CN118307684A - GLP-1-FGF21 fusion protein modified by fatty acid chain, and preparation method and application thereof - Google Patents
GLP-1-FGF21 fusion protein modified by fatty acid chain, and preparation method and application thereof Download PDFInfo
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- CN118307684A CN118307684A CN202410742771.1A CN202410742771A CN118307684A CN 118307684 A CN118307684 A CN 118307684A CN 202410742771 A CN202410742771 A CN 202410742771A CN 118307684 A CN118307684 A CN 118307684A
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Abstract
The invention provides a GLP-1-FGF21 fusion protein modified by a fatty acid chain, a preparation method and application thereof. In the fusion protein, after a specific mutation site is introduced into the C end of FGF21, the GLP-1-FGF21 end is modified by adopting a Sortase-A enzyme mediated molecular modification technology, and the fusion protein can be realized after fatty acid chain modification: GLP-1-FGF21 half-life is prolonged; fibroblast Activation Protease (FAP) slows down terminal degradation of FGF 21; operating in water solution at normal temperature, and preparing GLP-1-FGF21 fusion protein quickly at low cost. The GLP-1-FGF21 fusion protein provided by the invention adopts an enzyme-mediated molecular modification technology, and has remarkable advantages.
Description
Technical Field
The invention relates to a GLP-1-FGF21 fusion protein modified by a fatty acid chain, and a preparation method and application thereof, belonging to the technical field of biology.
Background
Human fibroblast growth factor 21 (Fibroblast growth factor, FGF 21) is a polypeptide consisting of 209 amino acids, wherein the N-terminus of the FGF21 protein contains a signal peptide consisting of 28 amino acids, so that mature FGF21 formed by cleavage of the signal peptide consists of 181 amino acids.
FGF21 is expressed in a variety of organs and tissues in humans, such as the liver, pancreas, adipose tissue, and the like. In humans, FGF21 can secrete into the blood circulation and induce a variety of signaling pathways and functional activities in liver, pancreas and adipose tissue, thereby achieving the physiological functions of regulation of glycolipid metabolism and protection of islet beta cells. Studies have shown that in obese mice produced by dietary induction or genetic manipulation, FGF21 protein injection can significantly reduce mouse body weight and blood glucose levels, as well as the Triglyceride (TRIGLYCERIDE) content in mouse serum. Further studies have shown that FGF21 can improve insulin sensitivity in the liver, thereby alleviating glucose intolerance. Studies in mammals more closely related to humans have shown that FGF21 can reduce body weight and body fat in experimental animals in a dose-dependent manner, such as administration of FGF21 to diabetic rhesus monkeys, with significant reductions in fasting plasma glucose and triglyceride levels. In addition, FGF21 also activates exocrine pancreatic cells and hepatocyte signaling pathways, inhibiting hepatic glycogen output.
FGF21 belongs to a family of FGF (fibroblast growth factor) members, however, unlike most FGFs which have a broad spectrum of mitogenic capacity, FGF21 does not have a significant capacity to promote cell proliferation. Studies show that FGF21 transgenic mice have no abnormal conditions such as tumor, tissue hyperplasia and the like in vivo in the whole life cycle. Meanwhile, pharmacokinetic and drug safety experiments show that the FGF21 with the pharmacological dose is not provided with hypoglycemia, which shows that the FGF21 is a potential ideal drug for treating diabetes, obesity and other diseases.
Unlike most FGF family members, FGF21 activation of downstream signaling pathways requires simultaneous activation by the FGF receptor (Fibroblast growth factor receptors, FGFRs) and the co-receptor β -klotho, neither β -klotho nor FGFR alone activates FGF21 signaling. Structural biology studies have shown that the structure of FGF21 can be divided into two parts, with the N-terminal domain involved in binding of the main FGFR and the C-terminal domain a relatively flexible sequence involved in binding of β -klotho. FGF21 activates downstream-associated signaling pathways only after formation of a ternary complex with β -klotho, FGFR, thereby exerting its biological effect.
