Soluble intermediate of vosoritide, method for preparing intermediate and method for preparing vosoritide

Abstract

Provided in the present invention are a soluble intermediate of vosoritide, a method for preparing the intermediate and a method for preparing vosoritide. The soluble intermediate of vosoritide comprises a Sumo tag protein, a cleavable site and vosoritide. The method can solve the problem of low yield in preparing vosoritide in the prior art, and is suitable for the field of the biosynthesis of polypeptide drugs.

Description

Soluble intermediate of vosolitide, preparation method of intermediate, and preparation method of vosolitide

[0001] This application is based on the Chinese application with CN application number 202410229257.8 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 field of polypeptide drug biosynthesis, and in particular to a soluble intermediate of vosolitide, a method for preparing the intermediate, and a method for preparing vosolitide. Background Art

[0003] Vosoritide is a peptide drug used to treat short skeletal growth in children and adolescents. It is a human growth hormone analog that acts on the growth plates of bones, promoting bone growth and thus helping patients increase their height.

[0004] At present, vosolitide is mainly prepared by chemical coupling synthesis or inclusion body expression. Among them, the process of chemical coupling synthesis is relatively complicated, and impurities are easily generated during the synthesis process, and the purity and recovery rate are relatively low. Although the inclusion body expression method can avoid protease degradation, the complex denaturation and renaturation process of the protein requires a large amount of denaturants, such as urea or guanidine hydrochloride, and the purification process is cumbersome, resulting in a low final yield. Although the prior art uses a variety of tags to express vosolitide variants by fusion, most of the fusion proteins obtained by expression are inclusion bodies, and the denaturation and renaturation process requires a large amount of denaturants such as urea or guanidine hydrochloride, the production cost is high, and the purification process is complicated, resulting in a very low final yield of vosolitide, which is difficult to meet the needs of industrial production.

[0005] Summary of the Invention

[0006] The main purpose of the present invention is to provide a soluble intermediate of vosolitide, a method for preparing the intermediate, and a method for preparing vosolitide, so as to solve the problem of low yield of vosolitide in the prior art.

[0007] In order to achieve the above-mentioned purpose, according to one aspect of the present invention, a soluble intermediate of wosolipide is provided, which comprises a Sumo tag protein, a detachable site and wosolipide; the Sumo tag protein is located at the N-terminal direction of the detachable site, and the wosolipide is located at the C-terminal direction of the detachable site.

[0008] Further, the separation site includes an aspartic acid.

[0009] Furthermore, the Sumo tag protein includes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, or a tag protein having more than 70% identity with the amino acid sequence as shown in SEQ ID NO: 1; the wosoritide includes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2, or a polypeptide having more than 70% identity with the amino acid sequence as shown in SEQ ID NO: 2 and containing a disulfide bond structure; preferably, the soluble intermediate of wosoritide is a polypeptide having an amino acid sequence as shown in SEQ ID NO: 3.

[0010] In order to achieve the above-mentioned purpose, according to the second aspect of the present invention, a method for preparing a soluble intermediate of vosolitide is provided, which comprises: constructing a recombinant plasmid, which contains a DNA sequence capable of expressing a Sumo tag protein, a DNA sequence capable of expressing a separable site, and a DNA sequence capable of expressing vosolitide, which are sequentially connected in the 5'-3' direction.

[0011] Furthermore, the Sumo tag protein includes a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, or a polypeptide having more than 70% identity with the amino acid sequence shown in SEQ ID NO: 1 and capable of promoting soluble expression of the protein; the wosoritide includes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, or a polypeptide having more than 70% identity with the amino acid sequence shown in SEQ ID NO: 2.

[0012] Furthermore, the DNA sequence capable of expressing the separable site consists of a codon expressing an aspartic acid.

[0013] Furthermore, the gene of the recombinant plasmid is the nucleotide sequence shown in SEQ ID NO:4.

[0014] Further, the recombinant plasmid expresses a soluble intermediate of wosolitide in a host cell; preferably, the host cell includes a eukaryotic cell or a prokaryotic cell; preferably, the prokaryotic cell includes Escherichia coli; preferably, the Escherichia coli includes BL21 (DE3), Origami B (DE3) or Shuffle T7-B.

