Method for preparing precursor peptide of semeglutide through intracellular enzyme digestion

Abstract

The invention discloses a method for preparing a simeglutide precursor peptide through intracellular enzyme digestion. The method comprises the following steps: (1) connecting a dissolution-promoting tag GST and the simeglutide precursor peptide together through protease specific recognition of amino acid sites to form a fusion protein; (2) integrating the coding nucleotide sequence of the fusion protein, the coding nucleotide sequence of KEN2 protease and the coding nucleotide sequence of carboxypeptidase B into the same expression vector to form a recombinant vector; (3) transforming the recombinant vector into engineering bacteria and carrying out fermentation production; and (4) centrifuging a fermentation product, collecting thalli, and purifying to obtain the precursor peptide powder of the semeglutide. The whole operation process is simpler, more convenient and smoother, tedious recovery steps are omitted, and the preparation and recovery difficulty of the precursor peptide of the semeglutide is remarkably reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of semaglutide production, and in particular to a method for preparing semaglutide precursor peptide by intracellular enzymatic cleavage. Background Art

[0002] Sermaglutide is a glucagon-like peptide-1 (GLP-1) derivative analog developed by Novo Nordisk. By modifying the structure of the GLP-1 molecule, such as introducing side chains and adjusting the amino acid sequence, the duration of its efficacy in the body is prolonged. The drug can not only significantly promote the secretion of insulin, but also inhibit the secretion of glucagon, thereby achieving the effect of lowering blood sugar.

[0003] Novo Nordisk, the original drug manufacturer, uses yeast fermentation to produce the precursor peptide chain of semaglutide, and further modifies and transforms the peptide chain to finally obtain the finished product of semaglutide. At present, relevant pharmaceutical companies mainly use chemical synthesis and semi-biological methods to produce semaglutide. The semi-biological method first uses Escherichia coli fermentation to produce the precursor peptide of semaglutide, and then attaches fatty acid side chains to the precursor peptide through chemical modification. In comparison, the technical threshold of chemical synthesis is lower, and most chemical companies can carry out related preparations. However, the chemical synthesis method has certain disadvantages in product purity and production cost, and usually requires the use of a large amount of toxic and harmful chemical reagents. For example, patents CN118666987A, CN118725078A and CN118834262A all involve the use of chemical reagents with environmental risks. In patent CN118666987A, chemical reagents such as isobutyric acid and histidine active ester are used to condense with peptide chains to prepare the precursor peptide of semaglutide; patent CN118725078A uses solid phase synthesis to prepare resin, and synthesizes semaglutide by adding cracking agents and ether reagents; in patent CN118834262A, 2-phenoxy-N-(3-pyridyl)acetamide and 1-phenyl-3-(cyclohexylmethylene)carbodiimide are used as activators and catalysts to prepare semaglutide intermediates. The use of these chemical reagents has certain adverse effects on the environment.

[0004] The semi-biosynthesis method through Escherichia coli fermentation has the advantages of simple fermentation process, convenient operation, low cost and easy scale-up. The production of semaglutide precursor peptide using this method usually involves the following main steps: (1) fusing the coding gene of semaglutide precursor peptide with the coding gene of other larger proteins to construct a new fusion protein coding gene and integrate it into the plasmid; (2) transforming the plasmid into Escherichia coli and inoculating the culture medium for fermentation production; (3) recovering the fusion protein in the inclusion body or extracting the soluble fusion protein from the supernatant, and then performing enzyme digestion after recovery treatment to obtain the crude semaglutide precursor peptide. In the above process, the recovery step is the key link to control the production cost. At present, the relevant semi-biosynthesis patents (such as patent CN118620091A) use a fusion protein expression system to produce semaglutide precursor peptide. Although this method improves the production efficiency to a certain extent, it still fails to completely solve the problem of operational complexity in the recovery process. For example, it is usually necessary to first obtain inclusion bodies, then perform renaturation treatment, then enzymatic digestion, and finally go through multiple steps such as secondary purification and recovery. These steps are cumbersome and pose challenges to production efficiency and cost control. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing semaglutide precursor peptide by intracellular enzymatic cleavage, aiming to achieve efficient preparation of semaglutide precursor peptide. The method makes the entire operation process simpler and smoother, omits cumbersome recovery steps, and significantly reduces the difficulty of preparing and recovering semaglutide precursor peptide.

[0006] The technical solution adopted by the present invention to solve its technical problem is: A method for preparing semaglutide precursor peptide by intracellular enzymatic cleavage, comprising the following steps: (1) The solubility-promoting tag GST and the semaglutide precursor peptide are linked together through the protease-specific recognition amino acid site to form a fusion protein; the front and rear ends of the semaglutide precursor peptide both have the protease-specific recognition amino acid site (RR); (2) Integrate the nucleotide sequence encoding the fusion protein, the nucleotide sequence encoding the KEX2 protease (NCBI: NC_001146.8), and the nucleotide sequence encoding the carboxypeptidase B (NCBI: NM_029706.2) into the same expression vector to form a recombinant vector; the nucleotide sequence encoding the fusion protein is shown in SEQ ID No. 1; (3) Transform the engineered bacteria with the recombinant vector and carry out fermentation production; (4) The fermentation product is centrifuged to collect the bacterial cells, the cells are ultrasonically disrupted, the supernatant is centrifuged and filtered, and the chromatography eluate is concentrated, desalted, and freeze-dried to obtain semaglutide precursor peptide powder.

