Phase separation polypeptide and GLP-1 RAs derivative containing same

Abstract

A segment of short-sequence polypeptide which can be separated in vitro is obtained through screening, and the short-sequence polypeptide is applied to polypeptide drugs such as analogues or derivatives of GLP-1, so that the stability of the polypeptide drugs can be improved, the action time of the polypeptide drugs can be prolonged, and the biological activity of the polypeptide drugs can be improved.

Description

Technical Field

[0001] The present invention relates to the field of polypeptide drugs, and in particular to a phase-separated polypeptide and a GLP-1RAs derivative comprising the polypeptide, as well as a preparation method and application of the GLP-1RAs derivative. Background Art

[0002] With the development of social science and economy, people's living standards have improved, and they are more likely to be exposed to high-sugar, high-salt and high-fat foods in their daily diet. Because such foods are easy for the human body to rely on, the number of diabetes patients has increased significantly. As of 2022, the number of diabetes patients in the world has reached 550 million. Diabetes has become a chronic disease that seriously threatens human life and health, alongside tumors and vascular diseases.

[0003] Diabetes is divided into type 1 and type 2, of which type 2 diabetes accounts for about 90% of the total number of people with diabetes. For type 2 diabetes, GLP-1RA (glucagon-like peptide 1 receptor agonist) is one of the main combination drugs besides metformin[1]. From the launch of the first GLP-1RA drug in 2005 to 2022, GLP-1RA drugs have accounted for 12% of the global hypoglycemic drugs in 17 years. However, the types of GLP-1RA drugs are limited, so the research and development of new GLP-1RA drugs is of great significance and has broad prospects.

[0004] GLP-1 can promote glucose-stimulated insulin secretion and maintain blood glucose homeostasis by reducing hepatic glucagon release, slowing gastric emptying and suppressing appetite [2]. GLP-1 is easily degraded by dipeptidyl peptidase (DPP4) 2-3 minutes after secretion in the human body, which greatly limits its application. Therefore, GLP-1 derivatives are developed through strategies such as amino acid replacement, fatty acid or polyethylene glycol modification, and protein fusion co-stabilization [4], which enhances the stability and half-life of GLP-1. GLP-1RA is an analog of GLP-1 and belongs to the class of incretin mimetics. It can enhance the hypoglycemic effect by enhancing the incretin effect [3].

[0005] CN104017062A discloses a new long-acting GLP-1 compound modified with non-proteinogenic amino acid residues; CN114958883A discloses a long-acting GLP-1 derivative modified by fatty acid acylation; CN109021093A provides a polyethylene glycol-modified GLP-1 derivative, which has excellent hypoglycemic effect and a long half-life in vivo; US7141547B2 and US7452966B2 disclose albiglutide, a GLP-1 containing albumin, in which the active main chain of GLP-1 is covalently fused to a large and stable carrier protein to achieve a prolonged action time.

[0006] The formation of biomolecular condensates to control spatial and temporal reactions is a widely used compartmentalization mechanism in cells. This process is called liquid-liquid phase separation (LLPS), which is related to various biological functions, such as the formation of membraneless organelles in cells, RNA metabolism, DNA damage signaling complexes, cytoskeleton and many other supramolecular assemblies. These discoveries and discussions triggered by LLPS have greatly enriched our understanding of cell biology [5]. Recently, phase separation has been shown to provide local high concentrations and selectively recruit components, while peptides with phase separation ability can encapsulate other molecules into the phase separation system to exert their effects [6]. However, natural phase separation peptides have the disadvantages of long sequences and difficulty in driving phase separation of exogenous amino acid sequences, which limits their application.

[0007] References

[0008] [1] Chinese Diabetes Society. Guidelines for the prevention and treatment of type 2 diabetes in China (2020 edition)[J]. Chinese Journal of Diabetes, 2021, 13(4):95.

[0009] [2]RODA VR, AHMANN A, CARIOU B, et al. Comparative efficacy, safety, and cardiovascular outcomes with once-weekly subcutaneous semaglutide in the treatment of type 2diabetes: Insights from the SUSTAIN 1-7trials[J].DiabetesMetab, 2019, 45(5):409-418.

[0010] [3] Zhang Yangyang, Li Lingling, Bai Jia, et al. Research progress on the non-hyperglycemic therapeutic effects of glucagon-like peptide 1 receptor agonists[J]. Shandong Medicine, 2022, 62(32): 84-88.

[0011] [4] Sang Yanxia. Strategies for long-acting glucagon-like peptide 1 analogs[J]. Chinese Journal of New Drugs and Clinical Medicine, 2021, 40(7): 481-488.

[0012] [5]Schneider N, Wieland FG, Kong D, et al. Liquid-liquid phaseseparation of light-inducible transcription factors increases transcription activation in mammalian cells and mice[J]. Science Advances, 2021, 7(1):eabd3568.

[0013] [6]SUN,YUE,LAU,SZE YI,LIM,ZHI WEI,et al.Phase-separating peptides fordirect cytosolic delivery and redox-activated release of macromoleculartherapeutics[J].2022,14(3):274-283. Summary of the invention

[0014] The present invention aims to screen out a short sequence polypeptide that can phase separate in vitro, and to realize engineering application of this phase separation, and to apply the phase separation polypeptide to polypeptide drugs and increase their activity.

