Recombinant escherichia coli efficient soluble expression semeglutide 9-37 aminopeptide and purification method

Abstract

The invention discloses a recombinant escherichia coli high-efficiency soluble expression semeglutide 9-37 amino peptide and a purification method, and belongs to the technical field of gene engineering. According to the invention, fusion protein mRNA folding is optimized, hairpin structures are reduced, and efficient soluble expression of fusion protein in escherichia coli is realized. The fusion protein is provided with an SUMO label and an enterokinase cleavage site, the fusion protein is purified through nickel ion affinity chromatography, the fusion protein is subjected to enterokinase specific recognition cleavage and then subjected to nickel ion affinity chromatography and gel filtration chromatography separation, Arg34GLP-1 (9-37) with the purity larger than or equal to 95% is obtained, the wet thallus amount is 150-200 g / L after fermentation culture, and the pure protein yield is 2-3 g / L. The invention provides a fusion expression and rapid purification method of the semeglutide Arg34GLP-1 (9-37) polypeptide, and a solid early-stage foundation is laid for industrial, efficient, green and low-cost preparation of the semeglutide.

Description

Technical Field

[0001] The invention relates to a method for efficiently solublely expressing semaglutide 9-37 aminopeptide by recombinant Escherichia coli and a purification method, and belongs to the technical field of genetic engineering. Background Art

[0002] Glucagon-like peptide-1 (GLP-1) is a peptide hormone produced by intestinal L cells. It acts on pancreatic β cells, promotes the synthesis and secretion of insulin, stimulates the proliferation and differentiation of pancreatic β cells, inhibits the apoptosis of pancreatic β cells, increases the number of pancreatic β cells, protects pancreatic function, promotes the secretion of somatostatin, and inhibits the secretion of glucagon. It acts on pancreatic α cells, inhibits the release of glucagon, and reduces the release of glycogen. It enhances insulin sensitivity, slows down gastric emptying, acts on the hypothalamus to suppress appetite, reduce weight, and achieve the effect of lowering blood sugar.

[0003] Glucagon-like peptide-1 receptor agonist (GLP-1RA) is a new type of hypoglycemic drug in recent years. It activates GLP-1 receptors, enhances insulin secretion in a glucose-dependent manner, inhibits glucagon secretion, delays gastric emptying, suppresses appetite and reduces food intake, thereby achieving the effects of lowering blood sugar and losing weight.

[0004] Semaglutide, also known as semaglutide, is a GLP-1 analog that can mimic the function of GLP-1 in the human body and is a GLP-1 receptor agonist. Structurally, the eighth alanine in GLP-1 (7-37) is replaced by α-aminoisobutyric acid, the thirty-fourth lysine is replaced by arginine, and the twenty-sixth lysine is acylated with a fatty acid side chain. Developed and produced by Novo Nordisk of Denmark, after these modifications, compared with other GLP-1 analogs, it has a higher affinity for albumin, masks the hydrolysis site of dipeptidyl peptidase (DDP-4), has good enzyme stability, a long half-life, and better blood sugar lowering effect. Semaglutide has significant hypoglycemic and weight loss effects, and is safe, and is currently the best drug for weight loss and treatment of type 2 diabetes.

[0005] Novo Nordisk's semaglutide is produced by semi-recombinant technology. The semaglutide precursor molecule (R34) GLP-1 (11-37) is heterologously expressed in Saccharomyces cerevisiae. The expression product is a fusion protein of the MFα1* leader sequence, the Lys-Arg cleavage site for the binary processing endopeptidase Kex2, and (R34) GLP-1 (11-37). After separation and purification, the precursor molecule is chemically coupled with the N-terminal amino acid protrusion containing non-protein amino acids and the acylated fatty acid side chain on the lysine, and semaglutide is separated and purified.

[0006] Other methods used in research, such as solid-phase chemical synthesis of peptides, form peptides by sequentially connecting amino acids, but have problems such as large feed ratio, high cost, side reactions, impure products, and large amounts of organic solvents. Biological synthesis of precursors has problems such as difficulty in expression, extremely low soluble expression levels, mostly inclusion bodies, long inclusion body renaturation time, large volume, low concentration, use of large amounts of urea, difficulty in separation and purification, and long culture time.

[0007] The series of semaglutide core 29 peptide fusion molecules based on the Escherichia coli expression system and their applications recorded in the publication number CN118620091A disclose that the inclusion body expression promoting label is used to increase the content of inclusion bodies containing semaglutide core 29 peptide fusion molecules, and inclusion bodies are water-insoluble protein particles, which are usually formed in prokaryotes such as Escherichia coli. Due to its high density and insolubility, inclusion bodies exist in the form of aggregates in cells and are difficult to be directly used for functional research or application. The proteins in the inclusion bodies are usually misfolded and cannot fully form the correct three-dimensional structure, so they have no biological activity or low biological activity. These proteins accumulate in cells and may interfere with normal cell metabolism. Although the inclusion body protein contains the correct amino acid sequence, it usually has no biological activity or very low biological activity due to misfolding. Even after renaturation treatment, its biological activity may be unstable or low; therefore, it is urgent to develop a system and technical method for soluble expression of semaglutide. Summary of the invention

[0008] Since the biological synthesis of semaglutide precursor polypeptide has problems such as difficult expression, extremely low soluble expression level, mostly inclusion bodies, long inclusion body renaturation time, large volume, low concentration, use of large amounts of urea, and difficulty in separation and purification, the present invention aims to express the semaglutide precursor polypeptide Arg34GLP-1 (9-37) efficiently and soluble through microbial heterologous expression technology, and to achieve separation and purification of the target protein.

[0009] The present invention provides a fusion protein comprising a semaglutide precursor polypeptide. The fusion protein comprises a lytic tag, an enzyme cleavage site and the semaglutide precursor polypeptide Arg34GLP-1(9-37).

[0010] In one embodiment of the present invention, the fusion protein uses His, Sumo, GST, DsbA, TrxA, Intein, ELP, NusA, Flag or MBP as a solubility-promoting tag.

[0011] In one embodiment of the present invention, the fusion protein uses the enterokinase site DDDDK, the thrombin site LVPRGS, the TEV protease site ENLYFQG, the SUMO protease site EQIGG or the HRV 3C site LEVLFQGP as the restriction site.

