CN115786426B

CN115786426B - High-specificity enterokinase enzyme digestion method and application of ethanol in improving enterokinase enzyme digestion specificity

Info

Publication number: CN115786426B
Application number: CN202211343921.9A
Authority: CN
Inventors: 吴玲; 屈智慧; 李岩; 潘海
Original assignee: Beijing Xianweida Biotechnology Co ltd; Sciwind Biosciences Co Ltd
Current assignee: Beijing Xianweida Biotechnology Co ltd; Hangzhou Xianweida Biotechnology Co ltd
Priority date: 2022-10-31
Filing date: 2022-10-31
Publication date: 2023-09-08
Anticipated expiration: 2042-10-31
Also published as: CN117467649A; CN115786426A; WO2024092879A1

Abstract

The application provides a high-specificity enterokinase enzyme digestion method, which comprises the following steps: and (3) carrying out enzyme digestion reaction on the substrate by utilizing enterokinase in the presence of enzyme digestion promoter, wherein the enzyme digestion promoter is one or more than two selected from ethanol, glycerol, sucrose and sodium chloride. The application also provides the use of ethanol in improving enterokinase enzyme cleavage specificity. The enterokinase enzyme digestion method provided by the application greatly reduces the content of nonspecific products compared with the prior art, thereby greatly improving the purity of the products. Meanwhile, the high cleavage rate can be kept, and the balance of enterokinase cleavage specificity and cleavage efficiency can be kept; simple operation and effectively reduced purification cost.

Description

High-specificity enterokinase enzyme digestion method and application of ethanol in improving enterokinase enzyme digestion specificity

Technical Field

The application belongs to the field of biological medicine, and in particular relates to a high-specificity enterokinase enzyme digestion method and application of ethanol in improving enterokinase enzyme digestion specificity.

Background

With the development of medicine, biomacromolecules, especially protein/polypeptide drugs, are increasingly being focused and favored by drug development enterprises due to their unique mechanism of action. However, biopharmaceuticals are more demanding in terms of manufacturing process than small molecule drugs. In the production or preparation of proteins/polypeptides, it is often necessary to cleave proteins in order to obtain active protein products, and it is currently common practice to use the sequence specificity of different proteases for cleavage substrates to cleave proteins. Enterokinase (EK) is a serine protease which has been widely used in biological engineering due to its strong specificity for Asp-Asp-Asp-Asp-Lys (D4K) sequences (Biochim. Biophys Acta 20 (1956) 443-434) and a broad range of activities (activity maintained at pH4.5-9.5 and at a temperature of 4-45 ℃), e.g., flag tag has been widely used as an important component in the production and purification of expressed proteins due to its Enterokinase cleavage site. In practice, it is described in CN101624588 that a recombinant enterokinase (rEK) variant having a possibility of enhancing the binding activity of EKL to DDDDK is selected by referring to the crystal structure of a complex of bovine enterokinase light chain EKL and its inhibitor VD 4K-chloromethyl (Code no:1EKB,Protein Dtabase Bank). Oi WahLiew et al (Protein expr. Purif.41 (2005) 332-340) describe a peptide sequence LKGDR that is more efficient than the D4K site in the enterokinase cleavage process. Although the prior art scheme has improved enzyme digestion activity, substrate specificity and other aspects compared with the traditional enterokinase enzyme digestion reaction, the technical problems of high enterokinase nonspecific product content, unbalanced enzyme digestion activity and specificity, complex improvement means and the like still exist in the face of the actual demands in the production and purification processes of protein/polypeptide, so that the enterokinase enzyme digestion process with higher enzyme digestion specificity, complete reaction, good industrial compliance, simple and convenient operation and economy is still needed.

Disclosure of Invention

The application provides a high-specificity enterokinase enzyme digestion method and application of ethanol in improving enterokinase enzyme digestion specificity.

In particular, the application relates to the following aspects:

1. a method of high specificity enterokinase cleavage, wherein the cleavage method comprises:

and (3) carrying out enzyme digestion reaction on the substrate by utilizing enterokinase in the presence of enzyme digestion promoter, wherein the enzyme digestion promoter is one or more than two selected from ethanol, glycerol, sucrose and sodium chloride.

2. The cleavage method according to item 1, wherein the cleavage promoter is ethanol.

3. The cleavage method according to item 2, wherein the volume concentration of ethanol used for the cleavage reaction is 1% to 50%, preferably 5% to 40%, further preferably 10% to 30%.

4. The cleavage method as recited in any one of items 1 to 3, wherein enterokinase, the cleavage promoter, and the substrate are subjected to cleavage reaction in a buffer.

5. The method according to item 4, wherein the pH of the buffer is 7.1 to 10, preferably 7.4 to 9.5, and more preferably 7.5 to 9.0.

6. The method according to item 4, wherein the buffer is one or more selected from the group consisting of phosphate buffer, tris-base buffer, organic acid buffer, borate buffer, and amino acid buffer,

Preferably selected from disodium hydrogen phosphate-citric acid buffer, citric acid-NaOH-HCl buffer, citric acid-sodium citrate buffer, acetic acid-sodium acetate buffer, phosphate Buffer (PBS), disodium hydrogen phosphate-sodium dihydrogen Phosphate Buffer (PB), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-NaOH buffer, barbital sodium-HCl buffer, NH ₄ HCO ₃ Buffer solution, sodium carbonate-sodium bicarbonate buffer solution and NaHCO ₃ Buffer solution, tris-HCl, glycine-NaOH buffer solution, boric acid-borax buffer solution and Na ₂ B ₇ O ₄ One or more than two buffers,

further preferred is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

7. The cleavage method according to claim 1, wherein the substrate concentration at which the cleavage reaction is carried out is 0.5 to 10mg/ml, preferably 1 to 8mg/ml, more preferably 1 to 4mg/ml.

8. The cleavage method according to claim 1, wherein the ratio of enterokinase enzyme activity to substrate for the cleavage reaction is 0.5 to 10U/mg, preferably 1.5 to 6U/mg, more preferably 2 to 5U/mg.

9. The cleavage method according to item 1, wherein the substrate is an amino acid sequence comprising DDDDK.

10. The cleavage method as recited in claim 1, wherein the reaction time of the cleavage reaction is 4 to 72 hours.

11. Use of ethanol for increasing enterokinase cleavage specificity.

12. The use according to item 11, wherein the substrate is subjected to an cleavage reaction using enterokinase in the presence of ethanol.

13. The use according to claim 12, wherein the ethanol volume concentration for the cleavage reaction is 1% -50%, preferably 5% -40%, further preferably 10% -30%.

14. The use according to item 12, wherein enterokinase, ethanol, and a substrate are subjected to an enzyme cleavage reaction in a buffer.

15. The use according to item 14, wherein the pH of the buffer is 7.1-10, preferably 7.4-9.5, further preferably 7.5-9.0.

16. The use according to item 14, wherein the buffer is one or more selected from phosphate buffer, tris-base buffer, organic acid buffer, borate buffer, amino acid buffer, preferably selected from disodium hydrogen phosphate-citric acid buffer, citric acid-NaOH-HCl buffer, citric acid-sodium citrate buffer, acetic acid-sodium acetate buffer, phosphate Buffer (PBS), disodium hydrogen phosphate-sodium dihydrogen Phosphate Buffer (PB), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-NaOH buffer, barbituric sodium-HCl buffer, NH ₄ HCO ₃ Buffer solution, sodium carbonate-sodium bicarbonate buffer solution and NaHCO ₃ Buffer solution, tris-HCl, glycine-NaOH buffer solution, boric acid-borax buffer solution and Na ₂ B ₇ O ₄ One or two or more buffers are more preferably disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

17. The use according to item 12, wherein the substrate concentration for performing the cleavage reaction is 0.5-10mg/ml, preferably 1-8mg/ml, further preferably 1-4mg/ml.

18. The use according to item 12, wherein the ratio of enterokinase enzyme activity to substrate for the cleavage reaction is 0.5 to 10U/mg, preferably 1.5 to 6U/mg, more preferably 2 to 5U/mg.

19. The use according to item 12, wherein the substrate comprises the amino acid sequence of DDDDK.

20. The use according to item 12, wherein the reaction time of the substrate cleavage reaction is 4 to 72 hours.

21. A method for producing a polypeptide, wherein the method comprises the high-specificity enterokinase cleavage method of any one of claims 1 to 10,

preferably, the method comprises performing an cleavage reaction on the substrate using enterokinase in the presence of ethanol.

