WO2022110499A1 - Application of signal peptide in expression of glp-1 fusion protein - Google Patents

Application of signal peptide in expression of glp-1 fusion protein Download PDF

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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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glp
amino acid
signal peptide
fusion protein
protein
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PCT/CN2020/141997
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French (fr)
Chinese (zh)
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王丁力
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广州汉腾生物科技有限公司
佛山汉腾生物科技有限公司
佛山普津生物技术有限公司
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Publication of WO2022110499A1 publication Critical patent/WO2022110499A1/en

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    • 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

Disclosed are an application of a signal peptide in the expression of GLP-1 or a GLP-1 fusion protein. The amino acid sequence of the signal peptide is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. The signal peptide can improve the expression, purity and biological activity of GLP-1 or a GLP-1 fusion protein.

Description

信号肽在表达GLP-1融合蛋白中的应用Application of signal peptide in expression of GLP-1 fusion protein 技术领域technical field
本发明涉及信号肽在表达GLP-1融合蛋白中的应用。The present invention relates to the application of signal peptide in expressing GLP-1 fusion protein.
背景技术Background technique
胰高血糖素样肽-1(glucagon-like peptide-1，GLP-1)是一种含30或31个氨基酸的多肽激素，来源于前胰高血糖素肽的组织特异性翻译后处理。它是由肠道内分泌L细胞和脑干孤束核内的某些神经元在进食时产生和分泌的。初始产物GLP-1(1-37)易被酰胺化和蛋白水解裂解，从而产生两种截短的等电位生物活性形式GLP-1(7-36)和GLP-1(7-37)。活性GLP-1由两个α-螺旋组成，分别位于氨基酸的13-20和24-35位，由一个连接区隔开。GLP-1与葡萄糖依赖性胰岛素原肽(GIP)一起，是一种肠促胰岛素，因此，它能够通过促进胰岛素的分泌以葡萄糖依赖的方式降低血糖水平。除了促胰岛素作用外，GLP-1还具有多种调节和保护作用。与GIP不同的是，GLP-1的作用在2型糖尿病患者中得以保留，因此大量的药物研究已被用于开发基于GLP-1的治疗方法。Glucagon-like peptide-1 (GLP-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 α-helices, located at amino acid positions 13-20 and 24-35, respectively, separated by a linker region. GLP-1, together with glucose-dependent proinsulin peptide (GIP), is an incretin, and thus, it is able to lower blood glucose levels in a glucose-dependent manner by promoting insulin secretion. In addition to its insulinotropic effects, GLP-1 has various regulatory and protective effects. Unlike GIP, the effects of GLP-1 are preserved in patients with type 2 diabetes, so a large amount of drug research has been used to develop GLP-1-based treatments.
除来自人体自身的内源性GLP-1外，有类似功效的多肽还有来自吉拉毒蜥(Heloderma suspectum)的外源性GLP-1(Extendin-4)，两者的氨基酸序列有一定的相似性，个别差异的氨基酸使得Extendin-4拥有比内源性GLP-1更长的体内半衰期。将天然GLP-1的第二位氨基酸Ala突变成与Extendin-4相同的Gly后，可以增加GLP-1对DDP-4的耐受性，从而延长半衰期。In addition to endogenous GLP-1 from the human body, 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.
哺乳动物细胞常被用于表达重组蛋白。信号肽位于分泌蛋白的N端。一般由15～30个氨基酸组成。包括三个区：一个带正电的N末端，称为碱性氨基末端：一个中间疏水序列。以中性氨基酸为主，能够形成一段α螺旋结构，它是信号肽的主要功能区；一个较长的带负电荷的C末端，含小分子氨基酸，是信号序列切割位点，也称加工区。当信号肽序列合成后，被信号识别颗粒(SRP)所识别，蛋白质合成暂停或减缓，信号识别颗粒将核糖体携带至内质网上，蛋白质合成重新开始。在信号肽的引导下，新合成的蛋白质进入内质网腔.而信号 肽序列则在信号肽酶的作用下被切除。不同的信号肽会导致不同的GLP-1融合蛋白N端截短比例，进而可能对纯度和活性产生影响，因此信号肽是影响GLP-1融合蛋白质量的重要因素，但现有技术还没有能够达到很好的纯度、活性结果。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 α-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 . 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.
