WO2022241831A1 - 多肽的制备方法 - Google Patents

多肽的制备方法 Download PDF

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WO2022241831A1
WO2022241831A1 PCT/CN2021/097429 CN2021097429W WO2022241831A1 WO 2022241831 A1 WO2022241831 A1 WO 2022241831A1 CN 2021097429 W CN2021097429 W CN 2021097429W WO 2022241831 A1 WO2022241831 A1 WO 2022241831A1
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polypeptide
preparation
hexafluoroisopropanol
acetonitrile
precipitation
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PCT/CN2021/097429
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English (en)
French (fr)
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洪浩
詹姆斯•盖吉
肖毅
张娜
焦学成
赵军旗
王磊
孟翔宇
张慕姣
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凯莱英医药集团（天津）股份有限公司
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Priority to US18/562,913 priority Critical patent/US20240182942A1/en
Priority to JP2023571760A priority patent/JP2024518625A/ja
Priority to KR1020237044417A priority patent/KR20240013779A/ko
Priority to EP21940283.1A priority patent/EP4328316A1/en
Publication of WO2022241831A1 publication Critical patent/WO2022241831A1/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
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • 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
    • 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
    • C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/22068—Ulp1 peptidase (3.4.22.68)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the invention relates to the technical field of biomedicine, in particular to a preparation method of a polypeptide.
  • Peptide drugs have attracted much attention because of their dual properties similar to proteins and small molecule drugs.
  • the sales of peptide drugs accounted for 5% of the drug market, reaching 50 billion US dollars, and increasing at a rate of 9-10% per year. Therefore, it is urgent to establish an efficient and low-cost peptide synthesis and purification technology.
  • Peptide synthesis mainly includes chemical methods, chemical/enzymatic methods and biological methods. Among them, the chemical method requires complex protection and deprotection processes, requires the use of a large amount of toxic reagents, and may produce racemates; chemical/biological methods usually use chemical methods to synthesize polypeptides, and further combine multiple peptides with specific peptide ligases.
  • Peptides are connected, the process is relatively complicated, and the connection efficiency and specificity are affected by the peptide sequence; biological methods include hydrolyzing plant or animal proteins with hydrolytic enzymes and then extracting specific functional peptides, but usually the yield is low and the cycle is long , heavy pollution, difficult to achieve industrial production; another common method is recombinant expression, that is, using genetic engineering techniques to introduce target gene expression elements into prokaryotic or eukaryotic organisms, and achieve efficient synthesis of target polypeptides through microbial fermentation.
  • recombinant expression that is, using genetic engineering techniques to introduce target gene expression elements into prokaryotic or eukaryotic organisms, and achieve efficient synthesis of target polypeptides through microbial fermentation.
  • the polypeptide itself is relatively short, it is difficult to form a specific spatial structure, which makes it very easy to be degraded by the protease of the expression host.
  • the complicated purification process reduces the product yield and increases the cost.
  • polypeptides produced by recombinant methods include polypeptide antibiotics, interferon, liraglutide precursor (GLP-1) and insulin, among which GLP-1 is developed by Novo Nordisk for the treatment of type II diabetes Peptide drugs, whose annual sales reach billions of dollars, are initially synthesized based on engineering strains secreted and expressed by Saccharomyces cerevisiae.
  • GLP-1 is developed by Novo Nordisk for the treatment of type II diabetes Peptide drugs, whose annual sales reach billions of dollars
  • GLP-1 is developed by Novo Nordisk for the treatment of type II diabetes Peptide drugs, whose annual sales reach billions of dollars
  • GLP-1 is developed by Novo Nordisk for the treatment of type II diabetes Peptide drugs, whose annual sales reach billions of dollars
  • GLP-1 is developed by Novo Nordisk for the treatment of type II diabetes Peptide drugs, whose annual sales reach billions of dollars
  • GLP-1 is developed by Novo Nordisk for the treatment of type II diabetes Peptide
  • GLP-1 is mainly expressed by fusion of inclusion bodies, such as KSI tag, endogenous peptide intein and signal peptide-enterokinase cleavage site-GLP-1, etc.
  • inclusion bodies such as KSI tag, endogenous peptide intein and signal peptide-enterokinase cleavage site-GLP-1, etc.
  • enterokinase is usually required, which is not only difficult to express but also causes non-specific cleavage.
  • the inclusion body expression constructed by using MFH fusion tag combined with formic acid cleavage and purification under denaturing conditions, avoids the renaturation process, but requires the use of urea, and further cleavage with Tev protease to obtain GLP-1 with a Gly at the N-terminal.
  • the Tev enzyme has low biological activity and produces non-specific cleavage, and the production steps are cumbersome and the cost is high.
  • Other fusion tags such as GST, MBP, TrxA, etc.
  • GLP-1 and its derivatives are mostly in the state of polymers, and purification needs to be carried out under denaturing conditions. The current purification steps are complicated and the yield is low.
  • the prior art mainly has the following problems: 1) chemical synthesis: complex process, toxic chemical reagents, racemates may be produced, long cycle, difficult product quality control; 2) inclusion body expression: complex The destructive purification process is complicated, and a large amount of urea is used.
  • the enterokinase or Tev used in the cleavage process is expensive and prone to non-specific cleavage; the complicated renaturation process makes the purification process very complicated and the yield is reduced;
  • 3) soluble Expression The soluble expression tags currently used mainly include TrxA, GST, and MBP. Restriction sites need to be added to the target peptides and tags. Commonly used enzymes are usually difficult to express and will cause non-specific cleavage. Sumo The tag was used for the study of liraglutide precursor fusion expression.
  • the present invention aims to provide a method for preparing a polypeptide to solve the technical problems of complicated polypeptide purification steps and low yield in the prior art.
  • a preparation method of a polypeptide comprises the following steps: constructing an engineering strain with a Sumo tag fused to express a polypeptide gene, and inducing the engineering strain to solublely express the polypeptide; purifying the crude protein containing a polypeptide precursor from the engineering strain; Ulp1 protease performs enzyme digestion to remove the Sumo tag; acetonitrile combined with heating precipitation method or hexafluoroisopropanol precipitation method is used to purify the enzyme digestion product of Ulp1 protease to obtain a polypeptide.
  • polypeptide is liraglutide precursor, nesiritide or teriparatide.
  • the Ulp1 protease is obtained by constructing an Ulp1 protease expression strain and inducing expression, wherein the Ulp1 protease is co-expressed with a chaperone protein.
  • the chaperone is GroEL/S chaperone.
  • the method of acetonitrile combined with heating and precipitation includes: adjusting the pH of the cleaved product of Ulp1 protease to 5.6, then adding acetonitrile, mixing and heat-treating at 60-80°C for 0.5-3h (preferably heat-treating at 70°C for 2h), and then centrifuging Separate the supernatant and pellet.
  • acetonitrile is an aqueous solution of acetonitrile with a mass percentage of 20-70%.
  • the purification of the crude protein containing the polypeptide precursor from the engineering strain includes: obtaining the crude enzyme solution after the engineering strain, ultrasonication, centrifugation, and membrane filtration, and then using affinity chromatography or anion column to purify to obtain the crude protein protein.
