WO2025179727A1 - Intermédiaire soluble de vosoritide, procédé de préparation de l'intermédiaire et procédé de préparation de vosoritide - Google Patents

Intermédiaire soluble de vosoritide, procédé de préparation de l'intermédiaire et procédé de préparation de vosoritide

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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)
Chinese (zh)
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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    • 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

La présente invention concerne un intermédiaire soluble de vosoritide, un procédé de préparation de l'intermédiaire et un procédé de préparation de vosoritide. L'intermédiaire soluble de vosoritide comprend une protéine de marquage Sumo, un site clivable et un vosoritide. Le procédé peut résoudre le problème de faible rendement dans la préparation de vosoritide dans l'état de la technique, et est approprié pour le domaine de la biosynthèse de médicaments polypeptidiques.
PCT/CN2024/100462 2024-02-29 2024-06-20 Intermédiaire soluble de vosoritide, procédé de préparation de l'intermédiaire et procédé de préparation de vosoritide Pending WO2025179727A1 (fr)

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