Recombinant fusion protein, polynucleotide, recombinant expression plasmid, engineered recombinant host cell and method for preparing target polypeptide

Abstract

The invention relates to the technical field of biology, in particular to a recombinant fusion protein, polynucleotide for coding the recombinant fusion protein, a recombinant expression plasmid containing the polynucleotide, an engineered recombinant host cell and a method for preparing target polypeptide by using the recombinant fusion protein. The recombinant fusion protein comprises tag polypeptide-target polypeptide-(linker 1-linker x-linker 2-target polypeptide) n from N terminal to C terminal, x is the number of linkers, x is 0 or 1, n is a positive integer not less than 1, the tag polypeptide is SEQ ID NO.1 or SEQ ID NO.2, the linker 1 contains a Kex2 protease enzyme cutting site and a carboxypeptidase B enzyme cutting site, and the linker 1 contains a Kex2 protease enzyme cutting site and a carboxypeptidase B enzyme cutting site. And the connecting peptide 2 contains a WELQ protease enzyme cutting site. The recombinant fusion protein disclosed by the invention is expressed in an inclusion body form, the synthesis process is simplified, the target polypeptide can be prepared with high yield and high purity by using the recombinant fusion protein disclosed by the invention, and the production cost can be reduced from multiple dimensions.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a recombinant fusion protein, a polynucleotide encoding the recombinant fusion protein, a recombinant expression plasmid containing the polynucleotide, an engineered recombinant host cell containing the recombinant expression plasmid or whose genome integrates the polynucleotide, and a method for preparing a target polypeptide using the recombinant fusion protein. Background Technology

[0002] Lowering the unit price of therapeutic proteins and peptides can significantly improve public health and reduce the cost burden of healthcare. In the past few years, the production capacity of therapeutic proteins and peptides has been measured in grams or kilograms. Due to their high unit price, while production costs have been a focus, progress in cost control, such as improvements in production processes, has been slow. Since last year, demand for oral semaglutide has increased, and hundreds of companies worldwide attempting to enter the market have begun expanding production to dilute and reduce labor, utilities, management, and plant depreciation costs. Previously, process costs, including materials and fillers, accounted for 5% to 30% of the total cost in grams or kilograms; after expansion, this proportion has increased to 50% to 85%. How to improve the process flow to reduce overall production costs has become an urgent problem to be solved.

[0003] CN106434717A describes a fusion protein design comprising three parts: an N-terminal soluble tag such as TrxA, DsbA, DsbC, Sumo, GST, or Intein; a soluble tag linked to an enzyme cleavage site (e.g., enterokinase, thrombin, or Sumo protease); and the target peptide. Due to the large molecular weight of the soluble tag, the target peptide constitutes a small proportion of the entire fusion protein, approximately 20%, with an expression level of 15%–50%, yielding about 10g of fusion protein per liter of fermentation. Furthermore, during purification, because of the soluble expression, the fusion protein needs to be captured, and the target peptide is produced after 4–12 hours of enzymatic digestion. Precipitation of the target peptide with 20% ethanol yields a final purity of approximately 70%. The target peptide prepared using the method disclosed in this patent application has low purity as a raw material or starting material for synthesis and requires further purification.

[0004] CN110128521B's fusion protein design comprises three parts: an N-terminus soluble and thermostable tag such as mTrA; a soluble and thermostable tag linked to an enzyme cleavage site such as enterokinase, thrombin, tobacco erosion virus protease, 3C protease, trypsin, or lysine endopeptidase, and the target polypeptide. Due to the large molecular weight of the mTrA tag, the target polypeptide accounts for a relatively small proportion of the entire fusion protein, approximately 20%, resulting in a 20% expression level of the fusion protein.

[0005] CN110128552B's fusion protein design comprises three parts: an N-terminus with an insoluble tag linking an enterokinase recognition site and the target peptide. Since the insoluble tag is approximately 7 kDa, and the target peptide constitutes about 30% of the entire fusion protein, the expression level of the fusion protein is unknown. High-density fermentation OD... 600 180. After washing the inclusion bodies, dissolving them in an alkaline solution easily produces epimers. Enzyme digestion at 37℃ for 12 hours is high in temperature and time, posing a high risk of contamination. Ion exchange chromatography purification yielded 92% purity of the target peptide and 87% yield, but there were impurity peaks before and after the target peak. The packing material loading was low, making it difficult to improve.

[0006] CN110305223B's fusion protein design comprises three parts: an N-terminal tag MHHHHEEAEAEARG for expression promotion and enzyme digestion, which links a Kex2 protease recognition site to a target polypeptide, with four repeats of the Kex2 protease recognition site and the target polypeptide. Based on its published SDS-PAGE diagram of recombinant engineered bacteria induced expression, the fusion protein expression level is approximately 20%–30%, and the high-density fermentation OD... 600 After cleaning the inclusion bodies, dissolve them in an alkaline solution (70-100°C). This process easily produces epimers. Add Kex2 protease and carboxypeptidase B for digestion at 25°C-35°C overnight. The long digestion time increases the risk of contamination. After Q anion exchange separation and purification, use a C4 reverse-phase column to purify to 99% purity. The process uses organic solvents, requiring increased explosion-proof requirements for the plant.