  • the hexafluoroisopropanol precipitation method includes: adjusting the pH of the cleaved product of Ulp1 protease to 5.6, then adding hexafluoroisopropanol, mixing and precipitating at room temperature for 1 hour, and then centrifuging to separate the supernatant and the precipitate.
  • the hexafluoroisopropanol is an aqueous solution of hexafluoroisopropanol with a mass percentage of 20-70%; preferably, the hexafluoroisopropanol is an aqueous solution of hexafluoroisopropanol with a mass percentage of 50%.
  • the preparation method includes the step of purifying the target polypeptide by HPLC.
  • the present invention establishes an efficient technology based on soluble recombinant expression of medicinal polypeptide or its precursor, establishes a simple purification process based on one-step precipitation purification, and further combines HPLC purification to achieve a purity of more than 97% of the polypeptide.
  • Figure 1 shows the SDS-PAGE electrophoresis figure of the target protein expressed by Sumo-Lira fusion protein and purified by affinity chromatography in Examples 1 and 5, wherein, Lane 1: protein molecular weight standard; Lane 2: Sumo-Lira soluble Expression part; Lane 3: flow-through sample; Lane 4: 60mM imidazole elution product; Lane 5: 300mM imidazole elution product;
  • Fig. 2 shows the SDS-PAGE electrophoresis figure of the target protein of Sumo-Nesi and Sumo-Teri fusion expression in embodiment 2 and 3, wherein, Lane 1: protein molecular weight standard; Lane 2,3: Sumo-Teri soluble expression Lane 4: Sumo-Teri precipitation fraction; Lane 5: Sumo-Nesi soluble expression fraction; Lane 6: Sumo-Nesi precipitation fraction;
  • Figure 3 shows the SDS-PAGE electrophoresis of the target protein during the expression optimization process of Ulp1 in Example 4, wherein, Lane 1: Ulp1 induced by IPTG, 37°C, 6h, soluble expression part; Lane 2: protein molecular weight standard; Lane 3: Ulp1 induced by IPTG, 37°C, 6h, precipitation part; Lane 4: Ulp1 and GroEL/S co-expression strain induced by IPTG, 37°C, 6h, soluble expression part; Lane 5: Ulp1 and GroEL/S co-expressed by IPTG Expression strain, 37°C, 6h, precipitation part;
  • Figure 4 shows the SDS-PAGE electrophoresis figure of the Sumo-Lira target protein purified by anion column chromatography in Example 5, wherein, Lane 1: protein molecular weight standard; Lane 2: Sumo-Lira broken liquid supernatant; Lane 3: stream Wear sample; Lane 4: 50mM NaCl elution; Lane 5: 100mM NaCl elution; Lane 6: 500mM NaCl elution; Lane 7: 700mM NaCl elution;
  • Figure 5 shows the SDS-PAGE electrophoresis figure of Ulp1 affinity chromatography purification target protein in embodiment 7, wherein, Lane 1: protein molecular weight standard; Lane 2: Ulp1 soluble expression part; Lane 3: flow-through sample; Lane 4: 500mM imidazole elution;
  • Figure 6 shows the SDS-PAGE electrophoresis of the target protein of liraglutide precursor precipitated by acetonitrile in Example 9, wherein, Lane1: 20% acetonitrile, pH5.6, 70 ° C treatment, supernatant; Lane 2: 20 % acetonitrile, pH5.6, treated at 70°C, precipitation part; Lane 3: 30% acetonitrile, pH5.6, treated at 70°C, supernatant part; Lane 4: 30% acetonitrile, pH5.6, treated at 70°C, precipitated part ; Lane 5: protein molecular weight standard; Lane 6: 40% acetonitrile, pH 5.6, 70 °C treatment, supernatant part; Lane 7: 40% acetonitrile, pH 5.6, 70 °C treatment, precipitation part; Lane 8: protein molecular weight Standard; Lane 9: 50% acetonitrile, pH5.6, treated at 70°C, supernatant; Lane 10: 50% acetonitrile, pH5.6,
  • Fig. 7 shows the SDS-PAGE electrophoresis figure of acetonitrile precipitation Nesi and Teri target protein in embodiment 9, wherein, Lane 1: protein molecular weight standard; Lane 2: Ulp1 cuts Sumo-Nesi; Lane 3: Precipitation method handles Sumo-Nesi Lane 4: Ulp1 digestion system of Sumo-Nesi by precipitation method, precipitation part; Lane 5: Ulp1 digestion of Sumo-Teri; Lane 6: Ulp1 digestion of Sumo-Teri by precipitation method System, supernatant part; Lane 7: Ulp1 digestion system of Sumo-Teri treated by precipitation method, precipitation part;
  • Figure 8 shows the SDS-PAGE electropherogram of the liraglutide precursor purified by HFIP precipitation method in Example 9, wherein, Lane 1, 18% HFIP selective precipitation supernatant; Lane 2, 18% HFIP selection Lane 3, the supernatant of 50% HFIP selective precipitation; Lane 4, the precipitation part of 50% HFIP selective precipitation; Lane 5, the supernatant of 70% HFIP selective precipitation; Lane 6, 70% HFIP selectively precipitated fraction;
  • Figure 9 shows the HPLC purification chromatograms for the preparation of liraglutide precursors in Examples 9 and 10;
  • Figure 10 shows the chromatogram of the purity analysis of liraglutide precursor in Example 10.
  • Fig. 11 shows Nesi preparation HPLC purification chromatogram among the embodiment 10
  • Fig. 12 shows Nesi purity analysis chromatogram among the embodiment 10
  • Fig. 13 shows Teri preparation HPLC purification chromatogram in embodiment 10
  • Fig. 14 shows Teri purity analysis chromatogram among the embodiment 10
  • Figure 15 shows the Liraglutide molecular weight of mass spectrometry analysis in embodiment 11
  • Figure 16 shows the Nesiritide molecular weight of mass spectrometry analysis in embodiment 11
  • FIG. 17 shows the molecular weight of Teriparatide analyzed by mass spectrometry in Example 11.
  • Polypeptide the English name polypeptide, is a peptide composed of 10-50 amino acid residues.
  • Liraglutide a human glucagon-like peptide-1 (GLP-1) analog
  • Liraglutide a human glucagon-like peptide-1 (GLP-1) analog
  • Lira Liraglutide
  • Nesiritide Nesiritide, Nesiritide, Nesi for short.
  • Teriparatide Teriparatide, Teri for short.
  • a polypeptide precursor refers to a polypeptide containing a Sumo tag in the present invention.
  • a preparation method of a polypeptide comprises the following steps: constructing an engineering strain with a Sumo tag fused to express a polypeptide gene, and inducing the engineering strain to solublely express the polypeptide; purifying the crude protein containing a polypeptide precursor from the engineering strain; Ulp1 protease performs enzyme digestion to remove the Sumo tag; acetonitrile combined with heating precipitation method or hexafluoroisopropanol precipitation method is used to purify the enzyme digestion product of Ulp1 protease to obtain a polypeptide.
  • the present invention can promote protein soluble expression based on the Sumo tag, and as a fusion tag, it can be efficiently excised by the Ulp1 protease (SUMO protease) that specifically recognizes its tertiary structure, and the target polypeptide can be obtained without causing non-specific cleavage, avoiding the To solve the problem of inclusion body denaturation, an engineering strain with Sumo tag fusion expressing polypeptide gene was constructed.
