Polypeptide synthesis and purification method and application thereof

Abstract

The application provides a polypeptide synthesis and purification method and application thereof, and specifically comprises the use of a compound shown in formula I or a salt thereof in protein purification. According to an embodiment of the application, the compound shown in formula I can be used as a precipitation tag, and the compound is used to effectively purify proteins by using the precipitation or dissolution state of the compound in different polar reagents, so that the purification process is simple, effective and environmentally friendly.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for polypeptide synthesis and purification and its application. Background Technology

[0002] Currently, peptides play an increasingly important role in the development of pharmaceuticals, biomaterials, diagnostic reagents, and cosmetics. The acquisition of peptides primarily relies on solid-phase synthesis technology. In solid-phase peptide synthesis, amino acids need to be sequentially condensed on a solid-phase support. Because incomplete reactions during condensation produce byproducts, additional separation and purification steps are required. High-performance liquid chromatography (HPLC) is used for the separation of peptides after solid-phase synthesis due to its high separation efficiency. However, this commonly used separation strategy also has drawbacks. First, single-phase HPLC cannot perform parallel separations, requiring significant equipment investment for large-scale production. Simultaneously, as peptide chain length increases, impurities also increase. Some impurities, if having the same retention time as the product, are difficult to purify. Some hydrophobic peptides also present purification challenges using HPLC; due to their hydrophobic properties, these peptides adhere to the chromatographic column, increasing system pressure and hindering separation and purification. Finally, the entire HPLC separation and purification process consumes large amounts of chromatographic-grade organic reagents, resulting in high costs and environmental pollution.

[0003] Therefore, it is still necessary to develop new peptide synthesis strategies based on solid-phase synthesis to avoid the use of HPLC separation, which has important economic and environmental value. Summary of the Invention

[0004] The purpose of this invention is to solve at least one of the above-mentioned technical problems.

[0005] In a first aspect, the invention provides for the use of the compound of Formula I or a salt thereof in the purification of proteins.

[0006]

[0007] According to embodiments of the present invention, the compound shown in Formula I or its salt can be dissolved in a solvent with low polarity and can precipitate in a highly polar solvent environment. Its aldehyde group and the naked amino group at the N-terminus of the polypeptide can react. Compared with the method of HPLC purification and separation of polypeptides, it is more effective for the separation and purification of longer polypeptide chains and hydrophobic peptides, and the purification process is simpler, reduces costs, and is more environmentally friendly.

[0008] In some embodiments of the present invention, the protein is a protein obtained through solid-phase synthesis.

[0009] In a second aspect, the present invention provides a method for purifying proteins. According to an embodiment of the present invention, the method includes: ligating a protein sample to be purified with a compound of Formula I or a salt thereof; precipitating the ligation product; and reconstituted the precipitated product to obtain the protein;

[0010]

[0011] According to embodiments of the present invention, the compound represented by Formula I or its salt can dissolve in a solvent with low polarity and precipitate in a highly polar solvent environment. Its aldehyde group and the naked amino group at the N-terminus of the polypeptide can react. Therefore, the method for purifying proteins described in this application is more effective for the separation and purification of longer polypeptide chains and hydrophobic peptides than the method for HPLC purification and separation of polypeptides. Moreover, the purification process is simpler, reduces costs, and is more environmentally friendly.

[0012] According to embodiments of the present invention, the method for purifying proteins may further include at least one of the following additional technical features:

[0013] According to an embodiment of the present invention, the mass ratio of the protein sample to be purified to the compound of Formula I or its salt is 1:1 to 3:2. When the mass ratio of the protein sample to be purified to the compound of Formula I or its salt is 1:1 to 3:2, the compound or its salt can fully contact and connect with the protein.

[0014] According to an embodiment of the present invention, the precipitation treatment is carried out in a highly polar solvent.

[0015] According to an embodiment of the present invention, the reconstitution treatment is performed in a low-polarity reagent.