The physiological functions of FGF21 in the aspects of controlling blood sugar, reducing weight and the like bring hope for treating related diseases, but wild FGF21 has the defects of easy hydrolysis by protease, too small molecular weight and the like, has half-life of only 0.5-2 hours, and is not suitable for being directly used as a medicine. Meanwhile, the flexible region contained at the C-terminal of FGF21 protein, which is responsible for binding to the beta-klotho receptor, is easily degraded by fibroblast activation protease (Fibroblast Activation Protein, FAP), and its main degradation site is around Pro 171. After cleavage of P171 by FAP protease, FGF21 molecules will no longer be able to bind to the β -klotho co-receptor, thereby resulting in inactivation of FGF21 molecules.
The major biologically active fragment of glucagon-like peptide-1 (GLP-1) is a 30 or 31 amino acid polypeptide fragment derived from post-translational processing of the pro-glucagon peptide (amino acids 7-36 or 7-37 of GLP-1). GLP-1 is a hormone produced mainly by intestinal cells in humans, belonging to the class of incretins. GLP-1 receptor agonists are novel hypoglycemic agents in recent years, and by activating GLP-1 receptor (GLP-1R), insulin secretion is enhanced in a glucose concentration dependent manner, glucagon secretion is inhibited, gastric emptying is delayed, and feeding amount is reduced through central appetite suppression, so that the effect of reducing blood glucose is achieved. Endogenous GLP-1 has only a half-life of about 2 minutes, so diabetics need to be supplemented with exogenous GLP-1 for hypoglycemic purposes.
In physiological function, GLP-1 and FGF21 have strong complementarity, and simultaneously, activating GLP-1R and FGFR can more effectively reduce blood sugar, weight, blood fat and the like, and Qi Pan and the like connect GLP-1 and FGF21 through Fc to obtain a GLP-1-Fc-FGF21 dual agonist (EBio medicine 2021 Jan;63:103202). However, the above-mentioned GLP-1-Fc-FGF21 dual agonist has complicated operation method, the constructed fusion protein has larger molecular weight, affects the molecular activity, and usually requires mammalian cell expression, and has higher cost.
After direct expression of the GLP-1 and FGF21 fusion proteins, the end of FGF21 can be modified by introducing appropriate mutation sites, e.g., using fatty acid chains or PEG modification, to achieve an extended half-life, and to shield FAP protease from degradation of the FGF21 end. Although the method can prepare GLP-1-FGF21 fusion protein with smaller molecular weight, chemical modification steps are required to be introduced in the process, the process is relatively complex, and a large number of mutation sites are required to be introduced in FGF21 or GLP-1 molecules so as to reduce nonspecific modification.
Therefore, there is a need for a GLP-1-FGF21 fusion protein which has simple development process, low cost, and can prolong the half-life of GLP-1-FGF21 and slow down the degradation of FAP to the end of FGF21, and a preparation method thereof.
Disclosure of Invention
The invention aims to prepare a novel GLP-1-FGF21 fusion protein by introducing a specific mutation site at the tail end of FGF21 and modifying the tail end of GLP-1-FGF21 by using a Sortase-A enzyme-mediated coupling method, wherein the fusion protein has the advantages of easy preparation, low cost and high coupling efficiency, and can effectively prolong the half-life of GLP-1-FGF21 and slow down the degradation of FAP to the tail end of FGF 21.
To achieve the above object, in a first aspect, the present invention provides a GLP-1-FGF21 fusion protein modified with a fatty acid chain, characterized in that the GLP-1-FGF21 fusion protein comprises GLP-1 or an active fragment thereof, a linker, a FGF21 variant or an active fragment thereof, the C-terminus of the FGF21 variant comprises a-LPXTG motif and the C-terminus is modified with a fatty acid chain, wherein X is any amino acid, and the linker is a connecting peptide.