发明内容SUMMARY OF THE INVENTION
基于此，有必要提供一种能够提高GLP-1蛋白表达纯度和生物活性的GLP-1表达蛋白及其生产方法。Based on this, it is necessary to provide a GLP-1 expression protein and its production method that can improve the expression purity and biological activity of the GLP-1 protein.
信号肽在表达GLP-1或GLP-1融合蛋白中的应用，其特征在于，所述信号肽的氨基酸序列如SEQ ID NO:1所示。The application of 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.
在其中一个实施例中，所述信号肽含有选自以下任意一种的氨基酸替换：In one embodiment, the signal peptide contains an amino acid substitution selected from any of the following:
第14位氨基酸替换为T；The 14th amino acid is replaced with T;
第19位氨基酸替换为S；Amino acid at position 19 is replaced with S;
第9位氨基酸替换为FW，第17位氨基酸替换为A。Amino acid at position 9 was replaced by FW and amino acid at position 17 was replaced by A.
在其中一个实施例中，所述GLP-1融合蛋白的融合标签为人免疫球蛋白Fc或白蛋白。In one embodiment, the fusion tag of the GLP-1 fusion protein is human immunoglobulin Fc or albumin.
一种多核苷酸，所述多核苷酸包括：A polynucleotide comprising:
编码信号肽的多核苷酸，和a polynucleotide encoding a signal peptide, and
编码GLP-1或GLP-1融合蛋白的多核苷酸；A polynucleotide encoding GLP-1 or a GLP-1 fusion protein;
所述信号肽的氨基酸序列如SEQ ID NO:1所示。The amino acid sequence of the signal peptide is shown in SEQ ID NO: 1.
在其中一个实施例中，所述信号肽含有选自以下任意一种氨基酸替换：In one embodiment, the signal peptide contains any amino acid substitution selected from the group consisting of:
第14位氨基酸替换为T；The 14th amino acid is replaced with T;
第19位氨基酸替换为S；Amino acid at position 19 is replaced with S;
第9位氨基酸替换为FW，第17位氨基酸替换为A。Amino acid at position 9 was replaced by FW and amino acid at position 17 was replaced by A.
在其中一个实施例中，所述GLP-1融合蛋白的融合标签为人免疫球蛋白Fc或白蛋白。In one embodiment, the fusion tag of the GLP-1 fusion protein is human immunoglobulin Fc or albumin.
一种表达载体，包含所述的多核苷酸。An expression vector, comprising the polynucleotide.
一种宿主细胞，所述宿主细胞含有所述的表达载体。A host cell containing the expression vector.
在其中一个实施例中，所述宿主细胞为哺乳动物细胞。In one embodiment, the host cell is a mammalian cell.
一种GLP-1或GLP-1融合蛋白的生产方法，使用所述的宿主细胞表达GLP-1或GLP-1融合蛋白。A method for producing GLP-1 or GLP-1 fusion protein, using the host cell to express GLP-1 or GLP-1 fusion protein.
本发明对GLP-1融合蛋白的体外宿主细胞表达进行研究，发现上述的信号肽可用于在宿主细胞中高效表达高活性的胰高血糖素样肽-1蛋白或其融合蛋白。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.
具体实施方式Detailed ways
为了便于理解本发明，下面将对本发明进行更全面的描述。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。In order to facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough and complete understanding of the present disclosure is provided.
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention.
本发明实施例提供一种表达蛋白，包括信号肽以及GLP-1。该信号肽用于引导GLP-1蛋白或GLP-1融合蛋白在宿主细胞中的表达。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.
