CN114075296A - Multifunctional variant protein and fusion protein thereof - Google Patents
Multifunctional variant protein and fusion protein thereof Download PDFInfo
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Abstract
The invention provides a multifunctional variant protein and a fusion protein thereof, which comprise Fibroblast growth factor 21(FGF21) protein or a mutant protein thereof and a Glucagon-like peptide-1 (GLP-1) receptor or a mutant protein thereof, wherein the pharmaceutical composition of the fusion protein can be used for treating diabetes, obesity, dyslipidemia, metabolic syndrome, nonalcoholic fatty liver disease, nonalcoholic fatty hepatitis and other related diseases.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a novel fibroblast growth factor 21(FGF21) mutant protein and a fusion protein of the novel fibroblast growth factor 21(FGF21) mutant protein and a Glucagon-like peptide-1 (GLP-1) receptor, and the novel fibroblast growth factor 21(FGF21) mutant protein can be used for treating diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, non-alcoholic fatty hepatitis and other related diseases.
Background
Fibroblast growth factor 21(FGF21) is a polypeptide consisting of 209 amino acids (SEQ ID NO:166) whose amino acid sequence has about 75% homology with mouse FGF 21. FGF21 contains a 28 amino acid signal peptide at the N-terminus, thus mature FGF21 consists of 181 amino acids (SEQ ID NO: 167). FGF21 is expressed primarily in the liver and pancreas, as well as in adipose and muscle tissue. FGF21 induces a variety of signaling pathways and functional activities in liver, pancreas, and adipose tissue, mediated by FCFR and aided by the transmembrane protein, beta klotho (klb), thereby testing the physiological functions of regulation of glycolipid metabolism and protection of islet beta cells. FGF21 regulates glucose uptake by adipocytes by activating non-insulin dependent glucose uptake. In addition, studies have shown that FGF21 can reduce body weight and systemic body fat dose-dependently. When FGF21 was administered to diabetic rhesus monkeys, significant reductions in fasting plasma glucose, triglyceride, and glucagon levels were found. Meanwhile, in FGF21 transgenic mice, the expression of white adipose tissue lipase is increased, and the levels of plasma beta-hydroxybutyric acid and free fatty acid are increased, which means that FGF21 can regulate lipid metabolism by promoting lipolysis and ketogenesis. FGF21 was able to inhibit glucose-mediated glucagon release, stimulate insulin production, and prevent islet cell apoptosis in islet cells and INS-1E cells, thereby improving pancreatic cell function. In addition, FGF21 also activates exocrine pancreatic cells and hepatocyte signaling pathways, inhibiting hepatic glycogen export.
FGF21 is a member of the FGF gene family, most FGFs have a spectrum of mitogenic properties, and research results indicate that FGF21 neither promotes cell proliferation nor antagonizes the function of other members of the FGF family. Experiments prove that in vivo abnormal conditions such as tumors and tissue hyperplasia are not found in FGF21 transgenic mice (the amount of FGF21 in vivo is about 150 times that of normal mice) in the whole life cycle. Meanwhile, the metabolic regulation effect of the compound is related to the metabolic level of the organism, the regulation effect only plays a role in abnormal metabolism, and hypoglycemia does not occur even if the dose of FGF21 exceeds the pharmacological dose. However, the main drugs (insulin, thiazolidinedione and the like) for treating diabetes on the market at present are easy to produce side effects when the dosage is not proper, such as hypoglycemia caused by large dose of insulin, liver function damage and edema caused by thiazolidinedione are not found in the animal experiment treated by FGF21, which is also enough to prove that FGF21 is an ideal drug for treating diabetes, obesity and other diseases.
The physiological functions of FGF21 in aspects of controlling blood sugar and reducing weight and the like bring hopes for treating related diseases, but wild FGF21 as a small molecular protein is easy to hydrolyze by protease and can be filtered by glomeruli, the half-life period is only 0.5-2h, and the effective drug action time is difficult to guarantee. In the face of the difficulty, the pharmaceutical industry improves the half-life of FGF21 by performing site-directed mutagenesis on the amino acids at the restriction sites to prepare long-acting fusion proteins or connecting polyethylene glycol to a polypeptide backbone. Furthermore, a significant challenge in developing FGF21 as a protein formulation also comes from instability due to its own aggregation. The desired effect of the therapeutic protein of interest is to increase the resistance to proteolysis and decrease the aggregation of the protein, thereby enhancing the half-life and stability of the FGF21 protein formulation, allowing for low frequency administration to the patient.
