WO2025179727A1 - 沃索利肽可溶性中间体、中间体制备方法及沃索利肽的制备方法 - Google Patents

沃索利肽可溶性中间体、中间体制备方法及沃索利肽的制备方法

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Publication number
WO2025179727A1
WO2025179727A1 PCT/CN2024/100462 CN2024100462W WO2025179727A1 WO 2025179727 A1 WO2025179727 A1 WO 2025179727A1 CN 2024100462 W CN2024100462 W CN 2024100462W WO 2025179727 A1 WO2025179727 A1 WO 2025179727A1
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Prior art keywords
vosolitide
seq
protein
amino acid
preparation
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PCT/CN2024/100462
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English (en)
French (fr)
Inventor
洪浩
詹姆斯•盖吉
张娜
赵军旗
焦学成
张法本
岳卓
安军伟
刘亚莉
李奕铭
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Tianjin Asymchem Biotechnology Co Ltd
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Tianjin Asymchem Biotechnology Co Ltd
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Publication of WO2025179727A1 publication Critical patent/WO2025179727A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin

Definitions

  • 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.
  • 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.
  • vosolitide is mainly prepared by chemical coupling synthesis or inclusion body expression.
  • 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.
  • 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.
  • 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.
  • a soluble intermediate of wosolipide 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.
  • the separation site includes an aspartic acid.
  • 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;
  • the soluble intermediate of wosoritide is a polypeptide having an amino acid sequence as shown in SEQ ID NO: 3.
  • a method for preparing a soluble intermediate of vosolitide 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.
  • 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 wosolitide 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.
  • the DNA sequence capable of expressing the separable site consists of a codon expressing an aspartic acid.
  • the gene of the recombinant plasmid is the nucleotide sequence shown in SEQ ID NO: 4.
  • the recombinant plasmid expresses a soluble intermediate of vosolitide 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.
  • a method for preparing vosolitide 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.
  • 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.
  • 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.
  • 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.
  • FIG2 shows the electrophoresis detection results of the eluted protein after purification of the crude enzyme solution containing the soluble intermediate of vosolitide according to Example 1 of the present invention.
  • 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.
  • FIG4 shows a graph showing the mass spectrometry results of LC-MS detection of vosolitide according to Example 1 of the present invention.
  • 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.
  • 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.
  • 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.
  • a soluble intermediate of wosoritide 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.
  • a protein tag refers to a technique that uses gene cloning to fuse a polypeptide, protein domain, or complete protein with a target protein to achieve expression, purification, detection, and tracing of the target protein.
  • Sumo (Small ubiquitin-like modifier) tag protein is a small molecule ubiquitin-like modifier protein.
  • Sumo tag protein can serve as a protein tag and molecular chaperone for recombinant protein expression, used to increase the solubility, stability, and purity of the target protein. This application achieves efficient soluble expression of a polypeptide containing a pair of disulfide bonds by fusing the Sumo tag protein with wosuoli peptide, thereby improving the soluble expression of wosuoli peptide.
  • the separation site comprises an aspartic acid.
  • the Sumo tag protein includes a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, or a tag protein having more than 70% identity with the amino acid sequence 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 wosoritide includes a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, or a tag protein having more than 70% identity with the amino acid sequence 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%, 9
  • SEQ ID NO: 1 (Sumo tag protein):
  • SEQ ID NO: 2 (woxolitide):
  • SEQ ID NO: 3 soluble intermediate of Vosolitide, Sumo-D-Vos fusion protein
  • the amino acid at position 107 in SEQ ID NO: 3 represents the aspartic acid cleavage site.
  • 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.
  • Identity refers to the "identity" between amino acid or nucleic acid sequences, i.e., the total ratio of identical amino acid residues or nucleotides within an amino acid or nucleic acid sequence.
  • the identity of amino acid or nucleic acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.
  • 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) consistency with the amino acid sequence shown in SEQ ID NO: 2 and have the same function.
  • the active sites, active pockets, active mechanisms, protein structures, etc. are most likely the same as those of the proteins provided by the above sequences.
  • 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).
  • Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;
  • hydrophobic amino acids with large side chains are replaced by other hydrophobic amino acids with large side chains;
  • Amino acids with positively charged side chains are replaced by other amino acids with positively charged side chains;
  • Amino acids with polar and uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar and uncharged side chains.
  • 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.
  • the inventors found that due to the presence of N-terminal proline, the efficiency of multiple enzymatic cleavages of wosolipide is very low, and it is difficult to separate wosolipide from the tag protein by enzymatic cleavage.
  • an aspartic acid is introduced between the Sumo tag protein and wosolipide, and a D-P 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.
  • a method for preparing a soluble intermediate of vosolitide 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.
  • 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 the soluble expression of the protein;
  • the vosol peptide 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 and capable of promoting the soluble expression of the protein.
  • the DNA sequence capable of expressing the separable site consists of a codon expressing an aspartic acid.
  • the gene of the recombinant plasmid is the nucleotide sequence of SEQ ID NO: 4.
