Efficient expression system of Exendin-4 polypeptide

Abstract

The invention relates to a fusion protein and application thereof in preparation of Exendin-4, and belongs to the technical field of biology. The invention provides a fusion protein for improving the expression quantity of Exendin-4. The fusion protein comprises a signal peptide, a fusion partner and the Exendin-4, in order to reduce the toxic and side effects of the Exendin-4 on the host cell, the signal peptide and the fusion partner are adopted to secrete the Exendin-4 fusion protein to the periplasmic space of the host cell and outside the host cell, the ultrahigh-yield Exendin-4 fusion protein is obtained, and the Exendin-4 fusion protein has great application prospects in preparation of Exendin-4.

Description

Technical Field

[0001] This invention relates to a high-efficiency expression system for the Exendin-4 peptide, belonging to the field of biotechnology. Background Technology

[0002] Exendin-4 (Ex-4) is a single-chain polypeptide derived from the saliva of the poison lizard (Heloderma suspectum), consisting of 39 amino acid residues. Its amino acid sequence shares approximately 50% homology with human glucagon-like peptide-1 (GLP-1). The N-terminal and mid-terminal regions of Exendin-4 retain key residues essential for binding to the GLP-1 receptor, while the nine amino acids at the C-terminus play a crucial role in maintaining high-affinity binding and receptor activation; the C-terminus is in an amidated form. Exendin-4 can specifically activate the GLP-1 receptor, thereby mimicking various biological effects of endogenous GLP-1, including promoting glucose-dependent insulin secretion, inhibiting glucagon release, delaying gastric emptying, increasing satiety, and improving pancreatic β-cell function. Compared to natural GLP-1, Exendin-4 exhibits stronger resistance to dipeptidyl peptidase IV (DPP-4) degradation, a longer half-life in vivo, and superior pharmacokinetic characteristics. Therefore, it has become an important lead molecule for the development of GLP-1 receptor agonist drugs and has been developed into drugs for the clinical treatment of type 2 diabetes, such as exenatide.

[0003] Currently, the preparation methods for Exendin-4 mainly fall into two categories: chemical solid-phase peptide synthesis and recombinant expression. Chemical solid-phase peptide synthesis can accurately synthesize the target sequence and achieve C-terminal amidation modification, but this method involves cumbersome synthesis steps, high costs, and significant difficulties in scaling up production, hindering industrial-scale production. Recombinant expression primarily utilizes hosts such as *E. coli* for exogenous protein expression, offering advantages in reducing production costs and increasing expression levels. However, Exendin-4 readily forms inclusion bodies in *E. coli*, resulting in limited yields of soluble protein. Furthermore, the inclusion body refolding process is complex and inefficient, and C-terminal amidation is difficult to achieve, making purification challenging. These factors collectively limit the yield and quality of the biologically prepared Exendin-4.

[0004] Therefore, existing technologies for the preparation of Exendin-4 still face problems such as low yield, poor refolding efficiency, difficulty in structural modification, and high production costs. Furthermore, compared to GLP-1, Exendin-4 has a nine-amino acid extension at its C-terminus. This C-terminal extension forms a "tryptophan cage" to support the secondary structure and improve the titer of the GLP-1 receptor. However, the rigid structure of the "tryptophan cage" is highly hydrophobic, leading to strong interactions with the cell membrane during expression in *E. coli*, affecting *E. coli* growth and resulting in very low yields of Exendin-4 peptides in *E. coli* expression systems. This severely limits the application of bio-fermentation methods in the preparation of Exendin-4. Therefore, there is an urgent need to develop an efficient, scalable method for preparing Exendin-4 with the correct chemical structure and biological activity to meet the needs of clinical and industrial applications. Summary of the Invention