Some mutants based on the human wild-type FGF21 polypeptide sequence have been described in WO2009/149171 and WO 2017/074117.
Glucagon-like peptide-1 (GLP-1) is a gastrointestinal tract regulating polypeptide containing 30 or 31 amino acid residues. GLP-1 secretion is regulated primarily by L-cells on the small intestine, depending on nutrient absorption and fluctuating blood glucose levels in the body. After food intake, the L-cells of the small intestine secrete large amounts of GLP-1 to enhance the endocrine function of the pancreas. GLP-1 polypeptides fulfill their physiological functions of controlling blood glucose and reducing appetite in vivo, primarily by activating GLP-1 receptors distributed on the cell membrane surface. The mechanism of GLP-1 for controlling blood sugar level in vivo is mainly to activate GLP-1 receptor distributed in islet beta cells so as to promote biosynthesis and secretion of insulin, and GLP-1 polypeptide can inhibit secretion of glucagon under the condition of high blood sugar level in vivo, gastric emptying and food intake and enhance degradation of glucose in vivo through specific nervous system action. Notably, the physiological function of GLP-1 polypeptides to promote insulin secretion is highly controlled by plasma glucose concentrations, so GLP-1 polypeptides do not induce severe and persistent hypoglycemia compared to other diabetes treatment drugs. In addition, the literature reports that GLP-1 polypeptide and analogs thereof have direct promotion effects on the growth, differentiation and proliferation of beta cells of experimental animals, and the GLP-1 polypeptide and analogs thereof can protect pancreatic islets, delay the physiological function of diabetes development and inhibit the apoptosis of the beta cells. GLP-1 polypeptides also have potential gastrin-inhibiting and feeding-stimulating gastric acid secretion properties, which means that GLP-1 polypeptides also have physiological effects in preventing digestive tract ulcers. GLP-1 polypeptides can also activate GLP-1 receptors distributed in the central nervous system of the brain to enhance satiety, reduce food intake, and achieve the physiological effects of maintaining or reducing body weight. Therefore, the extensive action mechanism and physiological function of the GLP-1 polypeptide and the analogues thereof mean that the GLP-1 polypeptide is an ideal medicament for treating non-insulin-dependent diabetes and obese diabetes.
The physiological functions of the GLP-1 polypeptide in aspects of controlling blood sugar, reducing weight and the like bring hopes for treating non-insulin-dependent diabetes/obese diabetes, but the natural GLP-1 of a human body has poor drug property, is easily degraded by dipeptidyl peptidase-IV (DPP-IV) in the body, so that the half life of the GLP-1 polypeptide in the human body is only 1-2 minutes. In the face of this difficulty, the pharmaceutical industry has constructed long-acting GLP-1 analogs and their derivatives by site-directed mutagenesis of the amino acids at the cleavage site, modification of the fatty acids in the polypeptide backbone, and conjugation of GLP-1 polypeptides to various proteins/polymer polymers. Long-acting GLP-1 analogs that are currently marketed and widely used clinically include canagliptin administered subcutaneously twice a day, liraglutide administered subcutaneously once a day, and dolacitide and somaglutide administered subcutaneously once a week, and the like.
Clinically, the side effects of the GLP-1 polypeptide and the derivatives thereof are mainly manifested by nausea, vomiting and diarrhea caused by gastrointestinal tracts; in addition, it has been found that GLP-1 polypeptides and derivatives thereof also induce an acceleration of the heartbeat in a subject and in certain cases increase the risk of pancreatitis in a patient. Thus, the administration dose of GLP-1 polypeptide and its derivatives is limited by the side effects it causes, and clinical use thereof is not effective in achieving glycemic control and weight loss in patients.
The present invention aims to provide fusion proteins of FGF21 mutant proteins with the GLP-1 receptor protein, which have significant advantages over FGF21 and the known FGF21 mutants disclosed in the art. These advantages include significant and long lasting hypoglycemic and weight-reducing effects. These features are advantageous for the preparation and formulation of therapeutic proteins and have potential therapeutic effects on diseases associated with diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, and the like.