  • the nucleotide sequence shown at positions 1 to 324 in the above SEQ ID NO: 4 is the gene capable of expressing Sumo tag protein.
  • the recombinant plasmid expresses a soluble intermediate of vosolitide 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.
  • a method for preparing vosolitide 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.
  • 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.
  • 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.
  • the Sumo tag protein and the target polypeptide sequence were fused and an expression vector was constructed in the pET-28a(+) plasmid.
  • the target peptide sequence is as shown in the amino acid sequence of SEQ NO: 2, with Asp introduced at its N-terminus for subsequent acid hydrolysis to remove the fusion tag:
  • 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 (where 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.
  • the electrophoresis diagram is shown in Figure 1, where lane M represents the marker band, lane 1 represents the band for electrophoresis detection of the supernatant, and lane 2 represents the band for electrophoresis detection of the precipitate. Among them, the band pointed by the arrow represents the bacteria in the precipitate containing the Sumo-D-Vos fusion protein.
  • 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 0.45 ⁇ m filter to obtain crude protein.
  • 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 the unbound protein was completely eluted.
  • binding buffer 50 mM Tris-Hcl, 200 mM NaCl, pH 8.0
  • the impurity protein was eluted for 4 column volumes using a buffer of 50 mM Tris-Hcl, 200 mM NaCl, 50 mM imidazole at pH 8.0.
  • the target protein was eluted in an elution buffer of 50 mM Tris-Hcl, 200 mM NaCl, 300 mM imidazole at pH 8.0.
  • lane M represents the marker band
  • lane 1 represents the band after electrophoresis detection of the eluted target protein
  • the band indicated by the arrow represents that the target protein contains Sumo-D-Vos fusion protein.
  • the detection diagram is shown in Figure 3, where 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 example is shown in Table 2.
  • the molecular weight of the prepared peptides was analyzed by LC-MS.
  • the sample was first separated by HPLC using an Agilent ZORBAX Edipse Plus C18 column, 4.6 ⁇ 100 mm, 3.5 ⁇ m.
  • Mobile phase A was 0.1% trifluoroacetic acid
  • 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
  • the flow rate was 0.3 mL/min.
  • the components separated by HPLC were analyzed using a Q Exactive HF 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.
  • the theoretical molecular weight of wosoritide was 4100.8 Da
  • the mass spectrometrically resolved molecular weight of wosoritide was 4100.1.
  • the mass spectrum is shown in Figure 4.
  • Example 1 The difference between this comparative example and Example 1 is that the label 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 is conducive to the binding of the protein endonuclease to the cleavage site.
  • SEQ ID NO: 6 (Trx tag protein nucleotide sequence):
  • Protein tag removal is done by endonuclease cleavage:
  • 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.
  • 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.
  • the KEX2 enzyme digestion reaction was carried out at 25°C. The specific steps were: 50mM Tris-HCl, 200mM NaCl, 300mM imidazole, pH 8.0, purified Trx-(G3S)3-KR-Vos fusion protein and KEX2 at a mass ratio of 200:1 (mg/mg), and enzyme digestion for 16h.
  • the target polypeptide was purified by acetonitrile precipitation, with the same specific steps as in Example 1.
  • 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.
  • Trx-(G3S)3-KR-Vos SEQ ID NO: 7
  • Table 1 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 removing the protein tag Trx in the fusion protein by enzymatic cleavage in this comparative example is shown in Table 2.
  • SEQ ID NO: 7 (Trx-(G3S)3-KR-Vos fusion protein amino acid sequence):
  • amino acids at positions 1 to 115 in SEQ ID NO: 7 are the amino acid sequence of the Trx tag protein, the amino acids at positions 116 to 127 are the amino acid sequence of the connecting peptide, and the amino acids at positions 128 to 129 are the amino acid sequence of the enzyme cleavage site.
  • SEQ ID NO: 8 (Trx-(G3S)3-KR-Vos fusion protein nucleotide sequence):
  • nucleotides from positions 1 to 345 in SEQ ID NO: 8 are the nucleotide sequence of the Trx tag protein
  • nucleotides from positions 346 to 381 are the nucleotide sequence of the connecting peptide
  • nucleotides from positions 382 to 387 are the nucleotide sequence of the enzyme cleavage site.
  • SEQ ID NO: 13 amino acid sequence of connecting peptide: GGGSGGGSGGGS.
  • SEQ ID NO: 14 (nucleotide sequence of connecting peptide): GGTGGCGGTTCTGGCGGTGGCAGCGGCGGTGGCAGC.
  • 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
  • the method and specific steps of protein tag removal are the same as those in comparative example 1.
  • SEQ ID NO: 9 (MBP tag protein amino acid sequence):
  • SEQ ID NO: 10 MBP tag protein nucleotide sequence
  • SEQ ID NO: 11 (MBP-(G3S)3-KR-Vos fusion protein amino acid sequence):
  • amino acids at positions 1 to 373 in SEQ ID NO: 11 are the amino acid sequence of the MBP tag protein, the amino acids at positions 374 to 385 are the amino acid sequence of the connecting peptide, and the amino acids at positions 386 to 387 are the amino acid sequence of the enzyme cleavage site.
