Preparation method of polypeptide for preparing GLP-1 analogue through tandem expression

Abstract

The invention belongs to the technical field of biomedical engineering, and particularly relates to a polypeptide preparation method for preparing a GLP-1 analogue through tandem expression. According to the method, a coding gene sequence of the GLP-1 analogue is repeatedly connected in series for a plurality of times, and then expression preparation is carried out on the basis of a genetic engineering technology. The inventor optimizes a series-connected polypeptide structure by adding a section of key peptide sequence. The key peptide serving as a leading peptide can greatly improve the expression quantity of the strain in the fermentation expression process; as a linked peptide, the key peptide sequence can effectively improve the recognition capability of enzyme digestion sites on one hand and can adjust the overall isoelectric point of the sequence on the other hand, so that the solubility, enzyme digestion yield and purity of a target product can be improved. Preliminary experiment results show that the expression quantity of the polypeptide prepared through expression is extremely high, the enzyme digestion efficiency is good, the good enzyme digestion efficiency can be achieved under the condition that the enzyme dosage is low, and the yield and purity of the final GLP-1 analogue are high.

Description

Technical Field

[0001] This application belongs to the field of biomedical engineering technology, specifically relating to a method for preparing GLP-1 analogs through tandem expression of peptides. Background Technology

[0002] In the human body, GLP-1 (glucagon-like peptide-1), an endogenous incretin hormone secreted by L cells in the small intestine, plays a crucial role in blood glucose control. In its natural source, proglucagon, a precursor to various gastrointestinal hormones, undergoes post-translational cleavage to yield active peptides such as glucagon and GLP-1. Endogenous GLP-1 exists in two main active forms: GLP-1(7-36)amide and GLP-1(7-37). Activated GLP-1 activates calmodulin by binding to its receptor, ultimately promoting a large release of insulin from pancreatic β cells in a glucose-dependent manner. The GLP-1 receptor (GLP1-receptor, GLP1R) is a member of the G protein-coupled receptor (GPCR) class B family.

[0003] However, existing studies suggest that GLP-1 is rapidly degraded and inactivated by dipeptidyl peptidase-4 (DPP-4) after being secreted into the bloodstream, with a very short half-life (generally considered to be only 2 minutes). Therefore, GLP-1 receptor agonists are used clinically for blood glucose regulation. In application, GLP-1 receptor agonists have similar effects to GLP-1, but due to their modified structure, their duration of action (half-life) can be significantly prolonged.

[0004] In existing technologies, GLP-1 and its analogues (GLP-1 receptor agonists) are mainly prepared using three technical pathways: natural extraction, artificial chemical synthesis, and genetic engineering. Natural extraction is only applicable to the preparation of GLP-1 (GLP-1 analogues are all artificially designed), and due to medical ethics issues, the source of raw materials is very limited. Coupled with the limited efficacy of GLP-1, this method is only suitable for experimental research applications. Artificial chemical synthesis mainly refers to the use of solid-phase synthesis to prepare peptide drugs such as GLP-1 and its analogues. However, because solid-phase synthesis is relatively complex and costly, it is mostly limited to experimental applications and cannot be well suited for industrial production.

[0005] Due to its advantages in safety, convenience, and yield, gene engineering technology is currently the main method for preparing related peptide drugs and even related chemical molecules. Based on the final expression strain, it can be divided into prokaryotic expression systems (represented by *E. coli*) and eukaryotic expression systems (represented by yeast). In practical applications, the choice of expression system generally depends on factors such as the molecular weight of the final expressed product, whether it can be secreted extracellularly, and whether its activity is affected after translation and expression. However, yeast generally has a relatively long culture cycle and high culture cost, resulting in relatively low yields. *E. coli*, on the other hand, is a more mainstream expression system due to its high yield, low cost, and relatively simple operation.

[0006] However, generally speaking, E. coli prokaryotic expression systems are not suitable for expressing small-molecule peptides or proteins (small peptides and proteins are easily degraded within the cells of E. coli). Therefore, when using E. coli for protein expression, it is often necessary to modify the target protein appropriately (e.g., by adding restriction enzyme sites) based on the genetic background of E. coli and the characteristics of the target protein to improve its expression efficiency. However, due to the different types of target proteins, the actual modification methods vary considerably, often requiring optimization and selection based on the actual technical difficulty of modification and the final preparation effect. Summary of the Invention

[0007] The purpose of this application is to provide a method for preparing GLP-1 analog peptides through tandem expression, thereby laying a certain technical foundation for the preparation of related drugs.

[0008] The technical solution adopted in this application is briefly described as follows.