[0015] The first aspect of the present invention is to provide a polypeptide having phase separation properties, which has 28-35 amino acids, and its amino acid sequence is X1-SGYSRGGSGYSRGGSGYSRGG-X2, wherein X1 is selected from: SGYSRGGSGYSRGG, SGYSRGG, GYSRGG, YSRGG, SRGG, RGG or RGGSGYSRGG, or, X1 is not present; X2 is selected from: SGYSRGGSGYSRGG, SGYSRGGSGYSRG, SGYSRGGSGYSR, SGYSRGGSGYS, SGYSRGGSGY, SGYSRGGSG, SGYSRGGS, SGYSRGG, SGYSRG, SGYSR or SGYS, or, X2 is not present; provided that X1 and X2 cannot be absent at the same time. That is, the polypeptide can be expressed as: X1-SGYSRGGSGYSRGGSGYSRGG, SGYSRGGSGYSRGGSGYSRGG-X2, and, , SRGG, RGG or RGGSGYSRGG;

[0016] In some embodiments of the present invention, in the polypeptide, X1 is preferably absent, and X2 is selected from the group consisting of: SGYSRGGSGYSRGG, SGYSRGGSGYSRG, SGYSRGGSGYSR, SGYSRGGSGYS, SGYSRGGSGY, SGYSRGGSG, SGYSRGGS, SGYSRGG. That is, the polypeptide is SGYSRGGSGYSRGGSGYSRGG-X2, and X2 is selected from the group consisting of: SGYSRGGSGYSRGG, SGYSRGGSGYSRG, SGYSRGGSGYSR, SGYSRGGSGYS, SGYSRGGSGY, SGYSRGGSG, SGYSRGGS, SGYSRGG.

[0017] In some embodiments of the present invention, in the polypeptide, X1 is RGG; X2 is selected from the group consisting of SGYSRGGSGYS, SGYSRGGSGY, SGYSRGGSG, SGYSRGGS, SGYSRGG, SGYSRG, SGYSR, SGYS. That is, the polypeptide is X1-SGYSRGGSGYSRGGSGYSRGG-X2, wherein X1 is RGG; X2 is selected from the group consisting of SGYSRGGSGYS, SGYSRGGSGY, SGYSRGGSG, SGYSRGGS, SGYSRGG, SGYSRG, SGYSR, SGYS.

[0018] In some embodiments of the present invention, in the polypeptide, X1 is preferably selected from the group consisting of GYSRGG, YSRGG, SRGG, and RGG; and X2 is SGYSRGG. That is, the polypeptide is X1-SGYSRGGSGYSRGGSGYSRGG-X2, wherein X1 is selected from the group consisting of GYSRGG, YSRGG, SRGG, and RGG; and X2 is SGYSRGG.

[0019] In some embodiments of the invention, the polypeptide is selected from: SGYSRGGSGYSRGGSGYSRGGSGYSRGGSGYSRGG; SGYSRGGSGYSRGGSGYSRGGSGYSRGGSGYSRG; GYSRGGSGYSRGGSGYSRGGSGYSRGGSGYSRG; YSRGGSGYSRGGSGYSRGGSGYSRGGSGYSRG ; SRGGSGYSRGGSGYSRGGSGYSRGGSGYSRG ; RGGSGYSRGGSGYSRGGSGYSRGGSGYSRG ; SGYSRGGSGYSRGGSGYSRGGSGYSRGGSGYSR ; GYSRGGSGYSRGGSGYSRGGSGYSRGGSGYSR ; YSRGGSGYSRGGSGYSRGGSGYSRGGSGYSR ;SRGGSGYSRGGSGYSRGGSGYSRGGSGYSR; RGGSGYSRGGSGYSRGGSGYSRGGSGYSR; SGYSRGGSGYSRGGSGYSRGGSGYSRGGSGYS; GYSRGGSGYSRGGSGYSRGGSGYSRGGSGYS; ;SRGGSGYSRGGSGYSRGGSGYSRGGSGYS; RGGSGYSRGGSGYSRGGSGYSRGGSGYS.

[0020] In some embodiments of the invention, the polypeptide is selected from: RGGSGYSRGGSGYSRGGSGYSRGGSGYSRGGSGYS; RGGSGYSRGGSGYSRGGSGYSRGGSGYSRGGSGY; GGSGYSRGGSGYSRGGSGYSRGGSGYSRGGSGY; GSGYSRGGSGYSRGGSGYSRGGSGYSRGGSGY ;SGYSRGGSGYSRGGSGYSRGGSGYSRGGSGY ;RGGSGYSRGGSGYSRGGSGYSRGGSGYSRGGSG ;GGSGYSRGGSGYSRGGSGYSRGGSGYSRGGSG ;GSYSRGGSGYSRGGSGYSRGGSGYSRGGSG ;SGYSRGGSGYSRGGSGYSRGGSGYSRGGSG ;RGGSGYSRGGSGYSRGGSGYSRGGSGYSRGGS;GGSGYSRGGSGYSRGGSGYSRGGSGYSRGGS;GSGYSRGGSGYSRGGSGYSRGGSGYSRGGS;SGYSRGGSGYSRGGSGYSRGGSGYSRGGS.

[0021] In one embodiment of the present invention, the amino acid sequence of the polypeptide is: SGYSRGGSGYSRGGSGYSRGGSGYSRGGSGYSRGG, which is represented by SEQ ID No. 1 (abbreviated as "P5") in the present invention.

[0022] In one embodiment of the present invention, the amino acid sequence of the polypeptide is: RGGSGYSRGGSGYSRGGSGYSRGGSGYSRGGSGYS.

[0023] The polypeptide of the present invention can be synthesized by methods known to those skilled in the art, such as solid phase synthesis, and purified by methods known to those skilled in the art, such as high performance liquid chromatography.

[0024] The second aspect of the present invention is to provide use of the phase separation polypeptide described in the first aspect of the present invention in preparing a fusion protein with phase separation properties.

[0025] The inventors of the present application have found that the polypeptide described in the first aspect of the present invention has phase separation properties when used alone, and the new fusion protein formed by further fusion with a heterologous protein can also have phase separation properties, especially when the number of amino acids in the heterologous protein is 25-40. The phase separation property is conducive to improving the stability of the protein, enriching it in the droplets formed by phase separation to increase the active concentration, thereby facilitating the improvement of the activity of the protein.

