Fusion protein and application thereof in preparation of precursor polypeptide of semeglutide

Abstract

The invention discloses a fusion protein and application thereof in preparation of a semeglutide precursor polypeptide, N (N is greater than or equal to 1) semeglutide fragments GLP-1 (11-37) are connected through KR, and then the semeglutide fragments GLP-1 (11-37), enterokinase enzyme cutting sites and fusion protein tags are subjected to fusion tandem expression in sequence, so that the semeglutide precursor polypeptide is obtained. The obtained fusion protein is a fusion protein tag-restriction enzyme cutting site-N-linked semeglutide fragment GLP-1 (11-37), and the fusion protein tag is an albumin affinity peptide (SEQ ID NO.1). The fusion protein is subjected to competent heterologous expression in escherichia coli BL21 (DE3), an engineering strain is constructed, and then fermentation expression is performed by using a shake flask, so that the thallus fermentation density is greatly improved, the expression of a target protein in an inclusion body is promoted, the yield of the target protein is improved, and the method has a good industrial application prospect. The fusion protein can be further successfully recognized by commercial enterokinase, KEX2 enzyme and carboxypeptidase B and cut to release the precursor polypeptide of the semeglutide.

Description

Technical Field

[0001] The present invention relates to a fusion protein and its application in preparing semaglutide precursor polypeptide, and specifically to a fusion protein sequence that can efficiently prepare semaglutide precursor peptide GLP-1, belonging to the technical field of genetic engineering and polypeptide preparation. Background Art

[0002] A protein tag is a polypeptide or protein that is fused and expressed with a target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and purification of the target protein. Commonly used protein tags include glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), streptavidin (SA), polyhistidine (Poly-His), small molecule ubiquitin-like modifier protein (SUMO) and hemagglutinin tag (HA). However, these fusion tags have large molecular weights and relatively low expression levels of fusion proteins. Albumin affinity peptide is a protein with a triple helical structure and high affinity for human serum albumin. Albumin affinity peptide has the characteristics of a small molecular weight and has great potential as a tag for fusion proteins in promoting protein expression.

[0003] Diabetes is a common chronic disease. With the improvement of people's living standards, the aging of the population and the increase in the incidence of obesity, the incidence of diabetes has been increasing year by year. Diabetes is mainly divided into type 1 diabetes, type 2 diabetes, gestational diabetes and other types of diabetes. Among them, 90% of patients suffer from type 2 diabetes, which can be treated by injection of insulin or oral hypoglycemic drugs. There are two ways of administering drugs for the treatment of diabetes on the market, namely injection drugs and oral drugs. Among them, oral drugs such as metformin have great side effects and long-term use will cause drug failure. Injectable drugs such as insulin will cause obesity or hypoglycemia at high doses or improper use. Polypeptide drugs such as glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP) and other incretins are considered to have no or very small side effects due to their natural secretion from the human body and their blood sugar dependence, and are receiving more and more attention.

[0004] Anti-semaglutide is a second-generation GLP-1 (glucagon-like peptide-1) analogue developed by diabetes giant Novo Nordisk. It is the seventh GLP-1 receptor agonist to be marketed in the world after exenatide, liraglutide, albiglutide, dulaglutide, lixisenatide, and benaglutide (approved in China), and is also the third long-acting GLP-1 weekly preparation in the world. As one of the representative drugs of GLP-1 analogues, semaglutide has a very prominent role in controlling blood sugar, reducing weight, and improving pancreatic β-cell function.

[0005] The main chain structure of semaglutide contains 27 amino acids of GLP-1. Currently, the main way to obtain GLP-1 is through chemical synthesis. However, chemical synthesis has many steps, uses a large amount of organic solvents, has low synthesis efficiency, is not conducive to large-scale production, and impurities generated during chemical synthesis bring certain risks to the use of drugs. The methods for preparing GLP-1 by biological methods mainly include soluble expression and inclusion body expression. Among them, the soluble expression (patent document with publication number CN104745597A, published in 2015) has a low expression level, which is not conducive to large-scale industrial production; the patent CN110498849A (published in 2019) involving inclusion body expression provides a method for preparing the main peptide chain of semaglutide with high purity and high yield, but because its preferred propeptide sequence KPSTYI belongs to a short peptide sequence, it cannot effectively increase the yield of the fusion protein; in addition, the patent document with patent publication number CN111378027A (published in 2020) carries out tandem expression of the intermediate polypeptide of semaglutide, but because the enzyme required is expensive, it does not have the significance of industrial scale-up.

[0006] There are many problems with the existing technology, so it is necessary to find a method with higher expression of fusion protein, simpler process steps and more suitable for industrial production. Summary of the invention

[0007] Technical problem solved by the present invention: The present invention discloses a fusion protein sequence and application thereof for efficiently preparing semaglutide precursor polypeptide GLP-1, wherein the fusion protein comprises a fusion protein tag, a protease cleavage site and a target main molecule sequence (semaglutide segment GLP-1 (11-37)), and based on an Escherichia coli expression system, a recombinant strain is constructed to obtain the fusion protein by fermentation, and then the fusion protein is enzymatically cleaved to obtain the semaglutide precursor GLP-1 polypeptide.