本发明的GLP-1蛋白是指内源性GLP-1或GLP-1类似物。The GLP-1 protein of the present invention refers to endogenous GLP-1 or a GLP-1 analog.
在一些实施方式中，所述信号肽的氨基酸序列如SEQ ID NO:1所示。In some embodiments, the amino acid sequence of the signal peptide is set forth in SEQ ID NO:1.
Figure PCTCN2020141997-appb-000001
Figure PCTCN2020141997-appb-000001
在一些实施方式中，所述信号肽的第14位氨基酸替换为T。该信号肽的氨基酸序列如SEQ ID NO:2所示。In some embodiments, 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.
Figure PCTCN2020141997-appb-000002
Figure PCTCN2020141997-appb-000002
在一些实施方式中，所述信号肽的第19位氨基酸替换为S。该信号肽的氨基酸序列如SEQ ID NO:3所示。In some embodiments, 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.
Figure PCTCN2020141997-appb-000003
Figure PCTCN2020141997-appb-000003
在一些实施方式中，所述信号肽的第9位氨基酸替换为FW，第17位氨基酸替换为A。该信号肽的氨基酸序列如SEQ ID NO:4所示。In some embodiments, 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.
Figure PCTCN2020141997-appb-000004
Figure PCTCN2020141997-appb-000004
在一些实施方式中，所述信号肽的第9位氨基酸替换为F，第14位氨基酸 替换为S,第17位氨基酸替换为A。该信号肽的氨基酸序列如SEQ ID NO:5所示。In some embodiments, 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.
Figure PCTCN2020141997-appb-000005
Figure PCTCN2020141997-appb-000005
在一些实施方式中，所述GLP-1融合蛋白包括依次连接的GLP-1蛋白和融合标签。在一些实施方式中，所述融合标签为人免疫球蛋白人免疫球蛋白Fc或白蛋白。一方面用于在细胞表达后的分离纯化，另一方面GLP-1融合蛋白类药物可通过将GLP-1与大分子蛋白如抗体的可结晶片段(Fc)或白蛋白融合来延长药物在体内的半衰期。In some embodiments, the GLP-1 fusion protein comprises a GLP-1 protein and a fusion tag linked in sequence. In some embodiments, the fusion tag is human immunoglobulin human immunoglobulin Fc or albumin. On the one hand, it is used for separation and purification after expression in cells, and on the other hand, 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.
在一些实施方式中，GLP-1融合蛋白的融合标签为人免疫球蛋白Fc，GLP-1融合蛋白的序列如SEQ ID NO:6所示。In some embodiments, 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为：SEQ ID NO:6 is:
Figure PCTCN2020141997-appb-000006
Figure PCTCN2020141997-appb-000006
本发明实施例还提供一种多核苷酸，所述多核苷酸编码所述的表达蛋白。即，该多核苷酸包含编码上述任一实施例的信号肽的多核苷酸及编码GLP-1或GLP-1融合蛋白的多核苷酸。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.
本发明的多核苷酸可通过常规的合成方法制备。The polynucleotides of the present invention can be prepared by conventional synthetic methods.
本发明的多核苷酸可添加于表达载体，用于GLP-1或GLP-1融合蛋白的分泌表达。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.
利用本发明的信号肽在宿主细胞中分泌表达GLP-1蛋白或其融合蛋白的方法为：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:
将编码本发明信号肽的多核苷酸与编码表达GLP-1蛋白或其融合蛋白的多核苷酸连接后克隆入宿主细胞表达载体，而后将该重组宿主细胞表达载体转染宿主细胞后表达目的GLP-1蛋白或其融合蛋白。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.
本发明实施例还提供一种表达载体，包含编码该多核苷酸。The embodiment of the present invention also provides an expression vector, comprising the polynucleotide encoding the same.