[0026] The third aspect of the present invention is to provide a fusion protein, which contains the phase separation polypeptide described in the first aspect of the present invention and a heterologous protein. The heterologous protein is any protein with biological activity. Preferably, the number of amino acids in the heterologous protein is 25 to 40.

[0027] Preferably, the fusion protein further comprises a connecting arm between the phase separation polypeptide and the heterologous protein. The connecting arm may use a flexible connecting sequence commonly used in the art, including but not limited to a GS connecting sequence, a DDK connecting sequence, or a combination of these connecting sequences. In one embodiment of the present invention, the amino acid sequence of the connecting arm is shown in SEQ ID No. 4.

[0028] Preferably, the heterologous protein is a GLP-1RAs derivative, which has GLP-1 receptor agonist activity, can promote insulin secretion, and lower blood sugar. The GLP-1RAs is GLP-1 (7-37) or its analogs, or derivatives of GLP-1 (7-37), or derivatives of GLP-1 (7-37) analogs, or exendin-4 (1-39) or its analogs, or derivatives of exendin-4 (1-39), or derivatives of exendin-4 (1-39) analogs. The analogs and derivatives can be described in patents such as CN1232470A, US5424286A, US2008 / 0200390A1, WO2006 / 097537A, US7141547B2, and US7452966B2.

[0029] For the convenience of description, herein, the "polypeptide" and "peptide" of the GLP-1RAs derivatives refer to a compound composed of at least 5 amino acids connected by peptide bonds. The constituent amino acids can be natural amino acids as well as synthetic amino acids. The "analog" of a polypeptide refers to a modified peptide, wherein one or more amino acid residues of the peptide have been substituted by other amino acid residues, and / or one or more amino acid residues have been deleted from the peptide, and / or one or more amino acid residues have been added to the peptide. The "derivative" of a polypeptide refers to a chemically modified peptide or an analog thereof, wherein at least one substituent is not present in the unmodified peptide or its analog, i.e., a peptide that has been covalently modified. Typical modifications are amides, sugars, alkyls, acyl groups, esters, and the like. A simple system is commonly used in the art to describe fragments and analogs of GLP-1RAs derivative peptides, for example, GLP-1 (1-37) refers to a GLP-1 polypeptide having all amino acids from the 1st (N-terminus) to the 37th (C-terminus); GLP-1 (7-37) refers to a GLP-1 polypeptide having all amino acids from the 7th (N-terminus) to the 37th (C-terminus), which has the amino acid sequence shown in SEQ ID No. 2; Gly8 -GLP-1(7-37) refers to a GLP-1 fragment formed by deleting amino acid residues 1 to 6 from GLP-1 and replacing the naturally occurring amino acid residue at position 8 (Ala) with Gly. 34 (Nε-tetradecanoyl)-GLP-1(7-37) represents GLP-1(7-37) in which the ε-amino group of the Lys residue at position 34 has been tetradecanoylated. 35 Exendin-4(1-39) represents exendin-4(1-39) lacking the Ala residue at position 35.

[0030] In some embodiments of the present invention, the analog of GLP-1 (7-37) is a GLP-1 (7-37) analog wherein the 8th amino acid of GLP-1 (7-37) is Ala, Gly, Val, Leu, Ile, Lys, Aib (α-aminoisobutyric acid), (1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl) carboxylic acid or (1-aminocyclooctyl) carboxylic acid; the 22nd amino acid is Gly, Glu or Aib; the 26th amino acid is Lys or Arg; the 34th amino acid is Lys, Glu, Asn or Arg; and the 36th amino acid is Arg, Gly or Lys. In one embodiment of the present invention, the analog of GLP-1 (7-37) is Gly 8 ,Glu 22 ,Gly 36 GLP-1(7-37), the amino acid sequence is shown in SEQ ID No.3.

[0031] An example of a derivative of GLP-1(7-37) is Lys 34 (Nε-tetradecanoyl)-GLP-1(7-37) and the like. An example of a derivative of a GLP-1(7-37) analog is Nε26-((4S)-4-(hexadecanoylamino)-carboxy-butyryl)[Arg 34 ,Lys 26 ]GLP-1-(7-37), etc.

[0032] The amino acid sequence of exendin-4(1-39) is shown in SEQ ID No. 7. Exendin-4(1-39) analogs such as exendin-4(1-31), Y 31 Exendin-4(1-31), desAla 35 exendin-4(1-39), des-Pro 36 exendin-4(1-39), des-Pro36 ,Pro 37 exendin-4(1-39), des-Pro 36 ,Pro 37 ,Pro 38 exendin-4(1-39), etc.

[0033] In some embodiments of the present invention, the fusion protein is from N-terminus to C-terminus: GLP-1RAs derivative polypeptide-connector-phase separation polypeptide, and the phase separation polypeptide is the polypeptide with phase separation properties described in the first aspect of the present invention. In a specific embodiment, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1. In a specific embodiment, the amino acid sequence of the connecting arm is shown in SEQ ID NO: 4. In a specific embodiment, the GLP-1RAs derivative polypeptide is selected from: GLP-1 (7-37), exendin-4 (1-39), exendin-4 (1-31), Y 31 exendin-4 (1-31), GLP-1 (7-37) analogs wherein the 8th amino acid is Ala, Gly, Val, Leu, Ile, Lys, Aib (α-aminoisobutyric acid), (1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl) carboxylic acid or (1-aminocyclooctyl) carboxylic acid; the 22nd amino acid is Gly, Glu or Aib; the 26th amino acid is Lys or Arg; the 34th amino acid is Lys, Glu, Asn or Arg; the 36th amino acid is Arg, Gly or Lys. In one embodiment, the GLP-1RAs derivative polypeptide is Gly 8 ,Glu 22 ,Gly 36 GLP-1(7-37), the amino acid sequence is shown in SEQ ID No.3.

