WO2022110499A1 - Application d'un peptide signal dans l'expression de la protéine de fusion de glp-1 - Google Patents
Application d'un peptide signal dans l'expression de la protéine de fusion de glp-1 Download PDFInfo
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- WO2022110499A1 WO2022110499A1 PCT/CN2020/141997 CN2020141997W WO2022110499A1 WO 2022110499 A1 WO2022110499 A1 WO 2022110499A1 CN 2020141997 W CN2020141997 W CN 2020141997W WO 2022110499 A1 WO2022110499 A1 WO 2022110499A1
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- WO
- WIPO (PCT)
- Prior art keywords
- glp
- amino acid
- signal peptide
- fusion protein
- protein
- Prior art date
Links
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- 101100337060 Caenorhabditis elegans glp-1 gene Proteins 0.000 title description 2
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- 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
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
Definitions
- the present invention relates to the application of signal peptide in expressing GLP-1 fusion protein.
- Glucagon-like peptide-1 is a 30- or 31-amino acid polypeptide hormone derived from tissue-specific post-translational processing of preglucagon peptides. It is produced and secreted by enteroendocrine L cells and certain neurons in the nucleus tractus solitarius of the brainstem during feeding.
- the initial product, GLP-1(1-37) is susceptible to amidation and proteolytic cleavage, resulting in two truncated isopotentially bioactive forms, GLP-1(7-36) and GLP-1(7-37).
- Active GLP-1 consists of two ⁇ -helices, located at amino acid positions 13-20 and 24-35, respectively, separated by a linker region.
- GLP-1 glucose-dependent proinsulin peptide
- GIP glucose-dependent proinsulin peptide
- polypeptides with similar efficacy also include exogenous GLP-1 (Extendin-4) from Heloderma suspectum, both of which have certain amino acid sequences. Similarities, individual differences in amino acids give Extendin-4 a longer in vivo half-life than endogenous GLP-1. After mutating the second amino acid Ala of native GLP-1 to Gly the same as Extendin-4, the tolerance of GLP-1 to DDP-4 can be increased, thereby prolonging the half-life.
- Mammalian cells are often used to express recombinant proteins.
- the signal peptide is located at the N-terminus of the secreted protein. Generally composed of 15 to 30 amino acids. Three regions are included: a positively charged N-terminus, called the basic amino-terminus: an intermediate hydrophobic sequence. Mainly neutral amino acids, can form an ⁇ -helix structure, which is the main functional area of the signal peptide; a longer negatively charged C-terminus, containing small molecular amino acids, is the signal sequence cleavage site, also known as the processing region .
- the signal peptide sequence When the signal peptide sequence is synthesized, it is recognized by the signal recognition granule (SRP), protein synthesis is suspended or slowed down, the signal recognition granule carries the ribosome to the endoplasmic reticulum, and the protein synthesis restarts. Under the guidance of the signal peptide, the newly synthesized protein enters the endoplasmic reticulum cavity. The signal peptide sequence is excised under the action of signal peptidase. Different signal peptides will lead to different N-terminal truncation ratios of GLP-1 fusion proteins, which may affect the purity and activity. Therefore, signal peptides are an important factor affecting the quality of GLP-1 fusion proteins, but the existing technology has not been able to Achieving good purity and activity results.
- SRP signal recognition granule
- signal peptide in expressing GLP-1 or GLP-1 fusion protein is characterized in that the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 1.
- the signal peptide contains an amino acid substitution selected from any of the following:
- the 14th amino acid is replaced with T;
- Amino acid at position 19 is replaced with S;
- Amino acid at position 9 was replaced by FW and amino acid at position 17 was replaced by A.
- the fusion tag of the GLP-1 fusion protein is human immunoglobulin Fc or albumin.
- a polynucleotide comprising:
- amino acid sequence of the signal peptide is shown in SEQ ID NO: 1.
- the signal peptide contains any amino acid substitution selected from the group consisting of:
- the 14th amino acid is replaced with T;
- Amino acid at position 19 is replaced with S;
- Amino acid at position 9 was replaced by FW and amino acid at position 17 was replaced by A.
- the fusion tag of the GLP-1 fusion protein is human immunoglobulin Fc or albumin.