[0007] It can be seen that although existing technologies disclose the molecular design of various fusion proteins, there are still problems such as complex process flow, low expression level of fusion proteins, high reaction temperature and long reaction time in the enzymatic digestion step, high risk of reaction contamination, low yield and purity of the target peptide, and high production cost. Summary of the Invention

[0008] To address the shortcomings of the existing technology, this invention provides a recombinant fusion protein, a polynucleotide encoding the recombinant fusion protein, a recombinant expression plasmid containing the polynucleotide, an engineered recombinant host cell containing the recombinant expression plasmid or whose genome integrates the polynucleotide, and a method for preparing a target polypeptide from the recombinant fusion protein. The target polypeptide has a high proportion in the recombinant fusion protein, the expression level of the recombinant fusion protein is high, and the process is simplified, reducing the cost of preparing the target polypeptide from the recombinant fusion protein from multiple dimensions.

[0009] The technical solution of this invention is as follows: This invention provides a recombinant fusion protein, wherein the recombinant fusion protein is, from N-terminus to C-terminus, a tag peptide-target peptide-(linker peptide 1-linker x-linker peptide 2-target peptide)n, wherein x is the number of linkers, x is 0 or 1, and n is a positive integer not less than 1. The tag peptide is SEQ ID NO.1 or SEQ ID NO.2. Linker peptide 1 contains a Kex2 protease cleavage site and a carboxypeptidase B cleavage site, and linker peptide 2 contains a WELQ protease cleavage site.

[0010] Preferably, the linker peptide 1 is KR or RR, and the linker peptide 2 is SEQ ID NO.5.

[0011] Preferably, the connector is SEQ ID NO.8.

[0012] Preferably, the target polypeptide is a precursor of smegglutinin or a precursor of liraglutinin, the amino acid sequence of the precursor of smegglutinin is shown in SEQ ID NO.9, and the amino acid sequence of the precursor of liraglutinin is shown in SEQ ID NO.4.

[0013] Preferably, n is 2 to 6.

[0014] Preferably, the amino acid sequence of the recombinant fusion protein is shown in SEQ ID NO.3.

[0015] The present invention also provides a polynucleotide that encodes the recombinant fusion protein of the present invention described above.

[0016] The present invention also provides a recombinant expression plasmid, which is formed by recombining a vector with the polynucleotides described above.

[0017] The present invention also provides an engineered recombinant host cell containing the above-mentioned recombinant expression plasmid, or having the above-mentioned polynucleotides integrated into its genome.

[0018] Preferably, the host cell is Escherichia coli.

[0019] The present invention also provides a method for preparing a target polypeptide, comprising the following steps: (a) Synthesize the polynucleotide encoding the recombinant fusion protein according to the amino acid sequence; (b) The polynucleotide is cloned into a vector to obtain a recombinant expression plasmid; (c) The recombinant expression plasmid is transformed into a host bacterium to obtain engineered recombinant host cells; (d) Use resistance plates to screen engineered recombinant host cells containing the recombinant expression plasmid; (e) Fermenting the engineered recombinant host cell containing the recombinant expression plasmid to induce the expression of the recombinant fusion protein in the form of intracellular insoluble inclusion bodies; (f) The fermented engineered recombinant host cells are homogenized under high pressure to break down the bacteria, the inclusion bodies are collected, and then the inclusion bodies are refolded. (g) The target polypeptide is obtained by enzymatic digestion, transformation and separation purification, wherein the proteases used for enzymatic digestion and transformation are Kex2 protease, carboxypeptidase B and WELQ protease.

[0020] Preferably, the inclusion bodies are refolded using a Tris-HCl buffer solution containing Triton X-100 at a concentration of 50 mM and a pH of 8.0.

[0021] Preferably, the host bacterium is Escherichia coli.

[0022] Preferably, the mass ratio of the Kex2 protease, carboxypeptidase B, and WELQ protease to the recombinant fusion protein is 1:800 to 1:1500, the enzyme digestion temperature is 10℃ to 37℃, and the enzyme digestion time is 2 to 6 hours.

[0023] More preferably, the mass ratio of the enzyme to the recombinant fusion protein is Kex2 enzyme:recombinant fusion protein = 1:1000, carboxypeptidase B:recombinant fusion protein = 1:1200, WELQ enzyme:recombinant fusion protein = 1:1500, the enzyme digestion temperature is 30℃, and the enzyme digestion time is 6 hours.

[0024] Preferably, the purification includes ion exchange resin purification and / or isoelectric point precipitation.