[0005] To address the above problems, this invention provides a fusion protein for increasing the expression level of a target peptide, the fusion protein comprising a signal peptide, a fusion chaperone, and a target peptide. The target polypeptide is selected from the GLP-1 peptide (e.g., GLP-1(7-37), GLP-1(7-36) and its analogues such as R34GLP-1(9-37), glucagon peptides such as glucagon(1-29), insulin precursors (e.g., A(1-21)-AAK-B(1-29)-human insulin) and exendin (e.g., exendin-3 and-4). The signal peptide includes the vesicle nucleation peptide VNP, and preferably the signal peptide is VNP6 or VNP15; Preferably, the fusion partner is selected from MrsB, DARP, TrxA, GB1, StefinA, P53TD / E2K, Trpzip4, sumo, GcN4-v4 or a combination thereof; more preferably, the fusion partner is selected from MrsB, DARP, GB1, TrxA, StefinA or a combination thereof; even more preferably, the fusion partner is selected from MrsB and DARP.

[0006] In one embodiment of the present invention, the signal peptide comprises: (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2; (b) A derivative polypeptide derived from (a) in which one or more amino acids are substituted, deleted or added in the amino acid sequence defined in (a) while retaining the biological function of the sequence from which it originated.

[0007] In one embodiment of the present invention, the fusion partner comprises: (c) A polypeptide with an amino acid sequence as shown in any one of SEQ ID NO.3 to SEQ ID NO.11; (d) A derivative polypeptide derived from (c) in which one or more amino acids are substituted, deleted or added in the amino acid sequence defined in (c) while retaining the biological function of the sequence from which it originated.

[0008] In one embodiment of the present invention, the target polypeptide comprises: (e) A polypeptide with an amino acid sequence as shown in SEQ ID NO.13-16; (f) A derivative polypeptide derived from (e) in which one or more amino acids are substituted, deleted or added in the amino acid sequence defined in (e) while retaining the biological function of the sequence from which it originated.

[0009] In one embodiment of the present invention, the amino acid sequence of the signal peptide is as shown in any one of SEQ ID NO.1 or SEQ ID NO.2; and the amino acid sequence of the fusion partner is as shown in any one of SEQ ID NO.3 to SEQ ID NO.11.

[0010] In one embodiment of the present invention, the fusion protein further includes enzyme cleavage sites; the enzyme cleavage sites include enterokinase cleavage sites EK and ulp1 cleavage sites.

[0011] In one embodiment of the present invention, the enzyme cleavage site is an enterokinase cleavage site EK; the amino acid sequence of the enterokinase cleavage site is shown in SEQ ID NO.12.

[0012] In one embodiment of the present invention, the restriction site is the ulp1 restriction site.

[0013] In one embodiment of the present invention, the fusion protein comprises a signal peptide, a fusion chaperone, an enzyme cleavage site, and Exendin-4, which are sequentially linked.

[0014] In one embodiment of the present invention, the fusion protein is composed of a signal peptide, a fusion chaperone, an enzyme cleavage site, and Exendin-4 connected in sequence.

[0015] In one embodiment of the invention, the fusion protein further comprises a purification tag or other tag.

[0016] In one embodiment of the present invention, the number of substituted, deleted, or added amino acids in the derived polypeptide does not exceed five.

[0017] In one embodiment of the present invention, the number of substituted, deleted, or added amino acids in the derived polypeptide does not exceed three.

[0018] The present invention also provides a nucleic acid molecule that encodes the above-mentioned fusion protein.

[0019] The present invention also provides a recombinant plasmid comprising a nucleic acid molecule expressing the above-described fusion protein or comprising the nucleic acid molecule described above.

[0020] In one embodiment of the present invention, the vector of the recombinant plasmid includes at least one of a viral vector or a non-viral vector; the viral vector includes at least one of a flavivirus vector, a retrovirus vector, a bacteriophage vector, adenovirus vector, adeno-associated virus vector, vaccinia virus vector, hybrid virus vector, baculovirus vector, herpes simplex virus vector, or lentivirus vector; the non-viral vector includes a plasmid vector.