In one embodiment of the present invention, the FGF21 variant is mutated based on the wild-type human FGF21 protein sequence, wherein the wild-type human FGF21 has the sequence shown in SEQ ID No. 1.
Further, in order to attenuate deamination reaction at position 121 and oxidation reaction at position 167, N is replaced by Q at position 121 and M is replaced by L at position 168 of the sequence shown in SEQ ID NO. 1, and the sequence of the FGF21 variant is shown in SEQ ID NO. 4, and the replacement of the amino acid does not affect activity or other functions of FGF 21.
Further, in order to enable the Sortase-A enzyme to recognize FGF21, any of the consecutive 7 amino acids 166-177 of the sequence shown in SEQ ID NO. 4 is replaced with LPXTGGG, wherein X represents any natural amino acid residue, and wherein the sequence after replacement is shown in SEQ ID NO. 5-8.
In a preferred embodiment of the invention, the FGF21 variant sequence is shown in SEQ ID NO. 8.
In a preferred embodiment of the invention, the GLP-1 polypeptide is selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 11 or SEQ ID NO. 13.
In a preferred embodiment of the invention, the fatty acid chain modification is coupling of the fatty acid chain to the side chain NH 2-of amino acid K at position 180 of the sequence shown in SEQ ID NO. 8.
In a preferred embodiment of the present invention, the linker peptide is GGGSGGGGS（SEQ ID NO:14）、（GGGGS）n、（GGGGS）n、SGGGGSGGGG（SEQ ID NO:15）、GGGGGSGGGGSSGGGGS（SEQ ID NO:16）、GGGGSGGGGSGGGG（SEQ ID NO:17）、GGGGSGGGGSGGGSGGGGS（SEQ ID NO:18）、GSPGSSSSGS（SEQ ID NO:19）、GSGSGSGS（SEQ ID NO:20）、GSGSGNGS（SEQ ID NO:21）、GGSGSGSG（SEQ ID NO:22）、GGSGSG（SEQ ID NO:23）、GGSG（SEQ ID NO:24）、GGSGNGSG（SEQ ID NO:25）、GGNGSGSG（SEQ ID NO:26）、GGNGSG（SEQ ID NO:27） or (GGGGGS) n, n may be an integer between 1 and 5.
In a preferred embodiment of the invention, the linker peptide is GGGGSGGGGS (SEQ ID NO: 28) or (GGGGS) 4 (SEQ ID NO: 29).
In a preferred embodiment of the invention, the sequence of the GLP-1-FGF21 fusion protein is shown as SEQ ID NO. 9 or SEQ ID NO. 12.
In a second aspect, the invention provides a method for preparing a GLP-1-FGF21 fusion protein modified by fatty acid chains, comprising the steps of: 1) Obtaining the GLP-1-FGF21 fusion protein, 2) obtaining a coupled polypeptide molecule modified by a fatty acid chain, and performing fatty acid chain modification on the GLP-1-FGF21 fusion protein by using a Sortase-A enzyme.
In one embodiment of the present invention, the step of modifying the fatty acid comprises: synthesizing a coupled polypeptide molecule, mixing the coupled polypeptide molecule with a fatty acid chain solution, and stirring at room temperature to obtain a coupled product A.
Further, the step of fatty acid modification further comprises: mixing the coupling product A with the GLP-1-FGF21 fusion protein, adding a Sortase-A enzyme solution, and stirring at room temperature to obtain the GLP-1-FGF21 fusion protein modified by fatty acid chains; optionally, the fatty acid chain modified GLP-1-FGF21 fusion protein is further purified.
In a preferred embodiment of the invention, the coupling polypeptide is shown in SEQ ID NO. 10, and the fatty acid chain modification is to couple a fatty acid chain to a side chain NH 2-of amino acid K at position 8 of the sequence shown in SEQ ID NO. 10.
In a third aspect the invention provides a host cell comprising said nucleic acid molecule or said expression vector.