22. The method according to claim 21, wherein the volume concentration of ethanol used for the cleavage reaction is 1% to 50%, preferably 5% to 40%, further preferably 10% to 30%.

23. The method according to item 21, wherein enterokinase, the cleavage promoter, and the substrate are subjected to a cleavage reaction in a buffer.

24. The method according to item 23, wherein the pH of the buffer is 7.1-10, preferably 7.4-9.5, further preferably 7.5-9.0.

25. The method of item 23, wherein,

the buffer solution is selected from one or more of phosphate buffer solution, tris buffer solution, organic acid buffer solution, borate buffer solution, and amino acid buffer solution, preferably selected from disodium hydrogen phosphate-citric acid buffer solution, citric acid-NaOH-HCl buffer solution, citric acid-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution, phosphate Buffer Solution (PBS), disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (PB), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, potassium dihydrogen phosphate-NaOH buffer solution, barbital sodium-HCl buffer solution, and NH ₄ HCO ₃ Buffer solution, sodium carbonate-sodium bicarbonate buffer solution and NaHCO ₃ Buffer solution, tris-HCl, glycine-NaOH buffer solution, boric acid-borax buffer solution and Na ₂ B ₇ O ₄ One or two or more buffers are more preferably disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

26. The method according to item 21, wherein the substrate concentration at which the cleavage reaction is carried out is 0.5-10mg/ml, preferably 1-8mg/ml, further preferably 1-4mg/ml.

27. The method according to claim 21, wherein the ratio of enterokinase enzyme activity to substrate for the cleavage reaction is 0.5 to 10U/mg, preferably 1.5 to 6U/mg, more preferably 2 to 5U/mg.

28. The method according to item 21, wherein the substrate is an amino acid sequence comprising DDDDK.

29. The method of claim 21, wherein the polypeptide is GLP-1, an analog or derivative thereof.

30. The method according to item 21, wherein the reaction time of the cleavage reaction is 4 to 72 hours.

31. A method for purifying a polypeptide, wherein the method comprises the high specificity enterokinase cleavage method of any one of claims 1 to 10,

32. The method according to item 31, wherein the volume concentration of ethanol used for the cleavage reaction is 1% -50%, preferably 5% -40%, further preferably 10% -30%.

33. The method according to item 31, wherein enterokinase, the cleavage promoter, and the substrate are subjected to a cleavage reaction in a buffer.

34. The method according to item 33, wherein the pH of the buffer is 7.1-10, preferably 7.4-9.0, further preferably 8.0-9.0.

35. The method of item 33, wherein the buffer

One or more selected from phosphate buffer, tris buffer, organic acid buffer, borate buffer, and amino acid buffer, preferably selected from disodium hydrogen phosphate-citric acid buffer, citric acid-NaOH-HCl buffer, citric acid-sodium citrate buffer, acetic acid-sodium acetate buffer, phosphate Buffer (PBS), disodium hydrogen phosphate-sodium dihydrogen Phosphate Buffer (PB), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-NaOH buffer, barbital sodium-HCl buffer, and NH ₄ HCO ₃ Buffer solution, sodium carbonate-sodium bicarbonate buffer solution and NaHCO ₃ Buffer solution, tris-HCl, glycine-NaOH buffer solution, boric acid-borax buffer solution and Na ₂ B ₇ O ₄ One or two or more buffers are more preferably disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

36. The method according to item 31, wherein the substrate concentration at which the cleavage reaction is carried out is 0.5-10mg/ml, preferably 1-8mg/ml, further preferably 1-4mg/ml.

37. The method according to item 31, wherein the ratio of enterokinase enzyme activity to substrate for the cleavage reaction is 0.5 to 10U/mg, preferably 1.5 to 6U/mg, more preferably 2 to 5U/mg.

38. The method according to item 31, wherein the substrate is an amino acid sequence comprising DDDDK.

38. The method of item 31, wherein the polypeptide is GLP-1, an analog or derivative thereof.

40. The method according to item 31, wherein the reaction time of the cleavage reaction is 4 to 72 hours.

41. A cleavage composition, wherein the cleavage composition is obtained by the cleavage method according to any one of claims 1 to 10,

preferably, the cleavage composition comprises the target polypeptide shown in SEQ ID NO. 1, wherein the mass content of the target polypeptide in the cleavage composition is 80% or more, preferably 85% or more, and more preferably 90% or more.

42. The cleavage composition of item 41, wherein the cleavage composition further comprises one or more of the polypeptides represented by SEQ ID NOs 4-7.

43. The cleavage composition according to item 41, wherein the total mass content of the polypeptide represented by SEQ ID NOs 4 to 7 in the cleavage composition is 10% or less, preferably 6% or less, more preferably 5% or less.

44. A cleavage impurity composition, wherein the cleavage impurity composition is obtained by the cleavage method according to any one of items 1 to 10,

Preferably, the enzyme-cleaved impurity composition comprises one or more than two polypeptides shown as SEQ ID NOs 4-7, wherein the total content of the polypeptides shown as SEQ ID NOs 4-7 in the enzyme-cleaved impurity composition is less than or equal to 10%, preferably less than or equal to 6%, and more preferably less than or equal to 5%.

45. The cleavage impurity composition of item 44, wherein the cleavage impurity composition further comprises the polypeptide of interest as set forth in SEQ ID NO. 1.

46. The cleavage impurity composition according to item 45, wherein the total mass content of the target polypeptide in the cleavage impurity composition is 80% or more, preferably 85% or more, and more preferably 90% or more.

According to the enterokinase enzyme digestion method provided by the application, the enzyme digestion promoter such as ethanol is added into the enzyme digestion system, so that the enzyme digestion specificity is improved, and the content of the nonspecific product obtained by the method is greatly reduced compared with that of the prior art, so that the purity of the product can be greatly improved. Meanwhile, the enzyme digestion method can keep the extremely high enzyme digestion rate of enterokinase, and ensures the balance of the enzyme digestion specificity and the enzyme digestion efficiency of the enterokinase; simple operation and effectively reduced purification cost. Meanwhile, through further research on various conventional parameters of enzyme and protein digestion systems, the enterokinase digestion method provided by the application is unexpectedly found to be less influenced by the conventional range of enzyme digestion parameters (such as enzyme digestion proportion, pH value, substrate protein concentration, enzyme digestion temperature, enzyme digestion time and the like) in the field, can be used for stably playing the application effect in the conventional non-extreme enterokinase digestion reaction, has the characteristics of wide applicable conditions, high method stability and excellent industrial applicability.

Detailed Description

The application will be further illustrated with reference to the following examples, which are to be understood as merely further illustrating and explaining the application and are not to be construed as limiting the application.

Unless defined otherwise, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the materials and methods are described herein below. In case of conflict, the present specification, including definitions therein, will control and materials, methods, and examples, will control and be in no way limiting.

In the process of enzyme digestion of expressed polypeptide products by enterokinase in the polypeptide recombinant production, the technical problem that a large number of various nonspecific cleavage products are generated after enzyme digestion is faced to the nonspecific cleavage by enterokinase, the occurrence of the nonspecific cleavage products greatly influences the purity and quality of the produced product, and the difficulty is brought to the subsequent polypeptide purification process, so that the related process is more complex, and therefore, a process method for the nonspecific cleavage products of enterokinase, which is simple, convenient, economical, stable and efficient to operate, is needed to be searched, so that the enzyme digestion specificity is improved and the purity of target polypeptide is increased on the premise of ensuring high-level enzyme digestion activity.

To this end, the present application provides a method for cleavage of enterokinase with high specificity, which comprises performing cleavage reaction on a substrate by enterokinase in the presence of a cleavage promoter. Wherein the enzyme digestion promoter is one or more selected from ethanol, glycerol, sucrose and sodium chloride.

Enterokinase is a serine protease that has cleavage specificity for an amino acid sequence comprising DDDDK. The method of the application is suitable for enterokinase of various sources. Enterokinase is commercially available or can be expressed by methods known in the art. Enterokinase includes, but is not limited to, natural animal-derived enterokinase, recombinant enterokinase, enterokinase light chain, mutants, fragments of the enterokinase or light chain activity described above, and protein products comprising the sequences described above. The animal is preferably a vertebrate, more preferably a mammal, more preferably a human, bovine or porcine.