[0021] In one embodiment of the present invention, the plasmid vector includes pET28a plasmid, pET9a plasmid, pET21a plasmid, pET24a plasmid, pET30a plasmid and / or pET32a plasmid.

[0022] In one embodiment of the present invention, the recombinant plasmid is prepared by inserting the above-mentioned nucleic acid molecule into a vector to obtain a recombinant plasmid.

[0023] The present invention also provides a host cell that expresses the above-mentioned fusion protein.

[0024] In one embodiment of the present invention, the host cell includes fungi, bacteria, plant cells and / or animal cells.

[0025] In one embodiment of the present invention, the bacteria include Escherichia coli, Bacillus subtilis, Lactococcus lactis and / or Corynebacterium glutamicum.

[0026] In one embodiment of the present invention, the host cell is *Escherichia coli*. Preferably, the *Escherichia coli* is selected from BL21(DE3), BL21star(DE3), and BL21(DE3)pLys.

[0027] In one embodiment of the present invention, the method for preparing the host cell is as follows: transforming the above-mentioned recombinant plasmid into the host cell.

[0028] The present invention also provides a formulation comprising the above-described fusion protein.

[0029] In one embodiment of the present invention, the pharmaceutical preparation includes an oral preparation, a microneedle preparation, or an antibody-drug conjugate.

[0030] In one embodiment of the present invention, the oral preparation is in the form of capsules, tablets, or oral liquid.

[0031] In one embodiment of the invention, the microneedle formulation comprises soluble microneedles or a biodegradable polymer microneedle carrier for transdermal delivery of Exendin-4.

[0032] In one embodiment of the present invention, the antibody-drug conjugate is a covalent conjugate of Exendin-4 with an antibody or antibody fragment, used to extend the half-life or achieve targeted delivery.

[0033] The present invention also provides a method for preparing a target polypeptide, wherein the host cells are first inoculated into a culture medium for fermentation to obtain a fermentation broth, and then the target polypeptide is separated from the fermentation broth.

[0034] The present invention also provides the application of the above-mentioned fusion protein, nucleic acid molecule, recombinant plasmid, host cell, or method in the preparation of Exendin-4.

[0035] The present invention also provides the use of the above-mentioned fusion protein or the above-mentioned nucleic acid molecule or the above-mentioned recombinant plasmid or the above-mentioned host cell or the above-mentioned method in the production of drugs for the prevention and / or treatment of type 2 diabetes and related metabolic diseases.

[0036] The technical solution of this invention has the following advantages: This invention provides a fusion protein for increasing the expression level of Exendin-4, comprising a signal peptide, a fusion chaperone, and Exendin-4. To reduce the toxic side effects of Exendin-4 on host cells, this invention uses the signal peptide and fusion chaperone to secrete the Exendin-4 fusion protein into the periplasmic space and extracellular space of host cells, obtaining ultra-high yields of the Exendin-4 fusion protein, which shows great promise for the preparation of Exendin-4 based on bio-fermentation.

[0037] This invention achieves efficient mass production of Exendin-4, a recombinant polypeptide that can be produced in various pharmaceutical formulations. Specifically, the high-yield Exendin-4 of this invention can be used to prepare oral formulations, overcoming the inconvenience of traditional injection administration; it can be used in microneedle patches to achieve transdermal drug delivery and improve patient compliance; and it can also be combined with antibodies or other drug carriers to form long-acting or targeted delivery systems, thereby expanding the application potential of Exendin-4 in the treatment of diabetes and related metabolic diseases. Attached Figure Description

[0038] Figure 1 The results of agarose gel electrophoresis of the fusion proteins s7 / s8 expressed in the BL21(DE3)pLys expression system are shown. Detailed Implementation

[0039] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0040] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0041] Example 1: Construction of a fusion protein that enhances Exendin-4 expression This embodiment provides a fusion protein for increasing the expression level of Exendin-4 (hereinafter referred to as E4). The fusion protein is composed of a signal peptide, a fusion chaperone, an enzyme cleavage site and Exendin-4 connected in sequence. The specific signal peptide, fusion chaperone and enzyme cleavage site used in the fusion protein can be seen in Table 1.