[0015] In order to achieve the above-mentioned purpose, according to the third aspect of the present invention, a method for preparing vosolitide is provided, which comprises: cutting the separable site in the vosolitide soluble intermediate obtained by any of the above-mentioned vosolitide soluble intermediates or the preparation method of any of the above-mentioned vosolitide soluble intermediates, removing the Sumo tag protein and the separable site sequence, and obtaining vosolitide.

[0016] Furthermore, the excision includes: using an acid solution to excise the Sumo tag protein and the detachable site at the detachable site to obtain vosolipid peptide.

[0017] The technical solution of the present invention is applied to provide a soluble intermediate comprising a Sumo tag protein, a detachable site and wosolipide, i.e., a fusion protein, wherein the Sumo tag protein is located at the N-terminal direction of the detachable site and the wosolipide is located at the C-terminal direction of the detachable site. The fusion protein has good solubility, and in the subsequent preparation of wosolipide, there is no need to add a protein denaturant and a renaturing agent for denaturing and renaturing wosolipide, and the yield of the final product wosolipide is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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:

[0019] FIG1 shows the electrophoresis detection results of the precipitate and supernatant of the strain expressing the soluble intermediate of wosoritide according to Example 1 of the present invention after induced centrifugation.

[0020] FIG2 shows the electrophoresis detection results of the eluted protein after purification of the crude enzyme solution containing the soluble intermediate of wosolitide according to Example 1 of the present invention.

[0021] FIG3 shows the electrophoresis detection results of the acid hydrolysis product of the soluble intermediate of wosoritide according to Example 1 of the present invention.

[0022] FIG4 shows a graph showing the mass spectrometry results of LC-MS detection of vosolitide according to Example 1 of the present invention. DETAILED DESCRIPTION

[0023] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0024] As mentioned in the background art, the prior art uses a chemical synthesis method for the preparation of wosolitide, which is not only complicated and costly, but may also produce racemates, resulting in a long preparation cycle and difficult product quality control. When wosolitide is prepared by enzyme synthesis, when it is expressed through fusion protein, the expression level is mostly low and inclusion bodies are easily formed. It is necessary to correct and purify the misfolding of the inclusion bodies by adding protein denaturants and renaturing agents, resulting in a complicated preparation process. In this application, the inventors attempted to develop a soluble intermediate of wosolitide, a method for preparing the intermediate, and a method for preparing wosolitide, and thus proposed a series of protection schemes for this application.

[0025] In a first typical embodiment of the present application, a soluble intermediate of wosoritide is provided, which comprises a Sumo tag protein, a detachable site and wosoritide; the Sumo tag protein is located at the N-terminal direction of the detachable site, and the wosoritide is located at the C-terminal direction of the detachable site.

[0026] Protein tag refers to the technology of using gene cloning to fuse polypeptides, protein domains or complete proteins with specific functions with target proteins to achieve application technologies such as expression, purification, detection and tracing of target proteins. Sumo (Small ubiquitin-like modifier) ​​tag protein is a small molecule ubiquitin-like modified protein. Sumo tag protein can be used as a protein tag and molecular chaperone for recombinant protein expression to increase the solubility, stability and purity of the target protein. This application achieves efficient soluble expression of polypeptides containing a pair of disulfide bonds by fusing Sumo tag protein with wosuoli peptide, thereby improving the soluble expression of wosuoli peptide.

[0027] In a preferred embodiment, the separation site comprises an aspartic acid.

[0028] In a preferred embodiment, the Sumo tag protein includes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, or a tag protein having more than 70% identity with the amino acid sequence as shown in SEQ ID NO: 1, including but not limited to 75%, 80%, 85%, 90%, 95%, 99% or more (such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more); the Vossoli peptide includes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2, or a tag protein having more than 70% identity with the amino acid sequence as shown in SEQ ID NO: 1. NO: 2 has an amino acid sequence with more than 70% identity and a polypeptide containing a disulfide bond structure, including but not limited to 75%, 80%, 85%, 90%, 95%, 99% or more (such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more); preferably, the soluble intermediate of wosoritide is a polypeptide having the amino acid sequence shown in SEQ ID NO: 3.