[0025] Reducing the cost of peptide production from recombinant fusion proteins is a multi-dimensional issue. For example, expressing recombinant fusion proteins as inclusion bodies reduces subsequent chromatography steps and simplifies the process, thus lowering costs. Increasing the yield of the target peptide in fermentation requires increasing the expression level of the recombinant fusion protein and its proportion within the total recombinant fusion protein. Since recombinant fusion protein expression is a burden on engineered bacteria, high expression levels often result in lower bacterial concentrations compared to low expression levels; therefore, increasing the concentration of high-density fermentation cultures is necessary. During production, reducing costly steps is crucial. This includes minimizing or eliminating the use of denaturing agents that dissolve inclusion bodies, avoiding extreme conditions such as high-pH alkaline solutions, and reducing the generation of epimers. Epimers are very similar to the product, leading to poor purification and increased purification costs. In enzymatic digestion, reaction time needs to be controlled, enzyme dosage reduced, and the enzymes used in the process highly specific to minimize mis-digestion. After 99% digestion of the fusion protein, the mis-digestion rate should be controlled below 2%. Achieving a certain level of purity for the target peptide after digestion allows the entire process to require only one purification step.

[0026] The recombinant fusion protein of this invention is expressed in the form of inclusion bodies, reducing subsequent capture chromatography steps, simplifying the production process, and significantly reducing production costs. The recombinant fusion protein of this invention inserts a tag peptide, including SEQ ID NO.1 (MSKIKR) or SEQ ID NO.2 (MSKIKWELQ), before the target peptide. This tag peptide can immediately prevent or release ribosome arrest during translation, thereby increasing the expression level of the recombinant fusion protein. Furthermore, the tag peptide makes the recombinant fusion protein more easily expressed in the form of inclusion bodies. On the other hand, this invention designs multiple target peptides tandemly in the recombinant fusion protein, increasing the proportion of the target peptide in the fusion protein. At the same expression level, a higher proportion of the target peptide in the fusion protein allows for the cleavage of more target peptides. The WELQ protease cleavage site in the recombinant fusion protein of this invention has fewer acidic amino acids compared to the enterokinase cleavage site (WELQ vs DDDDK), making the recombinant fusion protein more easily expressed in the form of inclusion bodies. WELQ exhibits good compatibility with the target peptides smegglutinin precursor and liraglutinin precursor, enhancing the expression of the recombinant fusion protein. Furthermore, the resulting recombinant fusion protein can be purified using simpler and more economical methods, simplifying subsequent purification steps and reducing purification costs. In addition, the recombinant fusion protein of this invention, when cleaved by WELQ protease, avoids miscleavage products of the target peptide due to enterokinase. The recombinant fusion protein of this invention can achieve a purity of over 96% after enzymatic digestion using a one-step ion exchange resin purification method, and a purity of over 95% can be achieved using isoelectric point precipitation, significantly reducing production costs compared to existing processes. The production process of this invention does not require ultrafiltration and reverse-phase purification; only one step or no chromatography purification is needed, achieving a purity of over 94%. Attached Figure Description

[0027] Figure 1 Molecular design I for the recombinant fusion protein in this invention; Figure 2 This is the molecular design II for the recombinant fusion protein in this invention; Figure 3 The recombinant expression plasmid used in Example 1, wherein GHB084 corresponds to the recombinant fusion protein; Figure 4 The SDS-PAGE map of the recombinant fusion protein produced after 8 hours of expression on the recombinant expression plasmid induced at 30°C. Figure 5 The SDS-PAGE profile of the recombinant fusion protein produced after 16 h of expression on the recombinant expression plasmid induced at 25°C. Figure 6 SDS-PAGE map of recombinant fusion protein expressed by high-density fermentation and induction of recombinant expression plasmid at 25°C; Figure 7 This is the HPLC chromatogram of the recombinant fusion protein before enzyme digestion in Example 4; Figure 8 The image shows the HPLC chromatogram of the target polypeptide after enzyme digestion in Example 4, where 8B is an enlarged version of 8A. Detailed Implementation

[0028] The present invention will be further explained below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention. Unless otherwise specified, the methods used in the present invention are conventional production methods; the raw materials used, unless otherwise specified, are conventional commercially available products.

[0029] like Figure 1 and 2 As shown, the recombinant fusion protein of the present invention, from N-terminus to C-terminus, is denoted as tag peptide-target peptide-(linker peptide 1-connector x-linker peptide 2-target peptide)n, where x is the number of connectors, x is 0 or 1, and n is a positive integer not less than 1. The tag peptide is SEQ ID NO. 1 (MSKIKR) or SEQ ID NO. 2 (MSKIKWELQ), which is linked to the target peptide, followed by one or more (linker peptide 1-connector x-linker peptide 2-target peptide) linkages. The tag peptide, linker peptide 1, and linker peptide 2 all contain protease cleavage sites, suitable for removing the tag peptide and linker peptide by protease cleavage after the recombinant fusion protein is expressed, thereby forming multiple free target peptides. The tag peptide may contain a Kex2 protease cleavage site, such as KR in MSKIKR. Furthermore, the tag peptide may also contain a WELQ protease cleavage site, such as WELQ in MSKIKWELQ. Linker peptide 1 contains a Kex2 protease cleavage site and a carboxypeptidase B cleavage site, and linker peptide 2 contains a WELQ protease cleavage site. In a preferred embodiment of the present invention, the linker peptide 1 is KR or RR, and the linker peptide 2 is WELQ (SEQ ID NO.5).