[0007] In the present invention, the semaglutide precursor peptide is located at the C-terminus of the GST protein ( Figure 1 ). In the present invention, the cleavage site of KEX2 protease (serine protease, Kex2 Protease) is: RR↓; the cleavage site of carboxypeptidase B is: ↓RR.

[0008] The present invention adopts a strategy of protease-specific recognition of the amino acid site RR. Specifically, the cleavage enzyme (such as KEX2 protease, carboxypeptidase, etc.) and the fusion protein are expressed together in the same host cell, and the cleavage enzyme can specifically recognize the enzyme cleavage site RR before and after the semaglutide precursor peptide in the fusion protein, and then perform an enzyme cleavage reaction to release the semaglutide precursor peptide. Through simple centrifugation and separation and purification steps, a high-purity semaglutide precursor peptide can be directly obtained, which significantly simplifies the recovery process. In addition, the present invention uses a solubilizing tag GST to effectively avoid the target protein from forming inclusion bodies. The C-terminus of the semaglutide precursor peptide is provided with an amino acid site RR that is specifically recognized by a protease, which cooperates with the RR site at the N-terminus to ensure the generation of accurate semaglutide precursor peptides, and the site also has the function of a stop codon.

[0009] The structure of the fusion protein of the present invention is: lytic tag GST+protease specific recognition amino acid site (RR)+semaglutide precursor peptide+protease specific recognition amino acid site (RR)+semaglutide precursor peptide+protease specific recognition amino acid site (RR). The fusion protein contains two semaglutide precursor peptides, thereby effectively improving the yield of fermentation production. However, if the number of semaglutide precursor peptides is too large, it may exceed the carrying capacity of the carrier. In some cases, the lytic tag GST+protease specific recognition amino acid site (RR)+semaglutide precursor peptide+protease specific recognition amino acid site (RR) can also be used. This construction method only contains one semaglutide precursor peptide. The amino acid sequence of the lytic tag GST is shown in SEQ ID No.3. The amino acid sequence of the semaglutide precursor peptide is shown in SEQ ID No.4.

[0010] The expression vector is selected from one of the pET series and pETDuet series expression vectors. Preferably, the pET series includes pET28, and the pETDuet series includes pETDuet-1.

[0011] The engineered bacteria is selected from one of Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum.

[0012] A fusion protein for preparing a semaglutide precursor peptide, comprising: a lytic tag GST+a protease-specific recognition amino acid site+a semaglutide precursor peptide+a protease-specific recognition amino acid site+a semaglutide precursor peptide+a protease-specific recognition amino acid site; the amino acid sequence of the fusion protein is shown in SEQ ID No. 2.

[0013] A recombinant vector for preparing a semaglutide precursor peptide, comprising: Part I: a nucleotide sequence encoding a fusion protein containing a semaglutide precursor peptide, wherein the nucleotide sequence encoding the fusion protein is shown in SEQ ID No. 1; Part II: a nucleotide sequence encoding the KEX2 protease; Part III: a nucleotide sequence encoding carboxypeptidase B; The above three parts are integrated into the same expression vector to form a recombinant vector.

[0014] The fusion protein is formed by linking the solubility-promoting tag GST and the semaglutide precursor peptide through a protease-specific recognition amino acid site.

[0015] The beneficial effects of the present invention are as follows: the present invention focuses on the recovery link, and by using molecular biological methods and genetic engineering technology, a new type of recombinant vector is constructed, which significantly simplifies the overall operation of the recovery process. The semaglutide precursor peptide can be directly separated and purified by a simple centrifugation step, omitting the complex steps of enzyme cleavage, acquisition of inclusion bodies and renaturation in the traditional method, greatly simplifying the preparation process of the semaglutide precursor peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the vector construction connection of the solubility-promoting tag GST and the semaglutide precursor in the present invention; Figure 2 It is a schematic diagram of the construction of the expression vector and sequence of the semaglutide precursor peptide of the present invention; Figure 3 is the HPLC detection chart of the eluate of Example 1; Figure 4 is the mass spectrum of semaglutide; Figure 5 is the HPLC detection chart of the eluate of Example 2; Figure 6 is the HPLC detection chart of the eluate of Example 3; Figure 7 It is the HPLC detection chart of the eluate of Example 4. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further described in detail below through specific embodiments.

[0018] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0019] In the present invention, the culture medium used in the embodiments is: The specific formula of LB liquid medium is: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L; The specific formula of LB solid medium is: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar 15g / L.

[0020] The protein fermentation medium used was a modified FCM medium with the following formula: yeast extract 50 g / L, glycerol 30 g / L, citric acid 2 g / L, KH2PO4 2 g / L, inorganic salt solution (200×) 6 mL (including MgCl2 300 g / L, KCl 100 g / L, CaCl2 100 g / L), trace element solution (1000×) 1.5 mL (including: FeCl3 54 g / L, MnCl2 16 g / L, ZnCl2 7.0 g / L, COCl2 4.8 g / L, CuSO4 2.2 g / L, H3BO 30.5 g / L, (NH4)6MO7O 24 0.02g / L).

[0021] In the present invention, all reagents used were of analytical grade, molecular cloning-related enzyme reagents were purchased from ThermoFisher, Escherichia coli strains were purchased from Miaoling Biotechnology, gene synthesis and gene sequencing were completed by Qingke Biotechnology, and the relevant steps in the embodiments were carried out according to the reaction conditions and experimental methods given in the product instructions recommended by the manufacturer.

[0022] In the present invention, molecular biology molecular cloning experiments are all referenced from Molecular Cloning Experiment Guide (Fourth Edition) (Edited by MR. Green and J. Sambrook, translated by He Fuchu, Beijing: Science Press, 2017).

