Fusion protein of lixisenatide precursor and use thereof

Abstract

Provided are a fusion protein of a lixisenatide precursor and the use thereof. The fusion protein comprises a lixisenatide precursor and a tag protein fused to the N-terminus of the lixisenatide precursor. The lixisenatide precursor has an amino acid sequence as set forth in SEQ ID NO: 1; and the tag protein is selected from any one of the following: a Fh8 tag protein, a Ffu209 tag protein, a CBM tag protein, a Sumo tag protein or a Trx tag protein. The lixisenatide precursor is fused with the tag protein, and the lixisenatide precursor with the tag protein has a relatively high expression level, and can be used to prepare a large number of high-purity lixisenatide precursors, thereby better conducting industrial production and application of lixisenatide.

Description

Fusion protein of lixisenatide precursor and its application

[0001] This application is based on the Chinese application with CN application number 202410229260.X and application date February 29, 2024, and claims its priority. The disclosed content of the CN application is again introduced as a whole into this application. Technical Field

[0002] The present invention relates to the technical field of protein expression, and in particular to a fusion protein of a lixisenatide precursor and applications thereof. Background Art

[0003] Lixisenatide has an NH2 residue at its C-terminus, while the C-terminus of the lixisenatide precursor obtained through recombinant microbial expression is COOH, requiring chemical modification to obtain the final lixisenatide. Currently, lixisenatide precursors are primarily obtained through chemical coupling synthesis and recombinant expression. Chemical coupling synthesis is complex and prone to impurities during the synthesis process. Chemical reagents are toxic and may produce racemates, resulting in relatively low purity and recovery rates. The synthesis cycle is long, making product quality control challenging. Recombinant expression utilizes genetic engineering techniques to introduce target gene expression elements into prokaryotes or eukaryotes. Microbial fermentation allows for efficient synthesis of the target peptide. Two methods are available: inclusion body expression and soluble expression. Inclusion body expression requires the use of large amounts of denaturants such as urea or guanidine hydrochloride during the denaturation and renaturation processes, resulting in complex purification processes and very low final yields. Soluble expression, however, can affect the solubility of the resulting fusion protein and the yield of the target peptide, depending on the type of tag protein introduced and the type of restriction sites linking the tag protein to the target peptide.

[0004] Summary of the Invention

[0005] The main purpose of the present invention is to provide a fusion protein of a lixisenatide precursor and its application, so as to solve the problem of low efficiency in preparing a lixisenatide precursor in the prior art.

[0006] In order to achieve the above-mentioned object, according to the first aspect of the present invention, a fusion protein of a lixisenatide precursor is provided, the fusion protein comprising a lixisenatide precursor and a tag protein fused to the N-terminus of the lixisenatide precursor; the lixisenatide precursor has an amino acid sequence as shown in SEQ ID NO: 1; the tag protein is selected from any one of the following: Fh8 tag protein, Ffu209 tag protein, CBM tag protein, Sumo tag protein or Trx tag protein.

[0007] Furthermore, the fusion protein has an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.

[0008] To achieve the above object, according to a second aspect of the present invention, a method for preparing a lixisenatide precursor is provided, the method comprising: preparing the above fusion protein; and removing the tag protein in the fusion protein by using an endonuclease to obtain a lixisenatide precursor.

[0009] Furthermore, the above-mentioned protein endonuclease is selected from Ulp1 enzyme or KEX2 enzyme; the mass ratio of Ulp1 enzyme to fusion protein is 1:20-100; the mass ratio of KEX2 enzyme to fusion protein is 1:40-500.

[0010] Furthermore, the above-mentioned preparation of the fusion protein includes: co-expressing the above-mentioned fusion protein with a chaperone protein to obtain the fusion protein; the expression plasmid of the chaperone protein includes: pGro7, pTF16, pKJE7 or pG-KJE8.

[0011] Furthermore, after the tag protein in the fusion protein is removed by using an endonuclease, the product after the endonuclease treatment is further purified to obtain a lixisenatide precursor; the purification includes: adjusting the pH value of the enzymatic cleavage system to the isoelectric point of the tag protein, adding an organic reagent and centrifuging, and the resulting supernatant is the lixisenatide precursor.