[0034] In one embodiment of the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID No. 5, hereinafter also referred to as "GLP-1-P5".

[0035] The fusion protein of the present invention containing the GLP-1RAs derivative polypeptide can overcome the shortcomings of natural GLP-1RAs such as GLP-1-(7-37) and exendin-4(1-39) in terms of short half-life and short duration of action, and improve their stability and biological activity.

[0036] A fourth aspect of the present invention is to provide:

[0037] Use of the fusion protein of the third aspect of the present invention in preparing a drug for preventing or treating a disease;

[0038] And, a pharmaceutical composition comprising the fusion protein of the third aspect of the present invention and a pharmaceutically acceptable carrier;

[0039] And, a method for preventing or treating a disease, comprising administering the fusion protein described in the third aspect of the present invention or a pharmaceutical composition comprising the fusion protein to a subject in need.

[0040] The pharmaceutical composition is in a form suitable for oral, parenteral (including subcutaneous (sc), intravenous (iv), intramuscular (im), epidural, direct intracerebral and intraperitoneal (ip)), rectal, intratracheal, intranasal, dermal, intravaginal, buccal, ocular or pulmonary administration, preferably in a form suitable for subcutaneous or oral administration. The pharmaceutical composition can be in conventional forms, such as capsules, tablets, sprays, topical forms, liquid or semi-liquid forms such as solutions, suspensions, dispersions, emulsions, micelles or liposomes. Liquid compositions are preferably suitable for subcutaneous administration. These pharmaceutical compositions can be prepared by methods well known to those skilled in the art.

[0041] In one embodiment of the present invention, the pharmaceutical composition of the present invention is administered subcutaneously. In another embodiment of the present invention, the pharmaceutical composition of the present invention is administered orally, in which case, the preferred administration form is a tablet or capsule.

[0042] The pharmaceutically acceptable carrier can be a conventional solid or liquid carrier. Examples of solid carriers include but are not limited to lactose, sucrose, cyclodextrin, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid or cellulose lower alkyl ethers, etc. Examples of liquid carriers include but are not limited to syrup, peanut oil, olive oil, phospholipids, sterols, fatty acids, fatty acid amines, polyoxyethylene, isotonic buffer and water, etc.

[0043] When the heterologous protein in the fusion protein is a GLP-1RAs derivative, the diseases prevented or treated by the drug include but are not limited to type I and type II diabetes, impaired glucose tolerance, insulin resistance, diabetic complications (nephropathy, retinopathy, neuropathy, cataracts), hyperglycemia, hypercholesterolemia, dyslipidemia, hyperlipidemia, obesity, hypertension, syndrome X, atherosclerosis, etc.

[0044] In one embodiment of the invention, the medicament is for use in delaying or preventing the development of type II diabetes.

[0045] In one embodiment of the invention, the medicament is for reducing food intake, reducing β-cell apoptosis, increasing β-cell function and β-cell mass and / or restoring glucose sensitivity of β-cells.

[0046] Treatment using a fusion protein comprising a GLP-1RAs derivative or a pharmaceutical composition thereof can also be combined with another one or more pharmacologically active substances, such as those selected from antidiabetic drugs, anti-obesity drugs, appetite regulators, antihypertensive drugs, preparations for treating and / or preventing complications caused by or associated with diabetes, and preparations for treating and / or preventing complications and disorders caused by or associated with obesity.Examples of such pharmacologically active substances are: insulin, sulfonylureas, biguanides, meglitinides, glucosidase inhibitors, glucagon antagonists, DPP-IV (dipeptidyl peptidase-IV) inhibitors, inhibitors of liver enzymes involved in the stimulation of gluconeogenesis and / or glycogenolysis, glucose uptake regulators, compounds regulating lipid metabolism such as antihyperlipidemic agents such as HMG CoA inhibitors (statins), gastric inhibitory polypeptide (GIP analogs), compounds that reduce food intake, RXR agonists, and agents that act on ATP-dependent potassium channels of beta cells; cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol, dextrothyroxine, neteglinide, repaglinide; beta blockers such as allelol, atenolol, timolol, indolol, naproxenol, and metoprolol; ACE (angiotensin-converting enzyme) inhibitors such as benazepril, captopril, enalapril, fosenopril, lisinopril, alatriopril il, quinapril and ramipril, calcium channel blockers such as nifedipine, diclofenac, nifedipine, isradipine, nimodipine, diltiazem and verapamil, and alpha blockers such as doxazosin, mefenamic acid, prazosin and terazosin; CART (cocaine amphetamine regulated transcript) agonists, NPY (neuropeptide Y) antagonists, PYY agonists, PYY2 agonists, PYY4 agonists, mixed PPY2 / PYY4 agonists, MC4 (melanocortin 4) agonists, orexin antagonists, TNF (tumor necrosis factor) agonists, CRF (corticotropin releasing factor) agonists, CRF BP (corticotropin releasing factor binding protein) antagonists, urocortin agonists, β3 agonists, MSH (melanocyte-stimulating hormone) agonists, MCH (melanocyte contracting hormone) antagonists, CCK (cholecystokinin) agonists, serotonin reuptake inhibitors, serotonin and norepinephrine reuptake inhibitors, mixed serotonin and norepinephrine compounds, 5HT (serotonin) agonists, bombesin agonists, ganglioside peptide antagonists, growth hormone, growth hormone releasing compounds, TRH (thyrotropin-releasing hormone) agonists, UCP 2 or 3 (uncoupling protein 2 or 3) modulators, leptin agonists, DA agonists (bromocriptine, doprexin), lipase / amylase inhibitors, RXR (retinoid X receptor) modulators, TRβ agonists; histamine H3 antagonists, gastric inhibitory polypeptide agonists or antagonists (GIP analogs), gastrin and gastrin analogs.

[0047] The present invention also provides a nucleic acid encoding the fusion protein described in the third aspect of the present invention.