- An expression vector comprising the polynucleotide.
- a host cell containing the expression vector A host cell containing the expression vector.
- the host cell is a mammalian cell.
- a method for producing GLP-1 or GLP-1 fusion protein using the host cell to express GLP-1 or GLP-1 fusion protein.
- the present invention studies the expression of GLP-1 fusion protein in host cells in vitro, and finds that the above-mentioned signal peptide can be used to efficiently express highly active glucagon-like peptide-1 protein or its fusion protein in host cells.
- the embodiment of the present invention provides an expressed protein, including a signal peptide and GLP-1.
- the signal peptide is used to direct the expression of GLP-1 protein or GLP-1 fusion protein in host cells.
- the GLP-1 protein of the present invention refers to endogenous GLP-1 or a GLP-1 analog.
- amino acid sequence of the signal peptide is set forth in SEQ ID NO:1.
- amino acid 14 of the signal peptide is replaced with a T.
- the amino acid sequence of the signal peptide is shown in SEQ ID NO:2.
- amino acid 19 of the signal peptide is replaced with S.
- the amino acid sequence of the signal peptide is shown in SEQ ID NO:3.
- amino acid 9 of the signal peptide is substituted with FW and amino acid 17 is substituted with A.
- the amino acid sequence of the signal peptide is shown in SEQ ID NO:4.
- the signal peptide has amino acid 9 replaced with F, amino acid 14 replaced with S, and amino acid 17 replaced with A.
- the amino acid sequence of the signal peptide is shown in SEQ ID NO:5.
- the GLP-1 fusion protein comprises a GLP-1 protein and a fusion tag linked in sequence.
- the fusion tag is human immunoglobulin human immunoglobulin Fc or albumin.
- GLP-1 fusion protein drugs can prolong the drug in vivo by fusing GLP-1 with macromolecular proteins such as antibody crystallizable fragments (Fc) or albumin. half-life.
- the fusion tag of the GLP-1 fusion protein is human immunoglobulin Fc, and the sequence of the GLP-1 fusion protein is shown in SEQ ID NO:6.
- SEQ ID NO:6 is:
- the embodiment of the present invention also provides a polynucleotide, the polynucleotide encodes the expressed protein. That is, the polynucleotide includes a polynucleotide encoding the signal peptide of any of the above embodiments and a polynucleotide encoding GLP-1 or a GLP-1 fusion protein.
- the polynucleotides of the present invention can be prepared by conventional synthetic methods.
- the polynucleotide of the present invention can be added to an expression vector for the secretory expression of GLP-1 or GLP-1 fusion protein.
- the method for secreting and expressing GLP-1 protein or its fusion protein in host cells using the signal peptide of the present invention is:
- the polynucleotide encoding the signal peptide of the present invention is connected with the polynucleotide encoding the expression GLP-1 protein or its fusion protein, and then cloned into the host cell expression vector, and then the recombinant host cell expression vector is transfected into the host cell to express the target GLP -1 protein or its fusion protein.
- the embodiment of the present invention also provides an expression vector, comprising the polynucleotide encoding the same.
- vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
- the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
- the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell.
- Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1 derived artificial chromosomes (PACs) ; Phage such as ⁇ phage or M13 phage and animal viruses.
- Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses Polyoma vacuolar virus (eg SV40).
- the vectors of the present invention contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (such as transcription termination signals, or multiple adenylation signals and poly U sequences, etc.).
- the vector of the present invention may also contain fragments of genes used for screening (eg, antibiotic resistance genes), nucleic acids used to generate fluorescent proteins, and the like.
- the fluorescent protein can be selected from green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein or red fluorescent protein.
- Common GFP can be used for green fluorescent protein, or modified GFP gene, such as enhanced GFP gene EGFP, etc.; blue fluorescent protein can be selected from EBFP, Azuritc, TagBFP, etc.; yellow fluorescent protein can be selected from EYFP, Ypct , PhiYFP, etc.; orange fluorescent protein can be selected from mKO, mOrange, mBanana, etc.; red fluorescent protein can be selected from TagRFP, mRuby, mCherry, mKate, etc.
- the polynucleotide encoding the signal peptide of the present invention is immediately preceding the GLP-1 or its fusion protein polynucleotide.