[0012] The present invention provides a nucleic acid molecule encoding a fusion protein containing semaglutide precursor polypeptide Arg34GLP-1 (9-37), and the sequence of the nucleic acid molecule is shown in SEQ ID NO.4.

[0013] The present invention also provides a method for increasing the expression amount of a fusion protein containing the semaglutide precursor polypeptide Arg34GLP-1 (9-37), the method comprising mutating a nucleic acid molecule of a fusion protein containing the semaglutide precursor polypeptide Arg34GLP-1 (9-37) having a nucleotide sequence as shown in SEQ ID NO.3 to obtain a nucleic acid molecule of a fusion protein containing the semaglutide precursor polypeptide Arg34GLP-1 (9-37) having a nucleotide sequence as shown in SEQ ID NO.4.

[0014] The present invention provides an expression cassette, wherein the expression cassette contains a nucleic acid molecule of a fusion protein of the semaglutide precursor polypeptide Arg34GLP-1(9-37) as shown in SEQ ID NO.4.

[0015] The present invention provides a recombinant vector, wherein the recombinant vector contains a nucleic acid molecule of a fusion protein of the semaglutide precursor polypeptide Arg34GLP-1(9-37) as shown in SEQ ID NO.4.

[0016] In one embodiment of the present invention, the recombinant vector is obtained by cloning the nucleic acid molecule into an expression vector.

[0017] In one embodiment of the present invention, the recombinant vector is an expression vector using pET series vectors, pCDF series vectors, pRSF series vectors or pGEX series vectors.

[0018] In one embodiment of the present invention, the recombinant vector is an expression vector using pET-28(a), pET-39(b), pET-26(b), pET-21(a), pRSFDuet-1, pCDFDuet-1 or pGEX-6P-1.

[0019] The present invention provides a recombinant strain, wherein the recombinant strain contains a nucleic acid molecule of a fusion protein of the semaglutide precursor polypeptide Arg34GLP-1(9-37) as shown in SEQ ID NO.4.

[0020] In one embodiment of the present invention, the recombinant strain is obtained by introducing the nucleic acid molecule or the expression cassette or the recombinant vector into Escherichia coli.

[0021] In one embodiment of the present invention, the recombinant cell uses Escherichia coli, Bacillus subtilis, Bacillus licheniformis or Pichia pastoris as a host cell.

[0022] The present invention also provides the use of the nucleic acid molecule with the sequence shown in SEQ ID NO.4 or the expression cassette or recombinant vector or recombinant strain or transgenic cell line in the preparation of semaglutide core 29 peptide; the amino acid sequence of the semaglutide core 29 peptide is shown in SEQ ID NO.1.

[0023] The present invention also provides a recombinant strain that solublely expresses a fusion protein containing the semaglutide precursor polypeptide Arg34GLP-1 (9-37), wherein the recombinant strain uses Escherichia coli as an expression host and expresses a nucleic acid molecule encoding the fusion protein containing the semaglutide precursor polypeptide Arg34GLP-1 (9-37); the sequence of the nucleic acid molecule is shown in SEQ ID NO.4.

[0024] In one embodiment of the present invention, the recombinant strain uses pET series vectors, pCDF series vectors, pRSF series vectors or pGEX series vectors as expression vectors.

[0025] In one embodiment of the present invention, the recombinant strain uses pET-28(a), pET-39(b), pET-26(b), pET-21(a), pRSFDuet-1, pCDFDuet-1 or pGEX-6P-1 as an expression vector.

[0026] In one embodiment of the present invention, the recombinant Escherichia coli is a fusion protein shown in SEQ ID NO.4 expressed in Escherichia coli BL21 (DE3) using pET-28a (+) as a vector.

[0027] The present invention also provides a method for constructing the recombinant Escherichia coli, comprising the following steps:

[0028] The fusion protein gene was connected to the vector pET-28a(+), and the obtained recombinant expression vector was transformed into E. coli BL21(DE3) to obtain the recombinant bacteria.

[0029] The present invention also provides a method for soluble expression of a fusion protein containing the semaglutide precursor polypeptide Arg34GLP-1 (9-37), which is prepared by fermenting the above-mentioned recombinant strain.

[0030] In one embodiment of the present invention, the recombinant cell uses E. coli BL21 (DE3) as an expression host.

[0031] In one embodiment of the present invention, the recombinant Escherichia coli is inoculated into a culture medium at a culture temperature of 37°C until 0D 600 When the concentration reached 0.6-0.8, the expression was induced under the conditions of 0.2 mM IPTG, 37°C induction temperature and 12 h induction time.

[0032] In one embodiment of the present invention, the culture medium formula is 10 g / L peptone, 5 g / L yeast powder, and 10 g / L sodium chloride.

[0033] The present invention also provides a method for purifying a fusion protein containing the semaglutide precursor polypeptide Arg34GLP-1 (9-37).

[0034] In one embodiment of the present invention, the recombinant cells after the induction culture are collected, and the impurity proteins are removed by affinity chromatography, reversible phase transition cycle or high temperature to obtain the pure fusion protein.

[0035] In one embodiment of the present invention, the elution conditions of nickel ion affinity chromatography are 50 mM imidazole for eluting impurities and 500 mM imidazole for eluting target proteins.

[0036] The present invention also provides a method for cutting a fusion protein containing the semaglutide precursor polypeptide Arg34GLP-1 (9-37).

[0037] In one embodiment of the present invention, the pure fusion protein is cleaved by a protease.

[0038] In one embodiment of the present invention, the protease is Sumo protease Ulp, enterokinase EK, thrombin, TEV protease or Prescission protease.

[0039] In one embodiment of the present invention, the amount of enterokinase added is 1 U / 50 mg, and the incubation is at 4° C. for 2 h.

[0040] The present invention also provides a method for purifying semaglutide precursor polypeptide Arg34GLP-1(9-37).

[0041] In one embodiment of the present invention, the sample after protease cleavage is subjected to affinity chromatography, ion exchange chromatography, gel filtration chromatography, Superdex peptide 10 / 300GL or high performance liquid chromatography to obtain pure Arg34GLP-1 (9-37) polypeptide.