Since enterokinase has cleavage specificity for amino acid sequence DDDDK, the method of the present application can be applied to any substance comprising amino acid sequence ddk, including but not limited to polypeptides, proteins, polypeptide complexes, protein complexes, polypeptide derivatives, protein derivatives, polypeptide analogs, protein analogs comprising amino acid sequence ddk, to increase cleavage specificity of enterokinase for the site of amino acid sequence DDDDK.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, the description for polypeptides applies equally to the description for peptides and the description for proteins, and vice versa. The term applies to naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the term encompasses amino acid chains of any length, including full length proteins, in which the amino acid residues are linked by covalent peptide bonds.

Wherein "complex" or "complexed" as used herein refers to a combination of two or more molecules that interact with each other through bonds and/or forces other than peptide bonds (e.g., van der Waals forces, hydrophobic forces, hydrophilic forces). In one embodiment, the complex is a heteromultimer. It is to be understood that the term "protein complex" or "polypeptide complex" as used herein includes complexes having a non-protein entity (e.g., including but not limited to a chemical molecule, such as a toxin or a detector) conjugated to a protein in the protein complex.

In the present application, the term "derivative" refers to a peptide fragment produced by chemical modification of one or more amino acid residues of a parent peptide, e.g., by alkylation, acylation, ester formation or amide formation.

The term "analogue" is used in the present application to refer to a peptide obtained by substituting one or more amino acid residues of a parent peptide with other amino acid residues and/or deleting one or more amino acid residues of a parent peptide and/or adding one or more amino acid residues to a parent peptide. Such addition may occur at the N-terminus and/or the C-terminus of the parent peptide.

In the present application the term "fusion protein" or "fusion polypeptide" is intended to mean a polypeptide comprising two or more polypeptides fused together, for example in order to constitute a non-naturally occurring polypeptide. The size of the fused polypeptide may vary and depends on the purpose of the fusion polypeptide. To enhance expression, fusion polypeptides are often used during recombinant expression of proteins to facilitate maintenance of soluble expression products, to facilitate excretion of the fusion polypeptide or portions thereof to extracellular medium, to protect the polypeptide from unintended treatment by proteases or peptidases, and the like. In such fusion polypeptides, one of the at least two constituent polypeptides is typically designated as the "polypeptide of interest" or "mature protein", i.e., the polypeptide to be produced by the recombinant expression process.

In a specific embodiment, the substrate is a fusion protein consisting of a Flag tag and a polypeptide or an analogue or derivative thereof. Such polypeptides include, but are not limited to, gastrin Inhibitory Peptide (GIP), glucagon-like peptide-1 (GLP-1), glucagon (Glucagon), and the like.

The Flag tag may be any tag comprising DDDDK or a tag comprising DDDDK + a linker peptide.

In a specific embodiment, the Flag tag has the sequence KETAAAKFE RQHMDS DDDDK (SEQ ID NO: 2)

In a specific embodiment, the Flag tag has the sequence HHHHHH DDDDK (SEQ ID NO: 3)

In a specific embodiment, the substrate is a fusion protein of glucagon-like peptide-1 (GLP-1) and a Flag tag, said GLP-1 meaning GLP-1, an analogue or derivative thereof.

In a specific embodiment, the substrate is a GLP-1 polypeptide analog Val ⁸ Glu ²² Lys ³⁰ Arg ^26，34 -fusion protein of GLP-1 (7-37) and Flag tag. Wherein the GLP-1 polypeptide analogue has an amino acid sequence of HVEGTFTSDV SSYLEEQAAR EFIKWLVRGR G (SEQ ID NO: 1).

In the enzyme digestion method, the addition of the enzyme digestion promoter can change the solvent environment of an enzyme digestion system, so that the enzyme digestion specificity is improved.

In a specific embodiment, the cleavage promoter is ethanol.

In a specific embodiment, the cleavage promoter is ethanol, and the volume concentration of ethanol in the cleavage system, i.e., the volume concentration of ethanol used in the cleavage reaction, is 1% -50%, and may be, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.

References herein to ethanol concentration refer to the volume concentration of ethanol unless otherwise specified.

In a specific embodiment, the cleavage promoter is ethanol, and the volume concentration of the ethanol in the cleavage system is 5% -40%.

In a specific embodiment, the cleavage promoter is ethanol, and the volume concentration of the ethanol in the cleavage system is 10% -30%.

In a specific embodiment, the cleavage promoter is ethanol, and the volume concentration of the ethanol in the cleavage system is 10% -20%.

In a specific embodiment, the cleavage promoter is ethanol, and the volume concentration of the ethanol in the cleavage system is 20% -30%.

In a specific embodiment, the cleavage promoter is ethanol, and the volume concentration of ethanol in the cleavage system is 10%.

In a specific embodiment, the cleavage promoter is ethanol, and the volume concentration of ethanol in the cleavage system is 20%.

In a specific embodiment, the cleavage promoter is ethanol, and the volume concentration of ethanol in the cleavage system is 30%.

In a specific embodiment, the cleavage promoter is glycerol.

In a specific embodiment, the cleavage promoter is glycerol, and the volume concentration of glycerol in the cleavage system is 1% -50%.

In a specific embodiment, the cleavage promoter is glycerol, and the volume concentration of glycerol in the cleavage system is 5% -40%.

In a specific embodiment, the cleavage promoter is glycerol, and the volume concentration of glycerol in the cleavage system is 10% -30%.

In the cleavage method of the present application, enterokinase, the cleavage promoter, and the substrate are subjected to cleavage reaction in a buffer having a pH of 7.1 to 10, i.e., the cleavage system of the present application may have a pH of 7.1 to 10, for example, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10. Those skilled in the art will fully appreciate that the above is only an example for the pH value range, and that the skilled person can adjust based on the actual situation in the application process.

In a specific embodiment, the pH of the buffer is 7.4-9.5.

In a specific embodiment, the pH of the buffer is 7.5-9.

In a specific embodiment, the pH of the buffer is 7.1-9.

In a specific embodiment, the pH of the buffer is 9-10.

In a specific embodiment, the pH of the buffer is 8-9.

Buffer solutions are a class of solutions that resist the effects of small amounts of external acids or bases or water when added and maintain the pH of the original system substantially unchanged. This effect of the solution against pH changes is called buffering. The buffer solution is usually a solution obtained by dissolving one or two compounds in a solvent (such as pure water), wherein the solute (compound) dissolved in the solution is called a buffer, and the buffer solution with different pH values can be prepared by adjusting the proportion of the buffer. The buffers of the present application may be any of a variety of buffers commonly used in the art, including but not limited to phosphates (H) ₂ PO ₄ ^- And HPO ₄ ^2- ) Buffers, tris-type buffers, organic acid buffers, borate buffers, amino acid buffers, or combinations thereof, preferably the buffers of the present application are selected from disodium hydrogen phosphate-citric acid buffers, citric acid-NaOH-HCl buffers, citric acid-sodium citrate buffers, acetic acid-sodium acetate buffers, phosphate Buffers (PBS), disodium hydrogen phosphate-sodium dihydrogen Phosphate Buffers (PB), disodium hydrogen phosphate-potassium dihydrogen phosphate buffers, potassium dihydrogen phosphate-NaOH buffers, barbites Tuna-HCl buffer, NH ₄ HCO ₃ Buffer solution, sodium carbonate-sodium bicarbonate buffer solution and NaHCO ₃ Buffer solution, tris-HCl, glycine-NaOH buffer solution, boric acid-borax buffer solution and Na ₂ B ₇ O ₄ And (3) a buffer solution. Those skilled in the art will fully appreciate that the above is only an example for buffers, and that the skilled person can adjust based on the actual situation in the application process.

In a specific embodiment, the buffer employed in the cleavage system is NH at pH7.1-10 ₄ HCO ₃ And (3) a buffer solution. Wherein in the buffer, NH ₄ HCO ₃ The concentration of (2) is not limited.

In a specific embodiment, the buffer used in the cleavage system is NaHCO pH7.1-10 ₃ And (3) a buffer solution. Wherein, in the buffer solution, naHCO ₃ The concentration of (2) is not limited.

In a specific embodiment, the buffer used in the digestion system is Tris-HCl buffer pH 7.1-10. Wherein, in the buffer, the concentration of Tris-HCl is not limited.