[0012] In order to achieve the above object, according to the third aspect of the present invention, a DNA molecule is provided, which encodes the above fusion protein.

[0013] Furthermore, the nucleotide sequence of the above DNA molecule is a codon-optimized nucleotide sequence.

[0014] Furthermore, the above DNA molecule has a nucleotide sequence as shown in SEQ ID NO: 3.

[0015] In order to achieve the above object, according to a fourth aspect of the present invention, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above DNA molecule.

[0016] In order to achieve the above object, according to a fifth aspect of the present invention, a host cell is provided, into which the above recombinant plasmid is transformed.

[0017] Furthermore, the host cell is a prokaryotic cell or a yeast cell.

[0018] In order to achieve the above object, according to the sixth aspect of the present invention, there is provided a use of the above fusion protein or the lixisenatide precursor prepared by the above method in the preparation of lixisenatide.

[0019] By applying the technical solution of the present invention, the lixisenatide precursor in the fusion protein of the present application is fused with a tag protein, has a high expression level, and can be prepared in large quantities and with high purity by a relatively simple method, thereby better carrying out the industrial production and application of lixisenatide. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0021] FIG1 shows the electrophoresis diagram of the fusion proteins Fh8-lixi, Ffu209-lixi, CBM-lixi, Trx-lixi, and Sumo-lixi expressed in E. coli in Example 1 of the present invention;

[0022] FIG2 shows an electrophoretic diagram of the fusion protein Sumo-lixi obtained by expression in E. coli and purification in Example 2 of the present invention;

[0023] FIG3 shows the electrophoresis diagram of the lixisenatide precursor obtained by acetonitrile precipitation and purification in Example 4 of the present invention;

[0024] FIG4 shows a schematic diagram of mass spectrometry detection of lixisenatide precursor in Example 5 of the present invention. DETAILED DESCRIPTION

[0025] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments in this application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0026] As mentioned in the background, existing methods for synthesizing lixisenatide precursors are unable to efficiently produce large quantities of lixisenatide precursor products. Chemical methods are complex and prone to impurities, while genetic engineering techniques can complicate inclusion body purification and result in low yields of soluble expression of large tagged proteins. Therefore, this application seeks to protect a lixisenatide precursor fused to a tagged protein that can be efficiently and efficiently expressed in large quantities.

[0027] In a first typical embodiment of the present invention, a lixisenatide fusion protein is provided, which comprises a lixisenatide precursor and a tag protein fused to the N-terminus of the lixisenatide precursor; the lixisenatide precursor has an amino acid sequence as shown in SEQ ID NO: 1; the tag protein is selected from any one of the following: Fh8 tag protein, Ffu209 tag protein, CBM tag protein, Sumo tag protein or Trx tag protein.

[0028] SEQ ID NO: 1: (amino acid sequence of lixisenatide precursor)

[0029] The fusion protein of the present invention is obtained by fusing the smaller molecular weight tag protein Sumo to the N-terminus of the lixisenatide precursor having the above-mentioned amino acid sequence. This allows for efficient soluble expression in host cells, providing a basis for subsequent processing to obtain large amounts of lixisenatide precursor. Furthermore, it is difficult to completely remove the tag protein after expression of the protein fused to the C-terminus.

[0030] In a preferred embodiment, the fusion protein has an amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. The fusion protein of the present application is a lixisenatide precursor fused to a tag protein at the N-terminus of the lixisenatide precursor, the amino acid sequence of the tag protein is the underlined sequence, and the italic portion (amino acid sequence at positions 76 to 96 in SEQ ID NO: 5, amino acid sequence at positions 164 to 184 in SEQ ID NO: 6, amino acid sequence at positions 171 to 191 in SEQ ID NO: 7 or amino acid sequence at positions 116 to 136 in SEQ ID NO: 8) is a connecting peptide sequence.