  • Ulp1 protease SUMO protease
  • the polypeptide in the present invention can be liraglutide precursor, nesiritide or teriparatide.
  • Ulp1 specifically cutting the Sumo tag. Ulp1 recognizes the spatial structure of the Sumo tag and only cuts at a specific site. ; In order to obtain an efficient Ulp1 engineering strain and facilitate the purification of Ulp1, construct a strain co-expressing Ulp1 and a chaperone.
  • the chaperone is a GroEL/S chaperone, which greatly increases the expression level of Ulp1.
  • the method of acetonitrile combined with heating precipitation comprises: adjusting the pH of the enzyme-cleaved product of Ulp1 protease to 5.6 (obtained in conjunction with the data of the isoelectric point), then adding acetonitrile, and mixing it at 60-80 °C heat treatment for 0.5-3 hours (preferably heat treatment at 70 °C for 2 hours), and then centrifuge to separate the supernatant and precipitate.
  • the acetonitrile is an aqueous solution of acetonitrile with a mass percentage of 20-70%, and the concentration of acetonitrile from 20-70% has a good preliminary purification effect.
  • the purification of the crude protein containing the polypeptide precursor from the engineering strain includes: obtaining the crude enzyme solution after the engineering strain, ultrasonication, centrifugation, and membrane filtration, and then using affinity chromatography or Anion column for purification to obtain crude protein.
  • the method for precipitating hexafluoroisopropanol comprises: adjusting the pH of the cleaved product of Ulp1 protease to 5.6, then adding hexafluoroisopropanol, mixing and precipitating at room temperature for 1 h, and then centrifuging Separation of supernatant and precipitation; preferably, hexafluoroisopropanol is an aqueous solution of hexafluoroisopropanol with a mass percentage of 20-70%; preferably, after selective precipitation with 50% hexafluoroisopropanol, the supernatant The concentration of liraglutide precursor in the solution is about 90%.
  • the preparation method further includes the step of purifying the target polypeptide by HPLC.
  • the precursor of liraglutide is easy to aggregate, and the method of organic solvent treatment and reverse-phase purification is adopted in the present invention, which can avoid the purification problem caused by the aggregation effect.
  • the synthesized gene fragment was digested with NdeI and XhoI from pUC57-Sumo-Lira, and then ligated with pET-28a(+) or pET-22b(+) that had undergone the same digestion at 16°C overnight, and then transformed into BL21 (DE3) Competent cells, single clones were sequenced and analyzed to obtain the correct clone expression vector pET-28a-Sumo-Lira or pET-22b-Sumo-Lira, and 3 correct clones were picked for each transformation and activated as seeds in 250mL shake Bottle pre-screening, from which an optimal strain was selected as the final expression strain.
  • Nesiritide a genetically engineered strain expressing Nesiritide fused with the Sumo tag
  • the vector construction strategy is the same as that in Example 1, and the synthetic gene sequence is as follows:
  • the gene was constructed in pET-28a(+) after enzyme digestion and ligation, and the correct expression plasmid pET-28a-Sumo-Nesi and the recombinant BL21(DE3) strain containing the expression were obtained by sequencing. The same method as Sumo-Lira was used to induce IPTG The latter fusion protein was soluble expressed ( Figure 2).
  • the vector construction strategy is the same as that in Example 1, and the synthetic gene sequence is as follows:
  • the gene was constructed in pET-28a(+) after NdeI and XhoI digestion and connection, and the correct expression plasmid pET-28a-Sumo-Teri and the recombinant BL21(DE3) strain containing the expression were obtained by sequencing, using the same expression as Sumo-Lira Conditions, the fusion protein was soluble expression after IPTG induction ( Figure 2).
  • Ulp1 protease D390 to K621 was directly amplified from the S. cerevisiae S288C genome by PCR, and the primers used were Ulp1-F (SEQ ID NO: 4): gggcatatgGATCTTAAAAAAAAGAAAGAACAATTGGCCAAGAAGAAACTTG and Ulp1-R (SEQ ID NO: 5 ): Gggctcgaggtattttaaagcgtcggttaaaatcaaatgggc, the gene sequence is as follows (or can be artificially synthesized according to the following sequence):
  • the amplified gene fragment was digested with NdeI and XhoI and ligated into pET-28a(+) to obtain the expression vector pET-28a-Ulp1, transformed into BL21(DE3), and the transformants were sequenced. Three clones with correct sequencing were selected as seeds for pre-screening in 250mL shake flasks, and an optimal one was selected as the final expression strain.
  • This sample can be directly digested with Ulp1 without desalting and imidazole removal.
  • 1g of sludge can obtain 35-40mg of purified Sumo-Lira fusion protein
  • 1g of sludge can obtain 15.2mg of purified Sumo-Nesi
  • 1g of sludge can obtain 18.3mg of purified Sumo-Teri (which can be further improved after condition optimization).
  • the expression of Sumo-Lira was purified by anion column.
  • the crude protein was obtained in the same way as affinity chromatography.
  • the AKTA system was equipped with anion column (Q FF, 5mL), the flow rate was 5mL/min, and the binding buffer was 50mM.
  • Tris-HCl pH8.0 wash with binding buffer after loading the sample until the unbound protein is completely eluted, then use 50mM Tris-HCl, pH8.0, 1M NaCl gradient elution to obtain the target protein under 500mM gradient, SDS-PAGE
  • the analysis shows that the target protein can reach a purity of more than 80% ( Figure 4), and the sample purified in this step can also be directly digested by Ulp1 without desalting.
  • the sludge obtained by inducing the expression of the recombinant strain was subjected to the same method as the Sumo-Lira sample to obtain the crude protein, and further purified by the same affinity chromatography to obtain the target fusion protein.
  • the purity of the target protein reached about 70%.
  • the obtained target protein can be directly used for the fusion proteins Sumo-Lira, Sumo-Teri and Digestion by Sumo-Nesi (products of Examples 5 and 6).
  • Ulp1 digestion reaction was carried out at 30°C, the specific process was: 50mM Tris-HCl, 10mM DTT, pH 8.0 Purified Sumo-Lira (the product of Example 5) and Ulp1 mass ratio is 10:1 ⁇ 1:1 (mg/ mg), samples were taken at different times, and Tricine was used to detect the cleavage efficiency, and to detect whether the target polypeptide was produced, and the cleavage efficiency could reach more than 80% within 24 hours.
  • the digestion conditions of Sumo-Teri and Sumo-Nesi are the same as those of Sumo-Lira.
  • Example 8 After the enzyme digestion in Example 8 is completed, adjust the pH to 5.6, then add different final concentrations of acetonitrile (20%, 30%, 40%, 50%, 60%, 70%) to the reaction system, and mix well at 70 degrees. After heat treatment for 2 hours, centrifuge at 12,000rpm to separate the supernatant and precipitate. Tricine detected the step-by-step situation of the peptide. It was found that under the combined conditions, the concentration of acetonitrile from 20-70% had a good preliminary purification effect. After the treatment, the precursor of liraglutide The purity of the crude product can reach more than 90% (Fig. 6), the purity of Nesi can reach 80%, and the purity of Teri can reach 50% (Fig. 7). The acetonitrile in the crude polypeptide was further removed by rotary evaporation or lyophilization, and then redissolved in 50mM Tris-HCl pH7.0, and purified by preparative HPLC.