In a specific embodiment, the buffer used in the cleavage system is Na at pH7.1-10 ₂ B ₇ O ₄ And (3) a buffer solution. Wherein, in the buffer, na ₂ B ₇ O ₄ The concentration of (2) is not limited.

In a specific embodiment, the buffer used in the cleavage system is PB buffer at pH 7.1-10. Wherein, in the buffer, the concentration of the phosphate is not limited.

In a specific embodiment, the buffer used in the cleavage system is PB buffer at pH 7.4-9.5. Wherein, in the buffer, the concentration of the phosphate is not limited.

In a specific embodiment, the buffer used in the cleavage system is PB buffer at pH 7.5-9. Wherein, in the buffer, the concentration of the phosphate is not limited.

In a specific embodiment, the buffer used in the cleavage system is PB buffer at pH 8-9. Wherein, in the buffer, the concentration of the phosphate is not limited.

In a specific embodiment, the buffer used in the cleavage system is PB buffer at pH 7.1. Wherein, in the buffer, the concentration of the phosphate is not limited.

In a specific embodiment, the buffer used in the cleavage system is PB buffer at pH 7.5. Wherein, in the buffer, the concentration of the phosphate is not limited.

In a specific embodiment, the buffer used in the cleavage system is PB buffer at pH 8. Wherein, in the buffer, the concentration of the phosphate is not limited.

In a specific embodiment, the buffer used in the cleavage system is PB buffer at pH 9. Wherein, in the buffer, the concentration of the phosphate is not limited.

In a specific embodiment, the cleavage system employs a PB buffer at pH7.4 at 20 mM.

In a specific embodiment, the cleavage system employs a PB buffer at pH8 at 20 mM.

The reaction temperature of the cleavage method of the present application may be any temperature at which the enzyme activity is acceptable, and preferably the cleavage reaction temperature may be 0 to 25℃and may be, for example, 0℃1℃2℃3℃4℃5℃6℃7℃8℃9℃10℃11℃12℃13℃14℃15℃16℃17℃18℃19℃20℃21℃22℃23℃24℃25 ℃. Those skilled in the art will fully appreciate that the above is only an example for a range of temperature values, and that the skilled person can adjust based on the actual situation in the application process.

In a specific embodiment, the reaction temperature of the cleavage process is 4-20 ℃.

In the enzyme digestion method, the reaction time can be adjusted according to different reaction substrates.

In a specific embodiment, the reaction time of the cleavage reaction is 4 to 72h, and may be, for example, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, 36h, 37h, 38h, 39h, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, 48h, 50h, 52h, 54h, 56h, 58h, 60h, 62h, 64h, 68h, 70h, 72h.

In the cleavage method of the present application, the concentration of the reaction substrate may be adjusted as required.

In a specific embodiment, the substrate concentration for performing the cleavage reaction is 0.5-10mg/ml, and may be, for example, 0.5mg/ml, 1mg/ml, 1.5mg/ml, 2mg/ml, 2.5mg/ml, 3mg/ml, 3.5mg/ml, 4mg/ml, 5mg/ml, 6mg/ml, 7mg/ml, 8mg/ml, 9mg/ml, 10mg/ml, preferably 1-4mg/ml.

In a specific embodiment, the substrate concentration for performing the cleavage reaction is 1-4mg/ml.

In a specific embodiment, the substrate concentration for performing the cleavage reaction is 2-4mg/ml.

The enzyme digestion ratio in the present application refers to the ratio of enterokinase enzyme activity to substrate mass.

In a specific embodiment, the ratio of enterokinase enzyme activity to substrate for the cleavage reaction is 1-10U/mg, for example 1U/mg, 2U/mg, 3U/mg, 4U/mg, 5U/mg, 6U/mg, 7U/mg, 8U/mg, 9U/mg, 10U/mg.

In a specific embodiment, the ratio of enterokinase enzyme activity to substrate for the cleavage reaction is 1-8U/mg.

In a specific embodiment, the ratio of enterokinase enzyme activity to substrate for the cleavage reaction is 1.5-6U/mg.

In a specific embodiment, the ratio of enterokinase enzyme activity to substrate for the cleavage reaction is 2-5U/mg.

In a specific embodiment, the cleavage method comprises: enterokinase, ethanol and a substrate are subjected to enzyme digestion reaction in a buffer solution, wherein the pH value of the buffer solution is 7.1-10, the volume concentration of the ethanol used for the enzyme digestion reaction is 1% -50%, the concentration of the substrate subjected to the enzyme digestion reaction is 0.5-10mg/ml, and the ratio of the enterokinase enzyme activity to the substrate subjected to the enzyme digestion reaction is 0.5-10U/mg.

In a specific embodiment, the cleavage method comprises: enterokinase, ethanol and a substrate are subjected to enzyme digestion reaction in a buffer solution, wherein the buffer solution is PB buffer solution with the pH of 7.1-10, the volume concentration of the ethanol for the enzyme digestion reaction is 10% -30%, the concentration of the substrate for the enzyme digestion reaction is 1-4mg/ml, and the ratio of the enterokinase enzyme activity to the substrate for the enzyme digestion reaction is 1.5-6U/mg.

In a specific embodiment, the cleavage method comprises: enterokinase, ethanol and a substrate are subjected to enzyme digestion reaction in a buffer solution, wherein the buffer solution is PB buffer solution with the pH of 8-9, the volume concentration of the ethanol for the enzyme digestion reaction is 20% -30%, the concentration of the substrate for the enzyme digestion reaction is 2-4mg/ml, and the ratio of enterokinase enzyme activity to the substrate for the enzyme digestion reaction is 2-5U/mg.

In a specific embodiment, the cleavage method comprises: enterokinase, ethanol and a substrate are subjected to enzyme digestion reaction in a buffer solution, wherein the buffer solution is PB buffer solution with the pH of 7.5-9, the volume concentration of the ethanol for the enzyme digestion reaction is 10% -30%, the concentration of the substrate for the enzyme digestion reaction is 1-4mg/ml, and the ratio of the enterokinase enzyme activity to the substrate for the enzyme digestion reaction is 1.5-6U/mg.

In a specific embodiment, the cleavage method comprises: the enterokinase, the ethanol and the substrate are subjected to enzyme digestion reaction in a buffer solution, wherein the pH of the buffer solution is 20mM PB buffer solution with the pH of 7.5-9, the volume concentration of the ethanol for the enzyme digestion reaction is 10% -30%, the concentration of the substrate for the enzyme digestion reaction is 1-4mg/ml, and the ratio of the enterokinase enzyme activity to the substrate for the enzyme digestion reaction is 1.5-6U/mg.

In a specific embodiment, the cleavage method comprises: the enterokinase, the ethanol and the substrate are subjected to enzyme digestion reaction in a buffer solution, wherein the pH of the buffer solution is 20mM PB buffer solution with the pH of 7.5-9, the volume concentration of the ethanol for the enzyme digestion reaction is 10% -30%, the concentration of the substrate for the enzyme digestion reaction is 1-4mg/ml, the ratio of the enterokinase enzyme activity to the substrate for the enzyme digestion reaction is 1.5-6U/mg, the enzyme digestion reaction temperature is 4-20 ℃, and the reaction time is 4-72h.

The application also provides the use of ethanol in improving enterokinase enzyme cleavage specificity.

Further, in this use, enterokinase is reacted in an enzyme digestion system containing ethanol.

Further, the ethanol concentration, the buffer used, the reaction temperature and time, the substrate concentration, the ratio of enterokinase enzyme activity to substrate in the enzyme digestion system are as described above.

The application also provides a method for producing a polypeptide, wherein the method comprises any one of the above-described methods of enzyme digestion.

In a specific embodiment, the method comprises performing an cleavage reaction on the substrate using enterokinase in the presence of ethanol.

In a specific embodiment, the cleavage promoter is ethanol, and the volume concentration of ethanol used for the cleavage reaction is 1% -50%.

In a specific embodiment, the cleavage promoter is ethanol, and the volume concentration of ethanol used for the cleavage reaction is 10% -30%.

In a specific embodiment, the cleavage promoter is ethanol, and the volume concentration of ethanol used for the cleavage reaction is 20% -30%.

Wherein enterokinase, the cleavage promoter and the substrate are subjected to cleavage reaction in a buffer solution.