[0009] A method for preparing GLP-1 analog peptides by tandem expression, wherein the coding gene sequence of GLP-1 analog is tandemly repeated several times, and then expressed and prepared based on genetic engineering technology; The GLP-1 analogue comprises two forms of amino acid sequences: 9-37 (Semaglutide, SEQ ID No. 1, 29aa): EGTFTSDVSSYLEGQAAKEFIAWLVRGRG; 11-37 (Smigratide, SEQ ID No. 2, 27aa): TFTSDVSSYLEGQAAKEFIAWLVRGRG; In the tandem connection of the GLP-1 analogues, the key peptide sequences can be linked in the following form: The key peptide (SEQ ID No. 3, 13aa): DVKPGQPLEDELG; The GLP-1 analog tandem process involves using key peptides to link multiple GLP-1 analogs into a single expression form; the specific tandem configuration is as follows: The three-tandem configuration is: M—key peptide—KR—GLP-1 analog—KR—key peptide—KR—GLP-1 analog—KR—key peptide—KR—GLP-1 analog; The specific amino acid sequence after tandem assembly is as follows: Tandem polypeptide 1: MDVKPGQPLEDELGKREGTFTSDVSSYLEGQAAKEFIAWLVRGRGKRDVKPGQPLEDELGKREGTFTSDVSSYLEGQAAKEFIAWLVRGRGKRDVKPGQPLEDELGKREGTFTSDVSSYLEGQAAKEFIAWLVRGRG; Tandem polypeptide 2: MDVKPGQPLEDELGKRTFTSDVSSYLEGQAAKEFIAWLVRGRGKRDVKPGQPLEDELGKRTFTSDVSSYLEGQAAKEFIAWLVRGRGKRDVKPGQPLEDELGKRTFTSDVSSYLEGQAAKEFIAWLVRGRG; The preparation process specifically includes the following steps: (I) Construction of recombinant plasmid vectors The tandem gene sequence was prepared, and the tandem gene sequence was recombined using pET plasmid as a vector. (ii) Transformation and Expression Transform the recombinant plasmid vector from step (I) into Escherichia coli BL21(DE3) strain and screen for correctly transformed strains; The correctly transformed strain was fermented at 37°C until OD200. 600 After reaching a concentration of ≥70-80, add IPTG to a final concentration of 0.05mM-0.5mM, and continue culturing at 37℃ for 8-10 hours to induce protein expression; (iii) Disruption of bacterial cells After induction and expression in step (II), centrifuge the bacterial culture at 8000 rpm for 10 minutes to collect the bacterial cells. Add lysis buffer to the bacterial cells at a ratio of 1g of bacterial cells to 5ml of lysis buffer, mix well, and then break the bacterial cells (the cells can be broken by high pressure homogenization, freeze-thaw or ultrasonic methods). The lysis buffer was a 0.74 g / L EDTA-2Na·2H2O aqueous solution with pH = 6.8 ± 0.1; (iv) Purification of the target protein After the bacterial cells were broken in step (iii), the pH of the mixture was adjusted to 10.0~11.0 (0.1M NaOH solution can be used for adjustment) to promote the dissolution of inclusion bodies (thereby facilitating subsequent ion exchange chromatography). After adjusting the pH, the sample is loaded for ion exchange chromatography (purification). During the ion exchange chromatography, an anion exchange chromatography column is used as the packing material. (The operation of ion exchange chromatography can be referenced as follows: after loading the sample, first equilibrate with buffer A for 2CV, and then elute with buffer B for 10CV.) The buffer solution A is: 20 mM Tris, pH = 7.25 ± 0.20; The buffer solution B is: 20 mM Tris, 0.5 M NaCl; (v) Enzyme digestion The elution buffer from step (iv) was subjected to double digestion with Kex2 enzyme and CPB enzyme; In the enzyme digestion system, the amount of Kex2 enzyme (based on enzyme activity of 14 U / mg) should be 1 / 1000-1 / 5000 (preferably 1 / 3000) of the total protein in the elution buffer. The recommended dosage of CPB enzyme (based on enzyme activity of 170 U / mg) is 1 / 1000 to 1 / 5000 of the total protein content in the eluent (preferably 1 / 5000). During enzyme digestion, the temperature is 10~40℃ (preferably 25℃) for 1~48h, and then the subsequent modification steps are carried out. (vi) Modification Take the reaction system after enzymatic digestion in step (5), add anhydrous sodium carbonate at a ratio of 0.1-0.3 times the amount of target protein (i.e., by mass ratio, target protein: anhydrous sodium carbonate = 1:0.1~0.3, preferably 1:0.1), and adjust the pH to 10.2±0.1 (pH adjustment can be done using 4mol / L sodium hydroxide solution). After the reaction system (suspension) becomes clear, add octadecanoic acid-glutamic acid-EAEA-active ester at a ratio of 0.7-1.0 times the amount of the target protein (i.e., by mass ratio, target protein: octadecanoic acid-glutamic acid-EAEA-active ester = 1:0.7~1.0), and adjust the pH to 10.5±0.2, stirring until the reaction is complete; After the reaction is complete, adjust the pH to 3.5±0.1 (6 mol / L hydrochloric acid can be used for adjustment), and let it stand to settle. After settling, centrifuge at 12000 rpm for at least 30 min, collect the solid precipitate, and directly enter the coupling program. (vii) Coupon Take the solid collected in step (vi) and dissolve it thoroughly in water at a mass ratio of about 8 times. After dissolution, add the following in sequence: His-Aib-BOC (0.2 to 0.5 times the amount of solid, i.e., by mass ratio, solid collected in step (vi): His-Aib-BOC = 1:0.2 to 0.5), and pre-cooled NMP (N-methylpyrrolidone, pre-cooled to about 2-8°C, the amount of NMP is 5 to 8 times the mass of the solid, i.e., by mass ratio, solid collected in step (vi): NMP = 1:5 to 8). Stir thoroughly and mix evenly. Subsequently, the following were added sequentially to the above solution system: HATU (added at 0.5 to 1 times the amount of solid, i.e., by mass ratio, the solid collected in step (VI) : HATU = 1: 0.5 to 1) and triethylamine (added at 0.5 to 1 times the amount of solid, i.e., by mass ratio, the solid collected in step (VI) : triethylamine = 1: 0.5 to 1). After adding the ingredients, stir thoroughly and allow the reaction to proceed at room temperature (at around 18-30°C, the coupling reaction will take at least 30 minutes to complete). After the reaction is complete, adjust the pH to 4.0 ± 0.1 (a 25% phosphoric acid solution can be used as a reference) and allow it to settle completely (this will take at least 1 hour). After sedimentation, centrifuge at 12,000 rpm for at least 30 minutes and collect the solid precipitate; Finally, the collected solid precipitate is added to trifluoroacetic acid at a mass ratio of 10 to 20 (i.e., solid precipitate: trifluoroacetic acid = 1:10 to 20 by mass), and stirred thoroughly (generally for at least 10 minutes) to react and remove the bound dipeptide protecting groups. After the reaction is complete, the resulting crude solution of GLP-1 analog is obtained. (viii) Reversed-phase chromatography purification The crude GLP-1 analog solution obtained in step (vii) was subjected to reversed-phase chromatography to obtain purified GLP-1 analog. Specifically, a C8 column was used for reversed-phase chromatography, and the procedure is as follows: First, equilibrate with solution A for 1.5~2 CV; then load the sample; after loading the sample, equilibrate with solution A again for 2.5~3 CV; finally, elute with solution B, using a gradient elution: first elute with 0~40% solution B for 1 CV, then elute with 40~60% solution B for 5 CV. Solution A consists of 0.1M ammonium bicarbonate, 10% acetonitrile, and pH = 6.50 ± 0.20. The B solution is: 100% acetonitrile; During the elution process, the eluent at a wavelength of 280 nm is collected in segments, and the result is the purified GLP-1 analog (i.e., a solution containing the purified GLP-1 analog). The purified product is then lyophilized to obtain the final product, smegglutinin.