  • Example 8 After the enzyme digestion in Example 8 is completed, adjust the pH to 5.6, add 10%-70% (v/v) hexafluoroisopropanol to the reaction system, precipitate at room temperature for 1 hour, collect the supernatant and precipitate by centrifugation, and use Tricine- SDS-PAGE detects the selective precipitation effect of different concentrations of hexafluoroisopropanol on the precursor of liraglutide, and the results are shown in Figure 9: after using 50% hexafluoroisopropanol for selective precipitation, the supernatant The liraglutide precursor concentration was about 90% ( Figure 8). The hexafluoroisopropanol in the crude polypeptide was further removed by rotary evaporation or lyophilization, and then redissolved in 50mM Tris-HCl pH7.0, and purified by preparative HPLC.
  • Lira adopts gradient elution method as follows: 0min 5% B, 5min 5% B, 25min 50% B, 27min 95% B, 33min 95% B, 38min 5% B, UV detector 210nm, flow rate 25mL/min, temperature 25°C, the results are shown in Figure 9: Lira was eluted around 24min, and then the samples were collected by rotary evaporation to remove most of the solvent and then freeze-dried. The purity was measured by HPLC, and the results showed that the purity of Lira reached more than 98% ( Figure 10 ).
  • Nesi The gradient elution method used by Nesi is as follows: 5min 5% B, 35min 30% B, 40min 95% B, UV detector 210nm, flow rate 25mL/min, temperature 25°C, the results are shown in Figure 11: Nesi was eliminated in 30min After elution, the sample was collected by rotary evaporation to remove most of the solvent and then freeze-dried. The purity was additionally measured by HPLC, and the results showed that the purity of Nesi reached more than 98% ( Figure 12).
  • the gradient elution method used by Teri is as follows: 5min 5% B, 35min 30% B, 47.8min 42.8% B, 50.5min 42.8% B, 57.7min 50% B, 67.7min 95% B, 40min 95% B, UV detection
  • the instrument was 210nm, the flow rate was 25mL/min, and the temperature was 25°C.
  • the results are shown in Figure 13: Teri was eluted in about 43 minutes, and then the samples were collected by rotary evaporation to remove most of the solvent and then freeze-dried. The purity was detected by HPLC.
  • Liraglutide precursor is prepared by this method, 3-4mg of liraglutide precursor with a purity of 98% can be obtained from 1g of shake flask fermentation sludge, and 1.26mg with a purity of 98% can be obtained by preparing 1g of bacteria sludge by this method % Nesi, 1.55 mg of Teri with a purity of 78% can be obtained by preparing 1 g of bacteria sludge by this method, and it is expected to further increase the yield of the target polypeptide through further fermentation optimization to improve the expression level of the fusion protein.
  • the molecular weight of the prepared polypeptide was analyzed by LC-MS.
  • the details are as follows: the sample is first separated by HPLC column: Agilent ZORBAX Edipse Plus C18, 4.6*100mm, 3.5 ⁇ m, mobile phase A: 0.1% trifluoroacetic acid, mobile phase B: 0.1% trifluoroacetic acid acetonitrile solution, using gradient elution : 0min 10% B, 9min 95% B, 12min 100% B, 12.1min 10% B, 15min 10% B, column temperature 40°C, UV detector 210nm, flow rate 1.5mL/min, components separated by HPLC Agilent 6200 series time-of-flight LC-MS was used for structural identification, using electrospray ion source (Dual AJS ESI), positive ion mode detection, spray pressure 35psig, ion source temperature 300°C, dry gas (N2) flow rate 10L/min , the scanning range is 100-2000m/z, the fragmentation voltage is