[0031] SEQ ID NO: 2: Amino acid sequence of Sumo-lixisenatide precursor

[0032] SEQ ID NO: 5: Amino acid sequence of Fh8-lixisenatide precursor

[0033] SEQ ID NO:6: Amino acid sequence of Ffu209-lixisenatide precursor

[0034] SEQ ID NO: 7: Amino acid sequence of CBM-lixisenatide precursor

[0035] SEQ ID NO: 8: Amino acid sequence of Trx-lixisenatide precursor

[0036] In a second typical embodiment of the present invention, a method for preparing a lixisenatide precursor is provided, comprising: preparing the above-mentioned fusion protein; and removing the tag protein from the fusion protein using an endonuclease to obtain a lixisenatide precursor. The above-mentioned fusion protein of the present application can be expressed in large quantities in a host cell, preferably Escherichia coli, to obtain the above-mentioned lixisenatide precursor with the tagged protein. To obtain the lixisenatide precursor, the tag protein needs to be removed. After treatment with an endonuclease that can specifically recognize a specific tagged protein, the lixisenatide precursor can be obtained, thereby achieving the purpose of large-scale and efficient production of the lixisenatide precursor.

[0037] The method for preparing the above-mentioned fusion protein includes: homologously ligating a codon-optimized lixisenatide precursor nucleotide sequence carrying a tagged protein with a vector capable of expressing the protein, and obtaining a recombinant plasmid with normal sequencing through positive screening. The correct plasmid is transferred into a suitable host cell for pre-screening, and the expression strain with the best expression level is selected for subsequent protein purification and culture. The expression of the protein is induced using the expression strain with the best expression level, the culture solution is centrifuged to collect the bacteria, resuspended and then disrupted, and the supernatant is purified by step-by-step elution to obtain the above-mentioned fusion protein.

[0038] In order to further increase the expression level of the lixisenatide precursor, ensure correct protein folding and aggregation, and prevent protein degradation, the fusion protein can be co-expressed with a chaperone protein. In a preferred embodiment, the preparation of the above-mentioned fusion protein includes: co-expressing the above-mentioned fusion protein with the chaperone protein to obtain a fusion protein; preferably, the expression plasmid of the chaperone protein includes pGro7 (containing the chaperone proteins groES and groEL), pTF16 (containing the chaperone protein tig), pKJE7 (containing the chaperone proteins danK, danJ and grpE), or pG-KJE8 (containing the chaperone proteins danK, dnaJ, grpE, groES and groEL). More preferably, it is pGro7 or pTF16.

[0039] The chaperone proteins groES and groEL are a pair of interacting proteins that form a complex within the cell, called the GroEL-GroES complex, involved in protein folding and repair. groES is a cofactor that provides a "lid" for groEL, allowing it to form a closed cavity that holds damaged or unfolded proteins and prevents them from nonspecifically interacting with other cellular components. groEL is a molecular chaperone protein with a large cylindrical structure that can accommodate unfolded proteins. By binding to and hydrolyzing ATP, groEL provides a suitable environment within the cavity, promoting proper protein folding.

[0040] Tig is another protein folding auxiliary factor. Its function in the GroEL / GroES system is similar to that of groES. It can form a complex with groEL to assist in protein folding.

[0041] danK, also known as DnaK, is a molecular chaperone protein that belongs to the Hsp70 family. DanK participates in the folding, repair, and degradation of proteins within cells, working in conjunction with other chaperone proteins to help proteins fold correctly and prevent aggregation. DanJ is a cofactor of danK, forming a complex with danK and supporting its role in protein folding and repair.

[0042] GrpE is another molecular chaperone protein that forms a complex with danK and participates in protein folding, repair, and degradation. GrpE plays a key role in regulating danK's ATPase activity, helping danK achieve correct protein folding.

[0043] Different tagged proteins require different types of endonucleases. In a preferred embodiment, the endonuclease is selected from Ulp1 enzyme or KEX2 enzyme, wherein the Ulp1 enzyme can specifically recognize the tertiary structure of the Sumo tag, thereby completely removing the Sumo-tagged protein from the target protein at the GH junction of the Sumo tag and the target protein; KEX2 can recognize the KR site of the connecting peptide and completely remove the Trx-tagged protein and the connecting peptide from the target protein. To further remove the tagged protein as cleanly as possible, in a preferred embodiment, the mass ratio of Ulp1 enzyme to fusion protein is 1:20-100, including 1:20, 1:40, 1:50, 1:60, 1:80, and 1:100; the mass ratio of KEX2 enzyme to fusion protein is 1:40-500, including 1:40, 1:80, 1:100, 1:120, 1:200, 1:300, 1:400, and 1:500; preferably, the enzyme cleavage time is 16-18 h.