[0030] Both the tag peptides MSKIKR and MSKIKWELQ contain the MSKIK sequence, which can immediately inhibit or release ribosome arrest during translation, thereby increasing the expression level of the recombinant fusion protein. Simultaneously, due to the short sequences of the tag peptides MSKIKR and MSKIKWELQ, they constitute a smaller proportion of the entire fusion protein, increasing the proportion of the target peptide. Therefore, the yield of the final target peptide can be increased. Furthermore, the tag peptides MSKIKR and MSKIKWELQ can also increase the probability of inclusion body formation. The amino acid sequence of the linker peptide 2 is WELQ, and the linker peptide 2 contains a WELQ protease cleavage site. The WELQ protease cleavage site has fewer acidic amino acids compared to the enterokinase cleavage site, making the recombinant fusion protein more easily expressed in the form of inclusion bodies. WELQ has good compatibility with the target peptides smegglutide precursor and liraglutide precursor, enhancing the expression of the recombinant fusion protein. The resulting recombinant fusion protein can be purified using simpler and more economical methods, simplifying subsequent purification steps and reducing purification costs. WELQ protease exhibits better specificity; the cleavage sites and linker peptides removed by enzyme cleavage do not occupy the loading capacity of ion exchange resins during separation, reducing the amount of packing material used and lowering costs. In this invention, the combined use of the tag peptide MSKIKR or MSKIKWELQ and the linker peptide 2 WELQ enables the recombinant fusion protein to be expressed in the form of inclusion bodies, simplifying subsequent processing and significantly reducing production costs.

[0031] The recombinant fusion protein of the present invention may further include a linker for connecting and spatially isolating linker peptide 1 and linker peptide 2 to avoid interference between different enzymes during subsequent digestion. Furthermore, by cooperating the linker sequence with sequences such as tag peptides, the overall properties of the recombinant fusion peptide can be regulated, making it more likely to be expressed in inclusion body form, enhancing expression, and simplifying subsequent purification processes. The preferred linker sequence is EAGSEA, where the presence of EA after the KR amino acid sequence can improve the digestion efficiency of the Kex2 enzyme.

[0032] In the recombinant fusion protein of this invention, n is a positive integer not less than 1, meaning that the fusion protein of this invention contains multiple target peptides tandemly, increasing the proportion of target peptides in the fusion protein and allowing for the cleavage of more target peptides after expression of the recombinant fusion protein. In a preferred embodiment of this invention, n is 2 to 6. When n is within the above range, the proportion of target peptides in the fusion protein is relatively high, and the repetitive sequences are suitable for expression by engineered bacteria. When n is greater than 6, the proportion of target protein in the fusion protein decreases, and the multiple repetitive sequences make expression difficult.

[0033] In a preferred embodiment of the present invention, the target polypeptide includes semaglutide precursor Arg34 GLP-1 (9-37) and liraglutide precursor, wherein the amino acid sequence of the semaglutide precursor is shown in SEQ ID NO.9 (EGTFTSDVSSYLEGQAAKEFIAWLVRGRG) and the amino acid sequence of the liraglutide precursor is shown in SEQ ID NO.4 (HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG).

[0034] In a preferred embodiment of the present invention, the amino acid sequence of the recombinant fusion protein is shown in SEQ ID NO. 3.

[0035] In one embodiment of the present invention, a polynucleotide encoding the recombinant fusion protein of the present invention is provided. In another embodiment of the present invention, a recombinant expression plasmid is provided, wherein the recombinant expression plasmid is formed by recombination of a vector and the polynucleotide. The vector used in the present invention can be a commonly used pET series expression vector in the art, such as pET30b vector, pET29b vector, etc.

[0036] Those skilled in the art can construct expression vectors containing the DNA sequence encoding the recombinant fusion protein of this invention and suitable transcription / translation control signals using well-known methods, including in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be effectively ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0037] One embodiment of the present invention provides an engineered recombinant host cell comprising the above-described recombinant expression plasmid or a genome encoding the recombinant fusion protein of the present invention. The host cell is preferably *Escherichia coli*, such as *Escherichia coli* BL21(DE3).