Disclosure of Invention
One embodiment of the invention is a polypeptide based on the sequence of wild-type human FGF21(SEQ ID NO:4), wherein:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) the Gly at the position 170 from the N end of the wild FGF21 protein is substituted by Thr;
(3) the Ser at position 172 from the N terminal of the wild-type FGF21 protein is replaced by Leu;
(4) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Glu, Trp, Leu, Tyr, or Ile;
and (5) replacement of Ala at position 180 from the N-terminus of wild-type FGF21 protein with Glu;
in yet another embodiment:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) the 163 th Ser from the N terminal of the wild-type FGF21 protein is replaced by one of Phe, Trp, Tyr, Leu, Gln or Thr;
(3) the Gly at the position 170 from the N end of the wild FGF21 protein is substituted by Thr;
(4) the Ser at position 172 from the N terminal of the wild-type FGF21 protein is replaced by Leu;
and (5) replacement of Ala at position 180 from the N-terminus of wild-type FGF21 protein with Glu;
a further preferred embodiment of the present invention, wherein:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) the 163 th Ser from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Trp, Tyr, Leu, Glu Ile, Asp or His;
(3) the Gly at the position 170 from the N end of the wild FGF21 protein is substituted by Thr;
(4) the Ser at position 172 from the N terminal of the wild-type FGF21 protein is replaced by Leu;
(5) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Glu, Trp, Leu, Tyr, or Ile;
(6) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
another preferred embodiment of the present invention, wherein:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) pro at position 171 from the N-terminus of wild-type FGF21 protein is replaced by Gly;
(3) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Glu, Trp, Leu, Tyr, or Ile;
(4) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
in another preferred embodiment of the present invention:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) the 163 th Ser from the N terminal of the wild-type FGF21 protein is replaced by one of Phe, Trp, Tyr, Leu, Gln or Thr;
(3) pro at position 171 from the N-terminus of wild-type FGF21 protein is replaced by Gly;
(4) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
in another preferred embodiment of the present invention:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) the 163 th Ser from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Trp, Tyr, Leu, Glu, Ile, Asp or His;
(3) pro at position 171 from the N-terminus of wild-type FGF21 protein is replaced by Gly;
(4) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Glu, Trp, Leu, Tyr, or Ile;
(5) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
in another preferred embodiment of the present invention:
(1) substitution of Arg for Leu at position 98 from the N-terminus of wild type F1.GF21 protein;
(2) the Gly-Pro-Ser at the position 170-172 from the N end of the wild FGF21 protein is substituted by Thr-Gly-Leu;
(3) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Glu, Trp, Leu, Tyr, or Ile;
(4) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
in yet another preferred embodiment of the present invention:
(1) substitution of Arg for Leu at position 98 from the N-terminus of wild type F1.GF21 protein;
(2) the 163 th Ser from the N terminal of the wild-type FGF21 protein is replaced by one of Phe, Trp, Tyr, Leu, Gln or Thr;
(3) the Gly-Pro-Ser at the position 170-172 from the N end of the wild FGF21 protein is substituted by Thr-Gly-Leu;
(4) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
in yet another preferred embodiment of the present invention:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) the 163 th Ser from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Trp, Tyr, Leu, Glu, Ile, Asp or His;
(3) the Gly-Pro-Ser at the position 170-172 from the N end of the wild FGF21 protein is substituted by Thr-Gly-Leu;
(4) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Glu, Trp, Leu, Tyr, or Ile;
(5) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
in another preferred embodiment of the present invention:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) pro at position 146 from the N-terminus of the wild-type FGF21 protein is replaced by Leu;
(3) the 166 th Leu is replaced by Phe from the N-terminal of wild FGF21 protein;
(4) pro at position 171 from the N-terminus of wild-type FGF21 protein is replaced by Gly;
(5) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by Trp;
(6) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
in another preferred embodiment of the present invention:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) pro at position 146 from the N-terminus of the wild-type FGF21 protein is replaced by Leu;
(3) the 166 th Leu is replaced by Phe from the N-terminal of wild FGF21 protein;
(4) the Gly-Pro-Ser at the position 170-172 from the N end of the wild FGF21 protein is substituted by Thr-Gly-Leu;