[0044] In order to further obtain a lixisenatide precursor with higher purity, the product treated with an endonuclease needs to be subjected to protein purification. In a preferred embodiment, after the tag protein in the fusion protein is removed by an endonuclease, the product treated with the endonuclease is further purified to obtain a lixisenatide precursor.

[0045] There are many methods for protein purification, including isoelectric point precipitation, salting out, column chromatography, membrane filtration, etc. Any protein purification method that can further separate the lixisenatide precursor from impurities is applicable to the present application. In a preferred embodiment, purification includes: adjusting the pH value of the enzymatic cleavage system to the isoelectric point of the tagged protein, adding an organic reagent, and centrifuging. The supernatant is the lixisenatide precursor. Preferably, the organic reagent includes methanol or acetonitrile. Among them, the isoelectric point of His-Sumo is 5.6. The tagged protein impurities precipitated by the isoelectric point are removed by precipitation after centrifugation, and finally the purified target product lixisenatide precursor is obtained in the supernatant. Among them, because the molecular weight of the lixisenatide precursor is smaller, it is more stable than protein, has good solubility in organic solvents, and is not easily precipitated.

[0046] In a third typical embodiment of the present invention, a DNA molecule is provided, which encodes the above-mentioned fusion protein.

[0047] When each host cell expresses a protein, it is a process of translating the codons. During the translation process, different host cells have certain preferences for codons. In order to obtain a protein product with a higher expression level in a specific host cell, the nucleotide sequence encoding the protein can be codon optimized for a specific host cell type. In a preferred embodiment, the nucleotide sequence of the DNA molecule is a codon-optimized nucleotide sequence; preferably, the DNA molecule has a nucleotide sequence as shown in SEQ ID NO: 3. The nucleotide sequence of the lixisenatide precursor encoding the Sumo-tagged protein of the present application is an optimized codon with a preference for Escherichia coli, and the nucleotide sequence of the tagged protein is the underlined sequence shown in SEQ ID NO: 3.

[0048] SEQ ID NO: 3: (Nucleotide sequence of fusion protein)

[0049] In a fourth typical embodiment of the present invention, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above-mentioned DNA molecule.

[0050] In a fifth typical embodiment of the present invention, a host cell is provided, wherein the host cell is transformed with the above-mentioned recombinant plasmid. The host cell is a prokaryotic cell or a yeast cell, preferably Escherichia coli.

[0051] The above-mentioned host cells can be used to replicate the recombinant plasmid in the host cells, and the DNA molecules carried on the recombinant plasmid can also be transcribed and translated to obtain a large amount of fusion protein. Using existing technologies, the host cells can be disrupted and purified by other methods to obtain the fusion protein, which is conducive to the subsequent preparation of lixisenatide using lixisenatide precursor.

[0052] In a sixth typical embodiment of the present invention, there is provided a use of the above-mentioned fusion protein or the lixisenatide precursor prepared by the above-mentioned method in the preparation of lixisenatide.

[0053] The present application is further described in detail below with reference to specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0054] Example 1 Construction of genetically engineered strains expressing lixisenatide precursors with different tags

[0055] The strategy for constructing a soluble expression polypeptide involved fusing multiple tag proteins with the lixisenatide precursor sequence and constructing it in the pET-28a(+) expression vector. A codon-optimized lixisenatide precursor sequence was synthesized by GeneWeiZhi. The nucleotide sequence of the codon-optimized lixisenatide precursor is as follows: SEQ ID NO: 4: CATGGCGAAGGCACCTTTACCAGCGATCTGAGCAAACAGATGGAAGAAGAAGCGGTGCGCCTGTTTATTGAATGGCTGAAAAACGGCGGCCCGAGCAGCGGCGCGCCGCCGAGCAAAAAAAAAAAAAAAAAATAA.