[0029] SEQ ID NO: 1 (Sumo tag protein):

[0030] SEQ ID NO: 2 (woxolitide):

[0031] SEQ ID NO: 3 (soluble intermediate of Vosolitide, Sumo-D-Vos fusion protein):

[0032] The amino acid at position 107 in SEQ ID NO: 3 represents an aspartic acid cleavage site.

[0033] Inclusion bodies refer to high-density, insoluble protein particles wrapped by membranes that are formed when exogenous genes are expressed in prokaryotic cells. Their formation is related to the rate of protein generation in the cytoplasm. The concentration of newly generated polypeptides is high and there is not enough time for folding, which makes it easy to form non-crystalline, amorphous protein aggregates. And because the N-terminus of wosoritide is proline, amino acids of this nature can promote the formation of inclusion bodies. Therefore, in existing methods, it is usually necessary to add protein denaturants and renaturing agents to correct and renature these misfolded inclusion bodies, and the target polypeptide wosoritide can be obtained only after purification and removal of excess reagents. In the present application, by connecting the Sumo tag to the wosoritide protein, a fusion protein with good solubility can be expressed and inclusion bodies will not be formed. Therefore, the problem of inclusion body denaturation and renaturation can be avoided, and the expression level is high, which can efficiently promote the soluble expression of the protein. Therefore, there is no need to add protein denaturants and renaturing agents, and the soluble intermediate of wosoritide can be directly prepared, and the target product wosoritide can be obtained in a high yield through a simple purification step.

[0034] Identity in this application refers to the "identity" between amino acid sequences or nucleic acid sequences, that is, the total ratio of amino acid residues or nucleotides of the same type in the amino acid sequence or nucleic acid sequence. The identity of amino acid sequences or nucleic acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0035] The above proteins have 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) identity with the amino acid sequence shown in SEQ ID NO: 2 and have the same function, and their active sites, active pockets, active mechanisms, protein structures, etc. are most likely the same as those of the proteins provided by the above sequences.

[0036] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0037] Substitution and replacement rules generally state that amino acids with similar properties will have similar effects after substitution. For example, conservative amino acid substitutions may occur in the homologous proteins mentioned above. "Conservative amino acid substitutions" include but are not limited to:

[0038] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0039] The hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with large side chains;

[0040] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0041] Amino acids with polar and uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar and uncharged side chains.

[0042] Those skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "BLOSUM62 scoring matrix" in the prior art.

[0043] In the prior art, protein tags commonly used for soluble expression in the preparation of vosolipids include Trx or MBP. These tags are prone to forming inclusion bodies, and even when they are absent, protein expression levels are low. Furthermore, when using existing endonucleases to cleave these tags and the target peptide, the cleavage efficiency is typically low, resulting in low yields of the pure peptide in the final product.

[0044] In the present application, the inventors found that due to the presence of N-terminal proline, the efficiency of multiple enzyme cleavages of wosolipide is very low, and it is difficult to separate wosolipide from the tag protein by enzymatic cleavage. In the present application, an aspartic acid is introduced between the Sumo tag protein and wosolipide, and a DP acid cleavage site is formed with the first amino acid proline of wosolipide. The Sumo tag protein is removed by acid cleavage to achieve efficient separation of the Sumo tag protein and wosolipide, and wosolipide is prepared. The preparation process is simple and efficient. The Sumo tag protein can help wosolipide to be expressed soluble. The acid cleavage site formed by aspartic acid and proline (the first amino acid of wosolipide) can further improve the efficiency and yield of purification, thereby improving the yield of wosolipide.

[0045] In a second typical embodiment of the present application, a method for preparing a soluble intermediate of vosolitide is provided, which comprises: constructing a recombinant plasmid containing a DNA sequence capable of expressing a Sumo tag protein, a DNA sequence capable of expressing a separable site, and a DNA sequence capable of expressing vosolitide, which are sequentially connected in the 5'-3' direction.

[0046] In a preferred embodiment, the Sumo tag protein includes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, or a polypeptide having more than 70% identity with the amino acid sequence as shown in SEQ ID NO: 1 and capable of promoting soluble expression of the protein; the wosoritide includes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2, or a polypeptide having more than 70% identity with the amino acid sequence as shown in SEQ ID NO: 2 and capable of promoting soluble expression of the protein.

[0047] In a preferred embodiment, the DNA sequence capable of expressing the separable site consists of a codon expressing an aspartic acid.