[0048] The present invention also provides an expression vector comprising the nucleic acid described above. In some embodiments, the expression vector is a plasmid vector. In some embodiments, the plasmid vector includes but is not limited to pGEX-4T-1, pGEX-6P-1, pET-GST, pSmart-I, pGEX-1λT, pGEX-2T, pGEX-2TK, pET-28a(+), etc.

[0049] The present invention also provides a host cell, the host cell comprising the expression vector described above. In some embodiments, the host cell is Escherichia coli, such as Escherichia coli shuffle T7, Escherichia coli BL21 (DE3), Escherichia coli Rosetta (DE3), Escherichia coli Mach1-T1 and / or Escherichia coli DH5α.

[0050] The present invention also relates to a method for preparing the fusion protein described in the third aspect using recombinant DNA technology, the method comprising the following steps: (a) introducing a nucleic acid sequence encoding the fusion protein into a host cell, (b) cultivating the host cell, and (c) isolating the fusion protein from the culture.

[0051] In the present invention, in order to improve the expression and purification efficiency of the fusion protein, an appropriate expression vector is used to express a fusion protein for expression containing the fusion protein, and its amino acid sequence is shown in SEQ ID No. 6. The fusion protein for expression is a GST tag and an enterokinase cleavage sequence fused to the N-terminus of the fusion protein. The amino acid sequence of the GST tag is MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGS. The enterokinase cleavage sequence is DDDDK.

[0052] The nucleic acid sequence encoding the fusion protein shown in SEQ ID No.5 is introduced into a host cell, the host cell is cultured, the expression fusion protein shown in SEQ ID No.6 is isolated from the culture, the expression fusion protein is cleaved with enterokinase, and then purified to obtain the fusion protein described in the third aspect of the present invention.

[0053] In some embodiments, the host cell is E. coli, such as E. coli shuffle T7, E. coli BL21 (DE3), E. coli Rosetta (DE3), E. coli Mach1-T1 and / or E. coli DH5α.

[0054] In some embodiments, the bacterial cells are suspended in a lysis / washing buffer, the bacterial cells are disrupted and then centrifuged to collect the fusion protein for expression, and the lysis / washing buffer contains 140-160 mM NaCl, 45-55 mM Tris-HCl, pH 7.6-8.4.

[0055] In some embodiments, the components of the digestion buffer for enterokinase digestion are 95-105 mM NaCl, 45-55 mM Tris-HCl, 1.5-2.5 mM DTT, pH 7.6-8.4.

[0056] In some embodiments, GST affinity chromatography is used for purification.

[0057] In one embodiment, the recombinant Escherichia coli expressing the expression fusion protein shown in SEQ ID No.6 is cultured at 35°C to 40°C for 10h to 12h to obtain a seed solution, and then inoculated into a culture medium at an inoculum amount of 0.1% to 2% (v / v) and cultured until the OD600 value of the fermentation liquid reaches 0.6 to 0.8, and IPTG with a final concentration of 0.05mM to 1mM is added for induction. After induction at 25°C to 40°C for 12h to 16h, the fermentation is terminated, the fermented bacteria are collected and crushed, the supernatant is purified by GST affinity chromatography after centrifugation, and after washing, enterokinase is added to the chromatography column and digested at 4°C to 25°C for 12h-16h, and then collected to finally obtain the fusion protein shown in SEQ ID No.5.

[0058] Beneficial effects of the present invention:

[0059] The present invention provides a short phase separation polypeptide, which has the advantages of short sequence, good phase separation effect and ability to drive phase separation of heterologous proteins relative to natural phase separation sequences; the present invention prolongs the half-life of heterologous proteins such as GLP-1-RAs derivatives relative to natural GLP-1 by fusing the above phase separation polypeptide, and greatly increases the action time; and by fusing the above phase separation polypeptide, heterologous proteins such as GLP-1-RAs derivatives increase the GLP-1 receptor activation activity through phase separation, have a higher potency than natural GLP-1, and increase drug activity.

[0060] The amino acid sequence of the present invention is as follows:

[0061] SEQ ID No.1 (abbreviated as "P5"): SGYSRGGSGYSRGGSGYSRGGSGYSRGGSGYSRGG

[0062] SEQ ID No.2 (GLP-1(7-37)): HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

[0063] SEQ ID No.3 (Gly 8 , Glu 22 , Gly 36 GLP-1(7-37)): HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG

[0064] SEQ ID No.4 (linker): GGGGSGGGGSGGGGS

[0065] SEQ ID No.5 (abbreviated as "GLP-1-P5"): HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGG GSGGGGSGGGGSSGYSRGGSGYSRGGSGYSRGGSGYSRGGSGYSRGG

[0066] SEQ ID No.6 (fusion protein with GST tag and enterokinase cleavage site): MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSDDDDKHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSSGYSRGGSGYSRGGSGYSRGGSGYSRGGSGYSRGG

[0067] SEQ ID No.7 (exendin-4(1-39)): HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS。

[0068] Definition:

[0069] “And / or” will be considered as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” used in phrases such as “A and / or B” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the term “and / or” used in phrases such as “A, B, and / or C” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0070] "Including" and "comprising" have the same meaning, are intended to be open ended and allow but not require the inclusion of additional elements or steps. When the terms "including" or "comprising" are used herein, the terms "consisting of" and / or "consisting essentially of" are also included and disclosed. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 :P5 peptide phase separation typical diagram

[0072] Figure 2 :Construction diagram of recombinant plasmid

[0073] Figure 3 :SDS-PAGE image of purified GLP-1-P5

[0074] Figure 4 :Serum stability of GLP-1-P5

[0075] Figure 5 :cAMP concentration response curves of GLP-1(7-37) and GLP-1-P5

[0076] Figure 6 :Photo of phase separation results of P3 peptide

[0077] Figure 7 :Photo of the phase separation results of GLP-1-P5

[0078] Figure 8 :Statistical chart of the results of in vivo hypoglycemic experiments of GLP-1-P5 and dulaglutide DETAILED DESCRIPTION

[0079] The present invention is further described below in conjunction with the examples. It should be noted that the examples cannot be used as a limitation on the protection scope of the present invention, and those skilled in the art understand that any improvements and changes made on the basis of the present invention are within the protection scope of the present invention.