[0056] The synthesized DNA fragments were ligated with the pET-28a-CBM, pET-28a-Fh8, pET-28a-Ffu209, pET-28a-Trx and pET-28a-Sumo vector backbones by homologous recombination. The ligation products were transformed into BL21 (DE3) competent cells. Monoclonal sequencing analysis was performed to obtain the correct cloning expression vector pET-28a-Tag-lixi (lixi is the abbreviation of lixisenatide precursor). Three correct clones were selected and activated and inoculated as seeds in 250 ml shake flasks for pre-screening. The best one was selected as the final expression strain. The tag proteins Fh8, Ffu209, and CBM, along with the tag proteins Sumo and Trx, all have relatively small molecular weights (7.5-18 kDa). Fh8 is a protein secreted by Fasciola hepatica during the early stages of infection, with a molecular weight of 7.5 kDa; Ffu209 is a protein derived from Bacillus salinarum, with a molecular weight of 17.75 kDa; CBM is a domain of β-fructosidase, with a molecular weight of 17.96 kDa; and Trx is a thioredoxin, with a molecular weight of 11.7 kDa. Sumo, with a molecular weight of 11.1 kDa, is a protein that can be fused to target proteins without a linker peptide. Its tertiary structure is directly recognized by specific enzymes, allowing Sumo to be completely cleaved and removed, yielding a relatively pure lixisenatide precursor.

[0057] 4 ml of the BL21 (DE3) strain containing the recombinant plasmid was inoculated into a 2 L Erlenmeyer flask containing 400 mL of LB medium and cultured at 37°C with shaking at 200 rpm until an OD600 of 1.0 was reached. IPTG was then added at a final concentration of 0.2 mM and induced overnight at 25°C. After induction, the cells were collected by centrifugation. The supernatant was collected by ultrasonic disruption and analyzed by 17% SDS-PAGE. The results showed that all fusion proteins were expressed in soluble form, as shown in Figure 1. Among them, the expression level of the lixisenatide precursor fused with the Sumo tag was the highest. Although the expression level of the lixisenatide precursor fused with the CBM tag was higher than that of the lixisenatide precursors fused with the other three tag proteins (Fh8, Ffu209, and Trx), the presence of two distinct bands in the electrophoresis results after CBM-lixi purification suggests that the CBM-lixi purification product contains not only CBM-lixi but also the CBM-tagged protein, which is not conducive to the subsequent collection and purification of the lixisenatide precursor.

[0058] Example 2Sumo-lixi purification

[0059] The bacterial slurry expressing Fh8-lixi, Ffu209-lixi, CBM-lixi, Trx-lixi and Sumo-lixi was resuspended at 20% bacterial concentration, ultrasonically disrupted (5s ultrasonication, 5s interval, 30% power), and then centrifuged. The supernatant was filtered through a 0.45μm filter to obtain a crude enzyme solution, which was then purified by affinity chromatography (AKTA system equipped with a 5ml HisTrap HP). The specific process is as follows: the filter sample was loaded at a flow rate of 5ml / min, and then washed with binding buffer (50mM Tris-HCl, 200mM NaCl, pH 8.0) until the unbound protein was completely eluted. Then, the impurities were eluted with a buffer of 50mM Tris-HCl, 200mM NaCl, 50mM imidazole, pH 8.0 for 4 column volumes, and finally the target protein was eluted with an elution buffer of 50mM Tris-HCl, 200mM NaCl, 300mM imidazole, pH 8.0 (as shown in Figure 2). 22.2 mg of purified Sumo-lixi fusion protein can be obtained from 1 g of wet cells, which is a significant increase in expression compared to Fh8-lixi, Ffu209-lixi, and Trx-lixi. As mentioned in Example 1, due to the similar size of CBM-lixi and CBM-tagged protein, the purified CBM-lixi contains a certain amount of impurities.