[0042] Table 1. Fusion proteins and their signal peptides, fusion chaperones, and restriction enzyme sites.

[0043] The experimental procedure is as follows: The nucleotide sequence genes encoding the fusion proteins s1~s10, s12, s13, s15, s46, s50, s51, and s56 were synthesized and inserted into the NcoI+XhoI positions of the pET28a plasmid, respectively, to obtain recombinant plasmids pET28a-s1~10, pET28a-s12, pET28a-s13, pET28a-s15, pET28a-s46, pET28a-s50, pET28a-s51, and pET28a-s56 (gene synthesis and insertion were commissioned to Shanghai Sangon Biotech Co., Ltd.). The recombinant plasmids pET28a-s1~10, pET28a-s12, pET28a-s13, pET28a-s15, pET28a-s46, pET28a-s50, pET28a-s51, and pET28a-s56 were transformed into Escherichia coli, respectively. Escherichia coli BL21(DE3) or BL21star(DE3) or BL21(DE3)pLys (purchased from Tiangen Biotech (Beijing) Co., Ltd.) were used to obtain transformation products; the numbers of each transformation product and the fusion protein expressed by it are shown in Table 2.

[0044] Table 2 Conversion Products

[0045] *E4s14 is the control group that does not contain VNP peptides and fusion partners. The transformation product was spread onto LB solid medium containing 50 μg / mL kanamycin (LB solid medium formulation: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride), and incubated upside down in a 37℃ incubator for 12 h to obtain single clones of recombinant Escherichia coli. Two single clones of recombinant Escherichia coli were picked and inoculated into 10 mL of LB liquid medium containing 50 μg / mL kanamycin (LB liquid medium formulation: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride), and cultured with shaking at 37℃ and 200 rpm for 16 h to obtain bacterial culture. Take a sterile 1.5 mL EP container, add 750 μL of bacterial solution, then add 250 μL of sterile 60% (v / v) glycerol, mix well to obtain a bacterial suspension; label the EP container and store it in a -80℃ refrigerator. After removing the bacterial suspension from the EP tank, transfer it to fresh LB liquid medium at a volume ratio of 1:50 (bacterial suspension:LB liquid medium = 1:50) and incubate with shaking at 37°C and 200 rpm until OD (dose retardation). 600 When the α=1.0, 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) was added to the culture system, and induction culture was carried out at 37℃ and 200 rpm to obtain fermentation broth. The fermentation broth was collected at 0 h, 6 h and 20 h of induction culture, respectively. The fermentation broth was centrifuged, and the cell pellet and supernatant were collected for SDS-PAGE detection (SDS-PAGE detection method is described in the literature "Green, MR, & Sambrook, J. (2001). Molecular cloning: a laboratory manual. Analytical Biochemistry, 186(1), 182-183."). The grayscale scanning analysis of the SDS-PAGE gel image of the fusion protein was performed using analysis software to obtain the proportion of the expressed fusion protein to the total protein. The expression level of the fusion protein increased with the increase of induction time, and the expression level was about 10%-40% or more.

[0046] Example 2: A method for preparing Exendin-4 This embodiment provides a method for preparing Exendin-4, the method comprising the following steps: 1. Primary Seed Preparation Plate preparation: After removing the bacterial suspension (E4s7 / E4s8 fusion protein expression strain) from the EP container, serially dilute it 10⁻⁶ times with sterilized LB liquid medium. -5 Take 100 μL of the diluted bacterial suspension and spread it evenly on LB solid medium. Incubate at 37°C for 16 h. After the single and uniform colonies have grown, store at 4°C.