[0048] In a preferred embodiment, the gene of the recombinant plasmid is the nucleotide sequence of SEQ ID NO: 4.

[0049] SEQ ID NO: 4:

[0050] The nucleotide sequence shown from position 1 to position 324 in the above SEQ ID NO: 4 is a gene capable of expressing Sumo tag protein.

[0051] In a preferred embodiment, the recombinant plasmid expresses a soluble intermediate of wosolitide in a host cell; preferably, the host cell includes a eukaryotic cell or a prokaryotic cell; preferably, the prokaryotic cell includes Escherichia coli; preferably, the Escherichia coli includes BL21 (DE3), Origami B (DE3) or Shuffle T7-B.

[0052] The above-mentioned host cells can be used to replicate the recombinant plasmid in the host cells, and the DNA molecules carried by the recombinant plasmid can also be transcribed and translated to obtain a large amount of soluble intermediates of wosoritide. Using existing technologies, the host cells can be disrupted, protein purification after disruption, or other methods can be used to obtain soluble intermediates of wosoritide. The host cells are not of plant origin.

[0053] In a third typical embodiment of the present application, a method for preparing vosolitide is provided, which comprises: cutting the separable site in vosolitide obtained by any of the above-mentioned vosolitide soluble intermediates or the preparation method of any of the above-mentioned vosolitide soluble intermediates, removing the Sumo tag protein and the separable site sequence, and obtaining vosolitide.

[0054] In a preferred embodiment, the excision comprises: using an acid solution to excise the Sumo tag protein and the detachable site at the detachable site to obtain vosolipid peptide.

[0055] Compared with the enzymatic cleavage method, the cleavage of the tagged protein by using an acid solution has low cost and fast reaction speed, and is not affected by factors such as enzyme activity, stability and purity. The reaction speed and degree of hydrolysis can be controlled by adjusting the acid concentration and reaction temperature. The reaction conditions are easier to control than those of the enzymatic cleavage method and are more suitable for large-scale production.

[0056] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0057] Example 1

[0058] A. Construction of a genetically engineered strain expressing Sumo-tagged protein fusion with wosuolitide

[0059] The Sumo tag protein and the target polypeptide sequence were fused and an expression vector was constructed in the pET-28a(+) plasmid.

[0060] 1. Ligate the Sumo-tagged protein to the pET-28a(+) plasmid to construct the pET-28a-Sumo vector backbone.

[0061] 2. The target polypeptide sequence is as shown in the amino acid sequence of SEQ NO: 2, and Asp is introduced at its N-terminus for subsequent acid hydrolysis to remove the fusion tag:

[0062] The target polypeptide fragment was ligated with the pET-28a-Sumo vector backbone via homologous recombination to obtain the recombinant vector pET-28a-Sumo-D-Vos (wherein Vos is the abbreviation for vosoritide). The DNA sequence capable of expressing the soluble intermediate of vosoritide on this recombinant vector is the nucleotide sequence set forth in SEQ ID NO: 4. The recombinant vector was transformed into BL21(DE3) competent cells (purchased from Quanshijin Biotechnology Co., Ltd., Catalog No. CD601-02), and single clone sequencing analysis was performed to screen for strains containing the correct cloned expression vector pET-28a-Sumo-D-Vos.

[0063] 3. Pick three of the above strains and inoculate them in 250mL shake flasks for screening. After the different clones are broken, centrifuged, and the precipitate is tested by SDS-PAGE. The group with better expression is screened by the depth of the electrophoretic band, and the final expression strain is selected. Take 4mL of the BL21 (DE3) strain containing the recombinant plasmid and inoculate it into a 2L Erlenmeyer flask containing 400mL LB medium. Shake and culture at 37℃ and 200rpm until the OD600 reaches 1.0. Add IPTG with a final concentration of 0.2mM and induce at 25℃ overnight. After the induction is completed, the bacteria are collected by centrifugation. The supernatant is collected by ultrasonic disruption and the results of 17% separation gel SDS-PAGE show that the target protein is mainly expressed in a soluble form. The electrophoresis diagram is shown in Figure 1, where lane M represents the marker band, lane 1 represents the band detected by electrophoresis of the supernatant, and lane 2 represents the band detected by electrophoresis of the precipitate. The band indicated by the arrow represents the bacteria in the precipitate containing the Sumo-D-Vos fusion protein.