[0010] For small protein molecules, tandem expression using prokaryotic expression systems is a good technical approach to avoid degradation by *E. coli*. However, current technologies present three major technical challenges regarding protein processing after tandem expression: 1. Too few repetitive sequences during expression are uneconomical, while too many may lead to ribosome arrest or reduced translation rate. Furthermore, long mRNAs are more susceptible to nuclease degradation, affecting expression levels. 2. Conventional methods for tandem protein expression often employ strong acid cleavage to break the peptide chain. However, this method lacks specificity. Therefore, during tandem peptide expression, enzyme cleavage sites are typically added, followed by enzymatic digestion after expression. However, the appropriate selection and design of these cleavage sites are crucial for practical application. The final results, such as peptide dissolution and purification, are also directly affected. If the restriction enzyme sites are not designed properly, they may be degraded by the host protease during expression. Even if known protease sites are avoided during design, accidental cleavage may still occur due to structural exposure, which will reduce the expression efficiency of tandem expression, or even make it less efficient than monomeric expression. 3. The restriction enzyme sites of tandem expression may be masked by other sites, affecting the restriction enzyme efficiency. In view of the characteristics of the target peptide in this application, since the target peptide is highly hydrophobic, the peptides expressed in tandem often cannot dissolve due to the increased hydrophobic surface of amino acids, which further affects the efficiency of subsequent purification and restriction enzyme digestion.