[0047] Primary activation: Pick a plump single colony from LB solid medium and inoculate it into an Erlenmeyer flask containing 50 mL of LB liquid medium, and add 50 μL of kanamycin stock solution (the concentration of kanamycin stock solution is 50 mg / mL).

[0048] Cultivation: After inoculation, place the Erlenmeyer flask in a constant temperature shaker, set the shaker speed to 250 rpm, and incubate at 30℃ for 9 h to obtain primary seed culture. Primary seed culture. OD 600 It is 1.4~1.6.

[0049] 2. Secondary seed preparation Inoculation: Remove the Erlenmeyer flask containing the primary seed culture from the constant temperature shaker, and inoculate 270 μL of the primary seed culture (inoculation amount 1‰, v / v) into the Erlenmeyer flask containing 270 mL of secondary seed culture medium in a clean workbench, and add 300 μL of kanamycin stock solution (the concentration of kanamycin stock solution is 50 mg / mL).

[0050] Cultivation: After inoculation, place the Erlenmeyer flask in a constant temperature shaker, set the shaker speed to 250 rpm, and incubate at 30℃ for 10 h to obtain secondary seed culture. Secondary seed culture. OD 600 The value is 3.9~4.1.

[0051] Microscopic examination: Remove the Erlenmeyer flask containing the secondary seed culture from the constant temperature shaker. Under clean bench conditions, aseptically sample 1 mL of the secondary seed culture, take 10 μL, and spread it on a glass slide for microscopic examination. Observe the size and morphology of the bacteria within the microscopic field of view; there should be no contaminating bacteria. Gram staining and microscopic examination: the bacteria should appear red and have the typical short rod-shaped morphology of Escherichia coli.

[0052] 3. Fermentation Preparations before fermentation (check the pipes for leaks); Check the equipment status and confirm that the equipment is available; Check steam pipes, valves, motors, power supplies, air filters, water pipes, etc. for leaks or disconnections; Clean the inside and outside walls of the fermentation tank with distilled water; Fermentation medium was added (fermentation medium formula: tryptone 12g / L, yeast extract 24g / L, dipotassium hydrogen phosphate 12.54g / L, potassium dihydrogen phosphate 2.31g / L, ammonium sulfate 3.30g / L, sodium chloride 0.50g / L and glucose 6g / L), and the pH electrode and dissolved oxygen electrode (DO electrode) were calibrated. Among them, pH electrode calibration (4.01, 7.00): Connect the pH electrode to the fermentation control system. First, place the pH electrode probe in 7.00. After the pH in the system displays 7.00 and the calibration is stable, place the pH electrode probe in 4.01. After the pH in the system displays 4.01 and the calibration is stable, place the pH electrode probe in 4.01. Dissolved oxygen electrode calibration: Zero-point calibration: Connect the dissolved oxygen electrode to the fermentation control system, place the electrode tip in a 5% (w / v, g / 100 mL) saturated sodium sulfite solution, and the calibration is complete when the system displays 0% or stabilizes near zero. Dissolved oxygen 100% calibration: At a temperature of 37℃, set the aeration rate to 10.0 SLPM and the stirring to 1000 rpm, start calibrating to 100% dissolved oxygen, and the calibration is complete after stabilization. Tighten the top cover bolts and clamp all the pipes, leaving only the vent for ventilation; Insert the probe into the fermenter; Place the fermentation tank into an autoclave for off-site sterilization; After sterilization, tighten the vent, turn on the motor and all probes, and turn on the cold water to cool it down; Inoculation: Under flame protection, aseptically transfer 2.2 mL of fermentation medium, 1 mL of magnesium sulfate solution (1 M concentration) and 200 mL of secondary seed culture into the fermenter, and connect the acid (1 M hydrochloric acid), alkali (1 M ammonia water), antifoam 204 (purchased from Sigma, A6426-500G) and fed-batch medium (fed-batch medium formula: 40% glycerol, 40 g / L tryptone and 40 g / L yeast extract, where % refers to volume percentage). Set the fermentation parameters, enter the fermentation batch number, and start fermentation; Temperature control during fermentation is explained in the induction phase. Dissolved oxygen control: The rotation speed is always linked to the aeration rate. The initial rotation speed is 300 rpm, and the maximum rotation speed is 800 rpm. The dissolved oxygen is controlled at 35% before induction and at 25% after induction. Adjustment timing: When lowering the fermenter temperature, the dissolved oxygen is also adjusted to 25%. Feeding: Add trace elements (1‰, v / v) to the fed-batch culture medium before fermentation; Feeding method: When the cell count in the fermenter reaches OD... 600Feeding was initiated at a rate of 27.6 mL / h from 12:00 to 14:00; after IPTG induction, the feeding rate was adjusted to 30 mL / h; the feeding rate was finely adjusted in a timely manner according to dissolved oxygen (continuously rising or falling) during fermentation and induction until fermentation was completed.