[0064] B. Purification of Sumo-D-Vosoritide

[0065] 1. The expressed Sumo-D-Vos slurry was diluted to 20% of its concentration and resuspended, and ultrasonically disrupted (5s ultrasound, 5s interval, 30% power), then centrifuged, and the supernatant was filtered through a 0.45μm filter to obtain crude protein.

[0066] 2. Purify the crude protein using affinity chromatography (AKTA system equipped with 5 mL HisTrap HP):

[0067] The membrane filtered sample was loaded at a flow rate of 5 mL / min and then rinsed with binding buffer (50 mM Tris-Hcl, 200 mM NaCl, pH 8.0) until unbound protein was completely eluted. 4 column volumes of miscellaneous proteins were eluted using buffer 50 mM Tris-Hcl, 200 mM NaCl, 50 mM imidazole at pH 8.0. Finally, the target protein was eluted in elution buffer 50 mM Tris-Hcl, 200 mM NaCl, 300 mM imidazole at pH 8.0. The detection results are shown in Figure 2, where lane M represents the Marker band, lane 1 represents the band after electrophoresis detection of the eluted target protein, and the band indicated by the arrow represents that the target protein contains Sumo-D-Vos fusion protein.

[0068] It was calculated that 21 mg of purified Sumo-D-Vos fusion protein could be obtained from 1 gram of wet cells (this could be further increased through optimization of the conditions). The expression levels of the Sumo-D-Vos fusion protein are shown in Table 1.

[0069] C. Acid cleavage to remove fusion tags

[0070] The imidazole in the fusion protein Sumo-D-Vos was removed by ultrafiltration, and an acid cleavage solution (50% acetic acid / 30mM HCl) was added. The reaction was continued at 60°C for 20h, and then deionized water was added to dilute the solution 3 times. The pH in the solution was adjusted to 5.5 (the isoelectric point of His-sumo-D) using 5M NaOH. The supernatant was collected by centrifugation (12000rpm, 10min). The cleavage efficiency was detected by 17% SDS-PAGE, and the target polypeptide was generated. The detection diagram is shown in Figure 3, wherein lane M represents the Marker band, lane 1 represents the band diagram of the supernatant for electrophoresis detection, the band indicated by the arrow represents the presence of Vosoritide product in the supernatant, and lane 2 represents the band diagram of the precipitate for electrophoresis detection. The purity of the target polypeptide reached more than 80%. The yield of the pure product after the protein tag of the fusion protein was removed by the acid cleavage method in this embodiment is shown in Table 2.

[0071] D. Mass spectrometry detection of peptide molecular weight

[0072] The molecular weight of the prepared peptides was analyzed by LC-MS.

[0073] 1. The sample was first separated by HPLC using an Agilent ZORBAX Edipse Plus C18 column, 4.6×100 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 mode was as follows: 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.

[0074] 2. The components separated by HPLC were analyzed using a Q Exactive HF hybrid quadrupole-Orbitrap mass spectrometer with an electrospray ionization source (Dual AJS ESI) in positive ion mode, with a sheath gas flow rate of 35 arb, an auxiliary gas flow rate of 8 arb, a spray voltage of 3800 V, an ion transfer tube temperature of 320°C, and a scan range of 200-3000 m / z. The mass spectrometric data were processed using BioPharma Finder software. Finally, the theoretical molecular weight of wosoritide was 4100.8 Da, and the molecular weight of wosoritide analyzed by mass spectrometry was 4100.1. The mass spectrum is shown in Figure 4.

[0075] Comparative Example 1

[0076] The difference between this comparative example and Example 1 is that the tag protein in this comparative example is Trx (SEQ ID NO: 5), the protein endonuclease cleavage site is KR (Lys-Arg), and there is a connecting peptide sequence (SEQ ID NO: 13 and SEQ ID NO: 14) before the protein endonuclease cleavage site. The connecting peptide facilitates the binding of the protein endonuclease to the cleavage site.