[0008] In order to solve the technical problem of the present invention, the present invention provides the following technical solutions:

[0009] The first aspect of the present invention provides a semaglutide precursor fusion protein, wherein the semaglutide precursor is formed by a fusion protein tag, an enterokinase recognition sequence DDDDK and an N-linked semaglutide segment GLP-1 (11-37) in series, denoted as: fusion protein tag-DDDDK-N-linked GLP-1 (11-37), wherein:

[0010] The fusion protein tag is an albumin affinity peptide,

[0011] The N-linked semaglutide segment GLP-1 (11-37) is a polypeptide segment formed by connecting N semaglutide segments GLP-1 (11-37), where N≥1.

[0012] Optionally, in some embodiments of the present invention, the fusion protein tag is an albumin affinity peptide, and its amino acid sequence is derived from an amino acid sequence of an albumin domain. The amino acid sequence of the protein short peptide is shown in SEQ ID NO.1.

[0013] Optionally, in some embodiments of the present invention, when N≥2, the N semaglutide segments GLP-1(11-37) in the N-linked semaglutide segment are connected by KR.

[0014] Optionally, in some embodiments of the present invention, the amino acid sequence of the semaglutide precursor fusion protein comprises any one of SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

[0015] When N=1, the amino acid sequence of the semaglutide precursor fusion protein formed by a combined semaglutide segment is shown in SEQ ID NO.2.

[0016] When N=2, the amino acid sequence of the semaglutide precursor fusion protein formed by the double semaglutide segment is shown in SEQ ID NO.3.

[0017] When N=3, the amino acid sequence of the semaglutide precursor fusion protein formed by the triple semaglutide segment is shown in SEQ ID NO.4.

[0018] Optionally, in some embodiments of the present invention, the molecular weight of the semaglutide precursor fusion protein is lower than 25 KDa.

[0019] Further optionally, in some embodiments of the present invention, the molecular weight of the semaglutide precursor fusion protein is lower than 15 KDa.

[0020] Further optionally, in some embodiments of the present invention, the molecular weight of the semaglutide precursor fusion protein is lower than 10 KDa.

[0021] Further optionally, in some embodiments of the present invention, the molecular weight of the semaglutide precursor fusion protein is 9-16 KDa.

[0022] Further optionally, in some embodiments of the present invention, the molecular weight of the semaglutide precursor fusion protein is any one of 9.5 KDa, 12.74 KDa or 16 KDa.

[0023] Optionally, in some embodiments of the present invention, an enterokinase recognition sequence DDDDK is introduced at the N-terminus of the semaglutide precursor GLP-1(11-37).

[0024] Optionally, in some embodiments of the present invention, the enterokinase recognition sequence is DDDDK. The enterokinase recognition sequence DDDDK used in the present invention is a commercially available product.

[0025] Optionally, in some embodiments of the present invention, the Linker sequence for connecting multiple semaglutide precursors GLP-1 (11-37) in series is KR.

[0026] The present invention utilizes the enterokinase recognition sequence DDDDK to connect the fusion protein tag and the semaglutide segment GLP-1 (11-37), and utilizes the KEX2 enzyme and carboxypeptidase B to recognize the Linker sequence KR to connect multiple semaglutide precursor GLP-1 (11-37), so as to obtain the semaglutide precursor fusion protein, and after the semaglutide precursor fusion protein is expanded and cultured, the protease is used in the reverse direction to perform enzyme cleavage on each enzyme cleavage site, specifically, the enterokinase recognizes and cleaves DDDDK, and the KEX2 enzyme and carboxypeptidase B recognize and cleaves KR, so as to obtain the semaglutide segment GLP-1 (11-37) fragment with large-scale production.

[0027] The KEX2 enzyme and carboxypeptidase B recognition sequence KR used in the present invention are commercial products.

[0028] The second aspect of the present invention provides a recombinant gene, namely a nucleic acid molecule, which contains a gene sequence encoding the above-mentioned semaglutide precursor fusion protein and is used to encode any of the above-mentioned semaglutide precursor fusion proteins.

[0029] Optionally, in some embodiments of the present invention, the nucleotide sequence of the recombinant gene comprises any one of SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7.

[0030] The third aspect of the present invention provides a gene recombination expression plasmid, wherein the gene recombination expression plasmid comprises the gene sequence encoding the semaglutide precursor fusion protein as described above.

[0031] Optionally, in some embodiments of the present invention, the expression vector of the gene recombination expression plasmid includes but is not limited to any one of the pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K, pIC9K or pTrc series vectors.

[0032] Further optionally, in some embodiments of the present invention, the expression vector of the gene recombination expression plasmid is pET-22b(+).

[0033] The fourth aspect of the present invention provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium is obtained by transforming the above-mentioned gene recombination expression plasmid into competent cells for heterologous expression.