[0011] Based on the characteristics of the target peptide in this application, the inventors considered adding a lead peptide and a linker peptide to address this challenge during tandem expression. However, the selection and design of the lead peptide and linker peptide also required consideration of the following: 1. The peptide length should not be too long, otherwise it may affect the transport efficiency; 2. The lead peptide needs to have good compatibility with the E. coli host bacteria to induce E. coli recognition and increase expression levels; 3. Insufficient hydrophobic regions reduce protein expression efficiency; 4. While ensuring the presence of hydrophobic regions, increasing the surface area of ​​hydrophilic peptides is crucial for good protein solubility. Based on these technical considerations, after multiple screenings and optimizations of the lead peptide and linker peptide, the inventors discovered a key peptide sequence that can serve as both a lead peptide and a linker peptide. As a lead peptide, it can significantly improve the expression efficiency of the target protein, while as a linker peptide, it can increase the solubility of the current long chain without hindering enzymatic cleavage. Preliminary verification results show that it can ensure an enzymatic cleavage efficiency of 100%-110%.

[0012] In summary, this application optimizes the tandem peptide structure by adding a key peptide sequence, which serves as both a leader peptide and a linker peptide, based on the prokaryotic expression system and the characteristics of the target protein. During protein expression, the key peptide at the N-terminus acts as a leader peptide, significantly increasing the expression level of the strain during fermentation. In subsequent enzymatic digestion, the key peptide sequence, acting as a linker peptide, effectively enhances the recognition ability of the cleavage site and adjusts the overall isoelectric point of the sequence, thereby improving the solubility, digestion yield, and purity of the target product, ultimately facilitating subsequent digestion and purification. Based on this optimization approach, preliminary experimental results show that when preparing peptides using this method, the peptide expression level is extremely high, and the digestion efficiency is excellent. Good digestion efficiency can be achieved even with low enzyme dosage, and the final GLP-1 analog yield and purity are high. This result has significant practical value and technical significance for reducing the cost of glycemic control medications. Attached Figure Description

[0013] Figure 1 Electrophoresis results for the transformation of recombinant bacteria containing "tandem polypeptide 1" into protein expression after fermentation and induction; in the figure: band 1 is a marker; band 2 is the result of sampling after 2 hours of fermentation induction; band 3 is the result of sampling after 6 hours of fermentation induction; band 4 is the result of sampling after 8 hours of fermentation induction; band 5 is the result of sampling after 12 hours of fermentation termination; Figure 2 The electrophoresis results for tandem tripeptides lacking the key peptide are shown in the figure. In the figure: band 1 is the result of sampling and testing 2 hours after induction; band 2 is the result of sampling and testing 4 hours after induction; band 3 is the result of sampling and testing 6 hours after induction; band 4 is the result of sampling and testing 12 hours after induction; and band 5 is the result of detection of control bacteria transfected with an empty plasmid without the target gene. Figure 3 The results show the molecular weight of the "tandem polypeptide 1" expressed protein. Figure 4 The results are for molecular weight determination of the protein after enzyme digestion of "tandem polypeptide 1"; Figure 5 The liquid chromatography-mass spectra and deconvolution molecular weight spectra of the purified product of "tandem polypeptide 1" are shown. Detailed Implementation

[0014] The present application will be further explained below with reference to the embodiments.

[0015] Example 1 This application uses a GLP-1 analog (smegglutinin, corresponding to the GLP-1 (9-37) peptide) as the target product, and the preparation method of tandem expression of this polypeptide is described in detail below.

[0016] (a) Polypeptides and their encoding genes Taking a triplet polypeptide as an example, the specific triplet polypeptide (tandem polypeptide 1) sequence design is as follows: MDVKPGQPLEDELGKREGTFTSDVSSYLEGQAAKEFIAWLVRGRGKRDVKPGQPLEDELGKREGTFTSDVSSYLEGQAAKEFIAWLVRGRGKRDVKPGQPLEDELGKREGTFTSDVSSYLEGQAAKEFIAWLVRGRG.

[0017] During the design process, the key peptide (DVKPGQPLEDELG) was used as both a linker peptide and a signal peptide to improve the expression efficiency during subsequent protein expression. When designing the leader peptide, methionine was added to the N-terminus of the key peptide to maintain translation efficiency, stabilize the protein, and ensure compatibility with the host processing mechanism.