[0042] In one embodiment of the present invention, the sample after fusion protease cleavage is passed through a nickel column to bind to impurities and the flow-through is collected. The flow-through is separated by Superdex peptide 10 / 300GL to obtain pure Arg34GLP-1 (9-37) polypeptide.

[0043] The present invention also provides the use of the above fusion protein, or the above gene, or the above recombinant vector, or the above recombinant cell in preparing semaglutide or a drug containing semaglutide.

[0044] In one embodiment of the present invention, the method is used to efficiently synthesize the semaglutide precursor polypeptide Arg34GLP-1(9-37).

[0045] In one embodiment of the invention, the product is a polypeptide.

[0046] The present invention also provides a method for preparing semaglutide core 29 peptide by soluble expression, the method comprising the following steps: fermenting and culturing the above-mentioned recombinant strain, collecting the bacteria, crushing, purifying and enzymatically digesting them to obtain the semaglutide core 29 peptide; the amino acid sequence of the semaglutide core 29 peptide is shown in SEQ ID NO.1.

[0047] The present invention also provides a method for increasing the expression amount of a fusion protein containing semaglutide precursor polypeptide Arg34GLP-1 (9-37) soluble in Escherichia coli, the method comprising: mutating a nucleic acid molecule of a fusion protein containing semaglutide precursor polypeptide Arg34GLP-1 (9-37) having a nucleotide sequence as shown in SEQ ID NO.3 to obtain a nucleic acid molecule of a fusion protein containing semaglutide precursor polypeptide Arg34GLP-1 (9-37) having a nucleotide sequence as shown in SEQ ID NO.4; and expressing the nucleic acid molecule of a fusion protein containing semaglutide precursor polypeptide Arg34GLP-1 (9-37) having a nucleotide sequence as shown in SEQ ID NO.4 in Escherichia coli.

[0048] The present invention also provides the use of the above nucleic acid molecule, or the above method, or the above expression cassette, or recombinant vector, or recombinant strain, or transgenic cell line, or the above recombinant strain, or the fusion protein obtained by the above method in any of the following:

[0049] (a) improving the yield and / or production of the semaglutide backbone; and / or

[0050] (b) increasing the expression level of the semaglutide backbone; and / or

[0051] (c) preparing a product containing a semaglutide backbone.

[0052] In one embodiment of the present invention, (1) the amino acid sequence as shown in SEQ ID NO: 1; or

[0053] (2) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (1); or

[0054] (3) A sequence having a homology of 90% or more to the amino acid sequence shown in (1).

[0055] In one embodiment of the present invention, the semaglutide backbone includes but is not limited to the semaglutide core 29 peptide; the amino acid sequence of the semaglutide core 29 peptide is shown in SEQ ID NO.1.

[0056] Beneficial Effects

[0057] (1) The present invention obtains a fusion protein with high efficiency and soluble expression by expressing a fusion protein containing the semaglutide precursor polypeptide Arg34GLP-1 (9-37) in Escherichia coli, thereby solving the problem that small peptides are difficult to express soluble in bacteria and avoiding the problems of long inclusion body renaturation time, large volume, low concentration, and use of a large amount of urea;

[0058] (2) The fusion protein has an enterokinase cleavage site, which can be specifically recognized by enterokinase and cleaved by the C-terminus of DDDDK, thereby achieving the cleavage and separation of the semaglutide precursor polypeptide Arg34GLP-1(9-37) and the N-terminal fusion peptide, and does not contain any redundant amino acid residues.

[0059] (3) After enzyme cleavage, the N-terminal fusion peptide with His-tag can bind to the nickel column medium. The molecular weight of enterokinase and the fusion tag is very different from that of the small peptide, and can be separated by Superdex peptide10 / 300GL to achieve the separation and purification of semaglutide precursor peptide Arg34GLP-1(9-37). BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 : SDS-PAGE of fusion protein-TRXA expression; M: marker, 1-4: supernatants of cell disruption at different expression temperatures, 5-8: precipitates of cell disruption at different expression temperatures.

[0061] Figure 2: SDS-PAGE of fusion protein-GST expression; M: marker, 1, 3, 5, 7, 9: supernatants of cell disruption at different expression temperatures, 2, 4, 6, 8, 10: precipitates of cell disruption at different expression temperatures.

[0062] Figure 3 : SDS-PAGE of fusion protein-SUMO expression; M: marker, 1-4: supernatants of cells broken at different expression temperatures, 5-8: precipitates of cells broken at different expression temperatures.

[0063] Figure 4 : SDS-PAGE of fusion proteins expressed at different induction temperatures; M: marker, 1: supernatant of cell disruption induced at 17℃, 2: supernatant of cell disruption induced at 20℃, 3: supernatant of cell disruption induced at 28℃, 4: supernatant of cell disruption induced at 30℃, 5: supernatant of cell disruption induced at 37℃, 6: precipitate of cell disruption induced at 37℃, 7: precipitate of cell disruption induced at 30℃, 8: precipitate of cell disruption induced at 28℃, 9: precipitate of cell disruption induced at 20℃, 10: precipitate of cell disruption induced at 17℃.

[0064] Figure 5 : SDS-PAGE image of fusion protein expressed at different inducer concentrations; M: marker, 1: 0.1mM induced cell disruption supernatant, 2: 0.2mM induced cell disruption supernatant, 3: 0.3mM induced cell disruption supernatant, 4: 0.4mM induced cell disruption supernatant, 5: 0.5mM induced cell disruption supernatant, 6: 0.1mM induced cell disruption precipitate, 7: 0.2mM induced cell disruption precipitate, 8: 0.3mM induced cell disruption precipitate, 9: 0.4mM induced cell disruption precipitate, 10: 0.5mM induced cell disruption precipitate.

[0065] Figure 6 : SDS-PAGE of the fusion protein after nickel column purification; M: marker, 1: protein sample after nickel column purification.

[0066] Figure 7 : SDS-PAGE image of fusion protein digestion; M: low molecular weight marker, 1: pure fusion protein, 2: sample of fusion protein after enterokinase digestion.