[0038] In one embodiment of the present invention, the method for preparing the target polypeptide using the recombinant fusion protein of the present invention includes the following steps: (a) Synthesizing the encoding gene of the recombinant fusion protein of the present invention based on the amino acid sequence; (b) The encoding gene of the recombinant fusion protein is cloned into a vector to obtain a recombinant expression plasmid; (c) The recombinant expression plasmid is transformed into a host cell to obtain an engineered recombinant host cell; (d) Use resistance plates to screen engineered recombinant host cells containing the recombinant expression plasmid; (e) Fermentation of the engineered recombinant host cell containing the recombinant expression plasmid induces the expression of the recombinant fusion protein in the form of intracellular insoluble inclusion bodies; (f) The fermented engineered recombinant host cells are homogenized under high pressure to break down the bacteria, the inclusion bodies are collected, and then the inclusion bodies are refolded. (g) The target polypeptide is obtained by enzymatic digestion, transformation and purification, wherein the proteases used for enzymatic digestion are Kex2 protease, carboxypeptidase B and WELQ protease.

[0039] Those skilled in the art can prepare recombinant expression plasmids using methods well-known in the art, transform the expression vector into host cells, and ferment the engineered recombinant host cells selected through resistance plate screening in a fermentation medium. After fermentation, the cells are homogenized under high pressure to obtain inclusion bodies. The inclusion bodies are refolded and purified using a buffer solution containing a nonionic surfactant to obtain the recombinant fusion protein. The recombinant fusion protein is then cleaved into multiple independent target peptides by enzymatic digestion. The recombinant fusion protein is digested using three enzymes: WELQ protease, Kex2 protease, and carboxypeptidase B. The obtained target peptides are then separated and purified using various methods, preferably ion exchange chromatography and / or isoelectric point precipitation. The preparation method of the present invention enables the preparation of target peptides with high yield, high purity, and low cost.

[0040] In a preferred embodiment of the present invention, the inclusion bodies are refolded using a Tris-HCl buffer solution containing Triton X-100 at a concentration of 50 mM and a pH of 8.0. In the preparation method of the present invention, the dissolution and refolding of the inclusion bodies do not require denaturing agents or other extreme conditions, such as high pH, ​​which can reduce the generation of epimers. In a preferred embodiment of the present invention, the host cell used in the preparation method of the present invention is *Escherichia coli* BL21(DE3). The mass ratio of Kex2 protease, carboxypeptidase B, and WELQ protease to the recombinant fusion protein is preferably 1:800 to 1:1500, more preferably Kex2 enzyme:recombinant fusion protein = 1:1000, carboxypeptidase B:recombinant fusion protein = 1:1200, and WELQ enzyme:recombinant fusion protein = 1:1500, reducing the amount of enzymes used in the process. The above-mentioned enzyme digestion temperature is preferably 10℃ to 37℃, and the enzyme digestion time is preferably 2-6 hours, more preferably the enzyme digestion temperature is 30℃ and the enzyme digestion time is 6 hours, improving the enzyme digestion efficiency. Example 1

[0041] The recombinant fusion protein of the present invention is described below using semaglutide precursor as the target polypeptide as an example. The synthesis of the polynucleotide sequence, plasmid construction, and sequencing analysis of the recombinant fusion protein in this embodiment were completed by Suzhou Hongxun Biotechnology Co., Ltd. The amino acid sequence of the recombinant fusion protein is shown in SEQ ID NO.3, and the nucleotide sequence of the editing gene encoding the recombinant fusion protein is shown in SEQ ID NO.6. The nucleotide sequence of the MSKIKR tag polypeptide can be designed by those skilled in the art based on the correspondence between amino acids and bases, without special limitations. In this embodiment, the nucleotide sequence of the MSKIKR tag polypeptide is ATGTCTAAAATCAAACGT, as shown in SEQ ID NO.7. The amino acid sequence of linker peptide 1 is KR, the amino acid sequence of linker peptide 2 is WELQ, and the amino acid sequence of the linker is EAGSEA, as shown in SEQ ID NO.8. The target polypeptide is semaglutide precursor Arg34 GLP-1(9-37), whose amino acid sequence is EGTFTSDVSSYLEGQAAKEFIAWLVRGRG, as shown in SEQ ID NO.9. The nucleotide sequence of the target polypeptide is shown in SEQ ID NO.10. Where x is 1 and n is 5.

[0042] SEQ ID NO.3: MSKIKREGTFTSDVSSYLEGQAAKEFIAWLVRGRGKREAGSEAWELQEGTFTSDVSSYLEGQAAKEFIAWLVRGRGKREAGSEAWELQEGTFTSDVSSYLEGQAAKEFIAWLVRGRGKRE AGSEAWELQEGTFTSDVSSYLEGQAAKEFIAWLVRGRGKREAGSEAWELQEGTFTSDVSSYLEGQAAKEFIAWLVRGRGKREAGSEAWELQEGTFTSDVSSYLEGQAAKEFIAWLVRGRG.