[0034] Optionally, in some embodiments of the present invention, the competent cells include but are not limited to any one of Escherichia coli, Bacillus subtilis or Pichia pastoris.

[0035] Further optionally, in some embodiments of the present invention, the Escherichia coli includes any one of Escherichia coli JM109 (DE3), Escherichia coli HMS174 (DE3), Escherichia coli BL21 (DE3), Escherichia coli Rostta2 (DE3), Escherichia coli Rosttagami (DE3), Escherichia coli DH5α, Escherichia coli W3110 and / or Escherichia coli K12.

[0036] Further optionally, in some embodiments of the present invention, the competent cell is Escherichia coli BL21 (DE3).

[0037] Optionally, in some embodiments of the present invention, the expression system of the recombinant engineered bacteria is a prokaryotic expression system or a eukaryotic expression system.

[0038] Further optionally, in some embodiments of the present invention, a prokaryotic expression system is preferred.

[0039] The fifth aspect of the present invention provides a method for producing semaglutide precursor GLP-1 by enzymatic hydrolysis of the above-mentioned semaglutide precursor fusion protein.

[0040] Optionally, in some embodiments of the present invention, the method includes separating and purifying the semaglutide precursor fusion protein, and then cleaving it with enterokinase, KEX2 enzyme and carboxypeptidase B to release the semaglutide precursor GLP-1 polypeptide fragment.

[0041] The sixth aspect of the present invention provides a method for producing semaglutide precursor by fermentation using the above-mentioned recombinant engineered bacteria.

[0042] Optionally, in some embodiments of the present invention, the method includes using the recombinant engineered bacteria to ferment and produce semaglutide precursor fusion protein, and after separation and purification, it is enzymatically cleaved with enterokinase, KEX2 enzyme and carboxypeptidase B to release the semaglutide precursor GLP-1 polypeptide fragment.

[0043] Optionally, in some embodiments of the present invention, the method comprises the following steps:

[0044] (1) The recombinant engineered bacteria were cultured in LB medium at 35-40°C for 8-12 hours to obtain a cell seed solution, and the cell seed solution was inoculated into a fermentation medium for further culture, and cultured at 37°C until logarithmic growth (OD 600=0.6-0.8), add IPTG with a final concentration of 0.05-1mM for induction, and end the fermentation after induction at 16-40°C for 8-48h, and collect the bacteria;

[0045] (2) crushing the bacterial cells collected in step (1), and obtaining a supernatant and inclusion bodies after centrifugation;

[0046] (3) separating and purifying the supernatant and inclusion bodies obtained in step (2) to obtain semaglutide precursor fusion protein;

[0047] (4) digesting the fusion protein obtained in step (3) with enterokinase, KEX2 enzyme and carboxypeptidase B at 20-35° C. for 0-24 h to obtain a mixture of semaglutide precursor peptide and fusion protein peptide, and separating the mixture to obtain semaglutide precursor peptide;

[0048] Further optionally, in some embodiments of the present invention, the LB culture medium for preparing the cell seed solution includes: 5-15 g / L of tryptone, 2-10 g / L of yeast extract, and 5-15 g / L of sodium chloride (NaCl). Preferably, in some examples, the LB culture medium for preparing the cell seed solution includes: 10 g / L of tryptone, 5 g / L of yeast extract, and 10 g / L of sodium chloride (NaCl).

[0049] Further optionally, in some embodiments of the present invention, the fermentation medium used in the fermentation culture is TB medium, and the TB medium includes: Tryptone 5-15 g / L; Yeast Extract 10-30 g / L; Glycerin 2-10 g / L.

[0050] Preferably, in some examples, the TB culture medium includes: 12 g / L Tryptone; 24 g / L Yeast Extract; and 5 g / L Glycerin.

[0051] Further optionally, in some embodiments of the present invention, in step (1), the inducing agent used to induce expression is isopropylthiogalactoside (IPTG).

[0052] Further optionally, in some embodiments of the present invention, in step (3), the separation and purification method includes nickel column affinity chromatography and ion exchange chromatography.

[0053] Further optionally, in some embodiments of the present invention, in step (4), the enterokinase digestion includes digestion in a 10-30mM Tris-HCl pH7.4-10.0 system, at a ratio of 1U enterokinase to 2-20mg semaglutide precursor fusion protein, and reaction at 20-35°C for 0-24h. Preferably, in some examples, the enterokinase digestion includes digestion in a 25mMTris-HCl pH 8.0 system, at a ratio of 1U enterokinase to 10mg semaglutide precursor fusion protein, and reaction at 25°C for 10h.

[0054] Further optionally, in some embodiments of the present invention, in step (4), the KEX2 enzyme and carboxypeptidase B digestion include digestion in a 10-30mM Tris-HCl pH7.4-10.0 system, so that the mass ratio of recombinant KEX2 protease or carboxypeptidase B: fusion protein is 1:20-1:1000, and the reaction is carried out at 20-35°C for 0-24h. Preferably, in some examples, the KEX2 enzyme and carboxypeptidase B digestion include digestion in a 25mM Tris-HCl pH 8.0 system, and the KEX2 enzyme and carboxypeptidase B enzyme digest 5mg of semaglutide precursor tandem polypeptide at a ratio of 1:500, and react at 25°C for 8h.