[0018] Simultaneously, protein expression was performed using a triplet form lacking the key peptide as a linker (as a control). The specific amino acid sequence design for the triplet form is as follows: EGTFTSDVSSYLEGQAAKEFIAWLVRGRGKREGTFTSDVSSYLEGQAAKEFIAWLVRGRGKREGTFTSDVSSYLEGQAAKEFIAWLVRGRG.

[0019] (II) Construction of recombinant plasmid vectors Based on the designed amino acid sequence, Nanjing GenScript was commissioned to synthesize the codon gene sequence in step (I) using chemical synthesis. Subsequently, the gene sequence was recombined using the pET plasmid as a vector to prepare a recombinant plasmid expression vector. The target gene sequence of tandem polypeptide 1 is as follows: CATATGGATGTAAAACCAGGACAACCCCTAGAAGACGAACTGGGTAAGCGCGAAGGCACCTTTACTTCAGACGTCTCGAGCTACTTAGAGGGCCAAGCGGCGAAAGAATTCATTGCATGGCTGGTGCGTGGTCGTGGTAAGCGCGATGTGAAACCGGGTCAGCCGTTGGAAGATGAGCTGGGCAAGCGCGAGGGCACCTTTACCAGCGATGTT AGCTCTTACTTGGAGGGGTCAGGCTGCTAAAGAATTTATCGCCTGGTTGGTTTCGTGGTCGTGGCAAGAGATGTTAAACCAGGTCAACCGCTGGAGGACGAGCTGGGTAAGCGTGAAGGTACGTTCACCTCCGACGTGTCCAGCTATCTGGAAGGCCAGGCAGCGAAAGAGTTCATCGCGTGGCTGGTACGTGGCCGTGGCTGATAAAAGCTT; (III) Transformation and Expression The recombinant plasmid vector from step (II) was transformed into Escherichia coli BL21(DE3) strain, and the correctly transformed recombinant strain was obtained through resistance screening and identification. After the recombinant bacteria were cultured and amplified at 37°C and 200 rpm to prepare a seed culture, they were transferred to a fermenter for further culture and cultured at 37°C until the OD reached [value missing]. 600 After reaching approximately 70%, add IPTG to a final concentration of 0.5 mmol / L, and then incubate at 37°C for approximately 12 hours to induce protein expression.

[0020] Samples were taken during and after the induction process for SDS-PAGE analysis. Results are as follows: Figure 1 As shown. Simultaneously, SDS-PAGE analysis was performed on the control sample, and the results are as follows. Figure 2 As shown.

[0021] Brightness comparison shows that protein expression levels increased significantly after using key peptides for linking. Gel electrophoresis analysis of the proportion of the target protein in the total protein bands showed that the expression level of the target protein in the three-segment tandem strain with key peptides accounted for more than 50% of the total protein, which was much higher than that in the three-segment tandem strain without key peptides.

[0022] Furthermore, referring to the above fermentation conditions, the amount of target protein (using key peptide linkage) harvested after different batches of induced expression in a 1000L fermenter was recorded, and the results are shown in Table 1 below.

[0023] Table 1. Expression levels of target proteins harvested from a 1000L fermenter High performance liquid chromatography was used to detect the amount of the target protein after fermentation-induced expression. It can be seen that the final protein yield is relatively stable and high, which shows good prospects for commercial development and application.

[0024] (iv) Cell disruption Take the bacterial culture after induction expression in step (III), centrifuge at 8000 rpm for 10 min to collect the bacterial cells, add the centrifuged bacterial cells to the lysis buffer (EDTA-2Na·2H2O 0.74 g / L, pH adjusted to 6.8±0.1 with hydrochloric acid), and perform high pressure homogenization (homogenization twice at 600~650 bar pressure). The discharge temperature after homogenization should not exceed 40℃.

[0025] (v) Purification of the target protein After homogenizing the mixture from step (IV), detect the protein content (inclusion bodies), and then add 5L of lysis buffer at a ratio of 1g inclusion body to 5L. After resuspending, adjust the pH to between 10.0 and 11.0 using 0.1M NaOH solution to promote the dissolution of inclusion bodies. After dissolution, purify using an ion exchange chromatography column. The relevant operating parameters are as follows: Buffer A: 20 mM Tris, pH 7.25 ± 0.20 Buffer B: 20mM Tris, 0.5M NaCl After loading the sample, equilibrate with 2CV of buffer A, and then elute with 10CV of buffer B.

[0026] Samples of the eluent were taken and tested, and the results are as follows: Figure 3 As shown in the figure. Analysis shows that the purified tandem protein has a purity greater than 90% and a molecular weight of approximately 15179.0, consistent with the theoretical result.