术语“载体(vector)”是指，可将多聚核苷酸***其中的一种核酸运载工具。当载体能使***的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、***瘤病毒、***多瘤空泡病毒(如SV40)。在一些实施方式中，本发明所述载体中包含基因工程中常用的调控元件，例如增强子、启动子、内部核糖体进入位点(IRES)和其他表达控制元件(例如转录终止信号，或者多腺苷酸化信号和多聚U序列等)。The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When 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 λ 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). In some embodiments, 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.).
在一些实施方式中，本发明所述载体中还可以包含筛选所用的基因(例如抗生素抗性基因)、用于生成荧光蛋白的核酸等片段。荧光蛋白可以选择绿色荧光蛋白、蓝色荧光蛋白、黄色荧光蛋白、橙色荧光蛋白或红色荧光蛋白。In some embodiments, 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.
绿色荧光蛋白可以采用常见的GFP，也可以采用经过改造后的GFP基因，例如增强型GFP基因EGFP等；蓝色荧光蛋白可以选自EBFP、Azuritc、TagBFP等；黄色荧光蛋白可以选自EYFP、Ypct、PhiYFP等；橙色荧光蛋白可以选自mKO、mOrange、mBanana等；红色荧光蛋白可以选自TagRFP、mRuby、mCherry、mKate等。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.
在一些实施方式中，表达载体中，编码本发明信号肽的所述多核苷酸紧接在所述GLP-1或其融合蛋白多核苷酸的前端。In some embodiments, in the expression vector, the polynucleotide encoding the signal peptide of the present invention is immediately preceding the GLP-1 or its fusion protein polynucleotide.
在一些实施方式中，表达载体可为商品化质粒pXC17.4。In some embodiments, the expression vector may be the commercial plasmid pXC17.4.
本发明实施例还提供一种宿主细胞，为所述的表达载体所转染得到。即，该宿主细胞中具有该表达载体，在该宿主细胞中可表达上述表达蛋白。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.
优选的，宿主细胞选自哺乳动物细胞。Preferably, the host cells are selected from mammalian cells.
在一些实施方式中，所述哺乳动物细胞为啮齿类动物细胞，例如大鼠、小鼠、仓鼠。In some embodiments, the mammalian cells are rodent cells, eg, rat, mouse, hamster.
在一些实施方式中，所述哺乳动物细胞为灵长类动物细胞，优选为人。In some embodiments, the mammalian cells are primate cells, preferably human.
在一些实施方式中，所述哺乳动物细胞为原代细胞，例如肿瘤细胞、肝细胞、心肌细胞、神经元、内皮细胞、干细胞等。In some embodiments, the mammalian cells are primary cells, such as tumor cells, hepatocytes, cardiomyocytes, neurons, endothelial cells, stem cells, and the like.
在一些实施方式中，所述哺乳动物细胞为细胞系；In some embodiments, the mammalian cell is a cell line;
常见的细胞系例如：Common cell lines such as:
来源于人的细胞系：Cell lines derived from humans:
293、IMR-90、W1-38、A549、A431、BHL-100、BeWo、Caco-2、Chang、HCT-15、HeLa、HEp-G2、HEp-2、HT-1080、HT-29、JEG-2、MCF7、KB、Saos-2、WI-38、WISH、WS1、HUVEC、EB-3、Raji、IM-9、Daudi、H9、HL-60、Jurkat、K-562、U937、KG-1；293, IMR-90, W1-38, A549, A431, BHL-100, BeWo, Caco-2, Chang, HCT-15, HeLa, HEp-G2, HEp-2, HT-1080, HT-29, JEG- 2. MCF7, KB, Saos-2, WI-38, WISH, WS1, HUVEC, EB-3, Raji, IM-9, Daudi, H9, HL-60, Jurkat, K-562, U937, KG-1;
来源于小鼠的细胞系：Cell lines derived from mice:
McCoy、BALB/3T3、3T6、A9、AtT-20、Clone M-3、I-10、Y-1、WEHI-3b、ES-D3、F9；McCoy, BALB/3T3, 3T6, A9, AtT-20, Clone M-3, I-10, Y-1, WEHI-3b, ES-D3, F9;
来源于仓鼠的细胞系：Cell lines derived from hamsters:
BHK-21、HaK、CHO-K1；BHK-21, HaK, CHO-K1;
来源于大鼠的细胞系：Cell lines derived from rats:
AR42J、BRL3A、Clone 9、H4--Ⅱ-E-C3、GH1、GH3、IEC-6、L2、XC、LLC-WRC 256、Jensen、Rat2(TK-)、PC12、L6；AR42J, BRL3A, Clone 9, H4--Ⅱ-E-C3, GH1, GH3, IEC-6, L2, XC, LLC-WRC 256, Jensen, Rat2(TK-), PC12, L6;
来源于其他动物的细胞系：Cell lines derived from other animals:
D-17、BT、MARC-145、CV-1、COS-1、COS-3、COS-7、Vero、B95-8、CRFK。D-17, BT, MARC-145, CV-1, COS-1, COS-3, COS-7, Vero, B95-8, CRFK.