[0023] In the present invention, the nucleic acid sequence identity percentage refers to the comparison result obtained by sequence alignment using biology-related software or databases well-known in the biological field, such as Blast, Align, DNAman, VectorNTI and other software.

[0024] In the present invention, the protein expression system refers to the well-known expression frame in biology, that is, the promoter and terminator in the plasmid and the target gene sequence contained therein. This complete sequence can be integrated into the plasmid and then transformed into the host cell to achieve the expression of the protein encoded by the target gene sequence.

[0025] In order to solve the problem of multiple process steps in the existing Escherichia coli fermentation process for preparing semaglutide precursor peptide, and to focus on simplifying the production operation of semaglutide, this embodiment provides the following technical solutions: Example 1

[0026] 1. Construction and connection of vectors of soluble tag GST and semaglutide precursor Figure 1 As shown, the semaglutide precursor peptide expression vector and sequence construction are as follows Figure 2 shown.

[0027] The whole gene sequence (SEQ ID No. 1) was synthesized by Qingke Biotechnology, and then integrated into the same pET28 plasmid by Qingke Biotechnology and then returned to the plasmid. This example uses Escherichia coli BL21 (DE3) as the host cell. A tube of Escherichia coli BL21 (DE3) competent state (purchased from Qingke Biotechnology, 100 μL / tube) was taken out from the -80°C refrigerator and placed in crushed ice. After the competent state was naturally melted at room temperature, 100-200 ng of the plasmid returned after the gene synthesis of Qingke Biotechnology was added. After being gently blown evenly with a pipette tip, it was placed in an ice bath for 15 minutes, and then heat-shocked at 42°C for 90 seconds, and then immediately placed in crushed ice for 5 minutes, 900 μL of LB liquid culture medium was added thereto, and placed in a shaker at 37°C, and the shaker speed was set to 220 rpm. After one hour, 50 μL was aspirated with a pipette and coated on an LB solid culture medium containing 50 μg / mL kanamycin.

[0028] Place the culture medium upside down in a 37℃ constant temperature culture phase. After culturing for 12~14h, pick up a single colony with a toothpick using tweezers, and then directly throw the toothpick into LB liquid culture medium containing 50μg / mL kanamycin for expansion. After expansion, store it in a -80℃ refrigerator with 20% final concentration of glycerol for later use. After being sent to Qingke Bio for sequencing verification, it is the expression engineering strain.

[0029] 2. Use a pipette to take 5 μL of glycerol strain and inoculate it into 5 mL LB liquid medium containing 50 μg / mL kanamycin. Cultivate it at 37°C and 220 rpm on a shaker for 14 hours. Inoculate it into 200 mL FCM liquid medium containing 50 μg / mL kanamycin at a volume ratio of 1:100 for bacterial liquid to medium. Cultivate it at 37°C and 220 rpm on a shaker until OD600 is about 0.8, then add IPTG to a final concentration of 0.5 mM. Lower the culture temperature to 30°C and continue to induce expression for 18 hours.

[0030] 3. Collect the bacteria by centrifugation, resuspend with 1 times the volume of culture medium 50mM Tris-HCl, ultrasonically disrupt the cells, centrifuge the supernatant and filter with a 0.45μm filter membrane. The common hydrophobic chromatography method is used to purify the precursor polypeptide: the mobile phase of the hydrophobic chromatography is: mobile phase A: ultrapure water; mobile phase B: NaCl 1.75g / L, NaH2PO40.06g / L, Na2HPO47.02g / L, and the relevant sodium salts are purchased from Sinopharm Group. Use Pheny1 SepharoseHP chromatographic column, balance the column with 5 column volumes of mobile phase B to ensure stability, inject 2mL, pre-wash the column with 4 column volumes of mobile phase A, and perform 0~100% gradient elution, and the elution volume is greater than 30 column volumes. Collect 1mL of sample. The gradient elution program was set to an elution time of 110 min, the initial value of the mixture of mobile phase A and mobile phase B was 0:100%, the percentage of mobile phase A increased by 5% every 5 minutes, and the final value was 100%; the percentage of mobile phase B decreased by 5% every 5 minutes, and the final value was 0.

[0031] The liquid phase results of the eluent are as follows Figure 3 As shown, the purity is 95%.

[0032] The eluate was concentrated, desalted, and freeze-dried to obtain semaglutide precursor peptide powder. The mass spectrometry results of semaglutide precursor peptide were as follows: Figure 4 The results showed that this scheme successfully prepared semaglutide precursor peptide that was cleaved intracellularly.

[0033] Example 2 The difference from Example 1 is that the vector uses pETDuet-1 plasmid instead of pET28 plasmid.

[0034] 1. Similar to Example 1, the whole gene sequence (SEQ ID No. 1) was synthesized by Qingke Biotechnology, and then integrated into the same pETDuet-1 plasmid by Qingke Biotechnology and then returned to the plasmid. This example uses Escherichia coli BL21 (DE3) as the host cell. A tube of Escherichia coli BL21 (DE3) competent state (purchased from Qingke Biotechnology, 100 μL / tube) was taken out from the -80°C refrigerator and placed in crushed ice. After the competent state was naturally melted at room temperature, 100-200 ng of the plasmid returned after the gene synthesis of Qingke Biotechnology was added. After being gently blown evenly with a pipette tip, it was ice bathed for 15 minutes, and then heat-shocked at 42°C for 90 seconds, and then immediately placed in crushed ice for 5 minutes, 900 μL of LB liquid culture medium was added thereto, and placed in a shaker at 37°C, and the shaker speed was set to 220 rpm. After one hour, 50 μL was aspirated with a pipette and coated on an LB solid culture medium containing 50 μg / mL ampicillin.