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Abstract

一种多肽的制备方法。该制备方法包括以下步骤：构建Sumo标签融合表达多肽基因的工程菌株，并诱导工程菌株可溶表达多肽；从工程菌株中纯化得到含有多肽前体的粗蛋白；对含有多肽前体的粗蛋白采用Ulp1蛋白酶进行酶切，切除Sumo标签；采用乙腈结合加热沉淀的方法或采用六氟异丙醇沉淀的方法纯化Ulp1蛋白酶的酶切产物，得到多肽。建立了高效的基于可溶性重组表达药用多肽或其前体的技术，建立了基于一步沉淀纯化的简单纯化工艺，进一步的结合HPLC精制即可达到97％以上多肽纯度。

Description

多肽的制备方法 技术领域
本发明涉及生物医药技术领域，具体而言，涉及一种多肽的制备方法。
背景技术
多肽类药物因其具有类似蛋白和小分子药物双重特性而备受关注，仅2015年多肽药物的销售就占据药物市场的5％，达到500亿美元，并且以每年9-10％的速度上涨，因此亟待建立高效的低成本的多肽合成和纯化技术。多肽合成主要包括化学法、化学/酶法和生物法。其中，化学方法需要复杂的保护和脱保护过程，需要用到大量的有毒试剂，且可能产生消旋体；化学/生物法通常是用化学方法合成多肽，进一步通过特定的肽连接酶将多个肽段进行连接，工艺相对复杂，连接效率和特异性受到多肽序列的影响；生物法包括采用水解酶水解植物蛋白或动物蛋白然后提取获得特定的有功能的多肽，但是通常产量较低，周期长，污染重，难实现工业化生产；另一种较为普遍的方法是重组表达，即采用基因工程的技术手段在原核或真核生物中引入目标基因表达元件，通过微生物发酵实现目标多肽的高效合成，然而由于多肽自身比较短，很难形成特定的空间结构，导致其非常容易被表达宿主的蛋白酶所降解，此外复杂的纯化工艺又降低了产物收率，增加了成本。
目前，利用重组法生产的多肽包括多肽类抗生素、干扰素、利拉鲁肽前体(GLP-1)和胰岛素等，其中的GLP-1是由诺和诺德开发的用于治疗II型糖尿病的多肽药物，每年的销售额达到数十亿美元，其最初的合成方式是基于酿酒酵母分泌表达的工程菌株，然而由于其胞外蛋白酶的作用导致产物容易被降解，产量较低，敲除蛋白酶后表达的最高水平只有59mg/L。毕赤酵母中融合表达GLP-1,发酵5天的表达量只有26mg/L。大肠杆菌作为一种常用的宿主，已被广泛用于多肽的生物合成，然而同样存在蛋白酶降解的风险，通过融合表达可以解决此类问题。目前GLP-1主要采用包涵体融合表达，如KSI标签，内涵肽intein和信号肽-肠激酶酶切位点-GLP-1等，虽然包涵体表达量较高，可以避免蛋白酶降解，但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，纯化工艺复杂导致最终得率非常低。同时为了将融合标签完全去除通常需要用到肠激酶，该酶不仅难表达且会造成非特异切割。近来采用MFH融合标签构建的包涵体表达，结合甲酸切割和变性条件下纯化的工艺，避免了复性过程，但需用到尿素，进一步通过Tev蛋白酶切割获得N端含有一个Gly的GLP-1，且Tev酶生物活性较低和产生非特异切割，生产步骤繁琐，成本高。其他的融合标签如GST，MBP，TrxA等多用作可溶表达的标签，且一些肽经过这些标签获得了可溶表达，但是这些标签的分子量相对某些目标肽来说都偏大，导致去除标签后目标肽的得率偏低。此外GLP-1及其衍生物多呈聚合体状态，纯化需要在变性条件下进行，目前的纯化步骤复杂，得率低下。
总的来说，现有技术主要存在以下问题：1)化学合成：工艺复杂，化学试剂有毒，可能产生消旋体，周期较长，产品的质量控制比较困难；2)包涵体表达：变复性纯化工艺复杂， 用到大量的尿素，切割过程中用到的肠激酶或Tev价格昂贵，易产生非特异性切割；复杂的变复性工艺导致纯化过程非常复杂，得率降低；3)可溶表达：目前用到的可溶表达标签主要包括TrxA，GST，MBP，在目标肽段和标签需要加上酶切位点，常用到的酶通常难表达，且会造成非特异切割，没有将Sumo标签用于利拉鲁肽前体融合表达的研究。
发明内容
本发明旨在提供一种多肽的制备方法，以解决现有技术中多肽纯化步骤复杂、得率较低的技术问题。
为了实现上述目的，根据本发明的一个方面，提供了一种多肽的制备方法。该制备方法包括以下步骤：构建Sumo标签融合表达多肽基因的工程菌株，并诱导工程菌株可溶表达多肽；从工程菌株中纯化得到含有多肽前体的粗蛋白；对含有多肽前体的粗蛋白采用Ulp1蛋白酶进行酶切，切除Sumo标签；采用乙腈结合加热沉淀的方法或采用六氟异丙醇沉淀的方法纯化Ulp1蛋白酶的酶切产物，得到多肽。
进一步地，多肽为利拉鲁肽前体、奈西利肽或特立帕肽。
进一步地，Ulp1蛋白酶为通过构建Ulp1蛋白酶表达菌株并诱导表达获得，其中，Ulp1蛋白酶与伴侣蛋白共表达。
进一步地，伴侣蛋白为GroEL/S伴侣蛋白。
进一步地，乙腈结合加热沉淀的方法包括：将Ulp1蛋白酶的酶切产物调整pH至5.6，然后加入乙腈，混匀后在60～80℃热处理0.5～3h(优选在70℃热处理2h)，之后离心分离上清和沉淀。
进一步地，乙腈为质量百分含量为20～70％的乙腈水溶液。
进一步地，从工程菌株中纯化得到含有多肽前体的粗蛋白包括：将工程菌株的、超声破碎、离心、过滤膜后获得粗酶液，然后采用亲和层析或阴离子柱进行纯化，得到粗蛋白。
进一步地，六氟异丙醇沉淀的方法包括：将Ulp1蛋白酶的酶切产物调整pH至5.6，然后加入六氟异丙醇，混匀后在室温沉淀1h，之后离心分离上清和沉淀。
进一步地，六氟异丙醇为质量百分含量为20～70％的六氟异丙醇水溶液；优选的，六氟异丙醇为质量百分含量为50％的六氟异丙醇水溶液。
进一步地，制备方法包括采用HPLC纯化目标多肽的步骤。
本发明建立了高效的基于可溶性重组表达药用多肽或其前体的技术，建立了基于一步沉淀纯化的简单纯化工艺，进一步的结合HPLC精制即可达到97％以上多肽纯度。
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示出了实施例1和5中Sumo-Lira融合蛋白表达、亲和层析纯化的目标蛋白的SDS-PAGE电泳图，其中，Lane 1：蛋白分子量标准；Lane 2：Sumo-Lira可溶表达部分；Lane 3：流穿样品；Lane 4：60mM咪唑洗脱产品；Lane 5：300mM咪唑洗脱产品；
图2示出了实施例2和3中Sumo-Nesi和Sumo-Teri融合表达的目标蛋白的SDS-PAGE电泳图，其中，Lane 1：蛋白分子量标准；Lane 2、3：Sumo-Teri可溶表达部分；Lane 4：Sumo-Teri沉淀部分；Lane 5：Sumo-Nesi可溶表达部分；Lane 6：Sumo-Nesi沉淀部分；
图3示出了实施例4中Ulp1的表达优化过程中目标蛋白的SDS-PAGE电泳图，其中，Lane 1：IPTG诱导Ulp1，37℃，6h，可溶表达部分；Lane 2：蛋白分子量标准；Lane 3：IPTG诱导Ulp1，37℃，6h，沉淀部分；Lane 4：IPTG诱导Ulp1和GroEL/S共表达菌株，37℃，6h，可溶表达部分；Lane 5：IPTG诱导Ulp1和GroEL/S共表达菌株，37℃，6h，沉淀部分；