具体的，所述的哺乳动物细胞可以是中国仓鼠卵巢细胞(Chinese hamster ovary，CHO)、幼年仓鼠肾细胞(baby hamster kidney，BHK)、小鼠骨髓瘤细胞(SP2/0)、小鼠乳腺肿瘤细胞(C127)、人胚肾293细胞(human embryonic kidney293，HEK293)等。Specifically, 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.
本发明实施例还提供一种GLP-1蛋白或其融合蛋白的生产方法，为在适合条件下，培养所述的含有该表达载体的宿主细胞，而后从培养物中分离出GLP-1蛋白或其融合蛋白。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.
以下为具体实施例。The following are specific examples.
细胞池构建：Cell pool construction:
通过全基因合成技术合成信号肽(SP)+GLP-1-Fc融合蛋白的基因并将其构建至商品化质粒pXC17.4中，构建质粒pXC17.4-SP-GLP-1-Fc。将pXC17.4-SP-GLP-1-Fc质粒通过电穿孔转染至经悬浮无血清驯化的CHO K1细胞中。转染后用含25μM MSX的CD CHO培养基对转染后的细胞进行加压筛选，每3-4天更换一次培养基，直至细胞活率恢复至90％以上，撤去MSX。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 μM MSX, and the medium was changed every 3-4 days until the cell viability recovered to more than 90%, and MSX was removed.
蛋白表达：Protein:
将筛选后的细胞池以约0.5×10 6cell/ml接种至含60ml Dynamis培养基的250ml三角摇瓶中，培养条件：37℃，140RPM,5％CO 2，85％湿度。从第3天起，每天流加补料培养基3％(v/v)Cell Boost 7a和0.3％(v/v)Cell Boost 7b，并将葡萄糖控制在5-8g/L的浓度。培养至第10天时终止培养。2000rmp离心10min收获上清，再经0.22μm滤膜过滤后保存于2-8℃。 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 μm filter, and stored at 2-8 °C.
表达量检测：Expression detection:
将GLP-1-Fc融合蛋白参考品用稀释液(含0.1％BSA的PBST(PBS+0.05％tween20))按梯度稀释为250，125，62.5，31.3，15.6，7.8，3.9ug/ml用于制备标准曲线，同将细胞池培养上清用稀释液稀释100倍。用Octet分子相互作用仪搭配Protein A生物传感器检测参考品和细胞培养上清的稀释样品，通过标准曲线可以计算得出培养上清中GLP-1-Fc融合蛋白的表达量，如表1所示。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. .