The fourth aspect of the invention provides a pharmaceutical composition comprising a safe and effective amount of the fatty acid chain modified GLP-1-FGF21 fusion protein or the fatty acid chain modified GLP-1-FGF21 fusion protein prepared by the preparation method, and a pharmaceutically acceptable carrier.
In a fifth aspect the present invention provides a method for the preparation of said fatty acid chain modified GLP-1-FGF21 fusion protein, said method of preparation, said nucleic acid molecule, said expression vector, said host cell for the preparation of a medicament for the treatment or prophylaxis of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type1 diabetes, obesity, metabolic syndrome and neurodegenerative diseases, in particular for the delay of progression or prophylaxis of disease in type 2 diabetes, for the treatment of metabolic syndrome, for the treatment of obesity or prophylaxis of overweight, for reducing food intake, for increasing energy expenditure, for reducing body weight, for delaying the progression from Impaired Glucose Tolerance (IGT) to type 2 diabetes; controlling blood sugar and blood fat; delay progression from type 2 diabetes to diabetes requiring insulin; regulating appetite; inducing satiety; preventing weight recovery after successful weight loss; treating a disease or condition associated with overweight or obesity; treating bulimia; treating binge eating; the application of the medicine for treating atherosclerosis, hypertension, IGT, dyslipidemia, coronary heart disease, fatty liver and beta-blocker poisoning in inhibiting gastrointestinal tract movement.
All amino acid sequence numbers of FGF21 referred to in the present invention are based on wild-type FGF21 (shown as SEQ ID NO: 1), the first histidine residue position number in SEQ ID NO:1 is 1, the second is 2, and so on to the last amino acid 181.
Advantageous effects
The preparation method provided by the invention can prepare the GLP-1-FGF21 fusion protein modified by the fatty acid chain through a simple Sortase-A enzyme mediated coupling technology, is simple to operate, has no non-specific modification, and is operated in an aqueous solution at normal temperature, so that the preparation cost is reduced, and the preparation time is saved; meanwhile, the GLP-1-FGF21 fusion protein modified by the fatty acid chain prepared by the preparation method prolongs the half-life of GLP-1-FGF21 and greatly slows down the degradation of FAP to the tail end of FGF 21.
Drawings
The conception, specific structure, and technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, features, and effects of the present invention.
FIG. 1 is a comparison of the C-terminal sequences of four different Sortase-A coupled FGF21 proteins of example 3, with the black bolded italics being the sequence after Sortase-A coupling.
FIG. 2 is a schematic representation of the fatty acid chain modification of GLP-1-FGF21 fusion protein using the enzyme Sortase-A in example 4.
Figure 3 is the change in body weight of mice following administration in example 7.
FIG. 4 shows the change in blood glucose levels of mice after administration in example 7.
FIG. 5 is a comparison of liver pathological sections of the administration-end mice in example 7, wherein A, B, C, D, E correspond to the group G1, group G2, group G3, group G4, and group G5 mice, respectively, and wherein white spots are adipose tissue.
FIG. 6 is a mass spectrum identification of a complete GLP-1-FGF21 fusion protein after coupling of fatty acid chains, having a molecular weight of 24128 Da.
Detailed Description
Definition of the definition
Unless otherwise indicated, the terms used in the present application have the following meanings. A particular term, unless otherwise defined, shall not be construed as being ambiguous or otherwise unclear, but shall be construed in accordance with the ordinary meaning in the art.
Table 1: list of 20 natural amino acid abbreviations
The numerical ranges in the present invention mean that each integer in the given range, for example (GGGGS) n, n may be an integer between 1 and 5, i.e. n is 1,2, 3, 4, 5.
As used herein, the term "fusion protein" refers to a protein that results from the fusion of two or more proteins or polypeptides.
As used herein, the term "linker" is a common linker known in the art or described in the present invention, and the common linker may be (GGGS) n, (GGGGS) n, (GGGGGS) n.