[0067] Figure 8 : SDS-PAGE of protein purification; M: marker, 1: fusion protein, 2: sample after enzyme digestion, 3-4: flow-through after nickel column digestion, 5-6: samples of corresponding molecular weight were collected after the flow-through was separated by Superdex peptide 10 / 300GL. DETAILED DESCRIPTION

[0068] Technical terms:

[0069] Semaglutide precursor polypeptide Arg34GLP-1(9-37): It is a GLP-1 analogue, in which the lysine at position 34 in GLP-1(9-37) is replaced with arginine. Acylation of a fatty acid side chain on the lysine at position 26 and connection of His-Aib to the N-terminus can form semaglutide. Semaglutide has significant hypoglycemic and weight loss effects and good safety. It is currently the best drug for weight loss and treatment of type 2 diabetes.

[0070] Expression: The term "expression" includes any step involved in the production of the fusion protein including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0071] Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a fusion protein of the present invention and is operably linked to control sequences that provide for its expression.

[0072] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0073] The host cell can be any cell useful in the recombinant production of the fusion protein, such as a prokaryotic cell or a eukaryotic cell.

[0074] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanic Bacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Mycobacterium, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0075] The host cell may also be a eukaryotic organism, such as a mammalian, insect, plant, or fungal cell.

[0076] The culture medium involved in the following examples is as follows:

[0077] LB liquid medium: sodium chloride 10 g / L, peptone 10 g / L, yeast powder 5 g / L.

[0078] TB liquid culture medium: peptone 12 g / L, yeast powder 24 g / L, glycerol 4 mL / L, potassium dihydrogen phosphate 2.2 g / L, and potassium dihydrogen phosphate 9.4 g / L.

[0079] TY liquid culture medium: peptone 12 g / L, yeast powder 8 g / L, tripotassium phosphate 4 g / L, sodium chloride 3 g / L, citric acid 2.1 g / L, ammonium ferric citrate 0.3 g / L, ammonium sulfate 2.5 g / L, magnesium sulfate 0.5 g / L, glycerol 10 mL / L.

[0080] Feed medium: peptone 25 g / L, yeast powder 50 g / L, glycerol 400 mL / L.

[0081] The method for detecting the expression level of the semaglutide precursor polypeptide Arg34GLP-1(9-37) protein involved in the following examples is as follows:

[0082] After the recombinant E. coli was cultured and expressed the fusion protein, the bacteria were collected, the cells were ultrasonically disrupted, the supernatant and the precipitate were separated by centrifugation, and the grayscale of the protein electrophoresis bands was calculated by SDS-PAGE electrophoresis using ImageJ to obtain the target protein content, and the target protein amount was calculated in combination with the total protein content.

[0083] The lytic tags involved in the following examples are shown in Table 1:

[0084] Table 1: Solubility-enhancing tags

[0085]

[0086] The present invention provides a first implementation case: use of a tag protein in preparing a semaglutide backbone; the semaglutide backbone has:

[0087] (1) the amino acid sequence shown in SEQ ID NO: 1; or

[0088] (2) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (1); or

[0089] (3) A sequence having a homology of 90% or more to the amino acid sequence shown in (1).

[0090] The label protein includes: the fusion protein uses His, Sumo, GST, DsbA, TrxA, Intein, ELP, NusA, SUMO label, Flag or MBP as a solubility-promoting label.

[0091] A preferred lytic tag is the SUMO tag.

[0092] The present invention provides a second implementation example: a nucleic acid molecule encoding a fusion protein containing the semaglutide precursor polypeptide Arg34GLP-1(9-37), the sequence of the nucleic acid molecule being shown in SEQ ID NO.4.

[0093] The present invention provides a third implementation example: an expression cassette or a recombinant vector or a recombinant strain or a transgenic cell line, wherein the expression cassette or the recombinant vector or the recombinant strain or the transgenic cell line contains a nucleic acid molecule shown in SEQ ID NO.4.

[0094] The recombinant vector is obtained by cloning the nucleic acid molecule into an expression vector;

[0095] Preferably, the recombinant vector is an expression vector using pET series vectors, pCDF series vectors, pRSF series vectors or pGEX series vectors;

[0096] Preferably, the recombinant vector is an expression vector using pET-28(a), pET-39(b), pET-26(b), pET-21(a), pRSFDuet-1, pCDFDuet-1 or pGEX-6P-1.

[0097] Preferably, the recombinant strain is obtained by introducing the nucleic acid molecule or the expression cassette or the recombinant vector into bacteria or fungi.

[0098] The present invention provides a fourth implementation case: use of a nucleic acid molecule with a sequence as shown in SEQ ID NO.4 or the above expression cassette or recombinant vector or recombinant strain or transgenic cell line in preparing a semaglutide backbone; the semaglutide backbone has:

[0099] (1) the amino acid sequence shown in SEQ ID NO.1; or

[0100] (2) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (1); or

[0101] (3) A sequence having a homology of 90% or more to the amino acid sequence shown in (1).

[0102] The present invention provides a fifth implementation example: a recombinant Escherichia coli strain, wherein the recombinant strain uses Escherichia coli as an expression host and expresses a nucleic acid molecule having a sequence as shown in SEQ ID NO.4;

[0103] Preferably, the recombinant strain uses pET series vectors, pCDF series vectors, pRSF series vectors or pGEX series vectors as expression vectors;

[0104] Preferably, the recombinant strain uses pET-28(a), pET-39(b), pET-26(b), pET-21(a), pRSFDuet-1, pCDFDuet-1 or pGEX-6P-1 as an expression vector.

[0105] The present invention provides a sixth implementation case: a method for soluble expression of a fusion protein containing the semaglutide precursor polypeptide Arg34GLP-1(9-37), the method comprising fermenting the recombinant strain in the third or fifth implementation case to obtain the fusion protein.

[0106] The present invention provides a seventh implementation case: a method for preparing semaglutide core 29 peptide by soluble expression, the method comprising the following steps: fermenting and culturing the recombinant strain in the third or fifth implementation case, collecting the bacteria, crushing, purifying, and enzymatically digesting them to obtain the semaglutide core 29 peptide; the amino acid sequence of the semaglutide core 29 peptide is shown in SEQ ID NO.1.

[0107] The present invention provides an eighth implementation case: a method for increasing the expression level of a fusion protein containing semaglutide precursor polypeptide Arg34GLP-1(9-37) soluble in Escherichia coli, the method comprising using Escherichia coli to express a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.4.