[0035] Place the culture medium upside down in a 37℃ constant temperature culture phase and culture for 12~14h. After a single colony grows, pick up a single clone with a toothpick using tweezers and directly throw the toothpick into LB liquid culture medium containing 50μg / mL ampicillin for expansion. After expansion, store it in a -80℃ refrigerator with 20% final concentration of glycerol for later use. After being sent to Qingke Bio for sequencing verification, it is the expression engineering strain.

[0036] 2. Use a pipette to take 5 μL of glycerol strain and inoculate it into 5 mL LB liquid culture medium containing 50 μg / mL ampicillin. Cultivate it at 37°C and 220 rpm on a shaker for 14 hours. Inoculate it into 200 mL FCM liquid culture medium containing 50 μg / mL ampicillin at a volume ratio of 1:100 for bacterial liquid to culture medium. Cultivate it at 37°C and 220 rpm on a shaker until OD600 is about 0.8. Add IPTG to a final concentration of 0.5 mM. Lower the culture temperature to 30°C and continue to induce expression for 18 hours.

[0037] 3. Collect the bacteria by centrifugation, resuspend with 50mM Tris-HCl with one volume of culture medium, ultrasonically disrupt the cells, centrifuge the supernatant and filter with a 0.45μm filter membrane. The precursor polypeptide was purified by the commonly used hydrophobic chromatography method. The mobile phase of the hydrophobic chromatography is: mobile phase A: ultrapure water; mobile phase B: NaCl 1.75g / L, NaH2PO40.06g / L, Na2HPO47.02g / L, using Pheny1 SepharoseHP column, balance the column with 5 column volumes of mobile phase B, ensure that the system is stable, and inject 2mL of sample. Pre-rinse with 4 column volumes of mobile phase A to clean the column. Perform a gradient elution from 0 to 100%, the elution volume should be greater than 30 column volumes, and collect 1mL of eluted sample. The gradient elution program was set as follows: the elution time was 110 min, the initial ratio of mobile phase A to mobile phase B was 0:100%, the ratio of mobile phase A increased by 5% every 5 min and eventually reached 100%, and the ratio of mobile phase B decreased by 5% every 5 min and eventually dropped to 0%.

[0038] The HPLC results of the eluate are as follows Figure 5 As shown, the purity is 95%.

[0039] The eluate was concentrated, desalted, and freeze-dried to obtain semaglutide precursor peptide powder. The mass spectrometry results of semaglutide precursor peptide were as follows: Figure 4 The results showed that this method successfully prepared semaglutide precursor peptide that was cleaved intracellularly.

[0040] Example 3 The difference from Example 1 is that Bacillus subtilis ( Bacillus subtilis 168) Escherichia coli BL21(DE3) was used as the host cell.

[0041] 1. Similar to Example 1, the whole gene sequence (SEQ ID No. 1) was synthesized by Qingke Bio, and then integrated into the same pP43NMK plasmid by Qingke Bio and returned to the plasmid. This example uses Bacillus subtilis as the host cell. A tube of Bacillus subtilis competent state (purchased from Qingke Bio, 100 μL / tube) was taken out from the -80°C refrigerator and placed in crushed ice. After the competent state was naturally melted at room temperature, 100~200ng of the plasmid returned after the gene synthesis of Qingke Bio was added. After being gently blown evenly with a gun tip, it was ice bathed for 15min, and then heat-shocked at 42°C for 90s, and then immediately placed in crushed ice for 5min, 900μL of LB liquid culture medium was added thereto, and placed in a shaker at 37°C, and the shaker speed was set to 220rpm. After one hour, 50μL was aspirated with a pipette and coated on an LB solid culture medium containing 50μg / mL kanamycin.

[0042] Place the culture medium upside down in a 37℃ constant temperature culture phase. After culturing for 12~14h, pick up a single colony with a toothpick using tweezers, and then directly throw the toothpick into LB liquid culture medium containing 50μg / mL kanamycin for expansion. After expansion, store it in a -80℃ refrigerator with 20% final concentration of glycerol for later use. Send it to Qingke Bio for sequencing verification, and it is the expression engineering strain.

[0043] Use a pipette to take 5 μL of glycerol bacteria from step 1 and inoculate it into 5 mL of LB liquid culture medium containing 50 μg / mL kanamycin. Incubate at 37°C with a shaker speed of 220 rpm for 14 hours. Inoculate it into 200 mL of optimized LB liquid culture medium containing 50 μL of kanamycin at a volume ratio of 1:100 between the bacterial liquid and the culture medium, which includes Na2SO4 2.0 g / L, (NH4)2SO4 20.0 g / L, NH4Cl 3.5 g / L, K2HPO4 14.0 g / L, NaH2PO4 4.0 g / L, MgSO4 7.0 g / L, glucose 20.0 g / L, and trace metal elements 3 mL / L (including CaCl2 0.5 g / L, ZnSO4 0.2 g / L, MnSO4 0.1 g / L, FeCl3 8.5 g / L, CuSO4 0.2 g / L, CoCl2 0.2 g / L).

[0044] The expression was carried out at 37°C with a shaker at 220 rpm and 30°C for 18 hours.