图4示出了实施例5中阴离子柱层析纯化Sumo-Lira目标蛋白的SDS-PAGE电泳图，其中，Lane 1：蛋白分子量标准；Lane 2：Sumo-Lira破碎液上清；Lane 3：流穿样品；Lane 4：50mM NaCl洗脱；Lane 5：100mM NaCl洗脱；Lane 6：500mM NaCl洗脱；Lane 7：700mM NaCl洗脱；
图5示出了实施例7中Ulp1亲和层析纯化目标蛋白的SDS-PAGE电泳图，其中，Lane 1：蛋白分子量标准；Lane 2：Ulp1可溶表达部分；Lane 3：流穿样品；Lane 4：500mM咪唑洗脱；
图6示出了实施例9中乙腈沉淀利拉鲁肽前体目标蛋白的SDS-PAGE电泳图，其中，Lane1：20％乙腈，pH5.6，70℃处理，上清部分；Lane 2：20％乙腈，pH5.6，70℃处理，沉淀部分；Lane 3：30％乙腈，pH5.6，70℃处理，上清部分；Lane 4：30％乙腈，pH5.6，70℃处理，沉淀部分；Lane 5：蛋白分子量标准；Lane 6：40％乙腈，pH5.6，70℃处理，上清部分；Lane 7：40％乙腈，pH5.6，70℃处理，沉淀部分；Lane 8：蛋白分子量标准；Lane 9：50％乙腈，pH5.6，70℃处理，上清部分；Lane 10：50％乙腈，pH5.6，70℃处理，沉淀部分；Lane 11：60％乙腈，pH5.6，70℃处理，上清部分；Lane 12：60％乙腈，pH5.6，70℃处理，沉淀部分；Lane 13：70％乙腈，pH5.6，70℃处理，上清部分；Lane 14：70％乙腈，pH5.6，70℃处理，沉淀部分；
图7示出了实施例9中乙腈沉淀Nesi和Teri目标蛋白的SDS-PAGE电泳图，其中，Lane 1：蛋白分子量标准；Lane 2：Ulp1切割Sumo-Nesi；Lane 3：沉淀法处理Sumo-Nesi的Ulp1酶切体系，上清部分；Lane 4：沉淀法处理Sumo-Nesi的Ulp1酶切体系，沉淀部分；Lane 5：Ulp1切割Sumo-Teri；Lane 6：沉淀法处理Sumo-Teri的Ulp1酶切体系，上清部分；Lane 7：沉淀法处理Sumo-Teri的Ulp1酶切体系，沉淀部分；
图8示出了实施例9中，HFIP沉淀法纯化利拉鲁肽前体的SDS-PAGE电泳图，其中，Lane  1，18％HFIP选择性沉淀的上清液；Lane 2，18％HFIP选择性沉淀的沉淀部分；Lane 3，50％HFIP选择性沉淀的上清液；Lane 4，50％HFIP选择性沉淀的沉淀部分；Lane 5，70％HFIP选择性沉淀的上清液；Lane 6，70％HFIP选择性沉淀的沉淀部分；
图9示出了实施例9和10中利拉鲁肽前体制备HPLC纯化色谱图；
图10示出了实施例10中利拉鲁肽前体纯度分析色谱图；
图11示出了实施例10中Nesi制备HPLC纯化色谱图；
图12示出了实施例10中Nesi纯度分析色谱图；
图13示出了实施例10中Teri制备HPLC纯化色谱图；
图14示出了实施例10中Teri纯度分析色谱图；
图15示出了实施例11中质谱解析的Liraglutide分子量；
图16示出了实施例11中质谱解析的Nesiritide分子量
图17示出了实施例11中质谱解析的Teriparatide分子量。
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。下面将参考附图并结合实施例来详细说明本发明。
名词解释
多肽，英文名称polypeptide，由10-50个的氨基酸残基组成的肽。
利拉鲁肽，一种人胰高糖素样肽-1(GLP-1)类似物，Liraglutide，简称Lira。
奈西利肽，Nesiritide，简称Nesi。
特立帕肽，Teriparatide，简称Teri。
多肽前体，在本发明中是指含有Sumo标签的多肽。
根据本发明一种典型的实施方式，提供一种多肽的制备方法。该制备方法包括以下步骤：构建Sumo标签融合表达多肽基因的工程菌株，并诱导工程菌株可溶表达多肽；从工程菌株中纯化得到含有多肽前体的粗蛋白；对含有多肽前体的粗蛋白采用Ulp1蛋白酶进行酶切，切除Sumo标签；采用乙腈结合加热沉淀的方法或采用六氟异丙醇沉淀的方法纯化Ulp1蛋白酶的酶切产物，得到多肽。
本发明基于Sumo标签可以促进蛋白可溶表达，并且其作为融合标签可以被特异识别其三级结构的Ulp1蛋白酶(SUMO蛋白酶)高效切除，而且不会造成非特异切割的方法获得目标多肽，避免了包涵体变性的问题，构建了Sumo标签融合表达多肽基因的工程菌株。
典型的，本发明中的多肽可以为利拉鲁肽前体、奈西利肽或特立帕肽。
此外针对融合表达标签去除所需要的蛋白酶比较昂贵，且可造成非特异切割的问题，采用了Ulp1特异切割Sumo标签的策略，Ulp1识别的是Sumo标签的空间结构，并且只在特异位点进行切割；为了获得高效的Ulp1工程菌株和方便Ulp1的纯化，构建Ulp1和伴侣蛋白共表达的菌株，优选的，伴侣蛋白为GroEL/S伴侣蛋白，大大提高了Ulp1的表达水平。
根据本发明一种典型的实施方式，乙腈结合加热沉淀的方法包括：将Ulp1蛋白酶的酶切产物调整pH至5.6(结合等电点的数据获得)，然后加入乙腈，混匀后在60～80℃热处理0.5～3h(优选在70℃热处理2h)，之后离心分离上清和沉淀。优选的，乙腈为质量百分含量为20～70％的乙腈水溶液，乙腈浓度从20～70％都有着较好的初步纯化效果。
在本发明一典型的实施方式中，从工程菌株中纯化得到含有多肽前体的粗蛋白包括：将工程菌株的、超声破碎、离心、过滤膜后获得粗酶液，然后采用亲和层析或阴离子柱进行纯化，得到粗蛋白。
根据本发明一种典型的实施方式，六氟异丙醇沉淀的方法包括：将Ulp1蛋白酶的酶切产物调整pH至5.6，然后加入六氟异丙醇，混匀后在室温沉淀1h，之后离心分离上清和沉淀；优选的，六氟异丙醇为质量百分含量为20～70％的六氟异丙醇水溶液；优选的，使用50％的六氟异丙醇选择性沉淀后，上清液中的利拉鲁肽前体浓度约90％。
优选的，制备方法进一步包括采用HPLC纯化目标多肽的步骤。利拉鲁肽前体容易聚集，本发明采用的是有机溶剂处理和反相纯化的方法，可以避免因聚集效应造成的纯化问题。
下面将结合实施例进一步说明本发明的有益效果。
实施例1
构建Sumo标签融合表达利拉鲁肽前体(简称Lira)的基因工程菌株
构建基于可溶表达多肽的策略，具体是采用Sumo标签和目标多肽序列进行融合，构建于pET-28a(+)或pET-22b(+)表达载体。由金唯智进行全基因合成经过密码子优化的Sumo-Lira序列，在序列5’端引入NdeI酶切位点，在序列3’端引入XhoI酶切位点，构建于pUC57克隆载体，序列如下：
SEQ ID NO：1，CATATG GGCGGCAGTCTGCAAGATAGCGAAGTGAATCAAGAAGCG AAGCCAGAAGTGAAACCGGAAGTTAAACCGGAGACCCACATCAATCTGAAGGTGAGCG ACGGCAGCAGCGAGATCTTCTTCAAGATCAAGAAGACGACCCCGCTGCGTCGTCTGATG GAAGCCTTCGCCAAACGCCAAGGCAAAGAAATGGACAGTCTGCGCTTTCTGTACGATGG TATCCGCATCCAAGCCGATCAAGCCCCGGAAGATCTGGACATGGAGGACAACGACATCA TCGAGGCGCATCGCGAACAGATCGGCGGCCATGCCGAAGGCACCTTCACCAGCGATGTTAGCAGCTATCTGGAAGGCCAAGCCGCCAAAGAATTCATCGCGTGGCTGGTTCGCGGCCGCGGTTAGCTCGAG。其中，具有下划线的部分为Sumo标签，后边的是利拉鲁肽前体序列。