[0016] According to an embodiment of the present invention, the mass ratio of the protein sample to be purified to the compound shown in Formula I or its salt is 57:46. When the mass ratio of the protein sample to be purified to the compound shown in Formula I or its salt is 57:46, the compound or its salt can more fully contact and connect with the protein.

[0017] According to an embodiment of the present invention, in the ligation process, the -NH2 at the N-terminus of the protein undergoes a ligation reaction with the aldehyde group of the compound.

[0018] According to an embodiment of the present invention, the ligation process is performed under conditions of pH < 7.0. The ligation process is performed under weakly acidic conditions, and the choice of acid is not particularly limited, as long as the pH can be adjusted.

[0019] According to an embodiment of the present invention, the connection process is carried out in a THF-DMF mixture.

[0020] According to an embodiment of the present invention, the mass ratio of THF to DMF in the THF-DMF mixture is 2:1 to 3:1.

[0021] According to an embodiment of the present invention, the THF-DMF mixture further contains acetic acid.

[0022] According to an embodiment of the present invention, the volume percentage of acetic acid in the THF-DMF mixture is 0.1%.

[0023] According to an embodiment of the present invention, the connection process is carried out at 50°C to 60°C for 4.5 to 5.5 hours.

[0024] According to an embodiment of the present invention, the mass-to-volume ratio of the ligation product to the highly polar solvent is 29 (mg): 2 (mL) to 15 (mg): 1 (mL).

[0025] According to an embodiment of the present invention, the mass-to-volume ratio of the ligation product to the highly polar solvent is 76 (mg): 5 (mL).

[0026] According to an embodiment of the present invention, the polarity of the highly polar solvent is not less than 5.5.

[0027] According to embodiments of the present invention, the highly polar reagent includes at least one selected from acetonitrile, acetic acid, and aniline.

[0028] According to an embodiment of the present invention, after the precipitation treatment and before the resolution treatment, the precipitation product is further subjected to a first centrifugation treatment.

[0029] According to an embodiment of the present invention, the first centrifugation is performed at 3000 rpm to 4000 rpm for 2-4 minutes, and the centrifugation is repeated 1 to 3 times.

[0030] According to an embodiment of the present invention, the first centrifugation process is performed at 3500 rpm for 3 minutes, and the centrifugation is repeated twice.

[0031] According to an embodiment of the present invention, the mass-to-volume ratio of the precipitated product to the low-polarity solvent is 45 (mg): 1 (mL) to 40 (mg): 1 (mL). At this ratio, the precipitated product can be sufficiently contacted with the low-polarity solvent to dissolve and break the linkages between the protein and the compound of Formula I or its salt in the precipitated product.

[0032] According to an embodiment of the present invention, the precipitated product and the low polarity solvent have a mass-to-volume ratio of 206 (mg): 5 (mL).

[0033] According to an embodiment of the present invention, the polarity of the low-polarity solvent is less than 3.5.

[0034] According to embodiments of the present invention, the low-polarity solvent includes those selected from diethyl ether and / or methyl tert-butyl ether.

[0035] According to an embodiment of the present invention, the reconstitution treatment is performed in a low-polarity reagent and TFA.

[0036] According to an embodiment of the present invention, the mass-to-volume ratio of the low-polarity reagent to TFA is 6 (mg): 1 (mL) to 4 (mg): 1 (mL).

[0037] According to an embodiment of the present invention, the mass-to-volume ratio of the low-polarity reagent to TFA is 5 (mg): 1 (mL).

[0038] According to an embodiment of the present invention, the reconstitution treatment is carried out in a low-polarity reagent, TFA, H2O and Tips.

[0039] According to an embodiment of the present invention, the mass-to-volume ratio of the low-polarity reagent, TFA, H2O and Tips is 40 (mL): 2 (mL): 2000 (mg): 1 (mL).

[0040] According to embodiments of the present invention, the Tips comprise triisopropylsilane. Those skilled in the art will understand that the Tips are not particularly limited.

[0041] According to an embodiment of the present invention, the protein comprises a protein obtained by a solid-phase polypeptide synthesis method.