[0045] 3. Collect the bacteria by centrifugation, resuspend with 50mM Tris-HCl with one volume of culture medium, ultrasonically disrupt the cells, centrifuge the supernatant and filter with a 0.45μm filter membrane. Use the commonly used hydrophobic chromatography method to purify the precursor polypeptide: the mobile phase of the hydrophobic chromatography is: mobile phase A: ultrapure water; mobile phase B: NaCl 1.75g / L, NaH2PO40.06g / L, Na2HPO47.02g / L, use Pheny1 SepharoseHP column, use 5 column volumes of mobile phase B to balance the column, ensure that the system is stable, and inject 2mL of sample. Pre-rinse with 4 column volumes of mobile phase A to clean the column. Perform a gradient elution from 0 to 100%, the elution volume should be greater than 30 column volumes, and collect 1mL of eluted sample. The gradient elution program was set as follows: the elution time was 110 min, the initial ratio of mobile phase A to mobile phase B was 0:100%, the ratio of mobile phase A increased by 5% every 5 min and eventually reached 100%, and the ratio of mobile phase B decreased by 5% every 5 min and eventually dropped to 0%.

[0046] The HPLC results of the eluate are as follows Figure 6 As shown, the purity is 95%.

[0047] The eluate was concentrated, desalted, and freeze-dried to obtain semaglutide precursor peptide powder. The mass spectrometry results of semaglutide precursor peptide were as follows: Figure 4 The results showed that this method successfully prepared semaglutide precursor peptide that was cleaved intracellularly.

[0048] Example 4 The difference from Example 1 is that Corynebacterium glutamicum ( Corynebacterium glutamicum ATCC 13032) was used as the host cell instead of Escherichia coli BL21(DE3).

[0049] Similar to Example 1, the entire gene sequence (SEQ ID No. 1) was synthesized by Qingke Biotechnology, integrated into the same pXMJ19 plasmid by Qingke Biotechnology, and then returned to the plasmid. In this example, Corynebacterium glutamicum was used as the host cell. A tube of Corynebacterium glutamicum competent cells (purchased from Qingke Biotechnology, 100 μL / tube) was taken out of the -80℃ refrigerator and placed in crushed ice. After the competent cells were naturally melted at room temperature, 1 μg of the plasmid returned by Qingke Biotechnology after gene synthesis was added. The cells were gently blown evenly with a pipette tip and added to a cold electric shock cup. The cell was shocked at 2500V for 5ms, and 900 μL of preheated TSB medium (TSB medium formula: glucose 5 g / L, yeast powder 5 g / L, soy peptone 9 g / L, KHPO4·3H2O 1 g / L, urea 3 g / L, succinic acid 0.5 g / L, MgSO4·7H2O 0.1 g / L, MOPS 20 g / L, biotin 10 μg / L, VB1 0.1 mg / L, pH adjusted to 7.2) was added. The cells were heat shocked at 46℃ for 6 min and cultured at 30℃ at 200rpm for 2 h. Take an appropriate amount of bacterial liquid and spread it on TSB solid culture medium containing 50 μg / mL chloramphenicol, and culture it upside down in a constant temperature incubator at 30°C for 24 hours.

[0050] 2. After a single colony grows, pick up a single clone with a toothpick using tweezers, and then directly throw the toothpick into TSB liquid culture medium containing 50μg / mL chloramphenicol for expansion. After expansion, store it in a -80℃ refrigerator with 20% final concentration of glycerol for later use. After being sent to Qingke Bio for sequencing verification, it is the expression engineering strain.

[0051] Use a pipette to take 5 μL of glycerol bacteria from step 1 and inoculate it into 5 mL of TSB liquid culture medium containing 50 μg / mL chloramphenicol. Incubate at 37°C with a shaker at 220 rpm for 14 h. Inoculate it into 200 mL of optimized NCM liquid culture medium containing 50 μL chloramphenicol at a volume ratio of 1:100, which includes 80 g / L glucose, 40 g / L glucose, 30 g / L corn steep liquor, 45 g / L (NH4)2SO4, 1.5 g / L MgSO4∙7H2O, 1.0 g / L KH2SO4, 0.02 g / L FeSO4, 0.02 g / L MnSO4, 8 mg / L nicotinamide, 850 μg / L biotin, and 450 μg / L thiamine).

[0052] The expression was carried out at 37°C with a shaker at 220 rpm and 30°C for 18 hours.

[0053] 3. Collect the cells by centrifugation, resuspend with 50mM Tris-HCl of one volume of culture medium, ultrasonically disrupt the cells, centrifuge and take the supernatant and filter with a 0.45μm filter membrane. Use the commonly used hydrophobic chromatography method to purify the precursor polypeptide: the mobile phase of the hydrophobic chromatography is: mobile phase A: ultrapure water; mobile phase B: NaCl 1.75g / L, NaH2PO40.06g / L, Na2HPO47.02g / L, use Pheny1 SepharoseHP chromatographic column, balance the column with 5 column volumes of mobile phase B to ensure stability, inject 2mL, pre-wash the column with 4 column volumes of mobile phase A, perform 0~100% gradient elution, and the elution volume is greater than 30 column volumes. Collect and sample 1mL. The gradient elution program was set to an elution time of 110 min, the initial value of the mixture of mobile phase A and mobile phase B was 0:100%, the percentage of mobile phase A increased by 5% every 5 minutes, and the final value was 100%; the percentage of mobile phase B decreased by 5% every 5 minutes, and the final value was 0.

[0054] The HPLC results of the eluate are as follows Figure 7 As shown, the purity is 95%.

[0055] The eluate was concentrated, desalted, and freeze-dried to obtain semaglutide precursor peptide powder. The mass spectrometry results of semaglutide precursor peptide were as follows: Figure 4 The results showed that this method successfully prepared semaglutide precursor peptide that was cleaved intracellularly.