合成的基因片段从pUC57-Sumo-Lira上经过NdeI和XhoI酶切，切胶回收之后和经过同 样酶切的pET-28a(+)或pET-22b(+)进行16℃过夜连接，之后转化BL21(DE3)感受态细胞，单克隆进行测序分析获得正确的克隆表达载体pET-28a-Sumo-Lira或pET-22b-Sumo-Lira，每种转化挑取3个正确的克隆活化后作为种子250mL摇瓶预筛选，从中选出一个最优的作为最终的表达菌株。取含有重组质粒的BL21(DE3)菌株4mL，接种于含600mL 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1mM IPTG，37℃诱导6h，诱导结束，4℃离心收集菌体。通过超声破碎，收集上清，12％分离胶SDS-PAGE结果表明目标蛋白主要呈可溶表达(图1)。
实施例2
构建Sumo标签融合表达Nesiritide(奈西利肽，简称Nesi)的基因工程菌株
同样采用Sumo标签融合表达的策略，载体构建策略同实施例1，合成基因序列如下：
SEQ ID NO：2，CATATG GGCGGCAGTCTGCAAGATAGCGAAGTGAATCAAGAAGCG AAGCCAGAAGTGAAACCGGAAGTTAAACCGGAGACCCACATCAATCTGAAGGTGAGCG ACGGCAGCAGCGAGATCTTCTTCAAGATCAAGAAGACGACCCCGCTGCGTCGTCTGATG GAAGCCTTCGCCAAACGCCAAGGCAAAGAAATGGACAGTCTGCGCTTTCTGTACGATGG TATCCGCATCCAAGCCGATCAAGCCCCGGAAGATCTGGACATGGAGGACAACGACATCA TCGAGGCGCATCGCGAACAGATCGGCGGCTCTCCGAAAATGGTTCAGGGTTCTGGTTGCTTCGGTCGTAAAATGGACCGTATCTCTTCTTCTTCTGGTCTGGGTTGCAAAGTTCTGCGTCGTCACTAGCTCGAG。其中，具有下划线的部分为Sumo标签，后边的是奈西利肽序列。
基因经过酶切连接构建于pET-28a(+)，测序获得正确的表达质粒pET-28a-Sumo-Nesi及含有该表达的重组BL21(DE3)菌株，采用和Sumo-Lira同样的方法，IPTG诱导后融合蛋白呈可溶表达(图2)。
实施例3
构建Sumo标签融合表达Teriparatide(特立帕肽，简称Teri)的基因工程菌株
同样采用Sumo标签融合表达的策略，载体构建策略同实施例1，合成基因序列如下：
SEQ ID NO：3，CATATG GGCGGCAGTCTGCAAGATAGCGAAGTGAATCAAGAAGCG AAGCCAGAAGTGAAACCGGAAGTTAAACCGGAGACCCACATCAATCTGAAGGTGAGCG ACGGCAGCAGCGAGATCTTCTTCAAGATCAAGAAGACGACCCCGCTGCGTCGTCTGATG GAAGCCTTCGCCAAACGCCAAGGCAAAGAAATGGACAGTCTGCGCTTTCTGTACGATGG TATCCGCATCCAAGCCGATCAAGCCCCGGAAGATCTGGACATGGAGGACAACGACATCA TCGAGGCGCATCGCGAACAGATCGGCGGCTCTGTTTCTGAAATCCAGCTGATGCACAACCTGGGTAAACACCTGAACTCTATGGAACGTGTTGAATGGCTGCGTAAAAAACTGCAGGACGTTCACAACTTCTAACTCGAG。其中，具有下划线的部分为Sumo标签，后边的是特立帕肽序列。
基因经过NdeI和XhoI酶切连接构建于pET-28a(+)，测序获得正确的表达质粒pET-28a-Sumo-Teri及含有该表达的重组BL21(DE3)菌株，采用和Sumo-Lira同样的表达条件，IPTG诱导后融合蛋白呈可溶表达(图2)。
实施例4
构建Ulp1蛋白酶表达菌株
Ulp1蛋白酶的C端部分(D390到K621)直接通过PCR方法从酿酒酵母S288C基因组扩增，所用到的引物是Ulp1-F(SEQ ID NO：4)：gggcatatgGATCTTAAAAAAAAGAAAGAACAATTGGCCAAGAAGAAACTTG和Ulp1-R(SEQ ID NO：5)：Gggctcgaggtattttaaagcgtcggttaaaatcaaatgggc，基因序列如下所示(或可以按照如下序列进行人工合成)：
Figure PCTCN2021097429-appb-000001
扩增的基因片段通过NdeI和XhoI酶切后连接在pET-28a(+)获得表达载体pET-28a-Ulp1，转化BL21(DE3)，转化子进行测序。对测序正确的克隆挑选3个作为种子进行250mL摇瓶预筛选，从中选出一个最优的作为最终的表达菌株。取含有重组质粒的BL21(DE3)菌株4mL，接种于含600mL 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1mM IPTG，37℃诱导6h，诱导结束，4℃离心收集菌体。通过超声破碎，收集上清，12％分离胶SDS-PAGE结果表明目标蛋白部分呈可溶表达。为了进一步提高Ulp1的表达水平，将表达载体pET-28a-Ulp1和伴侣蛋白GroEL/S的表达质粒pGRO7进行了共表达，SDS-PAGE结果表明，Ulp1的表达水平进一步提高(图3)。
实施例5
Sumo-Lira纯化
将表达的Sumo-Lira菌泥以20％菌浓重悬，超声破碎(5s超声，6s间隔，35％功率)，离心，过0.45μm滤膜后获得粗酶液，然后采用亲和层析进行纯化(AKTA系统装配5mL  HisTrap HP)。具体流程是：滤膜过滤样品以5mL/min流速上样，然后采用结合缓冲液(20mM Tris-HCl，500mM NaCl，pH 7.4)冲洗直至未结合蛋白完全洗脱，接着采用60mM咪唑洗脱杂蛋白4个柱体积，然后在500mM洗脱目标蛋白(图1)。该样品可以直接进行Ulp1酶切，不用脱盐和去除咪唑。1g菌泥可以获得纯化的Sumo-Lira融合蛋白35-40mg,1g菌泥可以获得纯化的Sumo-Nesi 15.2mg，1g菌泥可以获得纯化的Sumo-Teri 18.3mg(经过条件优化可以进一步提高)。
为了降低成本，表达Sumo-Lira尝试采用阴离子柱进行了纯化，粗蛋白的获取方式和亲和层析相同，采用AKTA系统装配有阴离子柱(Q FF，5mL)，流速5mL/min，结合缓冲液50mM Tris-HCl pH8.0，上样之后采用结合缓冲液冲洗至未结合蛋白完全洗脱，然后采用50mM Tris-HCl，pH8.0,1M NaCl梯度洗脱在500mM梯度下获得目标蛋白，SDS-PAGE分析表明目标蛋白可以达到80％以上纯度(图4)，经过试验该步纯化的样品同样可以被Ulp1直接进行酶切，不需进行脱盐。
实施例6
Sumo-Teri和Sumo-Nesi纯化
重组菌株诱导表达获得的菌泥经过和Sumo-Lira样品同样的方式获得粗蛋白，进一步同样采用相同的亲和层析的方式进行纯化获得目标融合蛋白。
实施例7
Ulp1纯化