[0042] According to an embodiment of the present invention, the resolution treatment is carried out by shaking reaction at 24℃~28℃ for 2~3 hours.

[0043] According to an embodiment of the present invention, the dissolution treatment is carried out by shaking the reaction at 26°C for 2.5 hours.

[0044] According to an embodiment of the present invention, the method further includes a reprecipitation treatment.

[0045] According to an embodiment of the present invention, the reprecipitation treatment is performed by contacting the reconstitution product with a precipitating reagent to obtain the protein. During the reprecipitation treatment, the compound of formula I or its salt in the reconstitution product will precipitate again, while the protein remains dissolved in the precipitating reagent, thereby separating and purifying the two.

[0046] According to an embodiment of the present invention, after the resolution treatment and before the precipitation treatment, the product of the resolution treatment is further concentrated.

[0047] According to an embodiment of the present invention, the volume of the concentrated product is 2 to 5 mL.

[0048] According to an embodiment of the present invention, during the reprecipitation process, the mass-to-volume ratio of the sample to be purified to the precipitating reagent is 8 (mg): 1 (mL) to 10 (mg): 1 (mL).

[0049] According to an embodiment of the present invention, in the reprecipitation process, the mass-to-volume ratio of the sample to be purified to the precipitating reagent is 228 (mg): 5 (mL).

[0050] According to an embodiment of the present invention, the process further includes a second centrifugation of the reprecipitated product to obtain the protein.

[0051] According to an embodiment of the present invention, the second centrifugation is performed at 3000-4000 rpm for 2-4 minutes.

[0052] According to an embodiment of the present invention, the second centrifugation is performed at 3500 rpm for 3 minutes.

[0053] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0054] Figure 1 This diagram illustrates a strategy for obtaining proteins using the Fmoc method in a solid-phase manner and by precipitation tag homogenization, according to an embodiment of the present invention.

[0055] Figure 2 The figure shows the HPLC detection results of unpurified protein synthesized by the Fmoc method in solid phase according to an embodiment of the present invention.

[0056] Figure 3 The figure shows the HPLC detection results of the protein purified by the tag after solid-phase synthesis of protein using the Fmoc method according to an embodiment of the present invention;

[0057] Figure 4 The image shows the mass spectrometry results of the protein obtained after purification using the tag following solid-phase synthesis of protein by the Fmoc method according to an embodiment of the present invention. Detailed Implementation

[0058] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0059] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0060] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0061] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0062] The abbreviations used in this invention are listed below:

[0063] Fmoc: fluorene methoxycarbonyl;

[0064] HPLC: High Performance Liquid Chromatography;

[0065] HCTU: Tetramethylurea hexafluorophosphate;

[0066] DIEA: N,N-diisopropylethylamine;

[0067] Oxyma: Ethyl 2-oxime cyanoacetate;

[0068] DCM: Dichloromethane;

[0069] DMF: N,N-dimethylformamide;

[0070] HOBt: 1-Hydroxybenzotriazole;

[0071] TFA: Trifluoroacetic acid;

[0072] Tips: Triisopropylsilane.

[0073] This invention primarily relies on the properties of the compound or its salt (precipitation tag) shown in Formula I, which is soluble in low-polarity solvents and precipitates in high-polarity solvents. By combining the compound or its salt with classical Fmoc solid-phase synthesis, the aldehyde group on the precipitation tag and the exposed amino group at the N-terminus of the peptide undergo a Schiff base intermediate under weakly acidic conditions. This precipitates the peptide under acetonitrile / water polar conditions, yielding a pure, fully protected intermediate. Post-processing then yields a lyophilized pure peptide, eliminating the need for HPLC purification. Furthermore, the inventors have conducted extensive experimental screening and optimization of various parameters and reagents in the purification method, enabling the method described in this application to purify proteins more efficiently. The strategy for synthesizing, separating, and purifying the protein or peptide using the strategy described in this application is as follows: Figure 1 As shown:

[0074] 1) Using the classic Fmoc solid-phase synthesis strategy, acetic anhydride is added to block unreacted amino groups after each amino acid condensation. This process continues until the peptide solid-phase synthesis is complete.