[0056] The comparative examples are described below. In comparative example 1, the strategy of separating the proteins and expressing them independently was adopted, so an additional in vitro enzyme digestion step was required.

[0057] Comparative Example 1 1. Gene synthesis was performed on the three parts of the whole system respectively. The first part: a nucleotide sequence encoding a fusion protein containing a semaglutide precursor peptide (SEQ ID No. 1); the second part: a nucleotide sequence encoding a KEX2 protease; and the third part: a nucleotide sequence encoding a carboxypeptidase B.

[0058] The three parts were integrated into the pET28 plasmid vector, and the gene sequence was synthesized by Qingke Biotechnology. After being integrated into the pET28 plasmid by Qingke Biotechnology, the plasmid was returned. Escherichia coli BL21 (DE3) was still used as the host cell. Three tubes of Escherichia coli BL21 (DE3) competent cells (purchased from Qingke Biotechnology, 100μL / tube) were taken out of the -80℃ refrigerator and placed in crushed ice. After the competent cells were naturally melted at room temperature, 100~200ng of the plasmid returned after the gene synthesis of Qingke Biotechnology was added to each tube. After being gently blown evenly with a pipette tip, ice bath was placed for 15min, heat shock was performed at 42℃ for 90s, and it was immediately placed in crushed ice for 5min. 900μL of LB liquid culture medium was added to it, and it was placed in a shaker at 37℃, and the shaker speed was set to 220rpm. After one hour, 50μL was taken out of each tube with a pipette and coated on three LB solid culture medium plates containing 50μg / mL kanamycin.

[0059] Place the culture medium upside down in a 37℃ constant temperature culture phase. After culturing for 12~14h, pick up a single colony with a toothpick using tweezers, and then directly throw the toothpick into LB liquid culture medium containing 50μg / mL kanamycin for expansion. After expansion, store it in a -80℃ refrigerator with 20% final concentration of glycerol for later use. Send it to Qingke Bio for sequencing verification, and it is the expression engineering strain.

[0060] 2. Use a pipette to take 5 μL of glycerol bacteria from step 1 and inoculate it into 5 mL of LB liquid culture medium containing 50 μg / mL kanamycin. Incubate at 37°C with a shaker speed of 220 rpm for 14 hours. Inoculate each of the three bottles into a separate bottle of 200 mL of FCM liquid culture medium containing 50 μL kanamycin at a volume ratio of 1:100 between the bacterial liquid and the culture medium. Incubate at 37°C with a shaker speed of 220 rpm until OD600 is about 0.8. Add IPTG to a final concentration of 0.5 mM and induce expression at 30°C for 18 hours.

[0061] 3. Collect the cells by centrifugation, resuspend with 1 volume of 50mM Tris-HCl, ultrasonically disrupt the cells, centrifuge the supernatant and purify the protein with a nickel column. The following proteins were obtained: fusion protein of GST and semaglutide precursor peptide, KEX protease and carboxypeptidase B. Then the three proteins were mixed at a ratio of 1:1:1. Filter with a 0.45μm filter membrane. The precursor peptide was purified by a commonly used hydrophobic chromatography method: the mobile phase was: mobile phase A: ultrapure water; mobile phase B: NaCl 1.75g / L, NaH2PO40.06g / L, Na2HPO47.02g / L, Pheny1 SepharoseHP column was used, and the column was balanced with 5 column volumes of mobile phase B to ensure stability, 2mL was injected, and the column was pre-washed with 4 column volumes of mobile phase A, and a gradient elution of 0-100% was performed, and the elution volume was greater than 30 column volumes. Collect and sample 1mL. The gradient elution program was set to an elution time of 110 min, the initial value of the mixture of mobile phase A and mobile phase B was 0:100%, the percentage of mobile phase A increased by 5% every 5 minutes, and the final value was 100%; the percentage of mobile phase B decreased by 5% every 5 minutes, and the final value was 0.

[0062] The comparative examples are described below. In comparative example 2, the strategy of separating the proteins for independent expression was adopted, and the GST tag was no longer used, so the semaglutide precursor peptide was directly attached behind the His tag.

[0063] Comparative Example 2 1. Gene synthesis was performed on the three parts of the whole system respectively. The first part: a nucleotide sequence encoding a fusion protein containing semaglutide precursor peptide (i.e., the sequence SEQ ID No. 2 does not include a GST solubilizing tag); the second part: a nucleotide sequence encoding a KEX2 protease; and the third part: a nucleotide sequence encoding a carboxypeptidase B.

[0064] Each was integrated into the pET28 plasmid vector. The gene sequence was synthesized by Qingke Biotechnology, and the plasmid was returned after being integrated into the pET28 plasmid by Qingke Biotechnology. Escherichia coli BL21 (DE3) was still used as the host cell. Three tubes of Escherichia coli BL21 (DE3) competent cells (purchased from Qingke Biotechnology, 100μL / tube) were taken out of the -80℃ refrigerator and placed in crushed ice. After the competent cells were naturally melted at room temperature, 100~200ng of the plasmid returned after the gene synthesis of Qingke Biotechnology was added to each tube. The cells were gently blown evenly with a pipette tip and then placed in an ice bath for 15min, then heat-shocked at 42℃ for 90s, and immediately placed in crushed ice for 5min. 900μL of LB liquid culture medium was added to it, and it was placed in a shaker at 37℃, and the shaker speed was set to 220rpm. After one hour, 50μL was taken out with a pipette and spread on three LB solid culture medium plates containing 50μg / mL kanamycin.