[0080] The conventional reagents used in the following examples are all commercially available, and the biological experiments performed are all conventional biological experiments in the art and can be performed according to the instructions in the corresponding experimental manual or kit instructions.

[0081] Example 1: Design of phase separation peptide P5 and verification of phase separation properties

[0082] The P5 polypeptide shown in SEQ ID No. 1 was prepared by solid phase synthesis by Kesheng Jingpeptide Company, and its phase separation ability was observed by confocal microscopy in KMEI buffer, the composition of which was 150 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10 mM imidazole, pH 6.5. Figure 1 As shown, in the 5 mg / mL P5 system in KMEI buffer containing 5% PEG, P5 and the solvent undergo phase separation to form two phases.

[0083] However, the polypeptide containing three sets of SGYSRGG repeat sequences (abbreviated as "P3") could not undergo phase separation. Figure 6 As shown, in the KMEI buffer containing 15% PEG and pH = 6.5, even if P3 is incubated at a high PEG concentration (2.5 mM) for one day, the system is still a homogeneous solution.

[0084] According to the experimental research results, the inventors of the present application found that a polypeptide containing at least three groups of SGYSRGG repeating sequences is required to produce phase separation, but a polypeptide longer than five groups of SGYSRGG repeating sequences aggregates instead of phase separation.

[0085] Example 2: Construction of recombinant engineering bacteria

[0086] Considering the N-terminal functional region of GLP-1(7-37), the phase separation peptide P5 sequence is connected to Gly 8 ,Glu 22 ,Gly 36 The C-terminus of GLP-1(7-37) was tagged with three sets of repeated glycine linker arms. Considering the solubility effect of the GST tag and the highly specific purification effect, the pGEX-4T-1 expression vector containing an N-terminal GST tag was selected, and the GST tag and Gly 8 ,Glu 22 ,Gly 36 An enterokinase cleavage site (DDDDK) was introduced between GLP-1 (7-37) to allow for enzymatic cleavage after protein expression and purification, thereby obtaining the target fusion protein shown in SEQ ID No. 5 (abbreviated as GLP-1-P5) with complete activity. The amino acid sequence of the fusion protein for expression is SEQ ID No. 6.

[0087] The recombinant plasmid was synthesized by a biological company. The plasmid map is shown in Figure 2 Take an appropriate amount of plasmid and transform it into expression competent Escherichia coli shuffle-T7 by heat shock method to construct recombinant engineering bacteria.

[0088] Example 3: Expression and purification of polypeptide fusion protein

[0089] The recombinant E. coli was inoculated into 5 mL LB medium and cultured at 37°C for 12 hours to obtain a seed solution, which was then inoculated into the LB medium at a 2% (v / v) inoculation amount and cultured until the OD600 value of the fermentation liquid reached 0.6-0.8, and IPTG with a final concentration of 0.5 mM was added for induction. The fermentation was terminated after induction at 20°C for 12 hours. The cells were collected, suspended in a lysis / washing buffer, crushed by a high-pressure homogenizer, and centrifuged to obtain a supernatant, wherein the lysis / washing buffer contained 150 mM NaCl, 50 mM Tris-HCl, pH 8.0. The supernatant after bacterial cell crushing and centrifugation is bound to the chromatography column according to 6mL / 1mL glutathione agarose gel filler, and rotated and mixed at 4°C for 30min; impurities are removed by washing with lysis / washing buffer; enterokinase is added to the enzyme cleavage buffer at a ratio of 1mg enterokinase / 100mg substrate, and the enzyme cleavage buffer composition is 100mM NaCl, 50mM Tris-HCl, 2mM DTT, pH 8.0. Diluted enterokinase is added to the chromatography column at a ratio of 1mL diluted enterokinase / 1mL glutathione agarose gel filler. After adding enterokinase, the enzyme is digested at 4°C for 16h and then collected. After centrifugation, the precipitate is dissolved in 0.05% TFA solution, and after centrifugation, the supernatant is lyophilized to obtain GLP-1-P5 (SEQ ID No.5). Figure 3 As shown, the purity of the purified target polypeptide GLP-1-P5 can reach 95%. From left to right, lanes 1-14 are marker, bacteria before induction, bacteria after induction, supernatant of broken bacteria, precipitate of broken bacteria, supernatant not bound to the chromatography column, washing solution, beads after washing, enzyme elution solution 1, beads after enzyme elution, enzyme elution solutions 2, 3, 4, and freeze-dried polypeptide.

[0090] Example 4: Phase separation behavior and stability test of polypeptide

[0091] The phase separation ability of GLP-1-P5 was observed by confocal microscopy in KMEI buffer. Buffer conditions: KMEI buffer containing 15% PEG, pH = 6.5. Figure 7 As shown, GLP-1-P5 has the ability to phase separate and gradually aggregates as the peptide concentration increases.

[0092] 200 μg of GLP-1 (7-37) or GLP-1-P5 were incubated with 300 μL of normal saline containing 10% human serum at 37°C. 20 μL of GLP-1 (7-37) was sampled at 0 min, 5 min, 10 min, 15 min, and 30 min, respectively; GLP-1-P5 was sampled at 0 h, 2.5 h, 5 h, 10 h, 24 h, and 48 h, respectively. HPLC analysis was used to separate and quantify peptide degradation products, where phase A was ultrapure water (containing 0.05% TFA) and phase B was 80% (v / v) acetonitrile-water solution (containing 0.05% TFA). All solutions were filtered with a 0.22 μm filter membrane and then ultrasonically degassed. The analysis flow rate was 1 mL / min; the detection wavelengths were 215 nm and 254 nm. Figure 4 As shown, GLP-1(7-37) decays to a lower concentration within half an hour, while the half-life of GLP-1-P5 is approximately 32.5h.