(5) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by Trp;
(6) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
in yet another preferred embodiment of the present invention:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) pro at position 146 from the N-terminus of the wild-type FGF21 protein is replaced by Leu;
(3) the 163 th Ser from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Trp, Asp, Leu, Glu or Ile;
(4) the 166 th Leu is replaced by Phe from the N-terminal of wild FGF21 protein;
(5) pro at position 171 from the N-terminus of wild-type FGF21 protein is replaced by Gly;
(6) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by Trp;
(7) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
in yet another preferred embodiment of the present invention:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) pro at position 146 from the N-terminus of the wild-type FGF21 protein is replaced by Leu;
(3) the 163 th Ser from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Trp, His, Leu, Glu or Ile;
(4) the 166 th Leu is replaced by Phe from the N-terminal of wild FGF21 protein;
(5) the Gly-Pro-Ser at the position 170-172 from the N end of the wild FGF21 protein is substituted by Thr-Gly-Leu;
(6) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by Trp;
(7) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
a preferred embodiment of the invention wherein the FGF21 variant protein is linked to the GLP-1 protein via a linker protein. Another preferred embodiment of the invention wherein the FGF21 variant protein is linked to the GLP-1 protein by a linker protein, and the linker protein comprises an Fc fragment of an immunoglobulin.
The invention also relates to a fusion protein, which has the following general formula:
F1-F2-F3
(I)
wherein: f1Is a GLP-1 receptor protein or a variant thereof; f2Is a connexin; f3Is FGF21 protein or a variant thereof.
Another embodiment of the present invention is a fusion protein of formula (II), F2The general formula of the connexin is:
F4F5(GGGGS)m
(II)
wherein: f4Is SEQ ID NO: 161; f5An Fc fragment that is an immunoglobulin; m is 1-20.
Another embodiment of the present invention is a fusion protein of formula (I), said F2The connexin of formula (II) wherein F5Is SEQ ID NO: 162.
another embodiment of the present invention is a fusion protein of formula (I), said F2The connexin of general formula (II) wherein m is 3.
Another embodiment of the present invention is a fusion protein of formula (I), said F2The connexin of (a) or its variant sequence is SEQ ID NO: 163.
another embodiment of the present invention is a fusion protein of formula (I), said F1The sequence of the GLP-1 receptor protein or the variant thereof is SEQ ID NO: 164-165.
Another embodiment of the present invention is a fusion protein of formula (I), said F3The sequence of FGF21 protein or a variant thereof of SEQ ID NO: 1-160.
Another embodiment of the invention is a fusion protein of general formula (I) having the sequence of SEQ ID NO: 168-171.
The invention also relates to a polynucleotide encoding a fusion protein comprising the general formula (I).
The invention also relates to an expression vector containing the polynucleotide as described above.
In addition, the present invention also relates to a host cell introduced or containing the expression vector as described above, wherein the host cell is a bacterium, preferably Escherichia coli; or the host cell is saccharomycete, preferably pichia pastoris; or the host cell is a mammalian cell, preferably a CHO cell or a HEK293 cell.
The present invention also relates to a method for producing a protein comprising the steps of:
1) culturing a host cell as described above;
2) isolating the protein from the culture;
3) and purifying the protein.
The invention further comprises a pharmaceutical composition comprising a fusion protein according to formula (I) together with a pharmaceutically acceptable excipient, diluent or carrier.
The invention also relates to application of the protein and/or the pharmaceutical composition in preparing a medicament for treating or preventing and treating diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease or non-alcoholic fatty hepatitis and other related diseases.
Detailed description of the invention
Unless stated to the contrary, terms used in the specification and claims have the following meanings.
The amino acid position changes in the FGF21 mutants of the invention are determined from the amino acid positions in the mature human wild-type FGF21(SEQ ID NO:4) polypeptide.
The amino acid sequences of the present invention contain the standard single or three letter codes for twenty amino acids.
The term "FGF 21 polypeptide" refers to a naturally occurring wild-type polypeptide expressed in vivo in humans. Including SEQ ID NO 166 constituting the full-length form encoded by SEQ ID NO 172 and SEQ ID NO 167 constituting the mature form encoded by SEQ ID NO 173.