[0027] (v) Enzymatic digestion and preparation of finished products Take the purified protein solution from step (III), detect the protein content, and add Kex2 enzyme (Shanghai Yaxin Company product, enzyme activity: 14U / mg) at a ratio of 1:3000 and CPB enzyme (Shanghai Yaxin Company product, enzyme activity: 170U / mg) at a ratio of 1:5000 based on the protein mass ratio, and carry out the enzymatic digestion reaction at 25℃ for 10h.

[0028] After enzyme digestion, samples were taken for testing, and the results were as follows: Figure 4 As shown in the figure. Analysis shows that the GLP-1 (9-37) precursor can be detected, with a molecular weight of approximately 3174.6 kDa, consistent with the theoretical value.

[0029] Further testing and calculations were performed on different batches of samples at a fermentation scale of 1000L in step (III), and the results are shown in Table 2 below. It can be seen that more than 5000g of GLP-1 (9-37) precursor can be obtained from all batches, showing good prospects for mass production application.

[0030] Table 2. GLP-1 (9-37) protein yield harvested in a 1000L fermenter. .

[0031] It should be noted that the enzyme digestion efficiency is calculated using the following formula: Enzyme digestion efficiency = [content of target protein after digestion / molecular weight of target protein after digestion] ÷ [content of target protein before digestion / molecular weight of target protein before digestion].

[0032] Based on the smegglutinin precursor prepared in step (iv) above, further modification and coupling steps can be performed to prepare the bioactive smegglutinin product. Specific steps are as follows.

[0033] (vi) Modification Take the enzyme digestion solution from step (iv) above, add anhydrous sodium carbonate at a ratio of 0.1 to the target protein (i.e., by mass ratio, target protein: anhydrous sodium carbonate = 1:0.1), and adjust the pH to 10.2±0.1 (using 4 mol / L sodium hydroxide solution for pH adjustment). After the reaction system (suspension) becomes clear, add octadecanoic acid-glutamic acid-EAEA-active ester at a ratio of 0.7 times the amount of the target protein (by mass ratio, target protein: octadecanoic acid-glutamic acid-EAEA-active ester = 1:0.7), adjust the pH to 10.5±0.2, and stir the reaction for ≥5 minutes to ensure a complete reaction. After the reaction was complete, the pH was adjusted to 3.5 ± 0.1 (using 6 mol / L hydrochloric acid), and the mixture was allowed to stand for about 1 hour to ensure complete sedimentation. After sedimentation, the mixture was centrifuged at 12000 rpm for 30 minutes, and the solid precipitate was collected and directly used for subsequent coupling treatment. It should be noted that "octadecanoic acid-glutamic acid-EAEA-active ester" can be synthesized using conventional methods based on existing techniques. The specific synthetic route is as follows: .

[0034] (vii) Coupon Take the solid collected in step (vi), add water at a ratio of 8 times its mass to dissolve it completely. After dissolution, add the following in sequence: His-Aib-BOC (0.2 times the amount of solid, i.e., by mass ratio, solid collected in step (vi): His-Aib-BOC = 1:0.2), and pre-cooled NMP (N-methylpyrrolidone, pre-cooled to about 2-8℃, the amount of NMP is 5 times the mass of the solid, i.e., by mass ratio, solid collected in step (vi): NMP = 1:5). Stir thoroughly to dissolve evenly. Subsequently, the following were added sequentially to the above solution system: HATU (added at 1 times the amount of solid, i.e., by mass ratio, the solid collected in step (VI): HATU = 1:1) and triethylamine (added at 1 times the amount of solid, i.e., by mass ratio, the solid collected in step (VI): triethylamine = 1:1). After adding the ingredients, stir thoroughly and allow the mixture to react fully at room temperature (the coupling reaction takes about 30 minutes to complete at around 18-30°C). After the reaction is complete, adjust the pH to 4.0 ± 0.1 (using 25% phosphoric acid solution) and allow it to settle completely (this will take at least 1 hour). After sedimentation, centrifuge at 12,000 rpm for 30 min and collect the solid precipitate; Finally, the collected solid precipitate was added to trifluoroacetic acid at a mass ratio of 20 (i.e., solid precipitate: trifluoroacetic acid = 1:20 by mass), and stirred thoroughly (generally for at least 10 minutes) to react and remove the bound dipeptide protecting groups; after the reaction was completed, the resulting product was the crude smegglutinin solution. It should be explained that the dipeptide His-Aib-BOC can be prepared using conventional techniques, or it can be prepared using the following synthetic route: .