As used herein, the term "functional variant" refers to a protein or polypeptide in which one or more amino acids are substituted, deleted, or added based on the amino acid sequence of a parent protein, while still substantially retaining at least one of the biological properties of the parent protein.
As used herein, the term "active fragment" refers to a protein or polypeptide that is truncated on the basis of a parent protein but that substantially retains at least one biological property of the parent protein.
As used herein, the term "fatty acid chain" refers to a long aliphatic hydrocarbon chain organic containing one carboxyl group at one end, of the formula C (n) H (2n+1) COOH. It can be classified into short chain (containing 4-6 carbon atoms) fatty acids according to the different carbon chain lengths; medium chain (containing 8-14 carbon atoms) fatty acids; long chain (containing 16 to 20 carbon atoms) fatty acids and very long chain (containing 20 or more carbon atoms) fatty acids. The "fatty acid chain" coupled to the recombinant protein in the present invention is preferably a fatty acid chain represented by formula I:
-1inker- (CH 2) x-COOH (formula I)
Wherein linker moiety includes but is not limited to PEG (polyethylene glycol) fragment, amino acids such as glutamic acid, etc., (CH 2) wherein the number of x is between 10-20; preferably x is between 16 and 20; further preferred are:
Wherein the wavy line shows the site of attachment to an FGF21 amino acid residue.
Further, the fatty acid chains described herein are coupled to a polypeptide sequence by chemical reaction, the sequence comprising "GGGX 1X2X3X4 SPSYKS" wherein X 1,X2,X3 and X 4 represent any amino acid or blank, but do not include lysine residues. The amino acid side chain is coupled to the side chain of the lysine residue at the second position from the C terminal through chemical reaction.
Further preferably, the polypeptide sequence comprising "GGGX 1X2X3X4 SPSYKS" is shown as SEQ ID NO. 10.
Various polypeptide drugs on the market at present are prepared by increasing fatty acid side chains to realize non-covalent binding with plasma albumin, such as liraglutide and cable Ma Lutai of Noand Nord company. The invention utilizes a purified FGF21 mutant (FGF 21-P4, SEQ ID NO: 8) with-GGGSPSYKS-to connect a polypeptide sequence which comprises a 'GGGX 1X2X3X4 SPSYKS' sequence and is modified with a fatty acid chain at a side chain of a lysine residue at the second position from the C terminal with the FGF21-P4 through a connecting reaction catalyzed by a transpeptidase Sortase-A enzyme, so as to form the FGF21 protein mutant modified by the fatty acid chain.
As used herein, "treating" an individual with a disease or condition means that the symptoms of the individual are partially or fully alleviated, or remain unchanged after treatment. Thus, treatment includes prophylaxis, treatment and/or cure. Prevention refers to preventing an underlying disease and/or preventing worsening of symptoms or disease progression. Treatment also includes any nucleic acid molecule provided and any pharmaceutical use of the compositions provided herein.
As used herein, a "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, block or partially block the symptoms of a disease or disorder.
As used herein, a "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, e.g., prevent or delay the onset or recurrence of a disease or symptom, reducing the likelihood of the onset or recurrence of a disease or symptom. The fully prophylactically effective dose need not occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.
As used herein, the term "subject" refers to mammals, such as humans, cows and dogs.
As used herein and unless otherwise indicated, the terms "comprising," "including," "having," "containing," and their grammatical equivalents are generally understood to be open-ended and not to be limiting, e.g., not to exclude other, unrecited elements or steps.
The conception, specific structure, and technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, features, and effects of the present invention.
The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.
EXAMPLE 1 expression of FGF21 protein and GLP-1-FGF21 fusion protein
1.1 Construction of expression plasmids
Genes capable of expressing proteins numbered FGF21-1414 (wild-like FGF21; mutation sites 121Q,168L; shown as SEQ ID NO: 4), FGF21-P1 (SEQ ID NO: 5), FGF21-P2 (SEQ ID NO: 6), FGF21-P3 (SEQ ID NO: 7), FGF21-P4 (SEQ ID NO: 8) and GLP-1-FGF21-P4 (SEQ ID NO: 9) were each synthesized by the Suzhou gold-only biotechnology Co., ltd.) and cloned into the pET21b vector and then transformed into BL21 (DE 3) (purchased from Merck) strain.