[0093] Example 5: Functional receptor cAMP response assay

[0094] RIN-m5F cells from the Cell Resource Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences were pre-plated in a white opaque round-bottom 96-well cell culture plate and placed in a 37°C, 5% carbon dioxide incubator for 2 days. The number of cells in the cell culture plate was 5000-8000 cells / well. The culture medium in the culture wells was aspirated, and 40 μL of the test sample was added to each well to stimulate the cells, with concentrations of 150 μM, 50 μM, 10 μM, 5 μM, 1 μM, 500 nM, 50 nM, 5 nM, 0.5 nM, and 0.1 nM, respectively. The concentration of the test sample mother solution was 250 μM, and it was diluted to the corresponding concentration with the complete induction buffer provided by the cAMP-GloTM Max Assay kit of Promega. The 96-well cell culture plate was then incubated at 37°C for 5 minutes, and the changes in intracellular cAMP levels after stimulation of the test sample were detected using the kit, and the relative chemiluminescence unit (RLU) was read using the chemiluminescence detection module of the microplate reader. Graphpad Prism 8 software was used to plot the cAMP concentration response curves of natural GLP-1 and the GLP-1 derivatives and calculate the IC50 values. Figure 5 The IC50 values ​​of GLP-1(7-37) and GLP-1-P5 were 37.4 nM and 0.8 nM, respectively.

[0095] Example 6: In vivo glucose-lowering activity of GLP-1-P5

[0096] 6-8 weeks old male SD rats were randomly divided into groups, 5 in each group. Each group was subcutaneously injected with 200μL normal saline, 1.25μM dulaglutide, and 0.5μM GLP-1-P5, and then fasted overnight. The fasting rat tail blood glucose was measured with a blood glucose meter and then a glucose tolerance test was performed. Oral infusion of glucose 2g / kg; rat tail blood glucose was measured 60min, 120min, and 180min after sugar infusion, and the blood glucose curve was drawn.

[0097] The results are as follows Figure 8 The in vivo glucose-lowering activity of the GLP-1-P5 of the present invention is comparable to that of dulaglutide.

[0098] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that It has 28-35 amino acids, and its amino acid sequence is X1-SGYSRGGSGYSRGGSGYSRGG-X2, wherein X1 is selected from: SGYSRGGSGYSRGG, SGYSRGG, GYSRGG, YSRGG, SRGG, RGG or RGGSGYSRGG, or, X1 does not exist; X2 is selected from: SGYSRGGSGYSRGG, SGYSRGGSGYSRG, SGYSRGGSGYSR, SGYSRGGSGYS, SGYSRGGSGY, SGYSRGGSG, SGYSRGGS, SGYSRGG, SGYSRG, SGYSR or SGYS, or, X2 does not exist; provided that X1 and X2 cannot be absent at the same time; Preferably, in the polypeptide, X1 is absent; X2 is selected from the group consisting of: SGYSRGGSGYSRGG, SGYSRGGSGYSRG, SGYSRGGSGYSR, SGYSRGGSGYS, SGYSRGGSGY, SGYSRGGSG, SGYSRGGS, SGYSRGG; Preferably, in the polypeptide, X1 is RGG; X2 is selected from: SGYSRGGSGYS, SGYSRGGSGY, SGYSRGGSG, SGYSRGGS, SGYSRGG, SGYSRG, SGYSR, SGYS; Preferably, in the polypeptide, X1 is selected from: GYSRGG, YSRGG, SRGG, RGG; and X2 is SGYSRGG.

2. The polypeptide according to claim 1, characterized in that Selected from: SGYSRGGSGYSRGGSGYSRGGSGYSRGGSGYSRGG; SGYSRGGSGYSRGGSGYSRGGSGYSRGGSGYSRG; GYSRGGSGYSRGGSGYSRGGSGYSRGGSGYSRG; YSRGGSGYSRGGSGYSRGGSGYSRGGSGYSRG; SRGGSGYSRGGSGYSRGGSGYSRGGSGYSRG; RGGSGYSRGGSGYSRGGSGYSRGGSGYSRG; SGYSRGGSGYSRGGSGYSRGGSGYSRGGSGYSR; GYSRGGSGYSRGGSGYSRGGSGYSRGGSGYSR; YSRGGSGYSRGGSGYSRGGSGYSRGGSGYSR; SRGGSGYSRGGSGYSRGGSGYSRGGSGYSR; RGGSGYSRGGSGYSRGGSGYSRGGSGYSR; SGYSRGGSGYSRGGSGYSRGGSGYSRGGSGYS; GYSRGGSGYSRGGSGYSRGGSGYSRGGSGYS; YSRGGSGYSRGGSGYSRGGSGYSRGGSGYS; SRGGSGYSRGGSGYSRGGSGYSRGGSGYS; RGGSGYSRGGSGYSRGGSGYSRGGSGYS; Preferably SGYSRGGSGYSRGGSGYSRGGSGYSRGGSGYSRGG.