[0077] SEQ ID NO: 5 (Trx tag protein amino acid sequence):

[0078] SEQ ID NO: 6 (Trx tag protein nucleotide sequence):

[0079] Protein tag removal is done by endonuclease cleavage:

[0080] The protein endonuclease in this comparative example is KEX2 enzyme (purchased from Beyotime Biotechnology Co., Ltd., product number: P4229); the mass ratio of KEX2 enzyme to wosuolitide fusion protein is 1:40-500, preferably 1:200, and the enzyme cleavage time is 16h.

[0081] The KEX2 digestion reaction was carried out at 25°C, with the following specific steps: 50 mM Tris-HCl, 200 mM NaCl, 300 mM imidazole, pH 8.0, purified Trx-(G3S)3-KR-Vos fusion protein and KEX2 at a mass ratio of 200:1 (mg / mg), and digestion for 16 h.

[0082] After enzyme digestion, the target polypeptide was purified by acetonitrile precipitation, with the same specific steps as in Example 1.

[0083] After the enzymatic cleavage, the pH of the product was adjusted to 5.7 (the isoelectric point of His-MBP and His-Trx), and then 60% acetonitrile was added to the reaction system. After mixing, the mixture was shaken at 30°C for 2 hours, and then centrifuged at 12000 rpm to separate the supernatant and precipitate. The supernatant was vosolitide.

[0084] The expression level of Trx-(G3S)3-KR-Vos (SEQ ID NO: 7) fusion protein in this comparative example is shown in Table 1; the yield of pure product after the protein tag Trx in the fusion protein was removed by enzyme cleavage in this comparative example is shown in Table 2.

[0085] SEQ ID NO: 7 (Trx-(G3S)3-KR-Vos fusion protein amino acid sequence):

[0086] In SEQ ID NO: 7, amino acids 1 to 115 are the amino acid sequence of the Trx tag protein, amino acids 116 to 127 are the amino acid sequence of the connecting peptide, and amino acids 128 to 129 are the amino acid sequence of the enzyme cleavage site.

[0087] SEQ ID NO: 8 (Trx-(G3S)3-KR-Vos fusion protein nucleotide sequence):

[0088] In SEQ ID NO: 8, nucleotides from positions 1 to 345 are the nucleotide sequence of the Trx tag protein, nucleotides from positions 346 to 381 are the nucleotide sequence of the connecting peptide, and nucleotides from positions 382 to 387 are the nucleotide sequence of the enzyme cleavage site.

[0089] SEQ ID NO: 13 (amino acid sequence of the connecting peptide): GGGSGGGSGGGS.

[0090] SEQ ID NO: 14 (nucleotide sequence of the connecting peptide): GGTGGCGGTTCTGGCGGTGGCAGCGGCGGTGGCAGC.

[0091] Comparative Example 2

[0092] The difference between this comparative example and comparative example 1 is that the tag protein in this comparative example is MBP (SEQ ID NO: 9), the protein endonuclease cleavage site is KR (Lys-Arg), the connecting peptide is the nucleotide sequence shown in SEQ ID NO: 13, and the method and specific steps of protein tag removal are the same as those in comparative example 1.

[0093] SEQ ID NO: 9 (MBP tag protein amino acid sequence):

[0094] SEQ ID NO: 10 (MBP tag protein nucleotide sequence):

[0095] SEQ ID NO: 11 (MBP-(G3S)3-KR-Vos fusion protein amino acid sequence):

[0096] In SEQ ID NO: 11, amino acids 1 to 373 are the amino acid sequence of the MBP tag protein, amino acids 374 to 385 are the amino acid sequence of the connecting peptide, and amino acids 386 to 387 are the amino acid sequence of the enzyme cleavage site.

[0097] SEQ ID NO: 12 (MBP-(G3S)3-KR-Vos fusion protein nucleotide sequence):

[0098] In SEQ ID NO: 12, nucleotides from positions 1 to 1119 are the nucleotide sequence of the MBP tag protein, nucleotides from positions 1120 to 1155 are the nucleotide sequence of the connecting peptide, and nucleotides from positions 1156 to 1161 are the nucleotide sequence of the enzyme cleavage site.

[0099] The expression level of the MBP-KR-Vos fusion protein in this comparative example is shown in Table 1; the yield of the pure product after the protein tag MBP in the fusion protein was removed by enzyme cleavage in this comparative example is shown in Table 2.