[0043] SEQ ID NO.6: ATGTCTAAAATCAAACGTGAAGGCACTTTTACCAGCGATGTTAGCAGCTACCTGGAAGGCCAGGCTGCTAAAGAATTCATTGCTTGGCTGGTGCGTGGTCGTGGTAAACGTGAAGCGGGCTCTGAGGCATGGGAGCTGCAGGAAGGTACCTTCACTTCCGACGTCTCTAGCTACCTGGAGGGCCAAGCAGCTAAAGAATTCATCGCTTGGCTGGTTCGTGGTCGCGGTAAACGTGAGGCCGGTTCTGAAGCTTGGGAACTGCAGGAAGGTACTTTCACTTCTGATGTAAGCAGCTATCTGGAAGGCCAAGCCGCAAAAGAATTCATCGCGTGGCTGGTACGCGGTCGTGGCAAACGTGAAGCTGGCAGCGAAGCGTGGGAACTGCAGGAGGGTACCTTTACCAGCGACGTCAGCAGCTACCTGGAAGGTCAGGCCGCGAAAGAATTTATTGCCTGGCTGGTACGTGGTCGTGGTAAGCGTGAGGCAGGTTCCGAGGCATGGGAACTGCAGGAGGGCACTTTCACTTCCGACGTTTCTTCTTATCTGGAAGGCCAGGCGGCGAAAGAGTTTATCGCATGGCTGGTGCGTGGCCGCGGCAAACGCGAAGCAGGTTCTGAAGCGTGGGAGCTGCAGGAAGGCACGTTCACGTCTGACGTAAGCTCCTACCTGGAGGGTCAGGCCGCTAAGGAATTTATCGCCTGGCTGGTTCGTGGCCGTGGTTAA。

[0044] SEQ ID NO.10: GAAGGCACTTTTACCAGCGATGTTAGCAGCTACCTGGAAGGCCAGGCTGCTAAAGAATTCATTGCTTGGCTGGTGCGTGGTCGTGGT。

[0045] The coding gene of the recombinant fusion protein was cloned onto the expression vector pET30b by conventional genetic engineering, and the constructed recombinant expression plasmid was as Figure 3As shown. The constructed recombinant expression plasmid was transformed into *E. coli* BL21(DE3) competent cells (purchased from Shanghai Sangon Biotech Co., Ltd.) using the heat shock method. Since the constructed plasmid contained kanamycin resistance, the genetically engineered bacteria expressing the semaglutide precursor were obtained through resistance plate selection. The cells were cultured in LB medium containing 50 μg / mL kanamycin for 8–12 hours at 37°C. OD 600 After reaching a final concentration of 1.0, the culture was stored at -80°C with 20% glycerol. After sequencing analysis confirmed its correctness, it was identified as the recombinant expression transformant and named strain #84. The culture solution containing 20% ​​glycerol was named strain #84. Example 2

[0046] The #84 bacterial culture from Example 1 was inoculated at a volume ratio of 1:100 into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking at 200 rpm for 8 h until OD. 600 Approximately 1, isopropyl-β-D-thiogalactoside (IPTG) was added to final concentrations of 0 mM, 1.0 mM, 1.5 mM, and 2.0 mM, respectively. The mixture was induced at 30°C for 8 h. SDS-PAGE analysis was used to analyze the expression level. The uninduced group (IPTG final concentration 0 mM) served as the control group, with expression levels of 0%, 69.09%, 64.79%, and 55.57%, respectively. The SDS-PAGE (electrophoresis apparatus, Beijing Liuyi, DYCZ-24DN) pattern of the recombinant fusion protein is shown below. Figure 4 As shown, M represents the protein standard (Thermo Scientific™ PageRuler™ pre-stained protein molecular weight standard 26616), and lanes 1-4 correspond to groups with IPTG final concentrations of 0mM, 1.0mM, 1.5mM, and 2.0mM, respectively. Example 3

[0047] The #84 bacterial culture from Example 1 was inoculated at a volume ratio of 1:100 into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking at 200 rpm for 8 h until OD. 600 Approximately 1, isopropyl-β-D-thiogalactoside (IPTG) was added to final concentrations of 0 mM, 1.0 mM, 1.5 mM, and 2.0 mM, respectively. The mixture was induced at 25°C for 16 h. SDS-PAGE analysis was used to analyze the expression level. The uninduced group (IPTG final concentration 0 mM) served as the control group, with expression levels of 0%, 78.36%, 79.11%, and 69.67%, respectively. The SDS-PAGE (electrophoresis apparatus, Beijing Liuyi, DYCZ-24DN) pattern of the recombinant fusion protein is shown below. Figure 5As shown, M represents the protein standard (Thermo Scientific™ PageRuler™ pre-stained protein molecular weight standard 26616), and lanes 1-4 correspond to groups with IPTG final concentrations of 0mM, 1.0mM, 1.5mM, and 2.0mM, respectively. Example 4