Enterokinase, type and amount of substrate, and type and pH of buffer are as described above.

In a specific embodiment, the pH of the buffer is 7.1-10.

In a specific embodiment, the buffer is PB buffer having a pH of 7.1-10.

In a specific embodiment, the buffer of the composition is 20mM PB buffer pH 7.4.

The polypeptides produced include, but are not limited to, gastrin Inhibitory Peptides (GIPs), glucagon-like peptide-1 (GLP-1), glucagon (Glucagon), and the like, as well as analogs and derivatives thereof.

In a specific embodiment, the resulting polypeptide is GLP-1, an analog or derivative thereof.

In a specific embodiment, the resulting polypeptide is produced as an analog of GLP-1.

The application also provides a method for purifying a polypeptide, wherein the method comprises any one of the above methods of cleavage. The method comprises the step of carrying out enzyme digestion reaction on a substrate by utilizing enterokinase in the presence of an enzyme digestion promoter. The enzyme digestion reaction can improve the yield of the target polypeptide and reduce the content of impurity polypeptide, thereby achieving the aim of purifying the target polypeptide.

In a specific embodiment, the pH of the buffer is 7.1-10.

In a specific embodiment, the buffer is PB buffer having a pH of 7.1-10.

The purified polypeptides include, but are not limited to, gastrin Inhibitory Peptides (GIPs), glucagon-like peptide-1 (GLP-1), glucagon (Glucagon), and the like, as well as analogs and derivatives thereof.

In a specific embodiment, the purified polypeptide is GLP-1, an analog or derivative thereof.

In a specific embodiment, the purified polypeptide is an analog of GLP-1.

The method for producing and purifying the polypeptide has high digestion efficiency, and reduces the impurity content of nonspecific digestion products, thereby providing the yield of the target polypeptide.

The application also provides a composition for high-specificity enterokinase enzyme digestion, which comprises a buffer solution and an enzyme digestion promoter, wherein the enzyme digestion promoter is one or more than two selected from ethanol, glycerol, sucrose and sodium chloride.

In a specific embodiment, the cleavage promoter is ethanol, and the concentration of ethanol in the composition is 1% -50% by volume. In a specific embodiment, the cleavage promoter is ethanol, and the concentration of ethanol in the composition is 10% -30% by volume.

In a specific embodiment, the pH of the buffer of the composition is from 7.1 to 10.

In a specific embodiment, the buffer of the composition is PB buffer having a pH of 7.1-10.

Specifically, a certain amount of enterokinase and a substrate may be added to the composition for cleavage of enterokinase of the present application for cleavage reaction of high specificity. The enterokinase and the substrate are as described above.

The application also provides a high-specificity enterokinase enzyme digestion kit which comprises a first kit and a second kit comprising enterokinase, wherein the first kit comprises a buffer solution and an enzyme digestion promoter, and the enzyme digestion promoter is one or more than two of ethanol, glycerol, sucrose and sodium chloride. Wherein, the types and the amounts of enterokinase are as described above.

In a specific embodiment, the cleavage promoter is ethanol and the volume concentration in the kit is 1% -50%.

In a specific embodiment, the cleavage promoter is ethanol and the volume concentration in the kit is 10% -30%.

In a specific embodiment, the pH of the buffer of the kit is 7.1-10.

In a specific embodiment, the buffer of the kit is PB buffer having a pH of 7.1-10.

In a specific embodiment, the buffer of the kit is 20mM PB buffer pH 7.4.

Specifically, when the high specificity enterokinase cleavage kit of the present application is used, the cleavage reaction can be performed by mixing the first kit, the second kit and the substrate. Wherein the type and amount of the substrate are as described above.

The application also provides an enzyme digestion composition, which is obtained by the enzyme digestion method of any one of the above or any method for preparing polypeptide or purifying polypeptide.

The enzyme digestion composition comprises target polypeptide shown in SEQ ID NO. 1. Wherein the mass content of the target polypeptide in the enzyme digestion composition is 80% or more, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% and the like.

The target polypeptide shown in SEQ ID NO. 1 is the compound 1 of the application.

SEQ ID NO:1：HVEGTFTSDV SSYLEEQAAR EFIKWLVRGR G

In a specific embodiment, the cleavage composition comprises the target polypeptide shown in SEQ ID NO. 1, wherein the mass content of the target polypeptide in the cleavage composition is 80% or more.

In a specific embodiment, the cleavage composition comprises the target polypeptide shown in SEQ ID NO. 1, wherein the mass content of the target polypeptide in the cleavage composition is greater than or equal to 85%.

In a specific embodiment, the cleavage composition comprises the target polypeptide shown in SEQ ID NO. 1, wherein the mass content of the target polypeptide in the cleavage composition is greater than or equal to 90%.

Further, the enzyme digestion composition also comprises one or more than two polypeptides shown in SEQ ID NOs 4-7.

SEQ ID NO. 4 is: HVEGTFTSDV SSYLEEQAAR

SEQ ID NO. 5 is: EFIKWLVRGR G

SEQ ID NO. 6 is: EFIKWLVR

SEQ ID NO. 7 is: HVEGTFTSDV SSYLEEQAAR EFIKWLVR

In a specific embodiment, the cleavage composition comprises a polypeptide of interest as set forth in SEQ ID NO. 1 and a polypeptide as set forth in SEQ ID NO. 4.

In a specific embodiment, the cleavage composition comprises a polypeptide of interest as set forth in SEQ ID NO. 1 and a polypeptide as set forth in SEQ ID NO. 5.

In a specific embodiment, the cleavage composition comprises a polypeptide of interest as set forth in SEQ ID NO. 1 and a polypeptide as set forth in SEQ ID NO. 6.

In a specific embodiment, the cleavage composition comprises a polypeptide of interest as set forth in SEQ ID NO. 1 and a polypeptide as set forth in SEQ ID NO. 7.

In a specific embodiment, the cleavage composition comprises the target polypeptide shown in SEQ ID NO. 1 and the polypeptides shown in SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.

The total mass content of the polypeptide represented by SEQ ID NOs 4-7 in the enzyme-digested composition may be 10% or less, for example, 10%, 9.5%, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1% or the like.

In a specific embodiment, the total mass content of the polypeptides represented by SEQ ID NOs 4-7 in the cleavage composition is 6% or less.

In a specific embodiment, the total mass content of the polypeptides represented by SEQ ID NOs 4-7 in the cleavage composition is 5% or less.

In a specific embodiment, the mass content of the target polypeptide shown in SEQ ID NO. 1 in the enzyme digestion composition is more than or equal to 80%, and the total mass content of the polypeptides shown in SEQ ID NOs 4-7 is less than or equal to 10%.

In a specific embodiment, the mass content of the target polypeptide shown in SEQ ID NO. 1 in the enzyme digestion composition is more than or equal to 85%, and the total mass content of the polypeptides shown in SEQ ID NOs 4-7 is less than or equal to 10%.

In a specific embodiment, the mass content of the target polypeptide shown in SEQ ID NO. 1 in the enzyme digestion composition is more than or equal to 90%, and the total mass content of the polypeptides shown in SEQ ID NOs 4-7 is less than or equal to 10%.

In a specific embodiment, the mass content of the target polypeptide shown in SEQ ID NO. 1 in the enzyme digestion composition is more than or equal to 80%, and the total mass content of the polypeptides shown in SEQ ID NOs 4-7 is less than or equal to 6%.

In a specific embodiment, the mass content of the target polypeptide shown in SEQ ID NO. 1 in the enzyme digestion composition is more than or equal to 80%, and the total mass content of the polypeptides shown in SEQ ID NOs 4-7 is less than or equal to 5%.

In a specific embodiment, the mass content of the target polypeptide shown in SEQ ID NO. 1 in the enzyme digestion composition is more than or equal to 85%, and the total mass content of the polypeptides shown in SEQ ID NOs 4-7 is less than or equal to 6%.

In a specific embodiment, the mass content of the target polypeptide shown in SEQ ID NO. 1 in the enzyme digestion composition is more than or equal to 90%, and the total mass content of the polypeptides shown in SEQ ID NOs 4-7 is less than or equal to 5%.

The cleavage composition of the present application may further comprise other components that may be generated during the preparation process, for example, a salt component contained in a buffer, etc.