[0053] 4. Induction The culture temperature before induction was 37℃, and OD was induced. 600 The value is 70~75, the induction temperature is 30℃, and the OD is measured before reaching the induction value. 600 At 65°C, the temperature was adjusted to 30°C and the dissolved oxygen was adjusted to 32%. The inducing agent was added as follows: 0.1 mM was added initially, followed by 0.1 mM at 4-hour intervals, for a total of four additions, and the induction lasted for 20-22 hours.

[0054] 5. Harvesting (reconstitution) of bacterial cells and supernatant Sample testing: Induce fermentation by adding an inducer. Keep a sample after 0 hours of induction. After induction, take 1 mL of fermentation broth sample every 2 hours. Cell count: Analyze the OD value of the bacteria. 600 Calculate the final OD of the bacterial suspension by adding an appropriate amount of purified water. 600 Set the concentration to 10, then add an appropriate amount of 5×SDS Loading buffer, incubate at 100℃ for 5 min, centrifuge at 9000 rpm for 3 min, and take 13 μL of sample for SDS-PAGE electrophoresis (for proteins below 15 KD, use a small molecule gel). Supernatant: The gel running method is the same as for bacterial cell detection.

[0055] Fermentation broth retention method: Take 1 L from each tank, centrifuge at 9000 rpm and 4℃ for 20 min, and store the supernatant and precipitate at -20℃.

[0056] Protein expression detection of Exendin-4 cells: Take 5 mL of fermentation broth, centrifuge at 9000 rpm, 4℃ for 30 min, and pour the supernatant into a clean 50 mL centrifuge tube for later use; add 30 mL of PBS buffer to the cells, mix well, and sonicate for 50 min, centrifuge at 9000 rpm, 4℃ for 30 min, and collect the supernatant. Samples are then analyzed using SDS-PAGE and Lorry assays to determine purity and concentration.

[0057] After fermentation is complete, the fermentation liquid is removed, and the fermentation tank and experimental environment are cleaned and disinfected.

[0058] The agarose gel electrophoresis results of the fusion proteins s7 / s8 expressed in the E. coli expression system are shown in the figure below. Figure 1 .Depend on Figure 1It can be seen that the expression level of the fusion protein in the bacterial cells has reached its highest level, and the secretion of the fusion protein in the culture medium begins to increase significantly, reaching its peak at 19-21 hours after induction.

[0059] The fermentation results obtained from sample testing are as follows: OD of the final harvested fermentation broth 600 The concentration of the fusion protein s7 / s8 in the culture supernatant was 137-157, the cell concentration in the fermentation broth was 280-300 g / L, the protein expression level of the fusion protein s7 / s8 in the cells was 20-25 g / L, and the protein expression level of the fusion protein s7 / s8 secreted into the culture supernatant was 6-9 g / L.