- the expression vector may be the commercial plasmid pXC17.4.
- the embodiment of the present invention also provides a host cell obtained by transfection of the expression vector. That is, the host cell has the expression vector, and the above-mentioned expression protein can be expressed in the host cell.
- the host cells are selected from mammalian cells.
- the mammalian cells are rodent cells, eg, rat, mouse, hamster.
- the mammalian cells are primate cells, preferably human.
- the mammalian cells are primary cells, such as tumor cells, hepatocytes, cardiomyocytes, neurons, endothelial cells, stem cells, and the like.
- the mammalian cell is a cell line
- mice derived from mice:
- McCoy BALB/3T3, 3T6, A9, AtT-20, Clone M-3, I-10, Y-1, WEHI-3b, ES-D3, F9;
- the mammalian cells can be Chinese hamster ovary cells (Chinese hamster ovary, CHO), baby hamster kidney cells (baby hamster kidney, BHK), mouse myeloma cells (SP2/0), mouse mammary tumor cells cells (C127), human embryonic kidney 293 cells (human embryonic kidney293, HEK293) and the like.
- Chinese hamster ovary cells Choinese hamster ovary, CHO
- baby hamster kidney cells baby hamster kidney, BHK
- mouse myeloma cells SP2/0
- mouse mammary tumor cells cells C127
- human embryonic kidney 293 cells human embryonic kidney293, HEK293
- the embodiments of the present invention also provide a method for producing a GLP-1 protein or a fusion protein thereof, which comprises culturing the host cell containing the expression vector under suitable conditions, and then isolating the GLP-1 protein or its fusion protein.
- the gene of the signal peptide (SP)+GLP-1-Fc fusion protein was synthesized by the total gene synthesis technology and constructed into the commercial plasmid pXC17.4, and the plasmid pXC17.4-SP-GLP-1-Fc was constructed.
- the pXC17.4-SP-GLP-1-Fc plasmid was electroporated into suspension serum-free acclimated CHO K1 cells. After transfection, the transfected cells were pressurized and screened with CD CHO medium containing 25 ⁇ M MSX, and the medium was changed every 3-4 days until the cell viability recovered to more than 90%, and MSX was removed.
- the screened cell pool was inoculated into a 250 ml Erlenmeyer flask containing 60 ml Dynamis medium at about 0.5 ⁇ 10 6 cells/ml, and the culture conditions were: 37° C., 140 RPM, 5% CO 2 , and 85% humidity. From day 3, the feed medium was fed daily with 3% (v/v) Cell Boost 7a and 0.3% (v/v) Cell Boost 7b, and glucose was controlled at a concentration of 5-8 g/L. The culture was terminated on the 10th day. The supernatant was harvested by centrifugation at 2000 rmp for 10 min, filtered through a 0.22 ⁇ m filter, and stored at 2-8 °C.
- the GLP-1-Fc fusion protein reference substance was serially diluted to 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9ug/ml with diluent (PBST containing 0.1% BSA (PBS+0.05% tween20)) for To prepare a standard curve, dilute the cell pool culture supernatant 100-fold with diluent.
- the Octet molecular interaction instrument was used with Protein A biosensor to detect the diluted samples of the reference substance and cell culture supernatant, and the expression of GLP-1-Fc fusion protein in the culture supernatant could be calculated from the standard curve, as shown in Table 1. .
- the expression level of the GLP-1-Fc fusion protein guided by the several signal peptides of the present invention is far greater than the expression level of the GLP-1 fusion protein guided by the signal peptide in the prior art.
- GLP-1-Fc fusion protein contains an Fc tag and is theoretically purified with Protein A packing, due to the special property of GLP-1 that is easy to aggregate, GLP-1-Fc fusion protein is also prone to protein aggregation during elution.
- size exclusion chromatography was used for purification.
- the main peak components with a signal response value above 100 mAU were collected and merged, and were tested for purity and activity.
- Sample pretreatment Dilute the sample to about 0.8 mg/mL with 0.1 mol/L sodium bicarbonate. Take 250 ⁇ L of sample diluent, add 670 ⁇ L of 8 mol/L guanidine hydrochloride solution, and then add 100 ⁇ L of 8 mol/L guanidine hydrochloride solution containing 50 mg/mL dithiothreitol, and incubate at 37°C for 30 minutes after mixing, as the test solution. Take 50uL of the test solution, inject it into a liquid chromatograph, record the chromatogram, and calculate the purity according to the area normalization method, as shown in Table 4.