FGF21-1414 is a mutant of wild-type FGF21, called "wild-type FGF 21-like" (121Q, 168L), the amino acid sequence of which is shown in SEQ ID NO:4, and mutation sites 121Q and 168L are used for weakening deamination reaction at 121 and oxidation reaction at 167, and do not affect the activity or other functions, so that the mutant FGF21-1414 is used as a control in the embodiment of the invention, and the experimental result represents the result of wild-type FGF21, so that all sequences in the invention have the two mutation sites. Table 2 shows the sequences and numbers of FGF21 and variants thereof, GLP-1-FGF21-P4, and conjugated polypeptides involved in the present invention.
TABLE 2 FGF21 and variants thereof, GLP-1-FGF21-P4, sequence and numbering of the conjugated polypeptide
1.2 Expression
The overnight cultured seeds were transferred to Kana-resistant 500 mL TB medium-containing Erlenmeyer flasks at a volume ratio of 1:50, and were grown to an OD600 of 2.0 at 37℃and 220: 220 rpm, and were harvested after overnight culture at 37℃and 220: 220 rpm with the addition of IPTG (final concentration of 0.5 mmol/L). The protein is expressed in the form of Inclusion Bodies (IB).
EXAMPLE 2 renaturation and purification of FGF21 protein and GLP-1-FGF21 fusion protein
2.1 Inclusion body denaturation and dissolution
FGF21-1414, FGF21-P1, FGF21-P2, FGF21-P3, FGF21-P4, GLP-1-FGF21-P4 inclusion bodies obtained by cell disruption and washing in example 1 were lysed using 8M urea and 10mM DTT. Inclusion body dissolution was performed at room temperature for 4 hours.
2.2 Renaturation
The above solubilized inclusion body solution was added to a renaturation solution containing 2M urea, 10 mM cysteine and 20 mM Tris-Cl buffer pH 8.0, and incubated overnight at room temperature. Continuous stirring is required during renaturation at a rotational speed of 200 revolutions per minute.
2.3 FGF21 protein and GLP-1-FGF21 fusion protein for hydrophobic interaction chromatography Phenyl purification
After adding Tris-Cl buffer at a final concentration of 2M NaCl and 20mM, the mixture was passed through a hydrophobic chromatography GE Capto Phenyl (from GE), followed by gradient elution with NaCl solution at a gradient of 2M to 0M. The collection tubes in which the target proteins are selected according to SDS-PAGE are pooled and stored, and this step can capture the target proteins and remove some of the contaminating proteins.
2.4 Ion exchange chromatography Source 30Q purification of FGF21 protein and GLP-1-FGF21 fusion protein
The combined proteins after hydrophobic interaction chromatography were 10-fold diluted and then subjected to Source 30Q ion exchange chromatography, eluting with a gradient of 0M to 1.0M using NaCl solution to further remove nucleic acids and some protein polymers and hybrid proteins.
2.5 Molecular sieve fine purification of FGF21 protein and GLP-1-FGF21 fusion protein
After ion exchange chromatography source 30Q purification, the combined proteins are concentrated, then molecular sieve Superdex 200 fine purification is carried out, PBS is used as buffer solution, and the purified proteins are used as final samples, and the serial numbers are FGF21-1414, FGF21-P1, FGF21-P2, FGF21-P3, FGF21-P4 and GLP-1-FGF21-P4 respectively.
Purification information as shown in Table 3, all proteins achieved electrophoretically pure purity.