[0100] Table 1

[0101] Table 2

[0102] As shown in Tables 1 and 2, the expression level of the Sumo-D-Vos fusion protein, a vosolipid peptide intermediate linked to a Sumo-tagged protein, was higher than that of the other two fusion proteins. Furthermore, the acid-cleavage method for cleaving the tagged protein resulted in a higher yield of pure vosolipid peptide, demonstrating that acid-cleavage is more efficient and effective than the conventional endonuclease-based excision method.

[0103] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the present invention prepares a soluble intermediate of wosolith peptide by utilizing Sumo tag protein. Compared with other protein tags used to enhance the solubility of target proteins in the prior art, the soluble intermediate of wosolith peptide containing Sumo tag protein in the present application is not limited by the problem of inclusion body renaturation, does not require the use of additional reagents to treat the inclusion bodies, and does not require cumbersome purification steps in subsequent processes. The expression level is high, and wosolith peptide can be expressed more efficiently and soluble. In addition, the present application introduces an aspartic acid into the target protein wosolith peptide and the Sumo tag protein, so that it forms a DP acid cleavage site with the proline at the N-terminus of wosolith peptide, which simplifies the step of removing the protein tag. Compared with the enzymatic cleavage method of the prior art, the cost is relatively low and it is more efficient, which can further improve the yield of wosolith peptide.

[0104] 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 soluble intermediate of vosolitide, characterized in that: In the direction from N-terminus to C-terminus, the soluble intermediate of vosolitide includes a Sumo tag protein, a detachable site and vosolitide connected in sequence.

2. The soluble intermediate of vosolitide according to claim 1, characterized in that The separation site includes an aspartic acid.

3. The soluble intermediate of vosolitide according to claim 1, characterized in that The Sumo tag protein includes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, or a tag protein having a homology of more than 70% with the amino acid sequence as shown in SEQ ID NO: 1; The wosoritide includes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2, or a polypeptide having a homology of more than 70% with the amino acid sequence as shown in SEQ ID NO: 2 and containing a disulfide bond structure.

4. The soluble intermediate of vosolitide according to claim 1, characterized in that The soluble intermediate of wosoritide is a polypeptide having an amino acid sequence shown in SEQ ID NO:

3.

5. A method for preparing a soluble intermediate of vosolitide, characterized in that: The preparation method comprises: A recombinant plasmid is constructed, which contains a DNA sequence capable of expressing Sumo tag protein, a DNA sequence capable of expressing a separable site and a DNA sequence capable of expressing vosolipid peptide, which are sequentially connected in the 5'-3' direction.

6. The preparation method according to claim 5, characterized in that The Sumo tag protein includes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, or a polypeptide having more than 70% identity with the amino acid sequence as shown in SEQ ID NO: 1 and capable of promoting soluble protein expression; The wosoritide includes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2, or a polypeptide having a homology of more than 70% with the amino acid sequence as shown in SEQ ID NO:

2.

7. The preparation method according to claim 6, characterized in that The DNA sequence capable of expressing the separable site consists of a codon expressing an aspartic acid.

8. The preparation method according to any one of claims 5 to 7, characterized in that The gene of the recombinant plasmid is the nucleotide sequence shown in SEQ ID NO:

4.

9. The preparation method according to claim 5, characterized in that The recombinant plasmid expresses the soluble intermediate of wosoritide in host cells.

10. The preparation method according to claim 9, characterized in that The host cell includes a eukaryotic cell or a prokaryotic cell.

11. The preparation method according to claim 10, characterized in that The prokaryotic cells include Escherichia coli.

12. The preparation method according to claim 11, characterized in that The Escherichia coli includes BL21(DE3), Origami B(DE3) or Shuffle T7-B.

13. A method for preparing vosolitide, characterized in that: The preparation method comprises: The vosolitide is obtained by cutting the detachable site in the vosolitide soluble intermediate according to any one of claims 1 to 4 or the vosolitide soluble intermediate prepared by the preparation method of the vosolitide soluble intermediate according to any one of claims 5 to 12, and removing the Sumo tag protein and the detachable site sequence.

14. The preparation method according to claim 13, characterized in that The excision comprises: using an acid solution to excise the Sumo tag protein and the detachable site at the detachable site to obtain the vosolipid peptide.

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