product was purified again by Ni-NTA affinity chromatography, and the flow-through yielded the target small molecule collagen peptide. Metal binding was terminated by adding 1 mM EDTA. Results are shown below. Figure 3 .

[0077] 6. Filter the gel.

[0078] Purification was performed using gel filtration (e.g., Superdex 75). The gel filtration buffer consisted of 50 mM phosphate (pH 6.5) and 150 mM NaCl, with a flow rate of 0.5 ml / min. Results are shown below. Figure 4 .

[0079] Fractions from peaks 1 through 4 were collected, and the molecular weight and purity of the substances in each peak were verified by Tricine-SDS-PAGE electrophoresis. Results are shown below. Figure 5The molecular weight of the peptides in peak 3 was consistent with the expected theoretical value, while the protein in peak 4 was an impurity.

[0080] 7. Identification of Expression Products

[0081] The expression products were detected using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The specific steps are as follows:

[0082] 7.1 Preparation of Calibration Standards

[0083] 7.1.1 Add 125 μL of 0.1% trifluoroacetic acid to a peptide calibration standard test tube.

[0084] 7.1.2 Vortex or gently shake for a few seconds to completely dissolve the solid.

[0085] 7.2 Preparation of HCCA matrix solution

[0086] 7.2.1 Prepare the solvent according to the TA30 system (3 mL acetonitrile + 7 mL 0.1% TFA).

[0087] 7.2.2 Add an appropriate amount of HCCA (α-cyano-4-hydroxycinnamicacid) to TA30 and dissolve it completely at room temperature until it is nearly saturated.

[0088] 7.2.3 If necessary, use ultrasound to assist in dissolution.

[0089] 7.2.4 Centrifuge the solution at 14000 rpm for 5 min, discard the precipitate, and keep only the clear and transparent supernatant for later use (only the homogeneous and transparent phase will be used subsequently).

[0090] 7.3. Standard Spotting (Target Plate Preparation)

[0091] 7.3.1 Take 1 μL of peptide calibration standard solution and mix it with 1 μL of HCCA matrix solution.

[0092] 7.3.2 Apply 1 μL of the mixture to the designated well in the stainless steel MALDI target plate.

[0093] 3.3 Allow the sample to stand at room temperature until it dries and crystallizes naturally.

[0094] 7.4. Sample Spotting

[0095] 7.4.1 Take 1 μL of the sample solution to be tested and mix it with 1 μL of HCCA matrix solution.

[0096] 7.4.2 Apply 1 μL of the mixture to the corresponding well in the target plate.

[0097] 7.4.3 Allow the sample to stand at room temperature until it is completely dry.

[0098] See results Figure 6 The molecular weight of the peptide in peak fraction 3 was measured to be approximately 2599 Da (consistent with the theoretical value); peak fraction 4 contained impurities. The detection results are consistent with those obtained by Tricine-SDS-PAGE.

[0099] The polypeptide in peak 3 was freeze-dried and weighed, yielding approximately 2g.

[0100] Comparative Example 1

[0101] The fusion protein framework was designed according to the method in Example 1, and the constructed fusion protein framework was TrX-His6-Linker-tev-colpep3, wherein the Linker was replaced by Linker2 (SEQ ID NO:6, GSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGS), Linker3 (SEQ ID NO:7, AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAK), Linker4 (SEQ ID NO:8, GGGGSGGGGSGGGGSGGGGSGGGGSGGGGGGS), Linker5 (SEQ ID NO:9, GG ...

[0102] See results Figure 7 The fusion protein framework constructed with Linker1 showed the highest concentration of colpep3 peptide in recombinant expression, while the concentrations of other linker peptides were very low. The reason for this may be that the different rigidity and flexibility of different linkers lead to different cleavage efficiencies. Excessive rigidity or flexibility can affect the exposure and recognition of cleavage sites. Only Linker1 in Example 1 had a suitable degree of rigidity and flexibility, while Linker2, Linker4, and Linker5 were all purely flexible, Linker3 was purely rigid, and Linker6 was a flexible negatively charged linker, all showing low recombinant expression levels of colpep3 peptide.

[0103] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fusion protein for collagen peptide expression, characterized in that, This includes the following linked fragments: soluble tags, purification tags, linker peptides, protease cleavage recognition sequences, and designed target collagen peptides; The amino acid sequence of the linker peptide is shown in SEQ ID NO:

1.

2. The fusion protein for collagen peptide expression according to claim 1, characterized in that, The target collagen peptides in the design are formed by tandem repeating collagen functional peptides. The collagen functional peptides include Gly-XY tripeptide, where X and Y represent two arbitrary amino acids.

3. The fusion protein for collagen peptide expression according to claim 1, characterized in that, The soluble label includes any one of the following: TRX, SUMO, MBP, and GST; The purification tag includes a 6×His tag; The protease includes any one of the following enzymes: TEV protease, HRV 3C protease, and thrombin.

4. A fusion gene encoding the fusion protein of any one of claims 1 to 3.

5. A recombinant vector containing the fusion gene of claim 4.

6. A recombinant strain expressing the fusion protein of any one of claims 1 to 3.

7. The use of the fusion gene of claim 4, the recombinant vector of claim 5, or the recombinant strain of claim 6 in the recombinant expression preparation of collagen peptides.

8. A method for preparing collagen peptides by recombinant expression, characterized in that, Includes the following steps: The fusion gene described in claim 4 is recombinantly expressed in a host bacterium. The recombinant expression product is purified based on a purification tag. The purified product is subjected to enzyme digestion. After further purification and refinement, peptides of the target molecular weight are separated. The peptides are sequence-identified to obtain the target collagen peptide.

9. The method according to claim 8, characterized in that, The purification method includes Ni-NTA affinity chromatography. The binding buffer used in the Ni-NTA affinity chromatography method is 20 mM pH 7.5–8.5 Tris-HCl, 100–300 mM NaCl, and 5–20 mM imidazole. The washing buffer used in the Ni-NTA affinity chromatography method is 20 mM pH 7.5–8.5 Tris-HCl, 100–300 mM NaCl, and 20–100 mM imidazole; The elution buffer used in the Ni-NTA affinity chromatography method is 20 mM pH 7.5-8.5 Tris-HCl, 100-300 mM NaCl, and 100-300 mM imidazole.

10. The method according to claim 8, characterized in that, The enzyme digestion treatment uses a digestion buffer of 20 mM pH 7.5–8.5 Tris-HCl and 100–300 mM NaCl; During the enzymatic digestion treatment, the mass ratio of the purified product to the protease is 20–100:

1. The enzyme digestion treatment time is 4 to 24 hours.

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