  • nucleotides from positions 1 to 1119 in SEQ ID NO: 12 are the nucleotide sequence of the MBP tag protein
  • nucleotides from positions 1120 to 1155 are the nucleotide sequence of the connecting peptide
  • nucleotides from positions 1156 to 1161 are the nucleotide sequence of the enzyme cleavage site.
  • 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.
  • the present invention prepares a soluble intermediate of wosolith peptide by utilizing Sumo tag protein.
  • 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.
  • the present application introduces an aspartic acid into the target protein wosolith peptide and the Sumo tag protein, so that it forms a D-P acid cleavage site with the proline at the N-terminus of wosolith peptide, which simplifies the step of removing the protein tag.
  • the cost is relatively low and it is more efficient, which can further improve the yield of wosolith peptide.

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Abstract

本发明提供了一种沃索利肽可溶性中间体、中间体制备方法及沃索利肽的制备方法。该沃索利肽可溶性中间体包括Sumo标签蛋白、可分离位点和沃索利肽。能够解决现有技术中制备沃索利肽收率低的问题，适用于多肽药物生物合成领域。

Description

沃索利肽可溶性中间体、中间体制备方法及沃索利肽的制备方法
本申请是以CN申请号为202410229257.8，申请日为2024年02月29日的中国申请为基础，并主张其优先权，该CN申请的公开内容再次作为整体引入本申请中。
技术领域
本发明涉及多肽药物生物合成领域，具体而言，涉及一种沃索利肽可溶性中间体、中间体制备方法及沃索利肽的制备方法。
背景技术
沃索利肽(Vosoritide)是一种肽类药物，用于治疗儿童和青少年中的骨骼短小症。该药物是一种人类生长激素类似物，作用于骨骼生长板，促进骨骼生长，从而帮助患者增加身高。
目前沃索利肽主要通过化学法偶联合成法或包涵体表达法进行制备。其中，化学偶联合成法的工艺相对复杂，且合成过程中易产生杂质，纯度和回收率相对较低。包涵体表达法虽然能够避免蛋白酶降解，但蛋白复杂的变性和复性的过程要用到大量变性剂，如尿素或盐酸胍，纯化工艺繁琐，导致最终得率低。虽然现有技术中利用多种标签对沃索利肽变体进行融合表达，但表达获得的多数融合蛋白为包涵体，变性和复性的过程要用到大量变性剂如尿素或盐酸胍，生产成本高，纯化工艺复杂，导致沃索利肽的最终得率非常低，难以满足工业生产的需求。
发明内容
本发明的主要目的在于提供一种沃索利肽可溶性中间体、中间体制备方法及沃索利肽的制备方法，以解决现有技术中制备沃索利肽收率低的问题。
为了实现上述目的，根据本发明的一个方面，提供了一种沃索利肽可溶性中间体，该沃索利肽可溶性中间体包括Sumo标签蛋白、可分离位点和沃索利肽；Sumo标签蛋白位于可分离位点的N端方向，沃索利肽位于可分离位点的C端方向。
进一步地，分离位点包括一个天冬氨酸。
进一步地，Sumo标签蛋白包括具有SEQ ID NO：1所示氨基酸序列的多肽，或与SEQ ID NO：1所示的氨基酸序列具有70％以上一致性的标签蛋白；沃索利肽包括具有SEQ ID NO：2所示氨基酸序列的多肽，或与SEQ ID NO：2所示的氨基酸序列具有70％以上一致性、且含有二硫键结构的多肽；优选地，沃索利肽可溶性中间体为具有SEQ ID NO：3所示的氨基酸序列的多肽。