[0060] Example 3 The fusion protein constructed in Example 1 was expressed and detected by shaking flask following the steps in Example 2. The results are shown in Table 3 below: Table 3. Expression results of fusion proteins

[0061] *E4s14 is the control group that does not contain VNP peptides and fusion partners. Table 1 shows that to obtain fusion proteins with high expression levels (>40%), the optimal choice is to use MrsB or DARP as fusion partners, binding to the VNP6 peptide, the EK restriction site, and the BL21(DE3)pLys strain. For fusion proteins with medium expression levels (>20% or >30%), GB1, TrxA, and StefinA can bind to VNP6 or VNP15, and the strains BL21(DE3)pLys or BL21star(DE3) are all suitable. The absence of a fusion partner (none) or fusion partners such as sumo, GcN4-v4, P53TD / E2K, Trpzip4, and VNP15 may result in low expression levels in some cases and are not recommended. For strains, BL21(DE3)pLys is particularly suitable for high expression systems. For restriction sites, EK is the preferred choice, while ulp1 is less effective.

[0062] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

[0063] Biological sequence: Signal peptide VNP6 (SEQ ID NO.1): MDVFKKGFSIADEGVVGAVEKTDQGVTEAAEKTKEGVM VNP15 (SEQ ID NO.2): MDVFKKGFSIADEGVVGAVE MrsB(SEQ ID NO.3): ANKPSAEELKKNLSEMQFYVTQNHGTEPPFTGRLLHNKRDGVYHCLICDAPLFHSQTKYDSGCGWPSFYEPVSEESIRYIKDLSHGMQRIEIRCGNCDAHLGHVFPDGPQPTGERYCVNSASLRFTDGENGEEING DARP(SEQ ID NO.4): SNDIGDEMQDAFVVEDFDTICKLILAGSSPNSADRRDGSPIILRAAVTRDLDMINFLIGQQADVDSRGPKGLTALHAAALYGFVEILQRLIEAGSDTNAKDAEGATPFALASKDQGATIEMAKILIAHGADKSVKNSQGRILLDVYKEITGKTYDGI TrxA(SEQ ID NO.5): SDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA GB1 (SEQ ID NO.6): YKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE StefinA (SEQ ID NO.7) IPGGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVVAGTNYYIKVRAGDNKYMHLKVFKSLPGQNEDLVLTGYQVDKNKDDELTGF sumo(SEQ ID NO.8): SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG Trpzip4 (SEQ ID NO.9): GEWTWDDATKTWTWTE GcN4-v4 (SEQ ID NO.10): RMKQLEDKVEELLSKNYHLENEVARLKKLVG P53TD / E2K (SEQ ID NO.11): GEYFTLQIRGRERRFEMFRKLNKALELKDAQA EK restriction site (SEQ ID NO.12): KR ulp1 restriction site: This restriction site is part of the three-dimensional structure of the entire SUMO.

[0064] E4 (SEQ ID NO.13): HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS Glucagon (1-29) (SEQ ID NO.14) HSQGTFTSDYSKYLDSRRAQDFVQWLMNT A(1-21)-AAK-B(1-29)-Human Insulin (SEQ ID NO.15) GIVEQCCTSICSLYQLENYCNAAKFVNQHLCGSHLVEALYLVCGERGFFYTPK Arg34GLP-l (9-37) (SEQ ID NO.16) EGTFTSDVSSYLEGQAAKEFIAWLVRGRG