[0108] The present invention provides a ninth embodiment: use of a nucleic acid molecule having a sequence as shown in SEQ ID NO.4, or an expression cassette or a recombinant vector or a recombinant strain or a transgenic cell line in the third embodiment, or a recombinant strain in the third or fifth embodiment, or a fusion protein obtained by the method in the sixth to seventh embodiments in any of the following:

[0109] (a) improving the yield and / or production of the semaglutide backbone; and / or

[0110] (b) increasing the expression level of the semaglutide backbone; and / or

[0111] (c) preparing a product containing a semaglutide backbone.

[0112] The semaglutide main chain has:

[0113] (1) the amino acid sequence shown in SEQ ID NO.1; or

[0114] (2) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (1); or

[0115] (3) A sequence having a homology of 90% or more to the amino acid sequence shown in (1).

[0116] Example 1: Screening of lytic tags

[0117] The specific steps are as follows:

[0118] 1. Preparation of fusion protein

[0119] The fusion protein obtained by fusing SUMO tag / GST tag / TRXA tag (sequence shown in Table 1), enterokinase cleavage site (sequence: DDDDK), and Arg34GLP-1 (9-37) amino acid sequence (SEQ ID NO.1) was sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis, and after codon optimization according to the preference of Escherichia coli, fusion protein-SUMO, fusion protein-GST, and fusion protein-TRXA were prepared respectively.

[0120] The amino acid sequence of Arg34GLP-1(9-37) is as follows (SEQ ID NO.1): EGTFTSDVSSYLEGQAAKEFIAWLVRGRG.

[0121] 2. Construction of recombinant vector

[0122] The stop codon TAA was added to the C-terminus of the gene of each fusion protein after codon optimization prepared in step 1, and cloned into the vector pET-28a(+), the cloning site was BamH I / Xho I, the vector resistance was kanamycin resistance, and the recombinant plasmids were obtained, respectively named: pET28a-GLP(9-37)-SUMO, pET28a-GLP(9-37)-GST, pET28a-GLP(9-37)-TRXA, and stored at -20°C. The His tag of the fusion protein is already on the vector.

[0123] 3. Preparation of recombinant strains and protein expression

[0124] (1) Take 1 μL of the recombinant plasmid obtained in step 2 and add it to 100 μL of E. coli BL21 (DE3) competent cell suspension. After ice bathing for 30 minutes, place it in a 42°C metal bath for heat shock for 90 seconds. After heat shock, quickly place it on ice for 3-5 minutes. Add 700 μL of LB liquid culture medium to the centrifuge tube and shake and culture it at 37°C, 200 rpm for 1 hour. Take 50 μL of bacterial liquid and spread it on an LB solid culture medium plate containing 50 μg / mL kanamycin sulfate. Invert it in a 37°C constant temperature incubator and culture it overnight (about 10 hours); obtain the following recombinant strains:

[0125] E.coli BL21(DE3) / pET28a-GLP(9-37)-SUMO, E.coli BL21(DE3) / pET28a-GLP(9-37)-GST, E.coli BL21(DE3) / pET28a-GLP(9-37)-TRXA.

[0126] (2) Pick the positive clones obtained in step (1) and place them in 5 mL LB liquid culture medium test tubes containing 50 μg / mL kanamycin sulfate, and culture them at 37° C., 200 rpm, with shaking for about 8 h to prepare seed solution.

[0127] The above seed solution was inoculated into a 100 mL LB liquid medium shake flask containing 50 μg / mL kanamycin sulfate at a 1% (v / v) inoculation rate and cultured at 37°C, 200 rpm, until the OD 600 After reaching 0.6-0.8 (about 2 hours), add 0.2mM IPTG and induce at 17-37°C. After about 10 hours of induction culture, collect the bacteria by centrifugation at 8,000×g for 10 minutes and wash twice with saline.

[0128] The cells were resuspended in protein purification buffer A (20 mM Tris, 150 mM NaCl, pH 8.0), disrupted with an ultrasonic disruptor in an ice bath, and the disrupted liquid was centrifuged at 10,000×g and 4°C for 30 min to obtain the supernatant.

[0129] The protein expression in the supernatant was verified by SDS-PAGE. Figure 1-Figure 3 shown.

[0130] The results showed that the protein was expressed soluble when it contained SUMO tag, and the target protein was not expressed when it contained TRXA or GST tag.

[0131] Finally, a fusion protein-SUMO (named as fusion protein in subsequent examples) containing a SUMO tag, an enterokinase cleavage site (sequence: DDDDK), and an amino acid sequence of Arg34GLP-1 (9-37) was obtained. The amino acid sequence of the fusion protein-SUMO is as follows (SEQ ID NO.2):

[0132] MGSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGGDDDDKEGTFTSDVSSYLEGQAAKEFIAWLVRGRG

[0133] The nucleotide sequence of the codon-optimized fusion protein-SUMO is as follows (SEQ ID NO.3):

[0134] ATGGGGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTG TACGACGGTATTAGAATTCAAGCTGATCAGGCCCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACCGCGAACAGATTGGAGGTGATGATGATGATAAAGAAGGTACCTTCACCTCTGATGTTTCTAGCTACCTGGAAGGTCAGGCGGCGAAAGAATTCATCGCGTGGCTGGTTCGTGGTCGTGGTTAA

[0135] Example 2: Construction of recombinant bacteria after optimization of fusion protein-SUMO nucleotide sequence

[0136] The codon-optimized fusion protein-SUMO nucleotide sequence obtained in Example 1 (SEQ ID NO. 3, named as fusion protein-SUMO-WT) was mutated to further optimize the sequence.