为了实现上述目的，根据本发明的第二个方面，提供了一种沃索利肽可溶性中间体的制备方法，该制备方法包括：构建重组质粒，重组质粒上含有5’-3’端方向顺次连接的能够表达Sumo标签蛋白的DNA序列、能够表达可分离位点的DNA序列和能够表达沃索利肽的DNA序列。
进一步地，Sumo标签蛋白包括具有SEQ ID NO：1所示的氨基酸序列的多肽，或与SEQ ID NO：1所示的氨基酸序列具有70％以上一致性、且能够促进蛋白可溶性表达的多肽；沃索利肽包括具有SEQ ID NO：2所示的氨基酸序列的多肽，或与SEQ ID NO：2所示的氨基酸序列具有70％以上一致性的多肽。
进一步地，能够表达可分离位点的DNA序列由表达一个天冬氨酸的密码子组成。
进一步地，重组质粒的基因为SEQ ID NO：4所示的核苷酸序列。
进一步地，重组质粒在宿主细胞中表达沃索利肽可溶性中间体；优选地，宿主细胞包括真核细胞或原核细胞；优选地，原核细胞包括大肠杆菌；优选地，大肠杆菌包括BL21(DE3)、Origami B(DE3)或Shuffle T7-B。
为了实现上述目的，根据本发明的第三个方面，提供了一种沃索利肽的制备方法，该制备方法包括：通过切割上述任一种沃索利肽可溶性中间体或上述任一种沃索利肽可溶性中间体的制备方法制备获得的沃索利肽可溶性中间体中的可分离位点，将Sumo标签蛋白和可分离位点序列切除，获得沃索利肽。
进一步地，切除包括：利用酸溶液在可分离位点切除Sumo标签蛋白和可分离位点，获得沃索利肽。
应用本发明的技术方案，通过提供一种包括Sumo标签蛋白、可分离位点以及沃索利肽的可溶性中间体，即一种融合蛋白，其中，Sumo标签蛋白位于可分离位点的N端方向，沃索利肽位于可分离位点的C端方向，该种融合蛋白的可溶性较好，在后续制备沃索利肽时不需通过添加蛋白变性剂和复性剂用于对沃索利肽进行变性和复性，终产物沃索利肽的收率高。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1示出了根据本发明实施例1的表达沃索利肽可溶性中间体的菌株经诱导离心后沉淀及上清液的电泳检测结果图。
图2示出了根据本发明实施例1的含有沃索利肽可溶性中间体的粗酶液纯化后洗脱蛋白的电泳检测结果图。
图3示出了根据本发明实施例1的沃索利肽可溶性中间体中经过酸水解后产物的电泳检测结果图。
图4示出了根据本发明实施例1的沃索利肽LC-MS检测质谱结果图。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。
如背景技术所提到的，现有技术对于沃索利肽的制备采用化学合成的制备方法进行制备，不仅过程复杂，成本过高，而且可能会产生消旋体，导致制备周期较长，产品的质量控制比较困难。而通过酶合成的方法对沃索利肽进行制备，通过融合蛋白进行表达时，大多表达量较低，且易形成包涵体，需要通过添加蛋白变性剂和复性剂对包涵体的错误折叠进行校正和纯化，导致制备过程复杂。在本申请中发明人尝试开发一种索利肽可溶性中间体、中间体制备方法及沃索利肽制备方法，因而提出了本申请的一系列保护方案。
在本申请第一种典型的实施方式中，提供了一种沃索利肽可溶性中间体，该沃索利肽可溶性中间体包括Sumo标签蛋白、可分离位点和沃索利肽；Sumo标签蛋白位于可分离位点的N端方向，沃索利肽位于可分离位点的C端方向。
蛋白标签(Protein tag)指的是利用基因克隆的手段，将具有特定功能的多肽、蛋白质结构域或完整的蛋白质与目标蛋白融合，以实现目标蛋白的表达纯化、检测和示踪等应用技术。Sumo(Small ubiquitin-like modifier)标签蛋白是一种小分子泛素样修饰蛋白，Sumo标签蛋白能够作为重组蛋白表达的蛋白标签和分子伴侣，用于增加目标蛋白的溶解性、稳定性和纯度。本申请通过将Sumo标签蛋白与沃索利肽进行融合表达的方法，实现了对于含有一对二硫键的多肽的高效可溶表达，提高了沃索利肽的可溶表达性。
在一种优选的实施例中，分离位点包括一个天冬氨酸。
在一种优选的实施例中，Sumo标签蛋白包括具有SEQ ID NO：1所示氨基酸序列的多肽，或与SEQ ID NO：1所示的氨基酸序列具有70％以上一致性的标签蛋白，包括但不限于75％、80％、85％、90％、95％、99％以上(比如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％以上，甚至99.9％以上)；沃索利肽包括具有SEQ ID NO：2所示氨基酸序列的多肽，或与SEQ ID NO：2所示的氨基酸序列具有70％以上一致性、且含有二硫键结构的多肽，包括但不限于75％、80％、85％、90％、95％、99％以上(比如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％以上，甚至99.9％以上)；优选地，沃索利肽可溶性中间体为具有SEQ ID NO：3所示氨基酸序列的多肽。