[0048] The #84 bacterial culture from Example 1 was inoculated at a volume ratio of 1:100 into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking at 200 rpm for 8 h until OD. 600 Approximately 1, yielding the first culture medium. This first culture medium was inoculated at a volume ratio of 1:35 into a fermentation medium containing 50 μg / mL kanamycin (1% soybean peptone, 1% yeast extract, 0.4% sodium chloride, 0.25% K₂HPO₄·3H₂O, 0.1% KH₂PO₄, pH adjusted to 7.0 with 20% NaOH solution). The medium was incubated at 37°C, maintaining dissolved oxygen above 30% by adjusting the agitator speed, aeration rate, and pure oxygen supply. The pH was adjusted with ammonia and maintained at 6.8 until the bacterial OD of the culture medium reached 1. 600 When the concentration reached 78.8%, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mM. Fermentation was continued for 17.6 hours, then stopped. The bacterial culture was collected, and the fermentation endpoint OD was measured. 600The concentration was 301.2. After centrifugation at 8000 rpm for 30 min at 4℃, bacterial cells were obtained. The supernatant was discarded, and the bacterial cells were collected and stored at -20℃ for later use. Each liter of fermentation broth yielded 290 g of wet bacterial cells. The wet bacterial cells were resuspended at a weight ratio of 1:10 in 50 mM Tris-HCl buffer (pH 8.0) (Tris is an abbreviation for tris(hydroxymethyl)aminomethane, AR grade, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.). Homogenize three times at 750 bar using an ATS (AH-PILOT 2018) high-pressure homogenizer, followed by lysis at 8000 rpm for 60 min. Inclusion bodies are then collected and refolded using a Tris-HCl buffer containing 50 mM of the nonionic surfactant Triton X-100 (AR grade, Shanghai Maclean Biotechnology Co., Ltd.) at pH 8.0. The volume of the buffer is the same as that used for resuspending the wet bacterial culture. The surfactant is removed by isoelectric point precipitation of the refolded sample. Specifically, 2N HCl is added to the refolded sample to adjust the pH to 4.58, and the sample is refluxed at 8000 rpm. Centrifuge for 15 minutes, collect the white precipitate, and dissolve it in a 50 mM Tris-HCl buffer solution (pH 8.0). The volume of the buffer solution is the same as the volume of the buffer solution used for resuspending the wet bacterial culture. More than 22.94 g of recombinant fusion protein can be obtained per liter of fermentation broth before enzyme digestion. SDS-PAGE (electrophoresis apparatus, Beijing Liuyi, DYCZ-24DN) and HPLC (HPLC analyzer, Waters, e2695+PDA) analysis showed that the expression level of the recombinant fusion protein was 83.16%, and the purity was 96.501%. The SDS-PAGE pattern is shown below. Figure 6 As shown, this chromatogram illustrates the leakage and expression status of the recombinant fusion protein from inoculation to the end of fermentation. M represents the protein standard (Thermo Scientific™ PageRuler™ pre-stained protein molecular weight standard 26616). Lanes 1-14 represent the expression levels at the shake-flask leakage state, 2h, 17h, 18h, 20h, 22h, 23.3h, 23.6h, 23.7h (induction), 24h, 26h, 28h, 29h, and 41.3h, respectively. HPLC data is as follows: Figure 7 As shown, the retention time of the recombinant fusion protein was 5.614 minutes. After dissolving the recombinant fusion protein, 100X digestion buffer (200mM CaCl2) was added, followed by the addition of WELQ protease (Beyotime), Kex2 protease (Paijin Biotechnology), and carboxypeptidase B (Paijin Biotechnology). The enzyme-to-fusion protein ratio was 1:1000 for Kex2 protease: fusion protein, 1:1200 for carboxypeptidase B: fusion protein, and 1:1500 for WELQ protease. The digestion temperature was 30℃, and the digestion time was 6 hours. The digested products were detected using HPCL. Figure 8As shown, no recombinant fusion protein peak was detected (retention time 5.614 min), the enzyme digestion efficiency was 100%, and the purity of the target peptide was 94.58%. Compared with recombinant fusion proteins designed using enterokinase digestion, the recombinant fusion protein of the present invention did not exhibit miscleavage products of smegglutinin precursor produced by enterokinase during the enzyme digestion process (retention time 2.1 min).