[0065] Place the culture medium upside down in a 37℃ constant temperature culture phase. After culturing for 12~14h, pick up a single colony with a toothpick using tweezers, and then directly throw the toothpick into LB liquid culture medium containing 50μg / mL kanamycin for expansion. After expansion, store it in a -80℃ refrigerator with 20% final concentration of glycerol for later use. After being sent to Qingke Bio for sequencing verification, it is the expression engineering strain.

[0066] 2. Use a pipette to take 5 μL of glycerol bacteria from step 1 and inoculate it into 5 mL of LB liquid culture medium containing 50 μg / mL kanamycin. Incubate at 37°C with a shaker speed of 220 rpm for 14 hours. Inoculate each of the three bottles into a separate bottle of 200 mL of FCM liquid culture medium containing 50 μL kanamycin at a volume ratio of 1:100 between the bacterial liquid and the culture medium. Incubate at 37°C with a shaker speed of 220 rpm until OD600 is about 0.8. Add IPTG to a final concentration of 0.5 mM and induce expression at 30°C for 18 hours.

[0067] 3. Collect the cells by centrifugation, resuspend them with 1 volume of 50mM Tris-HCl, and disrupt the cells by ultrasound. For the semaglutide precursor peptide, after collecting the inclusion bodies by centrifugation, soak the inclusion bodies in a protein refolding and renaturing solution. The formula of the protein refolding and renaturing solution is: 20mM Tris-HCl, 8M urea, pH8.0. The addition ratio of the inclusion bodies and the protein refolding and renaturing solution is 1g:40mL; slowly stir the mixture for half an hour until the solution is clear, centrifuge at 8000rpm for 15min, and take the supernatant.

[0068] For KEX enzyme protein and carboxypeptidase B, the cells were ultrasonically disrupted and centrifuged to obtain the supernatant and the protein was purified using a nickel column. The following proteins were obtained: semaglutide precursor peptide, KEX protease and carboxypeptidase B. The three proteins were then mixed at a ratio of 1:1:1. Filtered with a 0.45μm filter membrane. The precursor peptide was purified using a commonly used hydrophobic chromatography method: the mobile phase was: mobile phase A: ultrapure water; mobile phase B: NaCl 1.75g / L, NaH2PO40.06g / L, Na2HPO47.02g / L. A Pheny1 SepharoseHP column was used. The column was first equilibrated with 5 column volumes of mobile phase B to ensure stability. 2mL was injected, and the column was pre-washed with 4 column volumes of mobile phase A, and then a 0-100% gradient elution was performed, with an elution volume greater than 30 column volumes. 1mL of sample was collected. The gradient elution program was set to an elution time of 110 min, the initial value of the mixture of mobile phase A and mobile phase B was 0:100%, the percentage of mobile phase A increased by 5% every 5 minutes, and the final value was 100%; the percentage of mobile phase B decreased by 5% every 5 minutes, and the final value was 0.

[0069] The following is a description of the comparative examples. In comparative example 3, the methods in other related patents are adopted, and the common method in patent CN118620091A is referred to.

[0070] Comparative Example 3 1. Human SODC protein (NCBI: AAD42179.1) was linked to semaglutide precursor, and then integrated into pET28 plasmid by Qingke Biotechnology and returned the plasmid. A tube of E. coli BL21 (DE3) competent cells (purchased from Qingke Biotechnology, 100 μL / tube) was taken out of the -80℃ refrigerator and placed in crushed ice. After the competent cells were naturally thawed at room temperature, 100-200 ng of the plasmid returned after gene synthesis by Qingke Biotechnology was added to each tube. After being gently blown evenly with a pipette tip, the cells were placed in an ice bath for 15 minutes, heat-shocked at 42℃ for 90 seconds, and immediately placed in crushed ice for 5 minutes. 900 μL of LB liquid culture medium was added to the cells, and the cells were placed in a shaker at 37℃, with the shaker speed set at 220 rpm. After one hour, 50 μL of the cells were each pipetted and coated on a LB solid culture medium plate containing 50 μg / mL kanamycin.

[0071] Place the culture medium upside down in a 37℃ constant temperature culture phase. After 12~14h of culture, pick up a single colony with a toothpick using tweezers and directly throw the toothpick into LB liquid culture medium containing 50μg / mL kanamycin for expansion. After expansion, store it in a -80℃ refrigerator with 20% final concentration of glycerol for later use. After being sent to Qingke Bio for sequencing verification, it is the expression engineering strain.

[0072] 2. Use a pipette to take 5 μL of glycerol bacteria from step 1 and inoculate it into 5 mL of LB liquid culture medium containing 50 μg / mL kanamycin. Incubate at 37°C with a shaker speed of 220 rpm for 14 hours. Inoculate each of the three bottles into a separate bottle of 200 mL of FCM liquid culture medium containing 50 μL of kanamycin at a volume ratio of 1:100 between the bacterial liquid and the culture medium. Incubate at 37°C with a shaker speed of 220 rpm until OD600 is about 0.8. Add IPTG to a final concentration of 0.5 mM, and then induce expression at 30°C for 18 hours.

[0073] 3. Collect the cells by centrifugation, resuspend them with 1 volume of 50mM Tris-HCl, and disrupt the cells by ultrasound. For the semaglutide precursor peptide, after collecting the inclusion bodies by centrifugation, soak the inclusion bodies in a protein refolding and renaturing solution. The formula of the protein refolding and renaturing solution is: 20mM Tris-HCl, 8M urea, pH8.0. The addition ratio of the inclusion bodies and the protein refolding and renaturing solution is 1g:40mL; slowly stir the mixture for half an hour until the solution is clear, centrifuge at 8000rpm for 15min, and take the supernatant.