SEQ ID NO：1(Sumo标签蛋白)：
SEQ ID NO：2(沃索利肽)：
SEQ ID NO：3(沃索利肽可溶性中间体，Sumo-D-Vos融合蛋白)：
SEQ ID NO：3中第107位的氨基酸代表天冬氨酸酸切位点。
包涵体指的是外源基因在原核细胞中表达时，形成的由膜包裹的高密度、不溶性蛋白质颗粒，其形成与胞质内蛋白质的生成速率有关，新生成的多肽浓度高，无充足的时间进行折叠，从而易形成非结晶、无定形的蛋白质聚集体。且因沃索利肽N端为脯氨酸，此性质的氨基酸能够促进包涵体的形成，故现有方法中通常需要添加蛋白变性剂和复性剂对这些错误折叠的包涵体进行校正后复性，且需纯化后去除多余试剂后，才能够得到目标多肽沃索利肽。而在本申请中，通过将Sumo标签与沃索利肽蛋白连接时，能够表达获得可溶性较好的融合蛋白，不会形成包涵体，故能够避免包涵体变复性的问题，且表达量较高，能够高效地促进蛋白的可溶表达，因此不需要添加蛋白的变性剂和复性剂，就能够直接制备获得沃索利肽可溶性中间体，进而通过简单的纯化步骤即可以较高的产率获得目标产物沃索利肽。
本申请中的一致性(Identity)是指氨基酸序列或核酸序列之间的“一致性”，即氨基酸序列或核酸序列中的种类相同的氨基酸残基或核苷酸的比率的总计。氨基酸序列或核酸序列的一致性可以利用BLAST(Basic Local Alignment Search Tool)、FASTA等比对程序来确定。
上述与SEQ ID NO：2所示的氨基酸序列具有70％、75％、80％、85％、90％、95％、99％以上(比如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％以上，甚至99.9％以上)一致性且具有相同功能的蛋白质，其活性位点、活性口袋、活性机制、蛋白结构等均和上述序列提供的蛋白质大概率相同。
如本文所用，氨基酸残基缩写如下：丙氨酸(Ala；A)、天冬酰胺(Asn；N)、天冬氨酸(Asp；D)、精氨酸(Arg；R)、半胱氨酸(Cys；C)、谷氨酸(Glu；E)、谷氨酰胺(Gln；Q)、甘氨酸(Gly；G)、组氨酸(His；H)、异亮氨酸(Ile；I)、亮氨酸(Leu；L)、赖氨酸(Lys；K)、蛋氨酸(Met；M)、苯丙氨酸(Phe；F)、脯氨酸(Pro；P)，丝氨酸(Ser；S)、苏氨酸(Thr；T)、色氨酸(Trp；W)、酪氨酸(Tyr；Y)和缬氨酸(Val；V)。
取代、替换等规则，一般情况下，哪些氨基酸性质类似，替换后的效果也类似。例如，在上述同源蛋白中，可发生保守的氨基酸替换。“保守的氨基酸替换”包括但不限于：
疏水性氨基酸(Ala、Cys、Gly、Pro、Met、Val、Ile、Leu)被其他疏水性氨基酸取代；
侧链大的疏水性氨基酸(Phe、Tyr、Trp)被其他侧链大的疏水性氨基酸取代；
侧链带正电的氨基酸(Arg、His、Lys)被其他侧链带正电的氨基酸取代；
侧链有极性不带电的氨基酸(Ser、Thr、Asn、Gln)被其他侧链有极性不带电的氨基酸取代。
本领域技术人员也可以根据现有技术中的“BLOSUM62评分矩阵”等本领域技术人员熟知的氨基酸替换规则对氨基酸进行保守替换。
现有技术中，在制备沃索利肽时，常用的具有可溶表达功能的蛋白标签包括Trx或MBP等，在使用这些蛋白标签时，容易形成包涵体，即使不产生包涵体，蛋白表达量也很低。且利用现有技术中的蛋白内切酶对该种蛋白标签以及目标肽段进行切割时，酶切效率通常较低，导致终产品多肽纯品收率低下。
在本申请中，发明人发现沃索利肽因N端脯氨酸的存在，导致多种酶切效率很低，难以通过酶切的方法将沃索利肽与标签蛋白分离。本申请中通过在Sumo标签蛋白与沃索利肽的之间引入一个天冬氨酸，与沃索利肽的第一个氨基酸脯氨酸构成D-P酸切位点，通过酸切将Sumo标签蛋白切除，实现Sumo标签蛋白与沃索利肽的高效分离，制备获得沃索利肽，制备过程简单高效，Sumo标签蛋白能帮助沃索利肽可溶性表达，天冬氨酸和脯氨酸(沃索利肽的第一个氨基酸)形成的酸切位点能够进一步提高纯化的效率和收率，使得沃索利肽的收率提高。
在本申请第二种典型的实施方式中，提供了一种沃索利肽可溶性中间体的制备方法，该制备方法包括：构建重组质粒，重组质粒上含有5’-3’端方向顺次连接的能够表达Sumo标签蛋白的DNA序列、能够表达可分离位点的DNA序列和能够表达沃索利肽的DNA序列。
在一种优选的实施例中，Sumo标签蛋白包括具有SEQ ID NO：1所示氨基酸序列的多肽，或与SEQ ID NO：1所示的氨基酸序列具有70％以上一致性、且能够促进蛋白可溶性表达的多肽；沃索利肽包括具有SEQ ID NO：2所示氨基酸序列的多肽，或与SEQ ID NO：2所示的氨基酸序列具有70％以上一致性、且能够促进蛋白可溶性表达的多肽。