[0060] Example 3 Co-expression of chaperone proteins

[0061] To further increase fusion protein production, the chaperone protein expression plasmids pGro7, pTF6, pKJE7, and pG-KJE8 were co-transfected with pET28a-Sumo-lixi into BL21(DE3) competent cells to generate recombinant expression strains co-expressing the chaperone proteins. The induction conditions were optimized, and the optimal induction conditions were: When the bacterial liquid reached an OD600 of 1.0, a final concentration of 0.2 mM IPTG and 1 mg / mL arabinose were added, and induction was carried out at 25°C overnight. It can be seen that the yield of the fusion proteins co-expressed with the chaperone proteins was increased, and the promotion effect was even greater when expressed with the chaperone protein vectors pGro7 and pTF6.

[0062] Example 4 High-density fermentation culture

[0063] Fermentation basal medium (11 L) in a fermenter: 20 g peptone, 110 g yeast powder, and 77 g NaCl.

[0064] Carbon supplement solution: 30g magnesium sulfate heptahydrate, 800g glucose, 360mL glycerol.

[0065] Nitrogen replenishing solution: 400g yeast powder, 400g peptone, 400g ammonium sulfate.

[0066] In a 20-L fermentor, add 11 L of basal medium for fermentation, 50-100 mL of activated seed bacteria containing Sumo-lixi, and an appropriate amount of defoamer. Ferment at 37°C and pH 7.0. Early in fermentation, maintain aeration at 5 L / min and a rotational speed of 150 rpm. During the logarithmic growth phase, maintain a dissolved oxygen concentration of 30%-50%. If the dissolved oxygen concentration is insufficient, the rotational speed can be increased to 750 rpm. The pH is adjusted to 7.0 using automated feeds of 2N HCl or ammonia. During fermentation, sample the cells for density. As dissolved oxygen and pH increase, add carbon supplementation, followed by nitrogen supplementation half an hour later. When the cells reach the late logarithmic growth phase, induce with IPTG and arabinose at a single dose. Final concentrations of IPTG and arabinose are 0.2 mM and 1 mg / mL, respectively. Induction is completed at 25°C for 16 hours.

[0067] Each liter of fermentation broth yields over 180g of bacterial sludge, and each gram of sludge containing the chaperone protein yields 36.5mg of the fusion protein, meaning 6.57g of the fusion protein, Sumo-lixi, is produced per liter of fermentation broth. Since the theoretical proportion of polypeptides is 28.2%, the theoretical yield of lixisenatide precursor is 1.84g / L. In actual production, the yield of lixisenatide precursor can reach over 1.5g / L.

[0068] Example 5 Enzyme Digestion

[0069] The Ulp1 digestion reaction was carried out at 30°C using the following procedure: 50mM Tris-HCl, 200mM NaCl, 300mM imidazole, pH 8.0, with a mass ratio of 40:1 (mg / mg) of purified Sumo-lixi and Ulp1. The digestion lasted for 16 hours, and the contents of lixi (i.e., the lixisenatide precursor) and sumo-lixi in the digested system were determined by mass spectrometry. The digestion efficiency was found to be over 90%. Furthermore, when the mass ratio of the fusion protein to Ulp1 was 100:1, the digestion efficiency reached approximately 80%, and at a ratio of 20:1, the digestion efficiency reached 95%. However, the digestion efficiency could not be further improved by adding Ulp1.

[0070] Example 6: Purification of target polypeptide by acetonitrile precipitation

[0071] The pH of the product after enzymatic digestion in Example 5 was adjusted to 5.6 (the isoelectric point of His-sumo), and then 60% acetonitrile was added to the reaction system. After mixing, the mixture was shaken at 30°C for 2 h. The supernatant and precipitate were then centrifuged at 12000 rpm. The purity of the purified product was detected by 17% SDS-PAGE, and whether the target polypeptide was generated was detected (the purified product obtained from the enzymatic digestion product in Example 5 is shown in Figure 3). The purity of the crude lixi product after enzymatic digestion in Example 5 was 50%.