- HEK293/GLP-1R-CRE-luciferase cells were trypsinized and resuspended in assay medium (DMEM medium containing 0.5% BSA and 0.25% FBS) to approximately 5 x 10 5 cells/mL.
- assay medium DMEM medium containing 0.5% BSA and 0.25% FBS
- the cell suspension was evenly seeded into a white opaque flat-bottomed 96-well cell culture plate at 100 ⁇ L/well.
- GLP-1 fusion protein Take the GLP-1 fusion protein, use the detection medium (DMEM medium containing 0.5% BSA and 0.25% FBS), and dilute the gradient to 20, 6.6667, 2.2222, 0.7407, 0.2469, 0.0823, 0.0274ng/ml, each gradient with 50 ⁇ L/well was added to the cell culture plate, and 3 replicate wells were set for each dilution. Shake the cell culture plate on a plate shaker for 30 seconds, then place it in a 37°C, 5% CO 2 incubator for 4-6 hours. Add 100 ⁇ L/well of luciferase reagent, incubate at 20-25° C. for 40-70 minutes, read the chemiluminescence value with a microplate reader, and calculate the EC 50 by fitting the curve, as shown in Table 5.
- DMEM medium containing 0.5% BSA and 0.25% FBS DMEM medium containing 0.5% BSA and 0.25% FBS
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Abstract
L'invention concerne une application d'un peptide signal dans l'expression de GLP-1 ou d'une protéine de fusion de GLP-1. La séquence d'acides aminés du peptide signal est représentée par SEQ ID NO : 1, SEQ ID NO : 2, SEQ ID NO : 3 ou SEQ ID NO : 4. Le peptide signal peut améliorer l'expression, la pureté et l'activité biologique de GLP-1 ou d'une protéine de fusion de GLP-1.
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CN118240917A (zh) * | 2024-05-27 | 2024-06-25 | 正大天晴药业集团南京顺欣制药有限公司 | 一种检测glp—1、glp—1类似物或glp—1融合蛋白生物学活性的方法 |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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CN110452288A (zh) * | 2018-05-08 | 2019-11-15 | 鲁南制药集团股份有限公司 | 一种人工合成信号肽 |
CN110494565A (zh) * | 2016-12-02 | 2019-11-22 | 朱诺治疗学股份有限公司 | 工程化b细胞及相关组合物和方法 |
WO2020048494A1 (fr) * | 2018-09-06 | 2020-03-12 | 浙江柏拉阿图医药科技有限公司 | Protéine de recombinaison glp1-fc-cd47 à action prolongée, son procédé de préparation et son utilisation |
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CN110494565A (zh) * | 2016-12-02 | 2019-11-22 | 朱诺治疗学股份有限公司 | 工程化b细胞及相关组合物和方法 |
CN108103088A (zh) * | 2017-12-21 | 2018-06-01 | 广东东阳光药业有限公司 | 重组GLP-1类似物Fc融合蛋白的优化基因及其应用 |
CN110452288A (zh) * | 2018-05-08 | 2019-11-15 | 鲁南制药集团股份有限公司 | 一种人工合成信号肽 |
WO2020048494A1 (fr) * | 2018-09-06 | 2020-03-12 | 浙江柏拉阿图医药科技有限公司 | Protéine de recombinaison glp1-fc-cd47 à action prolongée, son procédé de préparation et son utilisation |
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---|---|---|---|---|
CN116102625A (zh) * | 2023-03-20 | 2023-05-12 | 杭州斯达特生物科技有限公司 | 一种信号肽及其应用 |
CN116102625B (zh) * | 2023-03-20 | 2023-09-22 | 杭州斯达特生物科技有限公司 | 一种信号肽及其应用 |
CN118240917A (zh) * | 2024-05-27 | 2024-06-25 | 正大天晴药业集团南京顺欣制药有限公司 | 一种检测glp—1、glp—1类似物或glp—1融合蛋白生物学活性的方法 |
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