[0055] Optionally, in some embodiments of the present invention, in step (4), the enzyme cleavage time is 4-10 hours, preferably 6-8 hours, and more preferably 7-8 hours.

[0056] The seventh aspect of the present invention provides a semaglutide precursor obtained by the above fermentation method.

[0057] Optionally, in some embodiments of the present invention, the expression form of the semaglutide precursor is: the expression form of connecting one GLP-1 (11-37) polypeptide (monovalent expression) is supernatant and inclusion body expression, and the expression form of connecting two and connecting three GLP-1 (11-37) polypeptides (divalent and triple expression) is inclusion body expression.

[0058] Optionally, in some embodiments of the present invention, the yield of the semaglutide precursor is higher than 300 mg / L. The present invention provides a method for producing a semaglutide precursor at the shake flask fermentation level, which increases the yield of the target protein and has good industrial application prospects.

[0059] Beneficial effect: The advantages of the present invention are that the present invention provides a method for constructing and fermenting the main peptide chain of semaglutide, the expression amount of the semaglutide precursor fusion protein obtained by gene recombination technology is significantly increased, the purification process is simple, and the purity of the purified protein is high; the efficiency of enzyme cleavage using enterokinase, KEX2 enzyme and carboxypeptidase B is high, the enzyme cleavage time is greatly shortened, and the conditions are mild, which reduces the cost and is conducive to the scale-up of the process.

[0060] Another advantage of the present invention is that the fusion protein tag of the present invention has a relatively small molecular weight and accounts for a small proportion of the molecular weight in the fusion protein, which can effectively increase the yield of the target protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is a diagram of the construction of a recombinant plasmid.

[0062] Figure 2 The following are SDS-PAGE images of prokaryotic recombinant expression of fusion proteins. (A) is the expression of a single fusion protein; (B) is the expression of a double fusion protein; (C) is the expression of a triple fusion protein.

[0063] Figure 3 The following are SDS-PAGE gel images of the nickel column affinity chromatography purification of the fusion protein expressed in inclusion bodies. (A) is the gel image of the purification of a single fusion protein; (B) is the gel image of the purification of a double fusion protein; (C) is the gel image of the purification of a triple fusion protein;

[0064] Figure 4 The different induction temperatures and bacterial OD 600 Effects on the expression of fusion proteins.

[0065] Figure 5 The effect of different culture media on the expression of fusion protein.

[0066] Figure 6 It is the concentration and enzyme cleavage efficiency of GLP-1 at different enzyme cleavage times.

[0067] Figure 7 It is the HPLC chromatogram after fusion protease cleavage.

[0068] Figure 8 It is the mass spectrum of the GLP-1 peak after fusion protease cleavage. DETAILED DESCRIPTION

[0069] Example

[0070] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the specific results described in the examples are only used to illustrate the present invention, and should not and will not limit the technical solutions of the present invention described in detail in the claims.

[0071] 1. Gene cloning and expression vector construction:

[0072] The fusion protein tag is an albumin affinity peptide, and its amino acid sequence is shown in SEQ ID NO.1.

[0073] The enterokinase recognition sequence is DDDDK.

[0074] The linker sequence KR that KEX2 enzyme and carboxypeptidase B recognize as the tandem linker between GLP-1(11-37) is called KR.

[0075] The polypeptide coding gene of the target protein, i.e., semaglutide precursor GLP-1 (11-37) (the amino acid sequence of the one-linked fusion protein is shown in SEQ ID NO.2, the amino acid sequence of the two-linked fusion protein is shown in SEQ ID NO.3, and the amino acid sequence of the three-linked fusion protein is shown in SEQ ID NO.4) was obtained by artificial synthesis and PCR amplification. The tandemly expressed semaglutide precursor gene was integrated with the plasmid pET-22b (+) using whole genome synthesis technology to obtain sequence cDNA, whose restriction enzyme cutting site is NdeI / HindⅢ, and the target gene sequences of all vectors were verified by nucleic acid sequence determination.

[0076] 2. Induced expression of fusion protein:

[0077] The relevant recombinant plasmid was transformed into the competent state of Escherichia coli BL21 (DE3) to obtain the target engineering strain, i.e., the recombinant engineering strain. The recombinant engineering strain was successfully constructed by strain PCR sequencing verification. Among them, the amino acid sequence of the fusion protein is shown in any one of SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4, and the corresponding nucleotide sequence of the encoding gene is shown in any one of SEQ ID NO.5 or SEQ ID NO.5 or SEQ ID NO.7.