TABLE 3 concentration table of purified FGF21 protein and GLP-1-FGF21 fusion protein
EXAMPLE 3 screening of cell Activity of different FGF21 mutants
In order to determine the appropriate method for coupling using the Sortase-a enzyme, the present invention first screened the activity of the different coupled sequences. The Sortase-A enzyme can connect the N end and the C end of two sections of polypeptides in an enzyme-mediated mode, and the specific principle is that when the C end of one section of polypeptide is provided with an-LPXTG (X is any amino acid) sequence, the Sortase-A enzyme can recognize the C end of the polypeptide, cut off the end G, and connect the other section of polypeptide with the N end initiated by GGG-to form an-LPXTGGG-sequence, so that the enzyme-mediated coupling of the two sections of polypeptides is realized. Four different class of Sortase-A coupled FGF21 protein C-terminal sequences were designed in the present invention (as shown in FIG. 1) and tested for cellular activity.
To further verify the activity of FGF21-1414 and its mutants in the present invention, the activation of ERK signaling pathway by FGF21-1414 and its mutants was tested using HEK293 cells overexpressing β -Klotho (cells were constructed from kanglos formation), the specific experimental procedure was to culture HEK293 cells overexpressing β -Klotho using dmem+10% fbs+1 x ps cell culture medium and to add 20 μl of cell suspension to white 384 assay plates at cell culture to exponential phase, adding appropriate medium and appropriate cell density (5000/well), and culturing at 37 ℃ at 5% CO 2. FGF21-1414 and mutants thereof were initiated in medium at 3. Mu.M/6. Mu.M/15. Mu.M, diluted 3-fold, and 10. Mu.l of compound/well was added to the cell assay plate. The cells after addition of the protein to be tested were placed in a 5% CO2 incubator at 37℃for 0.5 hours. After removing the cell supernatant, 16. Mu.L of a cell lysis buffer is added, and the mixture is shaken at room temperature for 30 to 60 minutes. The plates were then sealed by adding 4. Mu.L of the pre-mixed antibody solution prepared in the detection buffer, and after incubation overnight at room temperature, the fluorescence emissions at 665nm and 620nm were read. As shown in Table 4, among four different Sortase-A-coupled FGF21 proteins designed in the invention, FGF21-P4 has similar cell activities of wild-type FGF21, namely FGF21-1414, and can be used for further research.
TABLE 4 four different Sortase-A coupled FGF21 protein cell Activity assays
EXAMPLE 4 fatty acid chain modification of GLP-1-FGF21 fusion protein Using the Sortase-A enzyme
In this example, we used the Sortase-A enzyme to modify the fatty acid chain of GLP-1-FGF21-P4, with specific procedures shown in FIG. 2. The conjugated polypeptide molecule GGGSPSYKS is synthesized by Nanjing Jinsri company, and the specific modification operation steps are as follows: and (3) measuring a proper amount of polypeptide molecule GGGSPSYKS solution (10 mg/ml, pH value is 9.5) and fatty acid chain solution (10 mg/ml) according to a molar ratio of 1:1.2, and quickly and uniformly mixing the solution and the fatty acid chain solution according to a volume ratio of 1:1 (the insufficient volume of the polypeptide molecule is complemented by carbonate buffer solution with pH value of 9.5), and stirring the mixture at room temperature for reaction time of 15-30min to obtain a coupling product A. Taking a proper amount of GLP-1-FGF21-P4 recombinant protein solution (0.5-1.0 mg/ml, pH value is 8.0), adding a solution of a coupling product A (namely a fatty acid chain modified coupling polypeptide molecule) with the molar number being 10 times that of GLP-1-FGF21-P4, adding a solution of Sortase-A with the molar ratio being 50:1 (GLP-1-FGF 21-P4: sortase-A), finally adding 50mM CaCl 2 solution, enabling CaCl 2 to have the final concentration of 5mM in a coupling reaction system, stirring the coupling reaction at room temperature for 16-24 hours, and terminating the coupling reaction for anion chromatography (GE Q-FF) purification. The purity of the final product is more than 95% by iodine staining SDS-PAGE analysis. The mass spectrum identification result of GLP-1-FGF21 fusion protein after coupling fatty acid chain is shown in FIG. 6.