The term "FGF 21 mutant" refers to an FGF21 polypeptide modified based on the naturally occurring amino acid sequence of FGF21(SEQ ID NO: 4). Such modifications include, but are not limited to, substitution of one or more amino acids, including, but not limited to, protease resistant FGF21 mutants, aggregation reduced FGF21 mutants, and FGF21 combination mutants, as described herein.
The term "conservative amino acid mutation" refers to a substitution by a natural amino acid residue such that the polarity or charge of the amino acid residue at that position has little effect.
Amino acids can be classified into the following groups according to the properties of their side chains:
(1) hydrophobicity: ala, Ile, Leu, Met, Phe, Pro, and Trp;
(2) neutral hydrophilicity: cys, Ser or Thr;
(3) acidity: asp and Glu;
(4) alkalinity: arg, Asn, Gln, His, or Lys.
Conservative substitutions include the replacement of a member of one of these classes by another member of the same class; non-conservative substitutions involve the replacement of a member of one of these classes by a member of the other class.
The term "dyslipidaemia" refers to disorders of lipoprotein metabolism, including overproduction or underproduction of lipoproteins. May be manifested as an increase in blood total cholesterol, low density lipoprotein cholesterol and triglyceride concentrations and/or a decrease in high density lipoprotein cholesterol concentrations.
The term "patient" is a mammal, preferably a human.
The term "treating" refers to slowing, reducing, or reversing the progression or severity of a symptom, disorder, or disease.
The term "Fc fragment" refers to the constant region of the heavy chain of an immunoglobulin.
The term "vector" refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to convey encoded information to a host cell.
The term "expression vector" refers to a vector suitable for transformation of a host cell and containing nucleic acid sequences that direct and/or control the expression of an inserted heterologous nucleic acid sequence. Including but not limited to processes such as transcription, translation, and RNA splicing.
The term "host cell" is used to refer to a cell transformed with a nucleic acid sequence or capable of being transformed with said nucleic acid sequence and then capable of expressing a selected gene of interest. The term includes progeny of the parent cell, whether or not the progeny is identical in morphology or genetic makeup to the original parent, with the selected gene being predominantly present.
Detailed Description
The following specific embodiments are provided in order to explain the present invention in more detail, but the present invention is not limited thereto.
EXAMPLE 1 preparation of Compound No. 1
1.1 purpose of experiment: preparation of numbered Compound 1 by cellular expression and purification
1.2 protocol:
1.2.1 Main test reagents
| Name (R) | Brand | Goods number |
| NI Sepharose excel | GE | 17-3712-01 |
| NaOH | Shanghai test | 10019718 |
| Imidazole | Shanghai test | 10000218 |
| PBS | Dingguosheng Changsheng | BF-0012 |
| 75% ethanol | Shanghai test | 801769610 |
1.2.2 Main laboratory instruments
| Name (R) | Brand | Model number |
| Protein purification instrument | GE | AKTA-Pure150 |
| Liquid transfer device | Eppendorf | 100-1000μL |
| Liquid transfer device | Eppendorf | 20-200μL |
| Liquid transfer device | Eppendorf | 2-20μL |
| Magnetic stirrer | Shanghai plum Yingpu | H03-A |
| Micro-spectrophotometer | Hangzhou Osheng | Nano-300 |
| Balance with a movable handle | Sartorius | SQP QUINTIX124-1CN/120 |
| PH meter | Mettler | FE28 |
| Multifunctional enzyme mark instrument | Thermo | Lux |
1.2.3 test methods
The fusion protein was expressed using the ExpicHO system (Thermo Fisher # A29133).
Specifically, the DNA sequence encoding the protein sequence of the numbered compound with His tag at the C-terminal is cloned into a pCDNA3.1 vector to obtain a plasmid for expressing the fusion protein. The plasmid was transfected into expihcho-S cells using expifctamine reagent, and after culturing the cells in 100 ml of expihcho medium for 7 days, the supernatant was harvested; clarifying the fermentation liquor by adopting a centrifugal filtration or deep filtration method.