表1Table 1
信号肽signal peptide 细胞培养上清中GLP-1-Fc融合蛋白的表达量(g/L)Expression of GLP-1-Fc fusion protein in cell culture supernatant (g/L)
SEQ ID NO:5SEQ ID NO: 5 2.62.6
SEQ ID NO:1SEQ ID NO: 1 3.33.3
SEQ ID NO:2SEQ ID NO: 2 2.92.9
SEQ ID NO:4SEQ ID NO: 4 2.62.6
SEQ ID NO:3SEQ ID NO: 3 3.13.1
本发明的这几个信号肽引导的GLP-1-Fc融合蛋白的表达量远大于现有技术中信号肽引导的GLP-1融合蛋白的表达量。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.
蛋白纯化protein purification
尽管GLP-1-Fc融合蛋白含有Fc标签，理论上用Protein A填料进行纯化， 但因为GLP-1容易集聚的特殊性质，GLP-1-Fc融合蛋白洗脱时也会容易产生蛋白集聚。为避免纯化方法引入蛋白集聚而进入分析样品的后续分析，改用分子排阻色谱进行纯化。Although 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. In order to avoid the introduction of protein aggregation into the subsequent analysis of the analytical sample by the purification method, size exclusion chromatography was used for purification.
层析柱：Superdex 200 increase，10/300 GL，CV＝24mLChromatography column: Superdex 200 increase, 10/300 GL, CV=24mL
流动相：10mM Na-Citrate，pH6.5Mobile phase: 10mM Na-Citrate, pH 6.5
流速:0.5mL/minFlow rate: 0.5mL/min
样品:500uL上述细胞培养上清Sample: 500uL of the above cell culture supernatant
收集并合并信号响应值在100mAU以上的主峰组分，并对其进行纯度和活性检测。The main peak components with a signal response value above 100 mAU were collected and merged, and were tested for purity and activity.
SEC-HPLC检测：SEC-HPLC detection:
色谱柱：TSKgel G3000WXL(5)7.8×300Chromatographic column: TSKgel G3000WXL(5) 7.8×300
流动相:100mM磷酸钠盐,150mM氯化钠,pH7.0±0.2Mobile phase: 100mM sodium phosphate, 150mM sodium chloride, pH7.0±0.2
流速:0.5mL/minFlow rate: 0.5mL/min
运行时间:30minRunning time: 30min
柱温:25±2℃Column temperature: 25±2℃
检测波长:214nmDetection wavelength: 214nm
取纯化后的蛋白溶液50uL，注入液相色谱仪，记录色谱图，按面积归一化法计算纯度，如表2所示。Take 50uL of the purified protein solution, inject it into a liquid chromatograph, record the chromatogram, and calculate the purity according to the area normalization method, as shown in Table 2.
表2Table 2
信号肽序列signal peptide sequence SEC-HPLC主峰(％)SEC-HPLC main peak (%)
SEQ ID NO:5SEQ ID NO: 5 98.9998.99
SEQ ID NO:1SEQ ID NO: 1 98.0998.09
SEQ ID NO:2SEQ ID NO: 2 98.9998.99
SEQ ID NO:4SEQ ID NO: 4 98.9098.90
SEQ ID NO:3SEQ ID NO: 3 99.0299.02
RP-HPLC检测RP-HPLC detection
色谱柱：Ace3 C4-300(4.6×150mm,3um)Chromatographic column: Ace3 C4-300 (4.6×150mm, 3um)
流动相A:0.05％(v/v)TFA-20％(v/v)乙醇水溶液Mobile phase A: 0.05% (v/v) TFA-20% (v/v) ethanol in water
流动相B:0.05％(v/v)TFA-90％(v/v)乙醇水溶液Mobile phase B: 0.05% (v/v) TFA-90% (v/v) ethanol in water
流速:1.0mL/minFlow rate: 1.0mL/min
柱温:60℃Column temperature: 60℃
检测波长:214nmDetection wavelength: 214nm
洗脱梯度如下表3所示。The elution gradients are shown in Table 3 below.