[0074] Recombinant enterokinase was added at a mass ratio of 1: 2000, and the digestion was carried out overnight (12-18 hours) at 20°C. After digestion, the supernatant was collected by centrifugation. Filtered with a 0.45μm filter membrane. The precursor polypeptide was purified by a commonly used hydrophobic chromatography method. The mobile phases were: mobile phase A: ultrapure water; mobile phase B: NaCl 1.75g / L, NaH2PO4 0.06g / L, Na2HPO4 7.02g / L.

[0075] Comparative Example 4 This comparative example uses other cleavage auxiliary peptides, which is different from Example 1 in that the KEX2 enzyme is replaced by TEV protease (NCBI: MK238517.1).

[0076] Finally, no semaglutide precursor peptide was produced.

[0077] The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

[0078] The sequences involved in the present invention SEQ ID No.1: ATGCGCCGCAGCCCGATTCTGGGCTATTGGAAAATTAAAGGCCTGGTGCAGCCGACCCGCCTGCTGCTGGAATATCTGGAAGAAAAATATGAAGAACATCTGTATGAACGCGATGAAGGCGATAAATGGCGCAACAAAAAATTTGAACTGGGCCTGGAATTTCCGAACCTGCCGTATTATATTGATGGCGATGTGAAACTGACCCAGAGCATGGCGATTATTCGCTATATTGCGGATAAACATAACATGCTGGGCGGCTGCCCGAAAGAACGCGCGGAAATTAGCATGCTGGAAGGCGCGGTGCTGGATATTCGCTATGGCGTGAGCCGCATTGCGTATAGCAAAGATTTTGAAACCCTGAAAGTGGATTTTCTGAGCAAACTGCCGGAAATGCTGAAAATGTTTGAAGATCGCCTGTGCCATAAAACCTATCTGAACGGCGATCATGTGACCCATCCGGATTTTATGCTGTATGATGCGCTGGATGTGGTGCTGTATATGGATCCGATGTGCCTGGATGCGTTTCCGAAACTGGTGTGCTTTAAAAAACGCATTGAAGCGATTCCGCAGATTGATAAATATCTGAAAAGCAGCAAATATATTGCGTGGCCGCTGCAGGGCTGGCAGGCGACCTTTGGCGGCGGCGATCATCCGCCGAAACGCCGCACCTTTACCAGCGATGTGAGCAGCTATCTGGAAGGCCAGGCGGCGCTGGAATTTATTGCGTGGCTGGTGCGCGGCCGCGGCCGCCGCACCTTTACCAGCGATGTGAGCAGCTATCTGGAAGGCCAGGCGGCGCTGGAATTTATTGCGTGGCTGGTGCGCGGCCGCGGCCGCCGCTAA。

[0079] SEQ ID No.2: MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKRRTFTSDVSSYLEGQAALEFIAWLVRGRGRRTFTSDVSSYLEGQAALEFIAWLVRGRGRR。

[0080] Amino acid sequence of the solubility-enhancing tag GST: MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK (SEQ ID No.3).

[0081] Amino acid sequence of the semaglutide precursor peptide TFTSDVSSYLEGQAALEFIAWLVRGRGRRTFTSDVSSYLEGQAALEFIAWLVRGRG SEQ ID No.4.

Claims

1. A method for preparing semaglutide precursor peptide by intracellular enzymatic cleavage, characterized in that: The following steps are involved: (1) The solubility-promoting tag GST and the semaglutide precursor peptide are linked together through the protease-specific recognition amino acid site to form a fusion protein; (2) Integrating the nucleotide sequence encoding the fusion protein, the nucleotide sequence encoding the KEX2 protease, and the nucleotide sequence encoding the carboxypeptidase B into the same expression vector to form a recombinant vector; the nucleotide sequence encoding the fusion protein is shown in SEQ ID No. 1; (3) Transform the engineered bacteria with the recombinant vector and carry out fermentation production; (4) The fermentation product is centrifuged to collect the bacterial cells, the cells are ultrasonically disrupted, the supernatant is directly centrifuged and filtered, and the eluted solution is concentrated, desalted, and freeze-dried to obtain semaglutide precursor peptide powder.

2. The method according to claim 1, characterized in that The expression vector is selected from one of the pET series, pETDuet series, pP43NMK plasmid, and pXMJ19 plasmid expression vectors.

3. The method according to claim 2, characterized in that The pET series includes pET28, and the pETDuet series includes pETDuet-1.

4. The method according to claim 1, characterized in that: The engineered bacteria is selected from one of Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum.

5. A fusion protein for preparing a semaglutide precursor peptide, characterized in that: The composition is: lytic tag GST+protease specific recognition amino acid site+semaglutide precursor peptide+protease specific recognition amino acid site+semaglutide precursor peptide+protease specific recognition amino acid site; the amino acid sequence of the fusion protein is shown in SEQ ID No.

2.

6. A recombinant vector for preparing semaglutide precursor peptide, characterized in that: include: Part I: a nucleotide sequence encoding a fusion protein containing a semaglutide precursor peptide, wherein the nucleotide sequence encoding the fusion protein is shown in SEQ ID No. 1; Part II: a nucleotide sequence encoding the KEX2 protease; Part III: a nucleotide sequence encoding carboxypeptidase B; The above three parts are integrated into the same expression vector to form a recombinant vector.

7. The recombinant vector according to claim 6, characterized in that The fusion protein is formed by linking the solubility-promoting tag GST and the semaglutide precursor peptide through a protease-specific recognition amino acid site.

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