[0078] The specific embodiments of the present invention all adopt laboratory small-scale shake flask expression. The cells were cultured overnight in LB liquid medium containing ampicillin resistance at 37°C for 12 hours, and then the overnight culture was transferred to a new TB medium containing ampicillin resistance (about 100 ml) at a ratio of 1:100, and cultured at 37°C with shaking until the appropriate logarithmic growth phase (OD 600 =0.6-0.8), 25°C, final concentration of 0.1 mM IPTG to induce expression for 48 h.

[0079] Take 100 ml of fermentation broth, centrifuge at 6000 rpm for 5 minutes at room temperature, discard the supernatant, collect the bacteria, resuspend with 1 / 10 to 1 / 5 of the fermentation broth volume of Buffer A solution (10mM Tris-HCl, 300mM NaCl, pH 7.4), and ultrasonically disrupt for 20 minutes. Take the disrupted sample, centrifuge at 9000 rpm for 10 minutes at room temperature, and separate the supernatant and inclusion bodies.

[0080] 3. Protein electrophoresis (SDS-PAGE) and quantification:

[0081] SDS-PAGE gel was prepared according to the gel kit. The prepared uninduced whole bacteria sample, induced whole bacteria sample, induced supernatant sample, induced inclusion body sample were loaded with equal volumes and tested by SDS-PAGE. First, the electrophoresis was performed at a constant voltage of 90V for 30min, and then at a constant voltage of 120V. After the electrophoresis, Coomassie Brilliant Blue R-250 staining was performed, and the electrophoresis results were obtained after decolorization.

[0082] Grayscale analysis was used to analyze the proportion of the target band of the fusion protein, and the expression level of the fusion protein in the inclusion body was detected using a BCA detection kit to obtain the expression level of the fusion protein; the content of the fusion protein could also be quantitatively calculated using the fusion protein standard curve obtained by HPLC.

[0083] In order to more clearly understand the above-mentioned objectives, features and advantages of the present invention, the scheme of the present invention will be further described below. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0084] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all of the embodiments.

[0085] Example 1: Recombinant prokaryotic expression of semaglutide precursor fusion protein in a shake flask system:

[0086] The tandemly expressed semaglutide precursor gene was integrated with the plasmid pET-22b(+) using whole genome synthesis technology to obtain sequence cDNA, whose restriction enzyme cutting site is NdeI / HindⅢ, and the constructed plasmid was transferred into E.coli to prepare recombinant engineered bacteria. Figure 1 The plasmid maps of the single, double and triple combination strains are shown. The overnight activated bacterial solution was transferred to a new TB medium (100 ml) containing ampicillin resistance at a ratio of 1:100 and cultured at 37°C with shaking until the logarithmic growth phase (OD 600=0.6-0.8), induced at 25°C, 0.1 mM IPTG for 48 h. Samples were taken at different time points to measure OD 600 , observe the growth trend of the host bacteria, and take samples for SDS-PAGE electrophoresis. Figure 2 As shown, the fusion protein was expressed in both the supernatant and inclusion bodies of the monovalent strain, while it was only expressed in the inclusion bodies of the divalent and trivalent strains.

[0087] Collect the cells, centrifuge the fermentation broth at 6000 rpm for 5 minutes at room temperature, discard the supernatant, collect the cells, resuspend the cell pellet with 1 / 10-1 / 5 of the fermentation broth volume of Buffer A solution, and ultrasonically disrupt for 20 minutes. Centrifuge the disrupted sample at 9000 rpm for 10 minutes at room temperature to separate the supernatant and inclusion bodies.

[0088] Example 2: Isolation and purification of semaglutide precursor fusion protein in a shake flask system

[0089] Purification was performed using nickel column affinity chromatography, equilibrated with Buffer A (10 mM Tris-HCl, 300 mM NaCl, pH 7.4), and eluted with Buffer B (10 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole, pH 7.4).

[0090] The supernatant of the fusion protein was mixed with the inclusion body in a volume ratio of 3:1 and then purified. The nickel column was first equilibrated with 3-5 column volumes of Buffer A solution, and then the supernatant filtered with a 0.45μm filter membrane was loaded to allow the fusion protein to bind to the column. The nickel column was then equilibrated with 3-5 column volumes of Buffer A solution, and eluted with 5%, 20%, 50%, and 100% imidazole Buffer B solution, respectively. The eluate was collected and verified by SDS-PAGE. The results are shown in Figure 3 As shown, 100% imidazole Buffer B solution can elute all the fusion protein.

[0091] Purification of fusion proteins in the double and triple inclusion bodies: first dissolve the inclusion bodies with 8M urea pH 8.0, and stir with a magnetic stirrer at room temperature for 2-4 hours to fully dissolve the inclusion bodies. Equilibrate the nickel column with 3-5 column volumes of Buffer A solution, then take the fully dissolved inclusion bodies filtered with a 0.45μm filter membrane to load the sample, so that the fusion protein binds to the column, and then equilibrate the nickel column with 3-5 column volumes of Buffer A solution, and elute with 10%, 20%, 50%, and 100% imidazole Buffer B solution, respectively. Collect the eluate and perform SDS-PAGE verification. The results are shown in Figure 3 As shown, 100% imidazole Buffer B solution can elute all the fusion protein. The recovery rate of nickel column affinity chromatography purification can reach more than 85%.