[0035] (viii) Reversed-phase chromatography purification The crude semaglutide solution obtained in step (vii) was subjected to reversed-phase chromatography to obtain purified semaglutide. Specifically, a C8 column was used for reversed-phase chromatography, and the procedure is as follows: First, equilibrate with solution A for 1.5~2 CV; then load the sample; after loading the sample, equilibrate with solution A again for 2.5~3 CV; finally, elute with solution B, using a gradient elution: first elute with 0~40% solution B for 1 CV, then elute with 40~60% solution B for 5 CV. Solution A consists of 0.1M ammonium bicarbonate, 10% acetonitrile, and pH = 6.50 ± 0.20. The B solution is: 100% acetonitrile; During the elution process, the eluent is collected in segments at a wavelength of 280 nm. The eluent is then freeze-dried to obtain pure Smegglutinin.

[0036] The results of testing (using high-performance liquid chromatography) and calculations on different batches of products at a fermentation scale of 1000L in step (V) above are as follows: Figure 5 As shown in Tables 3 and 4 below. The results indicate that over 1500g of pure smegglutinin can be obtained, with a purity exceeding 99.5% (e.g., ...). Figure 5 As shown in the figure, mass spectrometry identification showed a molecular weight of 4112 kDa (Table 4 shows that the peptide coverage was 100%), indicating good prospects for industrial application.

[0037] Table 3. Yield of Smegglutinin protein from a 1000L fermenter Table 4, Peptide Coverage Table .

Claims

1. A method for preparing GLP-1 analog peptides through tandem expression, characterized in that, This method involves tandemly repeating the coding gene sequence of a GLP-1 analog and then expressing it using genetic engineering techniques. The GLP-1 analogue comprises two forms of amino acid sequences: 9-37, as shown in SEQ ID No. 1, has the following specific amino acid sequence: EGTFTSDVSSYLEGQAAKEFIAWLVRGRG; 11-37, as shown in SEQ ID No. 2, has the following specific amino acid sequence: TFTSDVSSYLEGQAAKEFIAWLVRGRG; In the tandem formation of the GLP-1 analogues, key peptide sequences are used for connection: The key peptide, shown in SEQ ID No. 3, has the following specific amino acid sequence: DVKPGQPLEDELG; The GLP-1 analog tandem process involves using key peptides to link several GLP-1 analogs into a single expression form. The preparation process specifically includes the following steps: (I) Construction of recombinant plasmid vectors Prepare the tandem gene sequence using a plasmid vector and recombine the tandem gene sequence. (ii) Transformation and Expression Transform the recombinant plasmid vector from step (I) into Escherichia coli strains and screen for correctly transformed strains; After fermenting the correctly transformed strain, IPTG was added to induce protein expression. (iii) Disruption of bacterial cells After inducing expression in step (II), the bacterial culture is centrifuged to collect the bacterial cells. The bacterial cells are then added to the lysis buffer, mixed thoroughly, and the bacterial cells are lysed. (iv) Purification of the target protein After the bacterial cells were broken in step (iii), the pH of the mixture was adjusted to 10.0-11.0 to promote the dissolution of inclusion bodies; After adjusting the pH, the sample was loaded for ion exchange chromatography; (v) Enzyme digestion The elution buffer from step (iv) was subjected to double digestion with Kex2 enzyme and CPB enzyme; (vi) Modification Take the reaction system after enzymatic digestion in step (5), add anhydrous sodium carbonate, and adjust the pH to 10.2 ± 0.1; After the reaction system is clear, add octadecanoic acid-glutamic acid-EAEA-active ester, adjust the pH to 10.5±0.2, and stir until the reaction is complete; After the reaction was complete, the pH was adjusted to 3.5 ± 0.1, and the mixture was allowed to settle. After settling, the solid precipitate was collected by centrifugation. (vii) Coupon Take the solid collected in step (six), dissolve it fully in water, and then add the following in sequence: His-Aib-BOC (protective group dipeptide) and pre-cooled NMP. Stir thoroughly to mix evenly. Subsequently, HATU and triethylamine were added sequentially to the above solution system; after the addition was complete, the mixture was stirred thoroughly and allowed to react completely at room temperature. After the reaction is complete, adjust the pH to 4.0 ± 0.1 and allow it to settle completely. After sedimentation, centrifuge and collect the solid precipitate; Finally, the collected solid precipitate was added to trifluoroacetic acid and stirred thoroughly to remove the bound dipeptide protecting groups. After the reaction was completed, the resulting crude solution of GLP-1 analog was obtained. (viii) Reversed-phase chromatography purification The crude GLP-1 analog solution obtained in step (vii) was subjected to reverse phase chromatography to obtain purified GLP-1 analog.