[0137] The specific steps are as follows:

[0138] (1) The 1st to 81st bases of the fusion protein SUMO-WT were mutated to obtain the optimized fusion protein mSUMO, the sequence of which is as follows (SEQ ID NO. 4):

[0139] ATGGGaTCaGAtTCaGAAGTCAATCAAGAAGCaAAGCCAGAaGTaAAGCCAGAAGTaAAaCCaGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTG TACGACGGTATTAGAATTCAAGCTGATCAGGCCCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACCGCGAACAGATTGGAGGTGATGATGATGATAAAGAAGGTACCTTCACCTCTGATGTTTCTAGCTACCTGGAAGGTCAGGCGGCGAAAGAATTCATCGCGTGGCTGGTTCGTGGTCGTGGTTAA

[0140] (2) Construction of recombinant plasmid

[0141] Add the stop codon TAA to the C-terminus of the gene of the fusion protein-SUMO-1 prepared in step (1), clone it into the vector pET-28a(+), the cloning site is BamH I / Xho I, the vector resistance is kanamycin resistance, and obtain the recombinant plasmid pET28a-GLP(9-37)-mSUMO;

[0142] The primers involved are shown in Table 2:

[0143] Table 2: Primers

[0144]

[0145] The reaction system for PCR amplification was: 2× PrimeSTAR 25 μL, pET28a-GLP(9-37) plasmid 1 μL, ddH2O 10 μL, and primers 2 μL each. PCR program: 98°C 5 min; 98°C 30 s, 55°C 30 s, 72°C 30 s, 30 cycles; 72°C 10 min, 16°C 10 min.

[0146] After PCR, add 5.5μL 10×Quickcut Buffer to each PCR tube and mix well. Use 1% agarose gel electrophoresis to verify whether the band size is correct, and use the kit to purify the product. Add 10μL of the product to 100μL E.coli BL21 (DE3) competent cells, ice bath for 30 minutes, place in a 42℃ metal bath for heat shock for 90s, and quickly place on ice for 3-5 minutes after heat shock. Add 700μL LB liquid culture medium to the centrifuge tube, and shake and culture at 37℃, 200rpm for 1h. Centrifuge at 5,000×g, 4℃ for 5min, collect the bacteria, pour out the culture medium, and resuspend the bacteria with about 50-100μL remaining. Take 50μL of the bacterial solution and spread it on the LB solid culture medium plate containing 50μg / mL kanamycin sulfate. Invert and culture overnight in a 37℃ constant temperature incubator (about 10h). A single colony was picked and placed in a 5 mL LB liquid culture medium test tube containing 50 μg / mL kanamycin sulfate, and cultured at 37°C, 200 rpm, with shaking for 10 to 12 h, and the plasmid was extracted for sequencing.

[0147] Finally, the recombinant plasmid pET28a-GLP(9-37)-mSUMO was obtained and named: pET28a-mGLP(9-37).

[0148] (3) Construction of recombinant bacterial strains

[0149] Take 1μL of plasmid and add it to 100μL of E.coli BL21 (DE3) competent cell suspension. After ice bath for 30min, place it in a 42℃ metal bath for 90s. After heat shock, quickly place it on ice for 3-5min. Add 700μL of LB liquid culture medium to the centrifuge tube and shake and culture it at 37℃, 200rpm for 1h. Take 50μL of bacterial liquid and spread it on the LB solid culture medium plate containing 50μg / mL kanamycin sulfate. Invert it in a 37℃ constant temperature incubator and culture it overnight (about 10h).

[0150] The recombinant strain E. coli BL21 (DE3) / pET28a-mGLP (9-37) was obtained.

[0151] Example 3: Expression of recombinant fusion protein

[0152] The specific steps are as follows:

[0153] (1) Pick the positive clone single colonies prepared in Examples 1 to 2 (the strain E. coli BL21 (DE3) / pET28a-GLP (9-37) -SUMO-WT expressing the protein before mutation, and the strain E. coli BL21 (DE3) / pET28a-mGLP (9-37) expressing the protein after mutation) respectively and place them in a 5 mL LB liquid culture medium test tube containing 50 μg / mL kanamycin sulfate, and culture at 37 ° C, 200 rpm, and shake for about 8 hours to prepare a seed solution.

[0154] (2) The above seed solution was inoculated into 100 mL LB liquid medium containing 50 μg / mL kanamycin sulfate at a 1% (v / v) inoculum volume and cultured at 37°C, 200 rpm, and shaken until OD 600 After reaching 0.6-0.8 (about 2 hours), 0.2 mM IPTG was added and induced at 37°C. After about 10 hours of induction culture, the cells were centrifuged at 8,000×g for 10 minutes, and the cells were collected and washed twice with physiological saline.

[0155] (3) Resuspend the cells in protein purification buffer A (20 mM Tris, 150 mM NaCl, pH 8.0), disrupt the cells with an ultrasonic disruptor in an ice bath, and centrifuge the disrupted liquid at 10,000 × g and 4°C for 30 min to obtain the supernatant.

[0156] The protein expression in the supernatant of E.coli BL21(DE3) / pET28a-mGLP(9-37) was verified by SDS-PAGE, and the protein expression level was detected. The results showed:

[0157] The expression level of E.coli BL21(DE3) / pET28a-GLP(9-37)-SUMO-WT protein was: 0.14g / L g wet bacteria.

[0158] The expression level of E.coli BL21(DE3) / pET28a-mGLP(9-37) protein was 0.32g / 1g wet bacteria.

[0159] The results showed that the nucleotide sequence of the codon-optimized fusion protein-SUMO obtained in Example 1 was mutated to a sequence (SEQ ID NO. 4), which increased the expression amount of the final protein and achieved soluble expression.

[0160] Example 4: Optimization of the induction expression temperature of the recombinant fusion protein

[0161] The specific steps are as follows:

[0162] (1) A single colony of the positive clone E. coli BL21 (DE3) / pET28a-mGLP (9-37) prepared in Example 2 was picked and placed in a 5 mL LB liquid culture medium test tube containing 50 μg / mL kanamycin sulfate, and cultured at 37 ° C, 200 rpm, and shaking for about 8 hours to prepare a seed solution.

[0163] (2) The above seed solution was inoculated into a 100 mL LB liquid medium shake flask containing 50 μg / mL kanamycin sulfate at a 1% (v / v) inoculation rate and cultured at 37°C, 200 rpm, with shaking until the OD 600 After reaching 0.6-0.8 (about 2 hours), the culture medium was placed in an ice bath for 10 minutes, 0.2 mM IPTG was added, and the cells were induced at 17°C, 20°C, 28°C, 30°C, and 37°C, respectively. After induction culture for about 10-16 hours, the cells were collected by centrifugation at 8,000 × g for 10 minutes and washed twice with saline.