The application also provides a digestion impurity composition, which is obtained by any one of the digestion methods or any one of the methods for preparing a polypeptide or purifying a polypeptide.

The enzyme-cutting impurity composition comprises one or more than two polypeptides shown as SEQ ID NOs 4-7, wherein the total content of the polypeptides shown as SEQ ID NOs 4-7 in the enzyme-cutting impurity composition is less than or equal to 10%, for example, 10%, 9.5%, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1% and the like.

Further, the enzyme-cleaved impurity composition also comprises a target polypeptide shown as SEQ ID NO. 1.

In a specific embodiment, the total mass content of the target polypeptide in the enzyme-cleaved impurity composition is 80% or more, preferably 85% or more, and more preferably 90% or more.

In a specific embodiment, the mass content of the target polypeptide shown in SEQ ID NO. 1 in the enzyme-digested impurity composition is more than or equal to 80%, and the total mass content of the polypeptides shown in SEQ ID NOs 4-7 is less than or equal to 10%.

In a specific embodiment, the mass content of the target polypeptide shown in SEQ ID NO. 1 in the enzyme-digested impurity composition is more than or equal to 85%, and the total mass content of the polypeptides shown in SEQ ID NOs 4-7 is less than or equal to 10%.

In a specific embodiment, the mass content of the target polypeptide shown in SEQ ID NO. 1 in the enzyme-digested impurity composition is more than or equal to 90%, and the total mass content of the polypeptides shown in SEQ ID NOs 4-7 is less than or equal to 10%.

In a specific embodiment, the mass content of the target polypeptide shown in SEQ ID NO. 1 in the enzyme-digested impurity composition is more than or equal to 80%, and the total mass content of the polypeptides shown in SEQ ID NOs 4-7 is less than or equal to 6%.

In a specific embodiment, the mass content of the target polypeptide shown in SEQ ID NO. 1 in the enzyme-digested impurity composition is more than or equal to 80%, and the total mass content of the polypeptides shown in SEQ ID NOs 4-7 is less than or equal to 5%.

In a specific embodiment, the mass content of the target polypeptide shown in SEQ ID NO. 1 in the enzyme-digested impurity composition is more than or equal to 85%, and the total mass content of the polypeptides shown in SEQ ID NOs 4-7 is less than or equal to 6%.

In a specific embodiment, the mass content of the target polypeptide shown in SEQ ID NO. 1 in the enzyme-digested impurity composition is more than or equal to 90%, and the total mass content of the polypeptides shown in SEQ ID NOs 4-7 is less than or equal to 5%.

In the present application, the cleaved products are detected using mass spectrometry and high performance liquid chromatography.

Wherein, the mass spectrometry is an analysis method for generating gaseous ions by a compound to be detected, separating and detecting the ions according to mass-to-charge ratio (m/z), and the detection limit can reach 10 ^-15 ～10 ^-12 On the order of mol. Mass spectrometry can provide information on molecular mass and structure, and quantitative determination can be carried out by an internal standard method or an external standard method.

At about 10 maintained by the pump ^-3 ～10 ^-6 Under Pa vacuum state, various positive ions (or negative ions) generated by the ion source are accelerated, enter a mass analyzer for separation, and are detected by a detector. The computer system is used for controlling the instrument, recording, processing and storing data, and when the standard spectrum library software is configured, the computer system can compare the measured mass spectrum with the spectrum in the standard spectrum library to obtain the composition and structure information of the possible compounds. The liquid chromatography-mass spectrometry technology uses liquid chromatography as a separation system and mass spectrometry as a detection system.

Calculation method of GLP-1 purity and cleavage rate

The cleavage rate of the application is 100 percent minus the percentage content of substrates which are not cleaved, namely 100 percent to the percentage content of substrates which are not cleaved.

Protein purity was determined using area normalization: taking a certain amount of sample to be detected, detecting the sample by using high performance liquid chromatography, and recording a chromatogram. The area of each peak and the total chromatographic peak area on the chromatogram excluding the solvent peak were measured, and the percentage of each peak area to the total peak area was calculated. GLP-1 purity can be directly recorded according to chromatographic detection results.

EXAMPLE 1 expression, purification and assay of GLP-1 polypeptide analogs

Cell construction and expression: preparing engineering bacteria by recombinant expression method and expressing fusion protein M-Flag-Val in soluble form ⁸ Glu ²² Lys ³⁰ Arg ^26，34 -GLP-1 (7-37) and further processing to obtain a GLP-1 polypeptide analogue of interest. GLP-1 polypeptide analog Val of interest ⁸ Glu ²² Lys ³⁰ Arg ^26，34 GLP-1 (7-37) has the amino acid sequence shown in SEQ ID NO. 1, flag tag sequence (Flag 1) shown in SEQ ID NO. 2, and specific vector construction and expression steps can be described with reference to examples 1-3 in WO 2019201328A. In general, the Flag tag and the target GLP-1 polypeptide analogue encoding gene are fused in series in sequence, and chemical synthesis is performed to obtain gene fragments, the fragments are inserted into a prokaryotic expression plasmid pET-24 (+) through BamHI and XhoI sites and are sequenced and verified to obtain an expression plasmid, the expression plasmid is introduced into BL21 DE3 or Tuner DE3 E.coli competent cells, and the expression fusion protein M-Flag-Val is obtained through screening ⁸ Glu ²² Lys ³⁰ Arg ^26，34- Transformed BL21 DE3 or Tuner DE3 engineering bacteria of GLP-1 (7-37).

Culturing the transformed BL21 DE3 or Tuner DE3 engineering bacteria in LB culture medium containing antibiotics overnight, inoculating the overnight bacterial liquid into LB culture medium containing antibiotics with larger volume, continuously culturing for 4 hours at 37 ℃ by using a shaking table, performing induced expression on the engineering bacteria by using IPTG, centrifuging the bacterial liquid overnight, obtaining bacterial cells, performing ultrasonic crushing after re-suspension, centrifuging, and obtaining supernatant.

Separating and enzyme cutting: the supernatant was subjected to ion exchange chromatography to obtain expressed M-Flag-Val ⁸ Glu ²² Lys ³⁰ Arg ^26，34 GLP-1 (7-37) fusion protein, the chromatography medium is Unigel-80Q (Soy micro technology), the equilibration buffer is 20mmol/LPB (pH 7.4), the wash buffer is 20mmol/L PB (pH 7.4) containing 50mmol/L NaCl, the elution buffer is 20mmol/L PB (pH 7.4) containing 200mmol/L NaCl.

The adopted chromatographic column is XK26 of GE, the chromatographic medium is G25 (Cytiva, cat# 17003303), the method is that after balancing by adopting corresponding replacement buffer solution, loading and eluting are carried out, and fusion protein samples are collected according to ultraviolet and conductivity changes.

The fusion protein sample was subjected to digestion, diluted to 2mg/ml with 20mmol/L PB (pH 7.4) buffer, and then added with recombinant enterokinase rEK (available from Shanghai Yaxin Biotechnology Co., ltd., product No. REK 08) in a digestion ratio of about 1.5U/mg, and the mixture was mixed uniformly, followed by digestion reaction at 4℃for 12 hours. And determining the types of the target object and the impurity by adopting RP-HPLC (reverse osmosis high Performance liquid chromatography) combined mass spectrometry, and then adopting RP-HPLC for determination, and calculating the digestion rate, the purity of GLP-1 and the content of non-specific impurities according to an area normalization method. Wherein the high performance liquid chromatography condition is that the flow rate is 0.8ml/min, the column temperature is 35 ℃, the detection wavelength is 215nm and 280nm, and the mobile phase A is 100% H ₂ O+0.1% TFA, mobile phase B was 100% ACN+0.1% TFA, elution gradient is shown in Table 1.

TABLE 1 liquid chromatography elution gradient List

After analysis, the mass spectrum of each product after cleavage is as follows:

table 2 summary of mass spectrometry results

As shown in Table 2, the structure of each corresponding compound is deduced from mass spectrum information, wherein each compound is represented by the corresponding amino acid of SEQ ID NO. 1 and its position, and as compound 1 is a polypeptide fragment corresponding to the 4 th G to 13 th Y of the polypeptide shown in SEQ ID NO. 1. Compound 8, as analyzed, corresponds to the target polypeptide Val shown in SEQ ID NO. 1 ⁸ Glu ²² Lys ³⁰ Arg ^26，34 GLP-1 (7-37), while compounds 4, 5, 6, 9 are the main cleavage impurity, and represent an absolute advantage of the total impurity content, and the other impurity compounds are only in very small amounts.