表3table 3
时间(min)time (min) 流动相A(％)Mobile phase A (%) 流动相B(％)Mobile phase B (%)
00 8383 1717
1.01.0 8383 1717
21.021.0 7676 24twenty four
21.121.1 00 100100
25.025.0 00 100100
25.125.1 8383 1717
32.032.0 8383 1717
样品预处理:用0.1mol/L碳酸氢钠将样品稀释至约0.8mg/mL。取250μL样品稀释液，加入670μL 8mol/L盐酸胍溶液，再加入含50mg/mL二硫苏糖醇的8mol/L盐酸胍溶液100μL，混匀后37℃孵育30分钟，作为供试品溶液。取供试品溶液50uL，注入液相色谱仪，记录色谱图，按面积归一化法计算纯度，如表4所示。Sample pretreatment: Dilute the sample to about 0.8 mg/mL with 0.1 mol/L sodium bicarbonate. Take 250 μL of sample diluent, add 670 μL of 8 mol/L guanidine hydrochloride solution, and then add 100 μL 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.
表4Table 4
信号肽序列signal peptide sequence RP-HPLC主峰(％)RP-HPLC main peak (%)
SEQ ID NO:5SEQ ID NO: 5 65.1265.12
SEQ ID NO:1SEQ ID NO: 1 78.3178.31
SEQ ID NO:2SEQ ID NO: 2 77.7877.78
SEQ ID NO:4SEQ ID NO: 4 77.2077.20
SEQ ID NO:3SEQ ID NO: 3 77.0477.04
体外生物学检测In vitro biological assays
GLP-1融合蛋白对HEK293/GLP-1R-CRE-荧光素酶细胞刺激后，胞内cAMP表达量增高，通过CRE启动子激活荧光素酶报告基因的表达。利用Promega公司生产的SteadyGlo荧光素酶检测***，检测荧光素酶催化底物产生的荧光值，该指标与结合至细胞膜上GLP-1受体的蛋白产量呈正相关，从而检测GLP-1融合蛋白的体外生物学活性。After GLP-1 fusion protein stimulated HEK293/GLP-1R-CRE-luciferase cells, the expression of intracellular cAMP increased, and the expression of luciferase reporter gene was activated through the CRE promoter. The SteadyGlo luciferase detection system produced by Promega was used to detect the fluorescence value of the luciferase-catalyzed substrate, which was positively correlated with the protein production bound to the GLP-1 receptor on the cell membrane, so as to detect the GLP-1 fusion protein. In vitro biological activity.
将HEK293/GLP-1R-CRE-荧光素酶细胞用胰酶消化后，用检测培养基(含0.5％BSA和0.25％FBS的DMEM培养基)重悬为约5×10 5个细胞/mL的细胞悬 液，将此细胞悬液以100μL/孔均匀接种到白色不透明平底96孔细胞培养板中。取GLP-1融合蛋白，用检测培养基(含0.5％BSA和0.25％FBS的DMEM培养基)，梯度稀释成20，6.6667，2.2222，0.7407，0.2469，0.0823，0.0274ng/ml，每个梯度以50μL/孔加入细胞培养板中，每一稀释度设3个复孔。将细胞培养板在样板振荡器上振荡30秒，后置于37℃，5％CO 2培养箱中培养4-6小时。加入荧光素酶试剂100μL/孔，于20-25℃下孵育40-70分钟，用酶标仪读取化学发光值，拟合曲线计算EC 50，如表5所示。 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. The cell suspension was evenly seeded into a white opaque flat-bottomed 96-well cell culture plate at 100 μL/well. 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 μL/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 μL/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.
表5table 5
信号肽序列signal peptide sequence EC 50(ng/mL) EC50 (ng/mL)
SEQ ID NO:1SEQ ID NO: 1 1.47001.4700
SEQ ID NO:2SEQ ID NO: 2 1.24501.2450
SEQ ID NO:4SEQ ID NO: 4 1.36791.3679
SEQ ID NO:3SEQ ID NO: 3 1.32681.3268
以上实验结果表明，本发明实施例中的这几个信号肽引导GLP-1融合蛋白分泌表达时，除了能得到较高的蛋白表达量，GLP-1融合蛋白的纯度和生物学活性也都达到较高水平。The above experimental results show that when the signal peptides in the embodiments of the present invention guide the secretion and expression of GLP-1 fusion protein, in addition to obtaining a higher protein expression, the purity and biological activity of the GLP-1 fusion protein also reach higher level.