[0092] Example 3: Calculation of the expression level of semaglutide precursor fusion protein in a shake flask system

[0093] The expression level of the fusion protein was determined according to the instructions of the BCA kit.

[0094] The results are shown in Table 1. Both the dual and triple fusion proteins are expressed in inclusion bodies with high expression levels and small molecular weights.

[0095] Table 1 Expression levels of fusion proteins with different protein tags

[0096]

[0097] Example 4: Comparison of expression levels of fusion proteins of GST, MBP tags and albumin affinity peptide tags

[0098] The same construction method as the above-mentioned recombinant engineering bacteria was used, and GST and MBP tags were used as fusion protein tags to construct single-linked recombinant engineering bacteria GST-DDDDK-GLP-1 and MBP-DDDDK-GLP-1, respectively.

[0099] The three groups of one-linked recombinant engineering bacteria were fermented and expressed at 25°C, separated and purified, and the protein concentration was determined by the BCA method to calculate the expression amount of the fusion protein.

[0100] BCA method: make the total volume of sample diluent 20μL, add 200μL of BCA working solution, mix thoroughly, place at 37℃ for 30 minutes, measure color at 595nm wavelength and record the absorbance value; then, according to the absorbance value of the measured sample, the corresponding protein concentration can be obtained on the standard curve, and finally, the expression level of each fusion protein is calculated.

[0101] The results are shown in Table 2. The expression level of the fusion protein with albumin affinity peptide tag was better than that of GST and MBP tags, and had a smaller molecular weight.

[0102] Table 2 Expression levels of fusion proteins with different protein tags

[0103]

[0104] From the results in Table 1 and Table 2, the molecular weight of the fusion protein is relatively small. The smaller molecular weight gives it the following advantages in the E. coli expression system, making the fusion protein easier to express and purify, and having a higher yield:

[0105] (1) Higher expression efficiency, faster transcription and translation rates, smaller mRNA molecules and polypeptide chains are more easily recognized and translated by the ribosomes of E. coli, reducing stagnation in the translation process and thus improving protein expression efficiency.

[0106] (2) Reduce ribosome crowding: Large molecular weight proteins can easily cause ribosome crowding and affect translation efficiency, while small molecular weight proteins reduce this possibility.

[0107] (3) Reduce the complexity of mRNA structure. Smaller mRNAs usually have lower secondary structure complexity and are less likely to form stem-loop structures that hinder ribosome movement, thereby improving translation efficiency.

[0108] (4) Lower cytotoxicity and reduced metabolic burden. The expression of large molecular weight proteins often brings a greater metabolic burden to E. coli and consumes more cell resources, thus affecting cell growth and protein expression. The expression of small molecular weight proteins reduces this burden.

[0109] (5) Reduce misfolding and aggregation. Large molecular weight proteins are more likely to misfold and aggregate, forming inclusion bodies, while small molecular weight proteins are easier to fold correctly, reducing the formation of inclusion bodies and reducing toxic effects on cells.

[0110] (6) More easily tolerated by cells. Small molecular weight proteins are usually less toxic to cells, and cells are more likely to tolerate their expression, thereby increasing the expression level.

[0111] (7) It is easier to express by secretion and easier to pass through the secretory pathway. Small molecular weight proteins are more likely to pass through the secretory pathway of Escherichia coli, such as the periplasmic space or culture medium, thereby reducing intracellular accumulation and reducing the formation of inclusion bodies.

[0112] (8) It is easier to purify secretory proteins, which reduces the steps of cell crushing and inclusion body separation and improves purification efficiency.

[0113] (9) It is easier to fold correctly. Fewer structural domains make the structure of small molecular weight proteins simpler, and fewer structural domains are required to fold, making it easier to fold correctly into active proteins.

[0114] (11) Shorter folding time. Small molecular weight proteins have a shorter folding time and are easier to quickly form the correct conformation.

[0115] (12) It is easier to purify and separate. Small molecular weight proteins are less interfered by other impurities during purification, making it easier to achieve high-purity separation.

[0116] (13) It is easier to use molecular sieves. Small molecular weight proteins are easier to purify and separate using molecular sieves and other methods.

[0117] Example 5: Optimization of the induction temperature of semaglutide precursor fusion protein in a shake flask system

[0118] Induction temperature is one of the key parameters affecting heterologous protein expression. Low-temperature induction can reduce protein misfolding, but it will also reduce bacterial metabolic activity, thereby extending the fermentation cycle. High-temperature induction can accelerate metabolic activity and shorten the fermentation cycle, but it will cause misfolding due to too fast protein folding, resulting in a large amount of insoluble protein (inclusion body).