Example 2 digestion buffer System

According to the method of example 1, although the cleavage efficiency was as high as 95% or more, the total content of nonspecific cleavage products was as high as 40% or more, and the demand for obtaining the objective GLP-1 polypeptide derivative could not be satisfied. In order to obtain more ideal cleavage effect, cleavage was performed according to the cleavage reaction protocol shown in Table 3 under conditions that the concentration of the fusion protein was 1.0mg/ml, the cleavage ratio was 1-1.5U/mg, and the cleavage reaction was carried out at 4℃for 12 hours:

TABLE 3 Table 3

The cleavage products obtained in each set of experimental protocols were separated and analyzed by the high performance liquid chromatography and mass spectrometry detection method described in example 1, and the cleavage rate, purity of the target GLP-1 polypeptide analogue, and content of non-specific products were calculated, and the results are shown in Table 4.

TABLE 4 Table 4

As shown in table 4, compounds 4, 5, 6, 9 are the main impurities for each group of cleavage, and the A4-A8 protocol reduced the non-specific impurity levels to some extent, but still at higher levels; the A9 and A10 experimental schemes can obviously reduce the total content of main nonspecific cleavage products to below 5%, however, the cleavage rate and the content of target GLP-1 polypeptide analogues are also obviously reduced, and the A1 and A3 experimental schemes keep the better content of target GLP-1 polypeptide analogues, and meanwhile, the nonspecific cleavage content is relatively small, but the cleavage rate is still below 95%; the A2 experimental scheme achieves higher balance in terms of digestion rate, nonspecific digestion product content and target GLP-1 polypeptide analogue content, namely PB and NH ₄ HCO ₃ 、NaHCO ₃ Tris-HCl and Na ₂ B ₇ O ₄ In the buffer systems, PB is adopted when the digestion rate is higher than 95%The purity of the enzyme-digested GLP-1 is highest by the buffer system, the content of non-specific enzyme-digested impurities is 13.5%, and when the enzyme-digested rate of other buffer systems is higher than 95% and lower than 98%, the content of non-specific enzyme-digested impurities is far higher than that of the non-specific enzyme-digested impurities.

EXAMPLE 3 cleavage Environment Studies

In view of the fact that more non-specific cleavage products are produced when rEK enzyme cleaves fusion protein substrates, attempts have been made to add different reagents to adjust the solvent environment to alter rEK the selectivity of different cleavage sites, the application compares the effect of ethanol, glycerol, sodium chloride and sucrose on cleavage. To verify the cleavage effect of each protocol, the cleavage reaction was performed at 4℃for 12 hours at a protein concentration of 1.0mg/ml and a cleavage ratio of 1-4U/mg in different buffers. The cleavage was performed according to the cleavage reaction scheme shown in Table 5:

TABLE 5

The cleavage products obtained from each set of protocols were separated and analyzed by HPLC and MS methods as described in example 1, and the cleavage rates, purity of the target GLP-1 polypeptide analog, and non-specific cleavage product content were calculated as shown in Table 6.

TABLE 6

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Research results show that sucrose (experimental scheme B1) and NaCl (experimental schemes B8-B12) can keep higher enzyme digestion activity to a certain extent, but can not effectively reduce the content of nonspecific enzyme digestion products; notably, glycerol (protocol B2-B7) and ethanol (protocol B15) also significantly reduced the enterokinase nonspecific reaction while maintaining high levels of cleavage efficiency and GLP-1 content, and reduced the total amount of nonspecific products to 10% and below 5%, respectively, indicating that both additions are beneficial for reducing the production of nonspecific cleavage impurities. More satisfactory and unwanted As a result, NH which has not been proven to reduce nonspecific cleavage ₄ HCO ₃ (pH 7.88) and Tris HCl (pH 8.60) buffers also have the same effect of changing enterokinase nonspecific cleavage, comparison with NH ₄ HCO ₃ The content of nonspecific enzyme-cleaved impurities can be reduced from 22.1% (experimental scheme A4) to 4.94% (experimental scheme B13) by adding 20% ethanol before and after the data; comparing the data before and after adding 20% ethanol to Tris-HCl, the content of nonspecific enzyme-cleaved impurities can be reduced from 41.8% (experimental scheme A7) to 14.2% (experimental scheme B14).

EXAMPLE 4 cleavage Effect under different ethanol conditions

The application compares the influence on enzyme digestion under different ethanol concentration conditions. The enzyme digestion reaction was performed according to the enzyme digestion reaction scheme shown in Table 7 under the conditions that the protein concentration is 1.0mg/ml, the enzyme digestion ratio is 1-4U/mg, the ethanol content is 10%, 20% and 30% and the temperature is 4 ℃ respectively, so as to verify the ethanol effect:

TABLE 7

The cleavage products obtained in each set of experimental protocols were separated and analyzed by the high performance liquid chromatography and mass spectrometry detection method described in example 1, and the cleavage rate, purity of the target GLP-1 polypeptide analogue, and content of non-specific products were calculated, and the results are shown in Table 8.

TABLE 8

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The results of the experimental protocols C1-C8 in Table 8 above are summarized: the addition of ethanol with any content can obviously reduce the generation of non-specific enzyme digestion impurities, and has extremely low content of non-specific enzyme digestion products while maintaining high enzyme digestion rate and target GLP-1 polypeptide derivative content.

EXAMPLE 5 cleavage temperature Studies

The application compares the influence of the temperature in the range of 4-25 ℃ on the enzyme digestion under different temperature conditions. The protein concentration was 1.0mg/ml, 20% ethanol was contained, the enzyme digestion ratio was 2U/mg, and the buffer was 20mM, PB buffer pH 7.4. The cleavage reaction was carried out at 4℃and 10℃and 15℃and 20℃and 25℃for 12 hours, respectively. The cleavage products obtained in each set of experimental protocols were separated and analyzed by the high performance liquid chromatography and mass spectrometry detection method described in example 1, and the cleavage rate, purity of the target GLP-1 polypeptide analog, and non-specific product content were calculated. The results are shown in Table 9.

TABLE 9

The results in Table 9 show that the digestion effect is within an acceptable range at 0-25 ℃, especially below 20 ℃, the purity of GLP-1 can reach more than 90%, and the content of non-specific digestion impurities is lower than 5%.

EXAMPLE 6 cleavage pH Studies

The application compares the effect of pH values of different reactions on cleavage in the pH range 6.5-10.0 (20 mM PB buffer). The protein concentration is 1.0mg/ml, 20% ethanol is contained, the enzyme digestion proportion is 2U/mg, and enzyme digestion reactions are respectively carried out for 12 hours under the conditions of pH of 6.5, 7, 7.5, 8, 8.5, 9, 9.5 and 10 at the temperature of 4 ℃. The cleavage products obtained in each set of experimental protocols were separated and analyzed by the high performance liquid chromatography and mass spectrometry detection method described in example 1, and the cleavage rate, purity of the target GLP-1 polypeptide analog, and non-specific product content were calculated. The results are shown in Table 10.

Table 10

The results show that the digestion rate increases with increasing pH value, wherein the digestion rate is lower than 90% under the conditions of pH6.5-pH 7.0; in particular, in the cleavage reaction, the cleavage reaction is performed at a pH of 7.0 or more and pH9.5 or less, which shows a favorable effect.

EXAMPLE 7 concentration study of cleaved proteins

The application compares the effect of 1-4mg/ml protein concentration on digestion. The cleavage reaction system contained 20% ethanol at a cleavage ratio of 2U/mg and a buffer of 20mM PB buffer at pH 8.5. The cleavage reaction was carried out at 10℃for 12 hours using protein concentrations of 1mg/ml, 2mg/ml, 3mg/ml, and 4mg/ml, respectively, in the cleavage system. The cleavage products obtained in each set of experimental protocols were separated and analyzed by the high performance liquid chromatography and mass spectrometry detection method described in example 1, and the cleavage rate, purity of the target GLP-1 polypeptide analog, and non-specific product content were calculated. The results are shown in Table 11.

TABLE 11

The result shows that the protein concentration has little influence on the digestion, and the digestion concentration above 1mg/ml can reduce the content of the enterokinase nonspecific digestion product to below 5% on the premise of keeping high-level digestion rate and purity of the target GLP-1 polypeptide derivative.