2. The method for preparing GLP-1 analog peptides by tandem expression as described in claim 1, characterized in that, The triplet tandem form of GLP-1 analogs is: M—key peptide—KR—GLP-1 analog—KR—key peptide—KR—GLP-1 analog—KR—key peptide—KR—GLP-1 analog; The tandem amino acid sequence is as follows: Tandem polypeptide 1: MDVKPGQPLEDELGKREGTFTSDVSSYLEGQAAKEFIAWLVRGRGKRDVKPGQPLEDELGKREGTFTSDVSSYLEGQAAKEFIAWLVRGRGKRDVKPGQPLEDELGKREGTFTSDVSSYLEGQAAKEFIAWLVRGRG; Tandem polypeptide 2: MDVKPGQPLEDELGKRTFTSDVSSYLEGQAAKEFIAWLVRGRGKRDVKPGQPLEDELGKRTFTSDVSSYLEGQAAKEFIAWLVRGRGKRDVKPGQPLEDELGKRTFTSDVSSYLEGQAAKEFIAWLVRGRG.

3. The method for preparing GLP-1 analog peptides by tandem expression as described in claim 1, characterized in that, In step (1), pET plasmid is used as the plasmid vector.

4. The method for preparing GLP-1 analog peptides by tandem expression as described in claim 1, characterized in that, In step (ii), *Escherichia coli* BL21(DE3) strain was used as the expression strain; during fermentation culture of the correctly transformed strain, fermentation was carried out at 37°C until OD... 600 After reaching ≥70-80%, add IPTG to a final concentration of 0.05mM-0.5mM, and continue culturing at 37℃ for 8-10 hours to induce protein expression.

5. The method for preparing GLP-1 analog peptides by tandem expression as described in claim 1, characterized in that, In step (iii), the bacterial cells are added to the lysis buffer at a ratio of 1g of bacterial cells to 5ml of lysis buffer; The lysis solution was a 0.74 g / L aqueous solution of EDTA-2Na·2H2O with a pH of 6.8 ± 0.

1.

6. The method for preparing GLP-1 analog peptides by tandem expression as described in claim 1, characterized in that, In step (iv), during ion exchange chromatography, an anion exchange chromatography column is used as the packing material; the ion exchange chromatography operation is as follows: After loading the sample, first equilibrate with 2CV of buffer A, then elute with 10CV of buffer B; The buffer solution A is: 20 mM Tris, pH = 7.25 ± 0.20; The buffer solution B is: 20mM Tris, 0.5M NaCl.

7. The method for preparing GLP-1 analog peptides by tandem expression as described in claim 1, characterized in that, In step (5), the enzyme activity of Kex2 enzyme in the enzyme digestion system is 14 U / mg, and the amount used is 1 / 1000-1 / 5000 of the total protein mass in the elution buffer. The enzyme activity of CPB enzyme is calculated as 170 U / mg, and the amount used is 1 / 1000-1 / 5000 of the total protein content in the elution buffer. During enzyme digestion, the temperature should be 10~40℃ and the digestion time should be 1~48h.

8. The method for preparing GLP-1 analog peptides by tandem expression as described in claim 1, characterized in that, In step (vi), anhydrous sodium carbonate is added at a ratio of 0.1-0.3 times the amount of the target protein; octadecanoic acid-glutamic acid-EAEA-active ester is added at a ratio of 0.7-1.0 times the amount of the target protein.

9. The method for preparing GLP-1 analog peptides by tandem expression as described in claim 1, characterized in that, In step (seven), The protecting group dipeptide His-Aib-BOC is added at 0.2 to 0.5 times the amount of solids. The amount of NMP used is 5 to 8 times the mass of the solid. Add HATU at a ratio of 0.5 to 1 times the solids amount; Triethylamine is added at 0.5 to 1 times the amount of solids.

10. The method for preparing GLP-1 analog peptides by tandem expression as described in claim 1, characterized in that, In step (eight), during the reversed-phase chromatography operation, a C8 column is used for the reversed-phase chromatography operation. The operation procedure is as follows: First, equilibrate with solution A for 1.5~2 CV; then load the sample; after loading the sample, equilibrate with solution A again for 2.5~3 CV; finally, elute with solution B, using a gradient elution: first elute with 0~40% solution B for 1 CV, then elute with 40~60% solution B for 5 CV. Solution A consists of 0.1M ammonium bicarbonate, 10% acetonitrile, and pH = 6.50 ± 0.

20. Solution B is 100% acetonitrile.

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