[0119] The recombinant strain of albumin affinity peptide-DDDDK-GLP-1 precursor fusion protein was induced and expressed at 16℃, 25℃, 30℃, and 37℃, respectively. The results are as follows Figure 4 As shown in the figure, the expression amount induced at 16°C was the largest, and the bacterial concentration and expression level induced at 37°C were inhibited, so the appropriate induction temperature was 16°C-30°C. Further comprehensive consideration of the fact that the induction temperature below 20°C is not suitable for actual production in terms of fermentation cycle and temperature control, so the more suitable induction temperature was 20°C-30°C, and further 25°C-30°C, so the 25°C induction temperature was selected as the optimal condition in subsequent experiments.

[0120] Example 6: Optimization of fermentation medium for semaglutide precursor fusion protein in shake flask system

[0121] Based on Example 5, this example selected 7 culture media for fermentation expression, which are:

[0122] Culture medium No. 1: 30.0 g / L glycerol, 5.9 g / L sodium citrate dihydrate, 5.2 g / L ammonium sulfate, 8.6 g / L disodium hydrogen phosphate dodecahydrate, 4.4 g / L sodium dihydrogen phosphate dihydrate, 4.0 g / L potassium chloride, 1.0 g / L anhydrous magnesium sulfate, 0.25 g / L anhydrous calcium chloride, adjust pH to 7.0 with 5 mol / L sodium hydroxide.

[0123] Culture medium No. 2: 30.0 g / L glycerol, 4.2 g / L citric acid, 5.0 g / L ammonium sulfate, 8.57 g / L disodium hydrogen phosphate dodecahydrate, 4.4 g / L sodium dihydrogen phosphate dihydrate, 1.0 g / L anhydrous magnesium sulfate, 0.25 g / L anhydrous calcium chloride, adjust pH to 7.0 with 5 mol / L sodium hydroxide.

[0124] Medium No. 3: 2.0 g / L glucose, 20.0 g / L glycerol, 7.5 g / L ammonium citrate, 22.5 g / L yeast extract, 4.0 g / L ammonium sulfate, 5.0 g / L sodium chloride, 8.0 g / L disodium hydrogen phosphate dodecahydrate, 4.0 g / L potassium dihydrogen phosphate, 0.51 g / L magnesium sulfate, 0.01 g / L calcium chloride, adjust the pH to 7.0 with 5 mol / L sodium hydroxide.

[0125] Medium No. 4: 20.0 g / L glucose, 5.0 g / L tryptone, 5.0 g / L yeast extract, 0.5 g / L sodium chloride, 17.2 g / L disodium hydrogen phosphate dodecahydrate, 2.4 g / L potassium dihydrogen phosphate, 0.3 g / L magnesium sulfate, 0.01 g / L calcium chloride, adjust the pH to 7.0 with 5 mol / L sodium hydroxide.

[0126] Medium No. 5: 10.0 g / L glycerol, 3.2 g / L trisodium citrate, 5.0 g / L yeast extract, 10.0 g / L ammonium sulfate, 17.2 g / L disodium hydrogen phosphate dodecahydrate, 3.0 g / L potassium dihydrogen phosphate, 0.51 g / L magnesium sulfate, 0.011 g / L calcium chloride, adjust the pH to 7.0 with 5 mol / L sodium hydroxide.

[0127] Medium No. 6: 0.5 g / L glucose, 5.0 g / L glycerol, 10.0 g / L lactose, 2.67 g / L ammonium chloride, 10.0 g / L yeast extract, 1.42 g / L sodium sulfate, 8.95 g / L disodium hydrogen phosphate dodecahydrate, 3.4 g / L potassium dihydrogen phosphate, 1.0 g / L magnesium sulfate, 0.25 g / L calcium chloride, adjust the pH to 7.0 with 5 mol / L sodium hydroxide.

[0128] TB medium: peptone 12 g / L; yeast powder 24 g / L; glycerol 5 g / L; K2HPO4·3H2O 16.4 g / L; KH2PO4 2.31 g / L.

[0129] The results are as follows Figure 5 As shown, through grayscale analysis and comparison, it is known that the fusion protein expression yield of TB culture medium is the highest, so the present invention preferably selects TB culture medium as the fermentation medium.

[0130] Example 7: Enzymatic cleavage of semaglutide precursor fusion protein

[0131] The conjugate fusion protein obtained in Example 2 was replaced into an enzyme cleavage system (25 mM Tris-HCl pH 8.0), enterokinase was added into the enzyme cleavage system at a ratio of 1 U enterokinase to 10 mg semaglutide precursor fusion protein, and enzymatic hydrolysis was performed at 25° C. to obtain a GLP-1 and protein tag mixed solution.

[0132] The results are as follows Figure 6 As shown, when the enzyme digestion time is 10 hours, the enzyme digestion efficiency of enterokinase can reach 94.93%.

[0133] The diplex and triplicate fusion proteins obtained in Example 2 were replaced into an enzyme cleavage system (25 mM Tris-HCl pH 8.0), and enterokinase was added into the enzyme cleavage system at a ratio of 1 U enterokinase to 10 mg semaglutide precursor fusion protein. The enzyme was hydrolyzed at 25° C. for 10 h to obtain a mixture of semaglutide precursor tandem polypeptides and protein tags.