在一种优选的实施例中，能够表达可分离位点的DNA序列由表达一个天冬氨酸的密码子组成。
在一种优选的实施例中，重组质粒的基因为SEQ ID NO：4的核苷酸序列。
SEQ ID NO：4：
上述SEQ ID NO：4中第1位至第324位所示的核苷酸序列即为能够表达Sumo标签蛋白的基因。
在一种优选的实施例中，重组质粒在宿主细胞中表达沃索利肽可溶性中间体；优选地，宿主细胞包括真核细胞或原核细胞；优选地，原核细胞包括大肠杆菌；优选地，大肠杆菌包括BL21(DE3)、Origami B(DE3)或Shuffle T7-B。
利用上述宿主细胞，能够在宿主细胞中进行重组质粒的复制，也能够将重组质粒上携带的DNA分子进行转录、翻译，获得大量的沃索利肽可溶性中间体。利用现有技术，对宿主细胞进行破碎、破碎后蛋白纯化或其他方式，能够获得沃索利肽可溶性中间体。该宿主细胞为非植物来源的宿主细胞。
在本申请第三种典型的实施方式中，提供了一种沃索利肽的制备方法，该制备方法包括：通过切割上述任一种沃索利肽可溶性中间体或上述任一种沃索利肽可溶性中间体的制备方法制备获得的沃索利肽中的可分离位点，将Sumo标签蛋白和可分离位点序列切除，获得沃索利肽。
在一种优选的实施例中，切除包括：利用酸溶液在可分离位点切除Sumo标签蛋白和可分离位点，获得沃索利肽。
利用酸溶液对标签蛋白进行切割，相对于酶切法成本低、反应速度快，且不受酶的活性、稳定性和纯度等因素的影响，能够通过调整酸的浓度和反应温度来控制反应速度和水解程度，反应条件相对酶切法来说更易于控制，更适用于大规模生产。
下面将结合具体的实施例来进一步详细解释本申请的有益效果。
实施例1
A.构建Sumo标签蛋白融合表达沃索利肽的基因工程菌株
将Sumo标签蛋白和目标多肽序列进行融合，于pET-28a(+)质粒中构建表达载体。
1、将Sumo标签蛋白与pET-28a(+)质粒连接，构建pET-28a-Sumo载体骨架。
2、目标多肽序列如SEQ NO：2的氨基酸序列所示，在其N端引入Asp，用于后续的酸水解以去除融合标签：
将目标多肽片段与pET-28a-Sumo载体骨架经同源重组进行连接，获得重组载体pET-28a-Sumo-D-Vos(其中Vos为Vosoritide的缩写)，该重组载体上的能够表达沃索利肽可溶性中间体的DNA序列为SEQ ID NO：4所示的核苷酸序列。将重组载体转化入BL21(DE3)感受态细胞(购自全式金生物技术股份有限公司，货号：CD601-02)，进行单克隆测序分析，筛选出含有正确的克隆表达载体pET-28a-Sumo-D-Vos的菌种。
3、挑取3个上述菌种接种于250mL摇瓶中进行筛选，不同克隆菌泥经破碎后，离心，沉淀进行SDS-PAGE检测，通过电泳条带深浅筛选表达较好的组别，从中筛选出最终的表达菌 株。取含有重组质粒的BL21(DE3)菌株4mL，接种于含400mL LB培养基的2L三角瓶，37℃200rpm振荡培养至OD600为1.0时，加入终浓度为0.2mM IPTG，25℃诱导过夜，诱导结束，离心收集菌体。通过超声破碎，收集上清，17％分离胶SDS-PAGE结果表明目标蛋白主要呈可溶表达，电泳图如图1所示，其中泳道M表示的是Marker条带，泳道1代表上清液进行电泳检测的条带，泳道2代表沉淀进行电泳检测的条带，其中，箭头指出的条带代表沉淀中的菌体包含有Sumo-D-Vos融合蛋白。
B.Sumo-D-Vosoritide纯化
1、将表达的Sumo-D-Vos的菌泥，稀释至其浓度的20％后重悬，进行超声破碎(5s超声，5s间隔，30％功率)，然后离心，上清过0.45μm滤膜后获得粗蛋白。
2、利用亲和层析对获得的粗蛋白进行纯化(AKTA系统装配5mL HisTrap HP)：
滤膜过滤样品以5mL/min流速上样，然后采用结合缓冲液(50mM Tris-Hcl，200mM NaCl，pH 8.0)冲洗，直至未结合的蛋白完全被洗脱。利用缓冲液50mM Tris-Hcl，200mM NaCl，50mM咪唑，在pH 8.0的条件下，洗脱杂蛋白4个柱体积。最后在洗脱缓冲液50mM Tris-Hcl，200mM NaCl，300mM咪唑，pH＝8.0条件下洗脱目标蛋白，其检测结果图如图2所示，其中泳道M表示的是Marker条带，泳道1代表洗脱下的目标蛋白进行电泳检测后的条带，箭头指出的条带代表该目标蛋白中含有Sumo-D-Vos融合蛋白。
据计算，1g湿细胞可以获得纯化的Sumo-D-Vos融合蛋白21mg(经过条件优化可以进一步提高)。Sumo-D-Vos融合蛋白的表达量如表1所示。
C.酸切移除融合标签