[0072] Example 7 Mass Spectrometry Detection of Polypeptide Molecular Weight

[0073] The molecular weight of the prepared peptides was analyzed using LC-MS. Specifically, the sample was separated by HPLC using an Agilent ZORBAX Edipse Plus C18 column, 4.6 x 100 mm, 3.5 μm. Mobile phase A was 0.1% trifluoroacetic acid, and mobile phase B was 0.1% trifluoroacetic acid in acetonitrile. The gradient elution pattern was: 10% B at 0 min, 95% B at 9 min, 100% B at 12 min, 10% B at 12.1 min, and 10% B at 15 min. The column temperature was 50°C, the UV detector was at 210 nm, and the flow rate was 0.3 ml / min. The components after HPLC separation were analyzed by a Q Exactive HF combined quadrupole-Orbitrap mass spectrometer using an electrospray ionization source (Dual AJS ESI), positive ion mode detection, sheath gas flow rate of 35 arb, auxiliary gas flow rate of 8 arb, spray voltage of 3800 V, ion transfer tube temperature of 320°C, scan range of 200-3000 m / z, and mass spectrometry data were processed by BioPharma Finder software (as shown in Figure 4). The theoretical molecular weight of the lixisenatide precursor is 4857.5 Da, and the molecular weight of the lixisenatide precursor analyzed by mass spectrometry is 4856.5 Da, indicating that the lixisenatide precursor prepared in the present application is not much different from the theoretical value, which is in line with expectations.

[0074] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: the chemical coupling synthesis of lixisenatide precursor is complex and prone to impurities during the synthesis process, making product quality control difficult and resulting in relatively low purity and recovery. The present invention achieves soluble expression of lixisenatide precursor through genetic recombination. Through tag screening and optimization of expression conditions, the fermentation yield of lixisenatide precursor can reach levels exceeding 1 g / L, achieving efficient synthesis of lixisenatide precursor.

[0075] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A fusion protein of lixisenatide precursor, characterized in that: The fusion protein includes a lixisenatide precursor and a tag protein fused to the N-terminus of the lixisenatide precursor; The lixisenatide precursor has an amino acid sequence as shown in SEQ ID NO: 1; The tag protein is selected from any one of the following: Fh8 tag protein, Ffu209 tag protein, CBM tag protein, Sumo tag protein or Trx tag protein.

2. The fusion protein according to claim 1, characterized in that The fusion protein has the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:

8.

3. A method for preparing a lixisenatide precursor, characterized in that: The method comprises: Prepare the fusion protein according to claim 1 or 2; The tag protein in the fusion protein is removed by using an endonuclease to obtain the lixisenatide precursor.

4. The method according to claim 3, characterized in that The protein endonuclease is selected from Ulp1 enzyme or KEX2 enzyme; The mass ratio of the Ulp1 enzyme to the fusion protein is 1:20-100; The mass ratio of the KEX2 enzyme to the fusion protein is 1:40-500.

5. The method according to claim 3, characterized in that The preparation of the fusion protein according to claim 1 or 2 comprises: co-expressing the fusion protein according to claim 1 or 2 with a partner protein to obtain the fusion protein; The expression plasmid of the chaperone protein includes: pGro7, pTF16, pKJE7 or pG-KJE8.

6. The method according to claim 3, characterized in that After the tag protein in the fusion protein is removed by using an endonuclease, the product treated with the endonuclease is further purified to obtain the lixisenatide precursor; The purification comprises: adjusting the pH value of the enzyme cleavage system to the isoelectric point of the tag protein, adding an organic reagent and centrifuging, and the obtained supernatant is the lixisenatide precursor.

7. A DNA molecule, characterized in that The DNA molecule encodes the fusion protein according to claim 1 or 2.

8. The DNA molecule according to claim 7, characterized in that The nucleotide sequence of the DNA molecule is a codon-optimized nucleotide sequence.

9. The DNA molecule according to claim 8, characterized in that The DNA molecule has a nucleotide sequence as shown in SEQ ID NO:

3.

10. A recombinant plasmid, characterized in that: The recombinant plasmid is connected to the DNA molecule according to any one of claims 7 to 9.

11. A host cell, characterized in that The host cell is transformed with the recombinant plasmid according to claim 10.

12. The host cell according to claim 11, characterized in that The host cell is a prokaryotic cell or a yeast cell.

13. Use of the fusion protein according to claim 1 or 2 or the lixisenatide precursor prepared by the method according to any one of claims 3 to 6 in the preparation of lixisenatide.

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