[0164] (3) Resuspend the cells in protein purification buffer A (20 mM Tris, 150 mM NaCl, pH 8.0), disrupt the cells with an ultrasonic disruptor in an ice bath, and centrifuge the disrupted liquid at 10,000 × g and 4°C for 30 min. SDS-PAGE was used to verify the protein expression. The results are as follows: Figure 4 shown.

[0165] The results showed that when the inducer concentration was the same and the induction temperature was 20℃~37℃, the soluble expression level of the fusion protein in E. coli was basically the same, and the soluble expression level decreased at 17℃. At the same time, as the induction temperature increased, the content of the inclusion bodies formed also increased.

[0166] Example 5: Optimization of inducible expression inducing agent concentration for recombinant fusion protein

[0167] The specific steps are as follows:

[0168] (1) A single colony of the positive clone E. coli BL21 (DE3) / pET28a-mGLP (9-37) prepared in Example 2 was picked and placed in a 5 mL LB liquid culture medium test tube containing 50 μg / mL kanamycin sulfate, and cultured at 37 ° C, 200 rpm, and shaking for about 8 hours to prepare a seed solution.

[0169] (2) The above seed solution was inoculated into a 100 mL LB liquid medium shake flask containing 50 μg / mL kanamycin sulfate at a 1% (v / v) inoculation rate and cultured at 37°C, 200 rpm, with shaking until the OD 600After reaching 0.6-0.8 (about 2 hours), add 0.1, 0.2, 0.3, 0.4, and 0.5 mM IPTG respectively, continue to culture at 37°C for about 10 hours, centrifuge at 8,000×g and 4°C for 10 minutes to collect the bacteria, and wash twice with saline.

[0170] (3) The cells were resuspended in protein purification buffer A (20 mM Tris, 150 mM NaCl, pH 8.0), disrupted with an ultrasonic disruptor in an ice bath, and the disrupted liquid was centrifuged at 10,000 × g and 4°C for 30 min.

[0171] The protein expression was verified by SDS-PAGE. Figure 5 shown.

[0172] The results showed that when the induction temperature was the same and the inducer concentration was 0.2-0.5 mM, the soluble expression level of the fusion protein in E. coli was basically the same, and the soluble expression level decreased at 0.1 mM.

[0173] Example 6: Purification of recombinant fusion protein

[0174] The protein in the supernatant prepared in Example 3 (strain containing the mutated sequence) was purified. The N-terminus of the fusion protein carried a His-Tag, which could bind to nickel ions. Using the nickel ion affinity chromatography method, the protein could be adsorbed on the nickel column medium, while other impurities could not or could only be weakly adsorbed on the medium, thereby achieving the purpose of separation and purification.

[0175] The specific steps are as follows:

[0176] (1) Resuspend the cells in protein purification buffer A (20 mM Tris, 150 mM NaCl, pH 8.0), disrupt the cells with an ultrasonic disruptor in an ice bath, centrifuge the disrupted liquid at 10,000 × g and 4°C for 30 min to remove cell debris, and filter the supernatant with a 0.22 μm water filter to obtain a crude protein sample.

[0177] (2) The crude protein sample was purified using a HisTrap HP column (GE Healthcare, Chicago, USA). The specific steps were as follows: 5 column volumes were balanced with protein purification buffer A. After loading, the impurities were washed with 5% protein purification buffer B (20 mM Tris, 150 mM NaCl, 1 M imidazole, pH 8.0), and then the target protein was eluted with 50% protein purification buffer B and collected.

[0178] (3) Desalting the target protein solution after nickel column purification with a Sephadex G-25 desalting column to remove imidazole in the enzyme solution. The specific steps are: equilibrate 3 column volumes with ultrapure water and protein purification buffer A, load the sample, and then elute the protein with protein purification buffer A to obtain a pure protein sample.

[0179] The protein purification was verified by SDS-PAGE. Figure 6 shown.

[0180] The results showed that the fusion protein could be well separated from the impurity proteins after being purified by nickel column affinity chromatography to obtain a pure fusion protein sample.

[0181] Example 7: Enzymatic cleavage of recombinant fusion protein

[0182] The fusion protein has an enterokinase cleavage site DDDDK at the N-terminus of Arg34GLP-1 (9-37), which can be specifically recognized by enterokinase and then cleaved at the C-terminus of the cleavage site.

[0183] The specific steps are as follows:

[0184] 1U / 50mg enterokinase was added to the purified fusion protein solution obtained in Example 6, and the solution was placed at 4°C for 2h for enzymatic digestion. The enzymatic digestion was verified by SDS-PAGE. The results were as follows: Figure 7 shown.

[0185] The results showed that the fusion protein could be cleaved by enterokinase, and the resulting small peptide was about 3.5 kDa in size, corresponding to the molecular weight of Arg34GLP-1(9-37).

[0186] Example 8: Purification of Semaglutide Precursor Molecule Arg34GLP-1(9-37)

[0187] The protein obtained after enzyme digestion in Example 7 was purified as follows:

[0188] After enzyme cleavage, the fusion protein Arg34GLP-1 (9-37) is a separate polypeptide chain, and the other part is a His tag, a SUMO tag and an enterokinase cleavage site. This part can still bind to the nickel column medium. The sample also contains enterokinase, which has a large molecular weight and a large difference in molecular weight from Arg34GLP-1 (9-37). It is separated by Superdex peptide10 / 300GL.

[0189] The protein purification was verified by SDS-PAGE. Figure 8 shown.

[0190] After the fusion protein is digested by enzymes, purified by nickel ion affinity chromatography, and separated by Superdex peptide 10 / 300GL, pure Arg34GLP-1 (9-37) can be obtained.

[0191] Example 9: Fermentation and fusion protein expression of recombinant strains under fermentation tank conditions

[0192] The specific steps are as follows:

[0193] (1) A single colony of the positive clone E. coli BL21 (DE3) / pET28a-mGLP (9-37) containing the mutation prepared in Example 2 was picked and placed in a 5 mL LB liquid culture medium test tube containing 50 μg / mL kanamycin sulfate, and cultured at 37 ° C, 200 rpm, and shaking for about 8 hours to prepare a primary seed solution.