3. The polypeptide according to claim 1, characterized in that Selected from: RGGSGYSRGGSGYSRGGSGYSRGGSGYSRGGSGYS; RGGSGYSRGGSGYSRGGSGYSRGGSGYSRGGSGY; GGSGYSRGGSGYSRGGSGYSRGGSGYSRGGSGY; GSGYSRGGSGYSRGGSGYSRGGSGYSRGGSGY; SGYSRGGSGYSRGGSGYSRGGSGYSRGGSGY; RGGSGYSRGGSGYSRGGSGYSRGGSGYSRGGSG; GGSGYSRGGSGYSRGGSGYSRGGSGYSRGGSG; GSGYSRGGSGYSRGGSGYSRGGSGYSRGGSG; SGYSRGGSGYSRGGSGYSRGGSGYSRGGSG; RGGSGYSRGGSGYSRGGSGYSRGGSGYSRGGS; GGSGYSRGGSGYSRGGSGYSRGGSGYSRGGS; GSGYSRGGSGYSRGGSGYSRGGSGYSRGGS; SGYSRGGSGYSRGGSGYSRGGSGYSRGGS. Preferred are: RGGSGYSRGGSGYSRGGSGYSRGGSGYSRGGSGYS.

4. A fusion protein, characterized in that Containing the polypeptide according to any one of claims 1 to 3 and a heterologous protein; Preferably, the number of amino acids in the heterologous protein is 25 to 40; Preferably, the fusion protein further comprises a linker sequence located between the polypeptide and the heterologous protein; Preferably, the fusion protein is heterologous protein-linker sequence-polypeptide from N-terminus to C-terminus; Preferably, the heterologous protein is a GLP-1RAs derivative selected from GLP-1(7-37) or its analogs, or derivatives of GLP-1(7-37), or derivatives of GLP-1(7-37) analogs, or exendin-4(1-39) or its analogs, or derivatives of exendin-4(1-39), or derivatives of exendin-4(1-39) analogs; Preferably, the GLP-1(7-37) analog is a GLP-1(7-37) analog wherein the 8th amino acid of GLP-1(7-37) is Ala, Gly, Val, Leu, Ile, Lys, Aib, (1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl)carboxylic acid, (1-aminocyclopentyl)carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic acid or (1-aminocyclooctyl)carboxylic acid; the 22nd amino acid is Gly, Glu or Aib; the 26th amino acid is Lys or Arg; the 34th amino acid is Lys, Glu, Asn or Arg; and the 36th amino acid is Arg, Gly or Lys; Preferably, the analog of GLP-1(7-37) is Gly 8 ,Glu 22 ,Gly 36 GLP-1 (7-37); Preferably, the linker sequence is GGGGSGGGGSGGGGS; Preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID No.

5.

5. A pharmaceutical composition, characterized in that Comprising the fusion protein of claim 4 and a pharmaceutically acceptable carrier.

6. Use of the fusion protein of claim 4 or the pharmaceutical composition of claim 5 in the preparation of a medicament for preventing or treating or delaying the progression or onset of the following diseases: type I and type II diabetes, impaired glucose tolerance, insulin resistance, diabetic complications, hyperglycemia, hypercholesterolemia, dyslipidemia, hyperlipidemia, obesity, hypertension, syndrome X, atherosclerosis; Preferably, the diabetic complications are nephropathy, retinopathy, neuropathy, and cataracts; Preferably, the medicament is used to delay or prevent the development of type II diabetes; Preferably, the medicament is for reducing food intake, reducing β-cell apoptosis, increasing β-cell function and β-cell mass and / or restoring glucose sensitivity of β-cells.

7. A nucleic acid, characterized in that The nucleic acid encodes the fusion protein of claim 4.

8. An expression vector, characterized in that The expression vector comprises the nucleic acid of claim 7; Preferably, the expression vector is a plasmid vector; Preferably, the plasmid vector is selected from pGEX-4T-1, pGEX-6P-1, pET-GST, pSmart-I, pGEX-1λT, pGEX-2T, pGEX-2TK, and pET-28a(+).

9. A host cell, characterized in that The host cell comprises the expression vector of claim 8; Preferably, the host cell is Escherichia coli; Preferably, the host cell is Escherichia coli shuffle T7, Escherichia coli BL21 (DE3), Escherichia coli Rosetta (DE3), Escherichia coli Mach1-T1 and / or Escherichia coli DH5α.

10. A method for preparing the fusion protein according to claim 4 using recombinant DNA technology, characterized in that: The method comprises the following steps: (a) introducing a nucleic acid sequence encoding the fusion protein into a host cell, (b) cultivating the host cell, and (c) isolating the fusion protein from the culture; Preferably, the nucleic acid sequence encoding the fusion protein shown in SEQ ID No.5 is introduced into a host cell, the host cell is cultured, the fusion protein for expression shown in SEQ ID No.6 is isolated from the culture, the fusion protein for expression is cleaved with enterokinase, and then purified to obtain the fusion protein shown in SEQ ID No.5; Preferably, the host cell is E. coli shuffle T7, E. coli BL21 (DE3), E. coli Rosetta (DE3), E. coli Mach1-T1 and / or E. coli DH5α; Preferably, the bacterial cells are suspended in a lysis / washing buffer, the bacterial cells are broken and then centrifuged to collect the fusion protein for expression, wherein the lysis / washing buffer comprises 140-160 mM NaCl, 45-55 mM Tris-HCl, pH 7.6-8.4; Preferably, the digestion buffer for enterokinase digestion comprises 95-105 mM NaCl, 45-55 mM Tris-HCl, 1.5-2.5 mM DTT, pH 7.6-8.4; Preferably, the recombinant Escherichia coli expressing the fusion protein for expression shown in SEQ ID No.6 is cultured at 35°C to 40°C for 10h to 12h to obtain a seed solution, and then inoculated into a culture medium at an inoculum amount of 0.1% to 2% (v / v) and cultured until the OD600 value of the fermentation liquid reaches 0.6 to 0.8, and IPTG with a final concentration of 0.05mM to 1mM is added for induction. After induction at 25°C to 40°C for 12h to 16h, the fermentation is terminated, the fermented bacteria are collected and crushed, the supernatant is purified by GST affinity chromatography after centrifugation, and after washing, enterokinase is added to the chromatography column and digested at 4°C to 25°C for 12h-16h, and then collected to finally obtain the fusion protein shown in SEQ ID No.5.

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