将表达的Ulp1菌泥以10％菌浓重悬，超声破碎(5s超声，6s间隔，35％功率)，离心，过0.45μm滤膜后采用亲和层析进行纯化(AKTA系统装配5mL HisTrap HP)。具体流程是：滤膜过滤样品以4mL/min流速上样，然后采用结合缓冲液(20mM Tris-HCl，500mM NaCl，pH 7.4)冲洗直至未结合蛋白完全洗脱，接着采用60mM咪唑洗脱杂蛋白4个柱体积，然后在500mM洗脱目标蛋白,结果如图5SDS-PAGE所示目标蛋白纯度达到70％左右，获得的目标蛋白可以不用脱盐，直接用于融合蛋白Sumo-Lira、Sumo-Teri和Sumo-Nesi的酶切(实施例5和6的产物)。
实施例8
酶切
Ulp1酶切反应在30℃进行，具体过程是：50mM Tris-HCl,10mM DTT,pH 8.0纯化的Sumo-Lira(实施例5的产物)和Ulp1质量比是10:1～1:1(mg/mg)，在不同时间取样，采用Tricine 检测其切割效率，并检测是否有目标多肽生成，24h切割效率就能到达80％以上。Sumo-Teri和Sumo-Nesi的酶切条件与Sumo-Lira相同。
实施例9
乙腈/加热沉淀法纯化目标多肽
实施例8的酶切完成之后，调整pH于5.6，然后在反应体系加入不同终浓度的乙腈(20％，30％，40％，50％，60％，70％)，混匀后在70度热处理2h，之后12000rpm离心分离上清和沉淀，Tricine检测多肽的分步情况，结果发现在组合条件下，乙腈浓度从20-70％都有着较好的初步纯化效果，处理后利拉鲁肽前体粗品的纯度可以达到90％以上(图6)，Nesi的纯度可以达到80％，Teri纯度达到50％(图7)。进一步采用旋转蒸发或冻干法去除多肽粗品中的乙腈，然后重溶于50mM Tris-HCl pH7.0,并进行制备级HPLC纯化。
六氟异丙醇(hexafluoroisopropanol，HFIP)沉淀利拉鲁肽前体
实施例8的酶切完成之后，调整pH于5.6，向反应体系中分别加入10％-70％(v/v)的六氟异丙醇，室温沉淀1h，离心收集上清和沉淀，使用Tricine-SDS-PAGE检测不同浓度六氟异丙醇对于利拉鲁肽前体的选择性沉淀效果，结果如图9所示：使用50％的六氟异丙醇选择性沉淀后，上清液中的利拉鲁肽前体浓度约90％(图8)。进一步采用旋转蒸发或冻干法去除多肽粗品中的六氟异丙醇，然后重溶于50mM Tris-HCl pH7.0,并进行制备级HPLC纯化。
实施例10
制备HPLC纯化目标多肽
实施例9的产物，采用UniSil AQ C18 10μm 21.5*250mm，所用的所用缓冲液为缓冲液A为0.1％TFA，缓冲液B为乙腈。Lira采用梯度洗脱方式如下：0min 5％B，5min 5％B，25min 50％B，27min 95％B，33min 95％B，38min 5％B，紫外检测器210nm，流速25mL/min，温度为25℃，结果如图9所示：Lira在24min附近被洗脱，之后将样品收集旋转蒸发去除大部分溶剂后冻干，纯度采用HPLC方法加测，结果表明Lira纯度达到98％以上(图10)。
Nesi采用的梯度洗脱方式如下：5min 5％B，35min 30％B,40min 95％B,紫外检测器210nm，流速25mL/min，温度为25℃，结果如图11所示：Nesi在30min被洗脱，之后将样品收集旋转蒸发去除大部分溶剂后冻干，纯度采用HPLC方法加测，结果表明Nesi纯度达到98％以上(图12)。
Teri采用的梯度洗脱方式如下：5min 5％B，35min 30％B,47.8min 42.8％B，50.5min 42.8％B，57.7min 50％B，67.7min 95％B,40min 95％B,紫外检测器210nm，流速25mL/min， 温度为25℃，结果如图13所示：Teri在43min左右被洗脱，之后将样品收集旋转蒸发去除大部分溶剂后冻干，纯度采用HPLC方法检测，分析柱：Eclipse plus C18 4.6×100mm 3.5μm,210nm检测，flow＝1.5ml/min,洗脱梯度是0min 10％B 9min95％B12min 100％，12.1min 10％B，15min 10％。结果表明Teri纯度达到～78％(图14)。
利拉鲁肽前体采用此方法制备，从1g摇瓶发酵菌泥中可以获得3-4mg纯度为98％的利拉鲁肽前体，通过此方法制备1g菌泥可以获得1.26mg纯度为98％的Nesi，通过此方法制备1g菌泥可以获得1.55mg纯度为78％的Teri，通过进一步发酵优化提高融合蛋白的表达水平有望进一步提高目标多肽的得率。
实施例11
质谱检测多肽分子量
制备的多肽(实施例10的产物)分子量采用LC-MS进行分析。具体如下：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40℃，紫外检测器210nm，流速1.5mL/min，经过HPLC分离后的组分采用安捷伦6200系列飞行时间液质联用仪进行结构鉴定，采用电喷雾离子源(Dual AJS ESI)，正离子模式检测，喷雾压力35psig，离子源温度300℃，干燥气(N2)流速10L/min，扫描范围100～2000m/z，碎裂电压70V，并通过Analyst软件采集和处理质谱数据。Lira的理论分子量为3383.7Da，质谱解析的分子量是3382.7(图15)，Nesi的理论分子量为3464.8，质谱解析分子量是3463.7(图16)，Teri的理论分子量是4117.7，质谱解析的分子量是4118.7(图17)。
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 (11)

  1. 一种多肽的制备方法，其特征在于，包括以下步骤：
    构建Sumo标签融合表达多肽基因的工程菌株，并诱导所述工程菌株可溶表达所述多肽；
    从所述工程菌株中纯化得到含有多肽前体的粗蛋白；
    对含有所述多肽前体的粗蛋白采用Ulp1蛋白酶进行酶切，切除所述Sumo标签；
    采用乙腈结合加热沉淀的方法或采用六氟异丙醇沉淀的方法纯化所述Ulp1蛋白酶的酶切产物，得到所述多肽。
  2. 根据权利要求1所述制备方法，其特征在于，所述多肽为利拉鲁肽前体、奈西利肽或特立帕肽。
  3. 根据权利要求1所述制备方法，其特征在于，所述Ulp1蛋白酶为通过构建Ulp1蛋白酶表达菌株并诱导表达获得，其中，所述Ulp1蛋白酶与伴侣蛋白共表达。
  4. 根据权利要求3所述制备方法，其特征在于，所述伴侣蛋白为GroEL/S伴侣蛋白。
  5. 根据权利要求1所述制备方法，其特征在于，所述乙腈结合加热沉淀的方法包括：将所述Ulp1蛋白酶的酶切产物调整pH至5.6，然后加入乙腈，混匀后在60～80℃热处理0.5～3h，之后离心分离上清和沉淀。
  6. 根据权利要求5所述制备方法，其特征在于，所述乙腈为质量百分含量为20～70％的乙腈水溶液。
  7. 根据权利要求1所述制备方法，其特征在于，从所述工程菌株中纯化得到含有多肽前体的粗蛋白包括：将所述工程菌株的、超声破碎、离心、过滤膜后获得粗酶液，然后采用亲和层析或阴离子柱进行纯化，得到所述粗蛋白。
  8. 根据权利要求1所述制备方法，其特征在于，所述六氟异丙醇沉淀的方法包括：将所述Ulp1蛋白酶的酶切产物调整pH至5.6，然后加入六氟异丙醇，混匀后在室温沉淀1h，之后离心分离上清和沉淀。
  9. 根据权利要求8所述制备方法，其特征在于，所述六氟异丙醇为质量百分含量为20～70％的六氟异丙醇水溶液。
  10. 根据权利要求9所述制备方法，其特征在于，所述六氟异丙醇为质量百分含量为50％的六氟异丙醇水溶液。
  11. 根据权利要求1所述制备方法，其特征在于，所述制备方法进一步包括采用HPLC纯化目标多肽的步骤。
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