[0075] 2) Perform fully protected cleavage of the polypeptide obtained in step 1).

[0076] 3) React the fully protected peptide with the precipitated tag, and after the reaction is complete, add icy ether to precipitate the peptide.

[0077] 4) Collect the precipitate, cut it, remove the precipitate tag and side chain protection, precipitate with diethyl ether, and obtain a preliminary pure peptide.

[0078] 5) After dissolving the precipitate in acetonitrile / water, reprecipitate it, filter out the precipitate label, freeze-dry the filtrate, and then interface to obtain freeze-dried peptides.

[0079] The protein purification method using the aforementioned precipitation tag eliminates the need for HPLC separation and purification of peptides, enabling green peptide synthesis and significantly reducing costs associated with HPLC equipment, reagents, and time.

[0080] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the invention in any way. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0081] Example 1 Synthesis of polypeptides

[0082] In this embodiment, 1100 mg of king resin (0.4 mmol) with a substitution degree of 0.36 mmol / g was weighed and placed in a peptide synthesis tube. 5 mL of DMF and 5 mL of DCM were added, and the resin was allowed to swell at room temperature for 30 min. 619 mg (1.6 mmol) of Fmoc-Phe-OH (phenylalanine) was weighed and placed in a 50 mL centrifuge tube. 10 mL of DMF was added to dissolve the Fmoc, followed by 528 μL (3.2 mmol) of DIEA. The mixture was transferred to a peptide synthesis tube and reacted overnight at 33°C with shaking. The resin was then dried and washed. 10 mL of a methanol / DMF (1:20) mixture was added to the resin, and the mixture was reacted at 33°C with shaking for 10 min. The resin was then dried and washed. 10 mL of 20% piperidine solution (0.1 M oxyma) was added to the peptide synthesis tube to remove Fmoc. The reaction was repeated twice at 33°C, for 5 min + 10 min. Weigh 565 mg (1.6 mmol) of Fmoc-Ile-OH (isoleucine) and 628 mg (1.52 mmol) of the condensing agent HCTU into a 50 mL centrifuge tube. Dissolve the HCTU in 10 mL of DMF, then add 528 μL (3.2 mmol) of DIEA. Transfer the mixture to a peptide synthesis tube and incubate at 33 °C with shaking for 1 h. After the reaction, dry the tube and wash the resin. Add 10 mL of a acetic anhydride:DIEA:DMF (1:1:8) mixture to the resin to block unreacted amino acids. Incubate at 33 °C with shaking for 15 min. Repeat the reaction twice. After each incubation, dry the tube and wash the resin. For the remaining amino acids, proceed with amino acid condensation, amino acid blocking, and Fmoc deprotection according to the peptide sequence. Protect the amino acids with 4 equivs of Fmoc, 3.8 equivs of HCTU, and 8 equivs of DIEA for 1 h.

[0083] 10 mL of 1% TFA / DCM solution was added to the resin for fully protected peptide cleavage. The reaction was carried out at 33°C with shaking for 1 h, followed by filtration and collection of the filtrate. Excess DCM was removed from the filtrate using a rotary evaporator. An appropriate amount of ice-cold diethyl ether was added to precipitate the peptide, which was then collected by centrifugation at 3500 rpm. The ether washing process was repeated once. The precipitate was collected, dried, and the crude peptide was obtained.

[0084] Example 2: Purification of the polypeptide

[0085] Weigh 10 mg of the crude peptide obtained in Example 1 and add 1 mL of cleavage reagent directly to deprotect the fully protected peptide. The reaction was carried out with shaking at 26 °C for 2.5 h. After the reaction was complete, ice-cold ether was added, and the precipitate was collected after centrifugation at 3500 rpm. The precipitate was dissolved in 5 mL of acetonitrile / water and analyzed by HPLC as a control for peptide purity before using the precipitate label. Specific experimental results are shown below. Figure 2As shown, there are obvious impurity absorption peaks on the side of the main peak absorption position.