[0134] Continue to digest 5 mg of semaglutide precursor tandem peptides in the enzyme digestion system at a ratio of 1U KEX2 enzyme and carboxypeptidase B, and perform enzymatic digestion at 25°C for 10 hours to obtain a mixed solution of GLP-1 and protein tags. The enzymatic digestion efficiency of KEX2 enzyme and carboxypeptidase B can reach more than 85%.

[0135] The HPLC chromatogram of GLP-1 after enzyme digestion is as follows: Figure 7 As shown, the GLP-1 peak of the enzyme cleavage chromatogram was subjected to mass spectrometry analysis as shown Figure 8 The results of mass spectrometry analysis were consistent with the molecular weight of GLP-1, 2989.28, indicating that the cleaved GLP-1 polypeptide was correct.

[0136] As shown in Table 3, the semaglutide precursor obtained by enzyme cleavage of the triple fusion protein had the highest yield.

[0137] Table 3 Yields of three fusion proteins and semaglutide precursor

[0138]

[0139] It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0140] The above description is only a specific embodiment of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments described herein, but should conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A semaglutide precursor fusion protein, characterized in that: The semaglutide precursor fusion protein is formed by a fusion protein tag, an enterokinase cleavage site and an N-linked semaglutide segment GLP-1 (11-37) connected in series, wherein: The fusion protein tag is an albumin affinity peptide, The N-linked semaglutide segment GLP-1 (11-37) is a polypeptide segment formed by connecting N semaglutide segments GLP-1 (11-37), where N≥1.

2. The semaglutide precursor fusion protein according to claim 1, characterized in that: The amino acid sequence of the albumin affinity peptide is shown in SEQ ID NO.

1.

3. The semaglutide precursor fusion protein according to claim 1, characterized in that: When N≥2, the N semaglutide segments GLP-1(11-37) in the N-linked semaglutide segment are connected by KR, and / or The amino acid sequence of the semaglutide precursor fusion protein comprises any one of SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4; and / or The molecular weight of the semaglutide precursor fusion protein is less than 25 KDa, preferably 9-16 KDa.

4. A recombinant gene, characterized in that The recombinant gene encodes the semaglutide precursor fusion protein according to any one of claims 1 to 3.

5. A gene recombination expression plasmid, characterized in that: The gene recombination expression plasmid comprises the recombinant gene as claimed in claim 4.

6. The gene recombination expression plasmid according to claim 5, characterized in that: The expression vector of the gene recombination expression plasmid includes but is not limited to any one of the pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K, pIC9K or pTrc series vectors.

7. A recombinant engineered bacterium, characterized in that: The gene is obtained by transforming the gene recombination expression plasmid described in claim 5 or 6 into competent cells for heterologous expression.

8. The recombinant engineered bacterium according to claim 7, characterized in that The competent cells include but are not limited to any one of Escherichia coli, Bacillus subtilis or Pichia pastoris; Preferably, the Escherichia coli includes any one of Escherichia coli JM109 (DE3), Escherichia coli HMS174 (DE3), Escherichia coli BL21 (DE3), Escherichia coli Rostta2 (DE3), Escherichia coli Rosttagami (DE3), Escherichia coli DH5α, Escherichia coli W3110 and / or Escherichia coli K12; Preferably, the expression system of the recombinant engineered bacteria is a prokaryotic expression system or a eukaryotic expression system, preferably a prokaryotic expression system.

9. A method for preparing semaglutide precursor GLP-1, characterized in that: The semaglutide precursor fusion protein produced by fermentation of the recombinant engineered bacteria as described in claims 1-3 or as described in claim 7 or 8 is hydrolyzed by enterokinase, KEX2 enzyme and carboxypeptidase B to release the semaglutide precursor peptide fragment.

10. The method according to claim 9, characterized in that The method comprises the following steps: (1) culturing the recombinant engineered bacteria in LB medium at 35-40° C. for 8-12 hours to obtain a cell seed solution, inoculating the cell seed solution into a fermentation medium for further culturing, culturing at 37° C. until logarithmic growth, adding IPTG with a final concentration of 0.05-1 mM for induction, and ending the fermentation after induction at 16-40° C. for 8-48 hours, and collecting the bacteria; (2) crushing the bacterial cells collected in step (1), and obtaining a supernatant and inclusion bodies after centrifugation; (3) separating and purifying the supernatant and inclusion bodies obtained in step (2) to obtain semaglutide precursor fusion protein; (4) digesting the fusion protein obtained in step (3) with enterokinase, KEX2 enzyme and carboxypeptidase B at 20-35° C. for 0-24 h to obtain a mixture of semaglutide precursor peptide and fusion protein peptide, and separating the mixture to obtain semaglutide precursor peptide; Preferably, in step (1), the fermentation medium is TB medium, and the TB medium comprises: 5-15 g / L tryptone; Yeast extract 10-30g / L; Glycerol 2-10g / L; Preferably, in step (4), the enzyme cleavage time is 4-10 h, more preferably 6-8 h, and more preferably 7-8 h.

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