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity, all possible combinations of the technical features in the above-described embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be regarded as the scope described in this specification.
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are more specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those skilled in the art, without departing from the concept of the present invention, several modifications and improvements can be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the appended claims.

Claims (10)

  1. 信号肽在表达GLP-1或GLP-1融合蛋白中的应用，其特征在于，所述信号肽的氨基酸序列如SEQ ID NO:1所示。The application of 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.
  2. 根据权利要求1所述的应用，其特征在于，所述信号肽含有选自以下任意一种的氨基酸替换：The application according to claim 1, wherein the signal peptide contains an amino acid substitution selected from any of the following:
    第14位氨基酸替换为T；The 14th amino acid is replaced with T;
    第19位氨基酸替换为S；Amino acid at position 19 is replaced with S;
    第9位氨基酸替换为FW，第17位氨基酸替换为A。Amino acid at position 9 was replaced by FW and amino acid at position 17 was replaced by A.
  3. 根据权利要求1或2所述的应用，其特征在于，所述GLP-1融合蛋白的融合标签为人免疫球蛋白Fc或白蛋白。The use according to claim 1 or 2, wherein the fusion tag of the GLP-1 fusion protein is human immunoglobulin Fc or albumin.
  4. 一种多核苷酸，其特征在于，所述多核苷酸包括：A kind of polynucleotide, it is characterised in that the polynucleotide comprises:
    编码信号肽的多核苷酸，和a polynucleotide encoding a signal peptide, and
    编码GLP-1或GLP-1融合蛋白的多核苷酸；A polynucleotide encoding GLP-1 or a GLP-1 fusion protein;
    所述信号肽的氨基酸序列如SEQ ID NO:1所示。The amino acid sequence of the signal peptide is shown in SEQ ID NO: 1.
  5. 根据权利要求4所述的多核苷酸，其特征在于，所述信号肽含有选自以下任意一种氨基酸替换：The polynucleotide of claim 4, wherein the signal peptide contains any amino acid substitution selected from the group consisting of:
    第14位氨基酸替换为T；The 14th amino acid is replaced with T;
    第19位氨基酸替换为S；Amino acid at position 19 is replaced with S;
    第9位氨基酸替换为FW，第17位氨基酸替换为A。Amino acid at position 9 was replaced by FW and amino acid at position 17 was replaced by A.
  6. 根据权利要求4或5所述的多核苷酸，其特征在于，所述GLP-1融合蛋白的融合标签为人免疫球蛋白Fc或白蛋白。The polynucleotide according to claim 4 or 5, wherein the fusion tag of the GLP-1 fusion protein is human immunoglobulin Fc or albumin.
  7. 一种表达载体，其特征在于，包含权利要求4-6任一项所述的多核苷酸。An expression vector, characterized in that it comprises the polynucleotide of any one of claims 4-6.
  8. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求7所述的表达载体。A host cell, characterized in that the host cell contains the expression vector of claim 7 .
  9. 根据权利要求8所述的宿主细胞，其特征在于，所述宿主细胞为哺乳动物细胞。The host cell according to claim 8, wherein the host cell is a mammalian cell.
  10. 一种GLP-1或GLP-1融合蛋白的生产方法，其特征在于，使用权利要求8或9所述的宿主细胞表达GLP-1或GLP-1融合蛋白。A method for producing GLP-1 or GLP-1 fusion protein, characterized in that the host cell according to claim 8 or 9 is used to express GLP-1 or GLP-1 fusion protein.
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