[0086] Weigh 228 mg of the crude peptide obtained in Example 1 and 2 equiv of precipitated tag (184 mg, prepared by the inventors) into a 50 mL EP tube. Dissolve in 10 mL of THF:DMF (7:3), then add 100 μL of acetic acid. Incubate at 55 °C for 5 hours to ligate the peptide to the precipitated tag. Remove the 50 mL EP tube, add 15 mL of acetonitrile, and after observing precipitation by shaking the EP tube appropriately, place the EP tube in a centrifuge at 3500 rpm for 3 min. Collect the precipitate after centrifugation, repeat twice, air dry at room temperature, and then crush. Add 10 mL of TFA / H2O / phenol / Tips (10 mL / 500 μL / 500 mg / 250 μL) to the above precipitate and incubate at 26 °C with shaking for 2.5 h. Purge with nitrogen at room temperature to concentrate the solution to below 5 mL. Add 25 mL of acetonitrile to the concentrate, shake until precipitation occurs, then centrifuge at 3500 rpm for 3 min. Collect the supernatant and analyze it using HPLC to identify the change in peptide purity after using the precipitation tag. Mass spectrometry was also used to detect the protein obtained by the tag purification. Specific experimental results are as follows: Figure 3 and Figure 4 As shown, the results indicate that the impurity absorption peaks on the side of the original main peak of the peptide after purification and labeling have been removed, and the overall chromatographic yield reaches over 95%. Furthermore, the molecular weight of the synthesized peptide was correctly characterized by ESI-MS as 1015.6 Da. After lyophilization, the weight was 54 mg, representing a yield of 36%.

[0087] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0088] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0089] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The use of the compound shown in Formula I or a salt thereof in the purification of proteins, characterized in that, include: After the protein to be purified is fully cleaved and protected, it is then linked to the compound shown in Formula I or its salt. The ligation products were precipitated. as well as The precipitate product is reconstituted to obtain the protein. The protein in the protein sample to be purified was obtained by solid-phase polypeptide synthesis, wherein acetic anhydride was added to block unreacted amino groups after each amino acid condensation. The mass ratio of the protein sample to be purified to the compound shown in Formula I or its salt is 1:1 to 3:2; The precipitation treatment is carried out in a highly polar reagent, which includes at least one selected from acetonitrile, acetic acid, and aniline; The mass-to-volume ratio of the ligation product to the highly polar solvent is 29 mg: 2 mL to 15 mg: 1 mL; The resolution treatment is carried out in a low-polarity solvent, which includes a selection of diethyl ether and / or methyl tert-butyl ether. The mass-to-volume ratio of the precipitated product to the low-polarity solvent is 45 mg: 1 mL to 40 mg: 1 mL; Formula I.

2. A method for purifying proteins, characterized in that, include: After the protein to be purified is fully cleaved and protected, it is then linked to the compound shown in Formula I or its salt. The ligation products were precipitated. as well as The precipitate product is reconstituted to obtain the protein. Formula I, The protein in the protein sample to be purified is obtained by solid-phase polypeptide synthesis, wherein acetic anhydride is added to block unreacted amino groups after each amino acid condensation. The mass ratio of the protein sample to be purified to the compound shown in Formula I or its salt is 1:1 to 3:2; The precipitation treatment is carried out in a highly polar reagent, which includes at least one selected from acetonitrile, acetic acid, and aniline; The mass-to-volume ratio of the ligation product to the highly polar solvent is 29 mg: 2 mL to 15 mg: 1 mL; The resolution treatment is carried out in a low-polarity solvent, which includes a selection of diethyl ether and / or methyl tert-butyl ether. The mass-to-volume ratio of the precipitated product to the low-polarity solvent is 45 mg:1 mL to 40 mg:1 mL.