Claims

1. A fusion protein for increasing the expression level of a target polypeptide, characterized in that, The fusion protein comprises a signal peptide, a fusion partner and a target polypeptide; The target polypeptide is selected from the group consisting of GLP-1 peptides (e.g. GLP-1 (7-37), GLP-1 (7-36) and analogs thereof such as R34GLP-l (9-37), glucagon peptides such as glucagon (1-29), insulin precursors (e.g. A(1-21)-AAK-B(1-29)-human insulin) and exendins (e.g. exendin-3 and -4); The signal peptide comprises a vesicle nucleating peptide VNP, preferably the signal peptide is VNP6, VNP15; Preferably, the fusion partner is selected from the group consisting of MrsB, DARP, TrxA, GB1, StefinA, P53TD / E2K, Trpzip4, sumo, GcN4-v4 or a combination thereof; preferably, the fusion partner is selected from the group consisting of MrsB, DARP, GB1, TrxA, StefinA or a combination thereof; further preferably, the fusion partner is selected from the group consisting of MrsB, DARP; Preferably, the signal peptide is selected from the group consisting of: (a) a polypeptide having an amino acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 2; (b) a derivative polypeptide derived from (a) by substitution, deletion or addition of one or more amino acids in the amino acid sequence defined in (a) and retaining the biological function of the original sequence; Preferably, the fusion partner is selected from the group consisting of: (c) a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NO. 3 to SEQ ID NO. 11; (d) a derivative polypeptide derived from (c) by substitution, deletion or addition of one or more amino acids in the amino acid sequence defined in (c) and retaining the biological function of the original sequence.

2. The fusion protein of claim 1, wherein, The target polypeptide is selected from the group consisting of: (e) a polypeptide having an amino acid sequence as set forth in SEQ ID NO. 13 to 16; (f) a derivative polypeptide derived from (e) by substitution, deletion or addition of one or more amino acids in the amino acid sequence defined in (e) and retaining the biological function of the original sequence; Preferably, the signal peptide has an amino acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 2; and the fusion partner has an amino acid sequence as set forth in any one of SEQ ID NO. 3 to SEQ ID NO. 11; Preferably, the fusion protein further comprises an enzyme cleavage site; preferably, the enzyme cleavage site comprises an enterokinase cleavage site EK, an upl1 cleavage site; Preferably, the enzyme cleavage site is an enterokinase cleavage site; the enterokinase cleavage site has an amino acid sequence as set forth in SEQ ID NO.

12.

3. The fusion protein of any of claims 1-2, wherein, The fusion protein comprises a signal peptide, a fusion partner, an enzyme cleavage site and Exendin-4 in sequence; preferably, the fusion protein consists of a signal peptide, a fusion partner, an enzyme cleavage site and Exendin-4 in sequence; Preferably, the fusion protein further comprises a purification tag or other tag. Preferably, the signal peptide comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO. 1; the fusion partner comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO. 3; and the Exendin-4 comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO.

13. Preferably, the signal peptide comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO. 1; the fusion partner comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO. 3; and the Exendin-4 comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO.

13.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein according to any one of claims 1-3.

5. A recombinant plasmid, characterized in that, The recombinant plasmid comprises the nucleic acid molecule expressing the fusion protein according to any one of claims 1-3 or comprises the nucleic acid molecule according to claim 4.

6. A host cell, characterized in that, The host cell expresses the fusion protein according to any one of claims 1-3.

7. A formulation characterized in that, The preparation comprises the fusion protein according to any one of claims 1-3; preferably, the pharmaceutical preparation comprises an oral preparation, a microneedle preparation or an antibody conjugated preparation.

8. A method of producing a polypeptide of interest, characterized by, The method comprises inoculating the host cell according to claim 6 into a culture medium for fermentation to obtain a fermentation liquor, and then isolating the target polypeptide from the fermentation liquor.

9. Use of the fusion protein according to any one of claims 1-3 or the nucleic acid molecule according to claim 4 or the recombinant plasmid according to claim 5 or the host cell according to claim 6 or the preparation according to claim 7 or the method according to claim 8 in the preparation of Exendin-4.

10. Use of the fusion protein according to any one of claims 1-3 or the nucleic acid molecule according to claim 4 or the recombinant plasmid according to claim 5 or the host cell according to claim 6 or the preparation according to claim 7 or the method according to claim 8 in the production of a drug for preventing and / or treating type 2 diabetes.

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