The EQ buffer (PBS, pH7.4) was prepared by taking a bag of PBS phosphate buffer powder, dissolving the powder in 2000ml of ultrapure water, and filtering the solution through a 0.22 μm filter for use. The solution of Elution buffer (500mM imidazole, pH7.4) was prepared by weighing 34g of imidazole, adding 450ml of EQ buffer, adjusting pH to 7.4, diluting to 500ml, and filtering with 0.22 μm filter. The harvested supernatant was purified by AKTA Pure instrument. Firstly, balancing an instrument by using an EQ buffer solution until the pH value and the electric conductivity value of an effluent liquid are consistent with those of the EQ buffer solution; samples were collected again with Elution buffer.
1.3 Experimental results:
the concentration of compound No. 1 was 847mg/L as determined by UV spectrophotometry, and the purity was 96%.
EXAMPLE 2 preparation of Compounds No. 2-160
2.1 purpose of experiment: preparation of numbered Compounds 2-160 by cellular expression and purification
2.2 Experimental methods: compounds 2-160 were prepared using the procedure of example 2.
2.3 Experimental results: the concentrations and purities of compounds No. 2-160 were determined by uv spectrophotometry as follows:
EXAMPLE 3 preparation of compound No. 168
3.1 purpose of experiment: compound number 168 was prepared by a method of cellular expression and purification.
3.2 Experimental protocol:
3.2.1 Main test materials
| Name (R) | Brand | Goods number |
| Eshmuno A | MERCK | 1.25161.0001 |
| NaOH | Shanghai test | 10019718 |
| Acetic acid | Shanghai test | 10000218 |
| Sodium acetate (trihydrate) | Shanghai test | 10018718 |
| PBS | Dingguosheng Changsheng | BF-0012 |
| 75% ethanol | Shanghai test | 801769610 |
3.2.2 Main laboratory instruments
| Name (R) | Brand | Model number |
| Protein purification instrument | GE | AKTA-Pure150 |
| Liquid transfer device | Eppendorf | 100-1000μL |
| Liquid transfer device | Eppendorf | 20-200μL |
| Liquid transfer device | Eppendorf | 2-20μL |
| Magnetic stirrer | Shanghai plum Yingpu | H03-A |
| Micro-spectrophotometer | Hangzhou Osheng | Nano-300 |
| Balance with a movable handle | Sartorius | SQP QUINTIX124-1CN/120 |
| PH meter | Mettler | FE28 |
| Multifunctional enzyme mark instrument | Thermo | Lux |
3.2.3 Experimental methods
The fusion protein was expressed using the ExpicHO system (Thermo Fisher # A29133).
Specifically, a DNA sequence encoding the protein sequence of compound 168 was cloned into the pCDNA3.1 vector to obtain a plasmid expressing the fusion protein. The plasmid was transfected into ExpCHO-S cells using ExpFectamine reagent, and after culturing the cells in 100 ml of ExpCHO medium for 7 days, the supernatant was harvested. Clarifying the fermentation liquor by adopting a centrifugal filtration or deep filtration method.
The EQ buffer (PBS, pH7.4) was prepared by taking a bag of PBS phosphate buffer powder, dissolving the powder in 2000ml of ultrapure water, and filtering the solution through a 0.22 μm filter for use. The solution buffer (50mM HAc-NaAc, pH3.5) was prepared by weighing 1.91g of sodium acetate trihydrate, adjusting the pH to 3.5 with glacial acetic acid, diluting to 1000ml, and filtering with 0.22 μm filter. The harvested supernatant was purified by AKTA Pure instrument. The instrument was first equilibrated with EQ buffer until the pH and conductivity of the effluent was consistent with EQ buffer. Samples were collected again with Elution buffer.
3.3 Experimental results:
the concentration of compound No. 168 was 680mg/L and the purity was 90% as determined by UV spectrophotometry.
EXAMPLE 4 in vivo efficacy of Compound 168, No. 4
4.1 purpose of experiment:
test for the body weight modulation of ob/ob mice by subcutaneous administration of test number Compound
4.2 Experimental methods:
male ob/ob mice of age 15-19 weeks of week, weighing 58-70 grams. The ob/ob mice were injected subcutaneously every 3 days with the numbered compound (2 mg/kg body weight). Mice were observed for body weight and random blood glucose before and on the ninth day after dosing. The specific method for measuring blood sugar value is to fix the mouse by physical method, expose the tail and cut off a little, squeeze the tail to bleed, discard the 1 st drop of blood and then measure the blood sugar by Roche vitality type blood sugar meter. Calculating the weight loss percentage according to the weight value, wherein the calculation method comprises the following steps: percent weight loss (% weight on day 0 to day 9)/weight on day 0 × 100.