利用超滤置换将融合蛋白Sumo-D-Vos中的咪唑去除，加入酸切溶液(50％乙酸/30mM HCl)，60℃反应20h，加入去离子水稀释3倍，利用5M NaOH将溶液中的pH至5.5(His-sumo-D等电点)，离心(12000rpm，10min)收集上清。利用17％ SDS-PAGE检测其切割效率，并检测是否有目标多肽生成，检测图如图3所示，其中，泳道M表示的是Marker条带，泳道1代表上清液进行电泳检测的条带图，箭头指出的条带代表该上清液中有Vosoritide产物，泳道2代表沉淀进行电泳检测的条带图。目标多肽的纯度达到80％以上。利用本实施例中的酸切的方式将融合蛋白的蛋白标签切除后的纯品收率如表2所示。
D.质谱检测多肽分子量
制备的多肽分子量采用LC-MS进行分析。
1、样品先经过HPLC柱子分离：Agilent ZORBAX Edipse Plus C18，4.6×100mm，3.5μm，流动相A：0.1％三氟乙酸，流动相B：0.1％三氟乙酸乙腈溶液，采用梯度洗脱方式：0min 10％B，9min 95％B，12min 100％B，12.1min 10％B，15min 10％B，柱温50℃，紫外检测器210nm，流速0.3mL/min。
2、经过HPLC分离后的组分采用Q Exactive HF组合型四极杆Orbitrap质谱仪，利用电喷雾离子源(Dual AJS ESI)，设置为正离子模式检测，鞘气流速35arb，辅助气流速8arb，喷雾电压3800V，离子传输管温度320℃，扫描范围200～3000m/z，并通过BioPharma Finder软件处理质谱数据。最后，沃索利肽的理论分子量为4100.8Da，质谱解析的沃索利肽分子量是4100.1，质谱图如图4所示。
对比例1
本对比例与实施例1的区别在于，本对比例中的标签蛋白为Trx(SEQ ID NO：5)，蛋白内切酶酶切位点为KR(Lys-Arg)，在内切酶酶切位点前还有一段连接肽序列(SEQ ID NO：13和SEQ ID NO：14)，连接肽有利于蛋白内切酶与酶切位点结合。
SEQ ID NO：5(Trx标签蛋白氨基酸序列)：
SEQ ID NO：6(Trx标签蛋白核苷酸序列)：
蛋白标签切除的方式为蛋白内切酶切：
本对比例中的蛋白内切酶为KEX2酶(购自碧云天生物技术公司，货号：P4229)；KEX2酶与沃索利肽融合蛋白的质量比为1:40-500，优选为1：200，酶切时间16h。
KEX2酶切反应在25℃进行，具体步骤是：50mM Tris-Hcl，200mM NaCl，300mM咪唑，pH 8.0纯化的Trx-(G3S)3-KR-Vos融合蛋白和KEX2质量比200：1(mg/mg)，酶切16h。
酶切后乙腈沉淀法纯化目标多肽，具体步骤同实施例1。
酶切完成之后的产物，调整pH于5.7(His-MBP和His-Trx等电点)，然后在反应体系加入60％的乙腈，混匀后在30℃振荡处理2h，之后12000rpm离心分离上清和沉淀，上清中即为沃索利肽。
本对比例中Trx-(G3S)3-KR-Vos(SEQ ID NO：7)融合蛋白表达量如表1所示；利用本对比例中的酶切的方式将融合蛋白中的蛋白标签Trx切除后的纯品收率如表2所示。
SEQ ID NO：7(Trx-(G3S)3-KR-Vos融合蛋白氨基酸序列)：
SEQ ID NO：7中第1位至第115位的氨基酸为Trx标签蛋白的氨基酸序列，第116至第127位的氨基酸为连接肽的氨基酸序列，第128至第129位的氨基酸为酶切位点的氨基酸序列。
SEQ ID NO：8(Trx-(G3S)3-KR-Vos融合蛋白核苷酸序列)：
SEQ ID NO：8中第1位至第345位的核苷酸为Trx标签蛋白的核苷酸序列，第346位至第381位的核苷酸为连接肽的核苷酸序列，第382位至第387位的核苷酸为酶切位点的核苷酸序列。
SEQ ID NO：13(连接肽的氨基酸序列)：GGGSGGGSGGGS。
SEQ ID NO：14(连接肽的核苷酸序列)：GGTGGCGGTTCTGGCGGTGGCAGCGGCGGTGGCAGC。
对比例2
本对比例与对比例1的区别在于，本对比例中的标签蛋白为MBP(SEQ ID NO：9)，蛋白内切酶酶切位点为KR(Lys-Arg)，连接肽为SEQ ID NO：13所示的核苷酸序列，蛋白标签切除的方式和具体步骤与对比例1一致。
SEQ ID NO：9(MBP标签蛋白氨基酸序列)：
SEQ ID NO：10(MBP标签蛋白核苷酸序列)：
SEQ ID NO：11(MBP-(G3S)3-KR-Vos融合蛋白氨基酸序列)：
SEQ ID NO：11中第1位至第373位的氨基酸为MBP标签蛋白的氨基酸序列，第374至第385位的氨基酸为连接肽的氨基酸序列，第386位至第387位的氨基酸为酶切位点的氨基酸序列。