3. The method according to claim 2, characterized in that, The mass ratio of the protein sample to be purified to the compound shown in Formula I or its salt is 57:

46.

4. The method according to claim 2, characterized in that, In the ligation process, the -NH2 group at the N-terminus of the protein undergoes a ligation reaction with the aldehyde group of the compound.

5. The method according to claim 2, characterized in that, The connection process was performed under conditions of pH < 7.

0.

6. The method according to claim 2, characterized in that, The connection process is carried out in a THF-DMF mixture.

7. The method according to claim 6, characterized in that, The mass ratio of THF to DMF in the THF-DMF mixture is 2:1 to 3:

1.

8. The method according to claim 6, characterized in that, The THF-DMF mixture further contains acetic acid.

9. The method according to claim 8, characterized in that, The volume percentage of acetic acid in the THF-DMF mixture is 0.1%.

10. The method according to claim 2, characterized in that, The connection process is carried out at 50℃~60℃ for 4.5~5.5 h.

11. The method according to claim 2, characterized in that, The mass-to-volume ratio of the ligation product to the highly polar solvent is 76 mg: 5 mL.

12. The method according to claim 2, characterized in that, After precipitation treatment and before resolution treatment, the process further includes subjecting the precipitated product to a first centrifugation treatment.

13. The method according to claim 12, characterized in that, The first centrifugation process is carried out at 3000rpm~4000rpm for 2-4 minutes, and centrifuged 1-3 times.

14. The method according to claim 12, characterized in that, The first centrifugation process was carried out at 3500 rpm for 3 minutes, and the centrifugation was repeated twice.

15. The method according to claim 2, characterized in that, The precipitated product and the low-polarity solvent have a mass-to-volume ratio of 206 mg: 5 mL.

16. The method according to claim 2, characterized in that, The reconstitution process was carried out in a low-polarity reagent and TFA.

17. The method according to claim 16, characterized in that, The mass-to-volume ratio of the low-polarity reagent to TFA is 6 mg:1 mL to 4 mg:1 mL.

18. The method according to claim 16, characterized in that, The mass-to-volume ratio of the low-polarity reagent to TFA is 5 mg: 1 mL.

19. The method according to claim 2, characterized in that, The reconstitution process was carried out in the presence of low-polarity reagents, TFA, H2O, and Tips.

20. The method according to claim 19, characterized in that, The mass-to-volume ratio of the low-polarity reagent, TFA, H2O, and Tips is 40 mL: 2 mL: 2000 mg: 1 mL.

21. The method according to claim 2, characterized in that, The resolution treatment involves a shaking reaction at 24℃~28℃ for 2~3 hours.

22. The method according to claim 2, characterized in that, The resolution treatment was carried out by shaking the reaction at 26°C for 2.5 hours.

23. The method according to claim 2, characterized in that, Further, it includes a reprecipitation process.

24. The method according to claim 23, characterized in that, The reprecipitation treatment is performed in the following manner: The reconstitution product is contacted with a precipitation reagent to obtain the protein.

25. The method according to claim 24, characterized in that, After the resolution treatment and before the precipitation treatment, the process further includes concentrating the product from the resolution treatment.

26. The method according to claim 25, characterized in that, The volume of the concentrated product is 2-5 mL.

27. The method according to claim 24, characterized in that, In the reprecipitation process, the mass-to-volume ratio of the sample to be purified to the precipitating reagent is 8 mg:1 mL to 10 mg:1 mL.

28. The method according to claim 24, characterized in that, In the reprecipitation process, the mass-to-volume ratio of the sample to be purified to the precipitating reagent is 228 mg: 5 mL.

29. The method according to claim 24, characterized in that, The process further includes a second centrifugation of the reprecipitated product to obtain the protein.

30. The method according to claim 29, characterized in that, The second centrifugation process is carried out at 3000-4000 rpm for 2-4 minutes.

31. The method according to claim 30, characterized in that, The second centrifugation process was carried out at 3500 rpm for 3 minutes.

Images