[0194] (2) The primary seed solution was inoculated into a 150 mL TB liquid culture medium shake flask containing 50 μg / mL kanamycin sulfate at an inoculum volume of 2% (v / v), and cultured at 37° C., 200 rpm, with shaking for about 8 h to prepare a secondary seed solution.

[0195] (3) The above 150 mL of secondary seed solution was inoculated into a 2 L TY liquid medium fermentation tank containing 50 μg / mL kanamycin sulfate, stirred at 600 rpm, pH was controlled at 7.4, and feed medium was added when dissolved oxygen increased, at a rate of 20-30 mL / h, until OD 600 After reaching 16-20, 0.2 mM IPTG was added and induced at 37°C. After about 10 h of induction culture, the bacteria were collected by centrifugation at 8,000 × g for 30 min and washed twice with physiological saline.

[0196] (4) The cells were resuspended in protein purification buffer A (20 mM Tris, 150 mM NaCl, pH 8.0), disrupted with an ultrasonic disruptor in an ice bath, and the disrupted liquid was centrifuged at 10,000 × g and 4°C for 30 min.

[0197] The expression level of the protein in the supernatant was detected according to the method of the above example, and the yield of the purified protein was detected.

[0198] The results showed that the method of the present invention can obtain a recombinant strain (E. coli BL21 (DE3) / pET28a-mGLP (9-37)) that solublely expresses Arg34GLP-1 (9-37), and the wet bacterial volume after fermentation in a tank is 150 to 200 g / L, and the yield of pure protein Arg34GLP-1 (9-37) is 2 to 3 g / L.

[0199] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A nucleic acid molecule encoding a fusion protein containing semaglutide precursor polypeptide Arg34GLP-1 (9-37), characterized in that: The sequence of the nucleic acid molecule is shown in SEQ ID NO.

4.

2. A method for increasing the expression level of a fusion protein containing semaglutide precursor polypeptide Arg34GLP-1(9-37) in a host cell, characterized in that: The method comprises mutating a nucleic acid molecule of a fusion protein containing the semaglutide precursor polypeptide Arg34GLP-1 (9-37) as shown in SEQ ID NO.3 to obtain a nucleic acid molecule of a fusion protein containing the semaglutide precursor polypeptide Arg34GLP-1 (9-37) as shown in SEQ ID NO.4, and expressing the mutated nucleic acid molecule in a host cell.

3. An expression cassette or a recombinant vector or a recombinant strain or a transgenic cell line, characterized in that: The expression cassette or recombinant vector or recombinant strain or transgenic cell line contains the nucleic acid molecule according to claim 1.

4. The expression cassette or recombinant vector or recombinant strain or transgenic cell line according to claim 3, characterized in that: The recombinant vector is obtained by cloning the nucleic acid molecule into an expression vector; Preferably, the recombinant vector is an expression vector using pET series vectors, pCDF series vectors, pRSF series vectors or pGEX series vectors; Preferably, the recombinant vector is an expression vector using pET-28(a), pET-39(b), pET-26(b), pET-21(a), pRSFDuet-1, pCDFDuet-1 or pGEX-6P-1; Preferably, the recombinant strain is obtained by introducing the nucleic acid molecule or the expression cassette or the recombinant vector into bacteria or fungi.

5. Use of the nucleic acid molecule according to claim 1 or the expression cassette or recombinant vector or recombinant strain or transgenic cell line according to claim 3 or 4 in the preparation of semaglutide core 29 peptide; the amino acid sequence of the semaglutide core 29 peptide is shown in SEQ ID NO.

1.

6. A recombinant strain that solublely expresses a fusion protein containing semaglutide precursor polypeptide Arg34GLP-1(9-37), characterized in that: The recombinant strain uses Escherichia coli as an expression host and solublely expresses a nucleic acid molecule encoding a fusion protein containing the semaglutide precursor polypeptide Arg34GLP-1(9-37); the sequence of the nucleic acid molecule is shown in SEQ ID NO.4; Preferably, the recombinant strain uses pET series vectors, pCDF series vectors, pRSF series vectors or pGEX series vectors as expression vectors; Preferably, the recombinant strain uses pET-28(a), pET-39(b), pET-26(b), pET-21(a), pRSFDuet-1, pCDFDuet-1 or pGEX-6P-1 as an expression vector.

7. A method for soluble expression of a fusion protein containing semaglutide precursor polypeptide Arg34GLP-1(9-37), characterized in that: The method comprises fermenting the recombinant strain or transgenic cell according to claim 3 or 4, or the recombinant strain according to claim 6 to obtain the product.

8. A method for preparing semaglutide core 29 peptide by soluble expression, characterized in that: The method comprises the following steps: fermenting and culturing the recombinant strain or transgenic cell according to claim 3 or 4 or the recombinant strain according to claim 6, collecting the bacterial cells, crushing, purifying and enzymatically digesting them to obtain the semaglutide core 29 peptide; the amino acid sequence of the semaglutide core 29 peptide is shown in SEQ ID NO.

1.

9. A method for increasing the expression level of a fusion protein containing semaglutide precursor polypeptide Arg34GLP-1(9-37) expressed soluble in Escherichia coli, characterized in that: The method comprises the following steps: mutating a nucleic acid molecule of a fusion protein containing the semaglutide precursor polypeptide Arg34GLP-1 (9-37) having a nucleotide sequence as shown in SEQ ID NO.3 to obtain a nucleic acid molecule of a fusion protein containing the semaglutide precursor polypeptide Arg34GLP-1 (9-37) having a nucleotide sequence as shown in SEQ ID NO.4; and using Escherichia coli to solublely express the nucleic acid molecule of the fusion protein containing the semaglutide precursor polypeptide Arg34GLP-1 (9-37) having a nucleotide sequence as shown in SEQ ID NO.

4.

10. Use of the nucleic acid molecule according to claim 1, or the method according to claim 2, or the expression cassette, recombinant vector, recombinant strain or transgenic cell line according to claim 3 or 4, or the recombinant strain according to claim 6, or the fusion protein obtained by any one of the methods of claims 7 to 9 in any one or more of the following: (a) improving the yield and / or production of the semaglutide backbone; and / or (b) increasing the expression level of the semaglutide backbone; and / or (c) preparing a product containing a semaglutide backbone.

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