SEQ ID NO：12(MBP-(G3S)3-KR-Vos融合蛋白核苷酸序列)：

SEQ ID NO：12中第1位至第1119位的核苷酸为MBP标签蛋白的核苷酸序列，第1120位至第1155位的核苷酸为连接肽的核苷酸序列，第1156至第1161位的核苷酸为酶切位点的核苷酸序列。
本对比例中MBP-KR-Vos融合蛋白表达量如表1所示；利用本对比例中的酶切的方式将融合蛋白中的蛋白标签MBP切除后的纯品收率如表2所示。
表1
表2
从表1和表2可知，连接有Sumo标签蛋白的沃索利肽中间体，即Sumo-D-Vos融合蛋白的表达量与其他两种融合蛋白的表达量更高。且利用酸切的切割标签蛋白的方法，使得沃索 利肽的纯品收率更高，证明酸切切除标签蛋白与现有技术中常见的蛋白内切酶酶切切除标签蛋白的方法相比，切除效率高且效果好。
从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：本发明通过利用Sumo标签蛋白，制备沃索利肽可溶性中间体，与现有技术中其他用于增强目标蛋白可溶性的蛋白标签相比，本申请中含有Sumo标签蛋白的沃索利肽可溶性中间体不受包涵体变复性的问题限制，不需要使用额外的试剂对包涵体进行处理，也不需进行后续工艺繁琐的纯化步骤，且表达量高，能够更为高效地可溶性表达沃索利肽。且本申请在目标蛋白沃索利肽以及Sumo标签蛋白中引入一个天冬氨酸，使其与沃索利肽N端的脯氨酸形成D-P酸切位点，简化了切除蛋白标签的步骤，与现有技术的酶切法相比成本相对较低，且更高效，能够进一步提高沃索利肽收率。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (14)

  1. 一种沃索利肽可溶性中间体，其特征在于，按照从N端到C端的方向，所述沃索利肽可溶性中间体包括顺序连接的Sumo标签蛋白、可分离位点和沃索利肽。
  2. 根据权利要求1所述的沃索利肽可溶性中间体，其特征在于，所述分离位点包括一个天冬氨酸。
  3. 根据权利要求1所述的沃索利肽可溶性中间体，其特征在于，
    所述Sumo标签蛋白包括具有SEQ ID NO：1所示氨基酸序列的多肽，或与SEQ ID NO：1所示的氨基酸序列具有70％以上一致性的标签蛋白；
    所述沃索利肽包括具有SEQ ID NO：2所示氨基酸序列的多肽，或与SEQ ID NO：2所示的氨基酸序列具有70％以上一致性、且含有二硫键结构的多肽。
  4. 根据权利要求1所述的沃索利肽可溶性中间体，其特征在于，所述沃索利肽可溶性中间体为具有SEQ ID NO：3所示氨基酸序列的多肽。
  5. 一种沃索利肽可溶性中间体的制备方法，其特征在于，所述制备方法包括：
    构建重组质粒，所述重组质粒上含有5’-3’端方向顺次连接的能够表达Sumo标签蛋白的DNA序列、能够表达可分离位点的DNA序列和能够表达沃索利肽的DNA序列。
  6. 根据权利要求5所述的制备方法，其特征在于，所述Sumo标签蛋白包括具有SEQ ID NO：1所示氨基酸序列的多肽，或与SEQ ID NO：1所示的氨基酸序列具有70％以上一致性、且能够促进蛋白可溶性表达的多肽；
    所述沃索利肽包括具有SEQ ID NO：2所示氨基酸序列的多肽，或与SEQ ID NO：2所示的氨基酸序列具有70％以上一致性的多肽。
  7. 根据权利要求6所述的制备方法，其特征在于，所述能够表达可分离位点的DNA序列由表达一个天冬氨酸的密码子组成。
  8. 根据权利要求5-7中任一项所述的制备方法，其特征在于，所述重组质粒的基因为SEQ ID NO：4所示的核苷酸序列。
  9. 根据权利要求5所述的制备方法，其特征在于，所述重组质粒在宿主细胞中表达所述沃索利肽可溶性中间体。
  10. 根据权利要求9所述的制备方法，其特征在于，所述宿主细胞包括真核细胞或原核细胞。
  11. 根据权利要求10所述的制备方法，其特征在于所述原核细胞包括大肠杆菌。
  12. 根据权利要求11所述的制备方法，其特征在于所述大肠杆菌包括BL21(DE3)、Origami B(DE3)或Shuffle T7-B。
  13. 一种沃索利肽的制备方法，其特征在于，所述制备方法包括：
    通过切割权利要求1-4中任一项所述沃索利肽可溶性中间体或利用权利要求5-12中任一项所述的沃索利肽可溶性中间体的制备方法制备获得的所述沃索利肽可溶性中间体中的可分离位点，将所述Sumo标签蛋白和可分离位点序列切除，获得所述沃索利肽。
  14. 根据权利要求13所述的制备方法，其特征在于，所述切除包括：利用酸溶液在所述可分离位点切除所述Sumo标签蛋白和可分离位点，获得所述沃索利肽。
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