4.3 Experimental results:
4.4 conclusion of the experiment:
in this experiment, at a dose of 2 mg/kg body weight, compound 168 of the present invention showed significant and long lasting glucose and weight lowering effects, suggesting its potential to be a long acting drug for regulating metabolism.
EXAMPLE 5 in vivo efficacy of Compound 168 No. 5
4.1 purpose of experiment:
test for modulating effects of compound No. s administered subcutaneously on body weight, liver weight and blood glucose in ob/ob mice 4.2 experimental methods:
male ob/ob mice weighing 35-50 grams and aged 8-12 weeks. Mice were injected subcutaneously with placebo and the numbered compound (2 mg/kg body weight) every 3 days. The body weights of the mice were observed before and on the ninth day after the administration, and the percentage of body weight loss was calculated from the body weight values by the calculation method: percent weight loss (% weight on day 0 to day 9)/weight on day 0 × 100. Overnight fasting (over 16 h) the day before the endpoint; on the end point day, materials are taken after euthanasia, whole blood is collected, serum is collected after centrifugation for measuring blood sugar, the specific method for measuring the blood sugar value is to fix a mouse by a physical method, expose a tail, cut off a little of the tail, squeeze the tail to cause bleeding, discard the 1 st drop of blood and detect the blood sugar by a Roche vitality type glucometer; collecting the liver weight, and calculating the weight ratio of the liver, wherein the calculation method comprises the following steps: liver weight (% liver weight on day 9/weight on day 9 × 100).
4.3 Experimental results:
4.4 conclusion of the experiment:
in this experiment, at a dose of 2 mg/kg body weight, compound 168 of the present invention showed significant and long lasting weight, blood glucose and liver weight lowering effects, suggesting its potential as a long acting drug for regulating metabolism and improving liver status.
Claims (10)
1. A fusion protein of the general formula:
F1-F2-F3
(I)
wherein:
F1is GLP-1 receptor protein or variant thereof, and has a specific sequence of SEQ ID NO: 164-165;
F2is a connexin;
F3is FGF21 protein or a variant thereof, and has a specific sequence shown as SEQ ID NO: 1-160.
2. The fusion protein of claim 1, wherein F is2The connexin of (a) or its variant sequence is SEQ ID NO: 163.
3. the fusion protein of claim 1, wherein the specific sequence is SEQ ID NO: 168-171.
4. A polynucleotide encoding a polypeptide comprising the protein of any one of claims 1-3.
5. An expression vector comprising the polynucleotide of claim 4.
6. A host cell into which the expression vector of claim 5 is introduced or which contains the expression vector.
7. The host cell according to claim 6, wherein said host cell is a bacterium, preferably E.coli;
or the host cell is saccharomycete, preferably pichia pastoris;
preferably, the host cell is a mammalian cell, preferably a CHO cell or a HEK293 cell.
8. A method of producing a protein comprising the steps of:
culturing the host cell of claim 6 or 7;
isolating the protein from the culture;
and purifying the protein.
9. A pharmaceutical composition comprising a protein according to any one of claims 1 to 3 and a pharmaceutically acceptable excipient, diluent or carrier.
10. Use of a protein according to any one of claims 1-3 and/or a pharmaceutical composition according to claim 9 for the preparation of a medicament for the therapeutic or prophylactic treatment of diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease or a non-alcoholic steatohepatitis-related disease.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116284441A (en) * | 2023-01-18 | 2023-06-23 | 上海民为生物技术有限公司 | Fusion proteins with triple activity and uses thereof |
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| CN116284441A (en) * | 2023-01-18 | 2023-06-23 | 上海民为生物技术有限公司 | Fusion proteins with triple activity and uses thereof |
| CN116284441B (en) * | 2023-01-18 | 2023-12-19 | 上海民为生物技术有限公司 | Fusion proteins with triple activity and uses thereof |
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