[0049] The comparison results between the present invention and the prior art are shown in Table 1 below: Table 1

[0050] Compared with existing technologies, this invention reduces the cost of preparing target peptides from recombinant fusion proteins in multiple dimensions. The target peptide in this invention accounts for 70%–80% of the recombinant fusion protein, and the expression level of the recombinant fusion protein is 50%–90%, both higher than existing technologies. The recombinant fusion protein of this invention has a high expression level; after high-density fermentation, 290–350 g of wet cells can be obtained per liter of fermentation broth, and more than 20 g of recombinant fusion protein can be obtained per liter of fermentation broth before enzyme digestion, higher than existing technologies. Furthermore, the technical solution of this invention increases the expression of the recombinant fusion protein in inclusion bodies, and the purity of the target peptide before enzyme digestion is not less than 90%, reducing downstream chromatography steps. The dissolution and refolding of inclusion bodies in this invention do not require denaturants or other extreme conditions, such as pH, reducing the generation of epimers and lowering subsequent purification costs. This invention significantly reduces the miscleavage rate by using the highly specific WELQ protease. After 99% digestion of the recombinant fusion protein, the miscleavage rate can be controlled to below 2%. This invention controls the digestion reaction time and reduces the amount of enzyme used. The downstream purification process only requires one purification step to obtain a product with a purity of not less than 94%. The quality of the target peptide of this invention is significantly improved, and the purification of subsequent products is greatly facilitated. The safety of the target peptide as a pharmaceutical peptide is significantly improved, and its immunotoxicity is significantly reduced.

[0051] This invention is not limited to the optional embodiments described above, and anyone can derive other various forms of products based on the inspiration of this invention. The specific embodiments described above should not be construed as limiting the scope of protection of this invention; the scope of protection of this invention should be determined by the claims, and the specification can be used to interpret the claims.

Claims

1. A recombinant fusion protein, characterized in that, The recombinant fusion protein is defined from N-terminus to C-terminus as tag peptide-target peptide-(linker peptide 1-linker x-linker peptide 2-target peptide)n, where x is the number of linkers, x is 0 or 1, and n is a positive integer not less than 1. The tag peptide is SEQ ID NO.1 or SEQ ID NO.

2. Linker peptide 1 contains a Kex2 protease cleavage site and a carboxypeptidase B cleavage site, and linker peptide 2 contains a WELQ protease cleavage site.

2. The recombinant fusion protein according to claim 1, characterized in that, The linker peptide 1 is KR or RR, and the linker peptide 2 is SEQ ID NO.

5.

3. The recombinant fusion protein according to claim 1, characterized in that, The connector is SEQ ID NO.

8.

4. The recombinant fusion protein according to claim 1, characterized in that, The target polypeptide is a precursor of semaglutide or liraglutide, the amino acid sequence of which is shown in SEQ ID NO.9 and the amino acid sequence of which is shown in SEQ ID NO.

4.

5. The recombinant fusion protein according to any one of claims 1-4, characterized in that, The value of n is 2 to 6.

6. The recombinant fusion protein according to claim 1, characterized in that, The amino acid sequence of the recombinant fusion protein is shown in SEQ ID NO.

3.

7. A polynucleotide, characterized in that, The polynucleotide encodes the recombinant fusion protein according to any one of claims 1-6.

8. A recombinant expression plasmid, characterized in that, The recombinant expression plasmid is formed by recombining the vector with the polynucleotide described in claim 7.

9. An engineered recombinant host cell, characterized in that, The recombinant host cell contains the recombinant expression plasmid of claim 8, or the genome is integrated with the polynucleotide of claim 7.

10. The recombinant host cell according to claim 9, characterized in that, The host cell is Escherichia coli.

11. A method for preparing a target polypeptide, characterized in that, Includes the following steps: (a) Synthesize a polynucleotide encoding the recombinant fusion protein according to any one of claims 1-6 based on the amino acid sequence; (b) The polynucleotide is cloned into a vector to obtain a recombinant expression plasmid; (c) The recombinant expression plasmid is transformed into a host cell to obtain an engineered recombinant host cell; (d) Use resistance plates to screen engineered recombinant host cells containing the recombinant expression plasmid; (e) Fermenting the engineered recombinant host cell containing the recombinant expression plasmid to induce the expression of the recombinant fusion protein in the form of intracellular insoluble inclusion bodies; (f) The fermented engineered recombinant host cells are homogenized under high pressure to break down the bacteria, the inclusion bodies are collected, and then the inclusion bodies are refolded. (g) The target polypeptide is obtained by enzymatic digestion, transformation and separation purification, wherein the proteases used for enzymatic digestion and transformation are Kex2 protease, carboxypeptidase B and WELQ protease.

12. The preparation method according to claim 11, characterized in that, The host bacterium is Escherichia coli.

13. The method according to claim 11, characterized in that, The mass ratio of the Kex2 protease, carboxypeptidase B, and WELQ protease to the recombinant fusion protein is 1:800 to 1:1500, the enzyme digestion temperature is 10℃ to 37℃, and the enzyme digestion time is 2 to 6 hours.

14. The method according to claim 13, characterized in that, The mass ratio of the enzyme to the recombinant fusion protein is as follows: Kex2 enzyme: recombinant fusion protein = 1:1000, carboxypeptidase B: recombinant fusion protein = 1:1200, WELQ enzyme: recombinant fusion protein = 1:1500. The enzyme digestion temperature is 30℃ and the enzyme digestion time is 6 hours.

15. The method according to any one of claims 11-14, characterized in that, The purification includes ion exchange resin purification and / or isoelectric point precipitation.

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