EXAMPLE 8 cleavage time study

The application examines the influence of enzyme digestion time. The concentration of protein in the reaction system is 2.0mg/ml, the enzyme digestion ratio is 2U/mg, the buffer solution is 20mM, and the pH value is 8.5. The reaction system carries out enzyme digestion reaction at 10 ℃ for 2 hours, 4 hours, 16 hours, 24 hours, 48 hours and 72 hours respectively.

Table 12

As shown in Table 12, when the cleavage reaction was performed for more than 4 hours, the cleavage efficiency was 90% or more, and particularly, when the cleavage reaction was performed for more than 8 hours, the cleavage efficiency was 99%, the cleavage reaction was substantially complete, and the cleavage efficiency was substantially stable with the lapse of time, while the nonspecific cleavage product content was maintained at a low level. Thus, the above results indicate that cleavage is preferably continued for more than 3 days at 4 to 72 hours, the purity of GLP-1 is reduced and the nonspecific cleavage product is increased.

EXAMPLE 9 restriction enzyme proportion study

The application compares the influence of the enzyme digestion proportion on the enzyme digestion effect. The concentration of protein in the reaction system was 2.0mg/ml, 20% ethanol was contained, and the buffer was 20mM PB buffer, pH 8.5. The reaction system adopts different enzyme digestion ratios to carry out enzyme digestion reaction for 12 hours at the temperature of 10 ℃. The cleavage products obtained in each set of experimental protocols were separated and analyzed by the high performance liquid chromatography and mass spectrometry detection method described in example 1, and the cleavage rate, purity of the target GLP-1 polypeptide analog, and non-specific product content were calculated. The results are shown in Table 13.

TABLE 13

The result shows that the content of the nonspecific enzyme digestion products can be effectively controlled at a low level in any enzyme digestion proportion, especially the enzyme digestion rate can be ensured to be more than 90% when the enzyme digestion proportion is more than 1.5U/mg, and the content of nonspecific enzyme digestion impurities is less than 5%.

Example 10 Effect of other tags on ethanol addition

The application compares the ethanol addition to enterokinase enzyme digestion M-Flag2-Val ⁸ Glu ²² Lys ³⁰ Arg ^26，34 Effect of GLP-1 (7-37) sequence, flag2 tag sequence as shown in SEQID NO: 3. The concentration of protein in the reaction system was 2.0mg/ml, the enzyme digestion ratio was 1.5U/mg, and the buffer was 20mM PB buffer at pH 8.5. The reaction system containing 20% ethanol and no ethanol is adopted to carry out enzyme digestion reaction for 12 hours at 10 ℃. The cleavage products obtained in each set of experimental protocols were separated and analyzed by the high performance liquid chromatography and mass spectrometry detection method described in example 1, and the cleavage rate, purity of the target GLP-1 polypeptide analog, and non-specific product content were calculated. The results are shown in Table 14.

TABLE 14

The results showed that the cleavage substrate was M-Flag2-Val ⁸ Glu ²² Lys ³⁰ Arg ^26，34 When GLP-1 (7-37) is used, the content of impurities of nonspecific enzyme digestion products can be reduced by adding ethanol.

Sequence listing

SEQ ID NO:1

HVEGTFTSDV SSYLEEQAAR EFIKWLVRGR G

SEQ ID NO:2

KETAAAKFE RQHMDS DDDDK

SEQ ID NO:3

HHHHHH DDDDK

SEQ ID NO:4

HVEGTFTSDV SSYLEEQAAR

SEQ ID NO:5

EFIKWLVRGR G

SEQ ID NO:6

EFIKWLVR

SEQ ID NO:7

HVEGTFTSDV SSYLEEQAAR EFIKWLVR

Claims

1. A method for cleavage of enterokinase with high specificity, comprising:

Performing an enzyme digestion reaction on a substrate by using enterokinase in the presence of ethanol or glycerol, wherein the substrate contains a DDDDK amino acid sequence; the volume concentration of ethanol or glycerol for enzyme digestion reaction is 10% -30%;

carrying out enzyme digestion reaction on ethanol or glycerol, a substrate and enterokinase in a buffer solution, wherein the pH value of the buffer solution is 7.4-9.5;

the ratio of enterokinase enzyme activity to substrate is 0.5-10U/mg, and the reaction time of enzyme digestion reaction is 4-72h.

2. The method of claim 1, wherein the buffer has a pH of 7.5-9.0.

3. The method according to claim 1, wherein the buffer is selected from the group consisting of disodium hydrogen phosphate-sodium dihydrogen Phosphate Buffer (PB), NH ₄ HCO ₃ One or more of buffer solution and Tris-HCl buffer solution.

4. The method of claim 3, wherein the buffer is disodium hydrogen phosphate-sodium dihydrogen Phosphate Buffer (PB).

5. The method according to claim 1, wherein the substrate concentration for performing the cleavage reaction is 0.5-10mg/ml.

6. The method according to claim 5, wherein the substrate concentration for performing the cleavage reaction is 1-8mg/ml.

7. The method according to claim 6, wherein the substrate concentration for performing the cleavage reaction is 1-4mg/ml.

8. The method according to claim 1, wherein the ratio of enterokinase enzyme activity to substrate for the cleavage reaction is 1.5-6U/mg.

9. The method according to claim 8, wherein the ratio of enterokinase enzyme activity to substrate for the cleavage reaction is 2-5U/mg.

10. Use of ethanol for improving cleavage specificity of enterokinase, characterized in that in the presence of ethanol, cleavage reaction is performed on a substrate by enterokinase, wherein the volume concentration of ethanol used for the cleavage reaction is 10% -30%;

enterokinase, ethanol and substrate are subjected to enzyme digestion reaction in a buffer solution, wherein the pH value of the buffer solution is 7.1-10.

11. The use according to claim 10, wherein the pH of the buffer is 7.4-9.5.

12. The use according to claim 10, wherein the pH of the buffer is 7.5-9.0.

13. The use according to claim 10, wherein the buffer is one or more selected from phosphate buffer, tris buffer, organic acid buffer, borate buffer, amino acid buffer.

14. The use according to claim 13, wherein the buffer is selected from the group consisting of disodium hydrogen phosphate-citric acid buffer, citric acid-NaOH-HCl buffer, citric acid-sodium citrate buffer, acetic acid-sodium acetate buffer, phosphate Buffer (PBS), disodium hydrogen phosphate-sodium dihydrogen Phosphate Buffer (PB), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-NaOH buffer, barbituric sodium-HCl buffer, NH ₄ HCO ₃ Buffer solution, sodium carbonate-sodium bicarbonate buffer solution and NaHCO ₃ Buffer solution, tris-HCl, glycine-NaOH buffer solution, boric acid-borax buffer solution and Na ₂ B ₇ O ₄ One or more buffers.

15. The use according to claim 14, wherein the buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

16. The use according to claim 10, characterized in that the substrate concentration for the cleavage reaction is 0.5-10mg/ml.

17. The use according to claim 16, characterized in that the substrate concentration for the cleavage reaction is 1-8mg/ml.

18. The use according to claim 17, characterized in that the substrate concentration for the cleavage reaction is 1-4mg/ml.

19. The use according to claim 10, characterized in that the ratio of enterokinase enzyme activity to substrate for the cleavage reaction is 0.5-10U/mg.

20. The use according to claim 19, wherein the ratio of enterokinase enzyme activity to substrate for performing the cleavage reaction is 1.5-6U/mg.

21. The use according to claim 19, wherein the ratio of enterokinase enzyme activity to substrate for performing the cleavage reaction is 2-5U/mg.

22. The use according to claim 10, characterized in that the substrate comprises the amino acid sequence of DDDDK.

23. Use according to claim 10, characterized in that the reaction time of the substrate cleavage reaction is 4-72h.

24. A method for producing a polypeptide, characterized in that the method comprises the high specificity enterokinase cleavage method of any one of claims 1 to 9.

25. The method of claim 24, wherein the polypeptide is GLP-1, an analog or derivative thereof.

26. A method of purifying a polypeptide, comprising the high specificity enterokinase cleavage method of any one of claims 1 to 9.

27. The method of claim 26, wherein the polypeptide is GLP-1, an analog or derivative thereof.