Example 5 cell Activity assay of GLP-1-FGF21 fusion protein modified with fatty acid chain
To further verify the cellular activity of GLP-1-FGF21-P4-FA (fa= FATTY ACID, fatty acid) modified with fatty acid chains, we examined the cellular activity of this molecule, with specific methods consistent with those described in example 3, and with specific results shown in table 5.
TABLE 5 GLP-1-FGF21-P4-FA Activity assay by fatty acid chain modification
The cell activity experiment shows that the GLP-1-FGF21-P4 fusion protein fused with GLP-1 has similar cell activity to FGF21-P4, but the GLP-1-FGF21-P4-FA activity after being modified by a fatty acid chain is greatly enhanced, and similar activity of wild-type FGF21, namely FGF21-1414 is achieved, probably because the modification of the fatty acid chain changes the conformation of FGF21 and enhances the affinity of the FGF21 to a receptor.
Example 6 enzyme assay of FAP protease on GLP-1-FGF21 fusion protein modified with fatty acid chain
FAP is a protease present in the body that specifically cleaves amino acids 171-172 of FGF21, causing the C-terminus of FGF21 to be injected or otherwise incomplete in the body and thus unable to bind to the beta-klotho receptor in the body, losing activity and tissue specificity. This example uses this property to conduct an in vitro FAP (commercially available from Baipase, cat# FAP-H5244) enzyme assay to test whether GLP-1-FGF21-P4 molecules have the property of slowing down the hydrolysis of FAP protease after modification with fatty acid chains. Experiments were performed according to literature (http:// dx. Doi. Org/10.1016/j. Molset. 2016.07.003) and the results are shown in Table 6, when the negative control FGF21-1414 was completely digested with FAP (i.e.100% digested), only about 20% -30% of GLP-1-FGF21-P4-FA was digested, and the process was not affected by the presence or absence of HSA (human serum albumin).
Table 4 FAP protease enzyme assay for wild type GLP-1-FGF21-1414 and mutant GLP-1-FGF21-P4-FA
Example 7 efficacy experiment of fatty acid chain modified GLP-1-FGF21-P4-FA in DIO mouse model
To further verify the activity of the GLP-FGF21 mutant fusion proteins of the present invention, we selected one of the FGF21 mutant fusion proteins GLP-1-FGF21-P4-FA and used ob/ob mice (SPF grade, kwanas laboratory animal Co., ltd.) of 6-7 weeks old, and after the end of the adaptation period, the high-fat diet was fed for 3 weeks, and the administration period was 4 weeks. After the animals entered the experimental period, the fat DIO mice model was established by feeding the high fat diet for 7 weeks. The specific dosing regimen is as follows:
the group G1 is animals (Lean Mice) fed with common feed, and the total number of the animals is 10 as a control.
Meanwhile, animals fed with the high-fat feed are grouped, day1 is taken as the first Day of administration, serum TC, LDL-C, fasting blood glucose and weight data are taken as grouping basis before administration, the animals are divided into 5 groups, and 10 animals in each group are grouped according to the following specific grouping information: group G1 model (vehicle control group, ob/ob-Neg Ctr), group G2 model (Semaglutide control group, 30nmol/kg, sc, q3 d), group G3 model (GLP-1-FGF 21-P4-FA experimental group, 10nmol/kg, sc, q3 d), group G4 model (GLP-1-FGF 21-P4-FA experimental group, 30nmol/kg, sc, q3 d), group G5 model (GLP-1-FGF 21-P4-FA experimental group, 60nmol/kg, sc, q3 d). And continuously monitoring the change of blood sugar and body weight, and detecting the liver pathological change of each group of mice on the 30 th day to test the influence of molecules on liver and lipid metabolism, wherein the results are shown in table 5 and figures 3-5.
TABLE 5 comparison of total cholesterol TC and Low Density lipoprotein LDL-C at 24 days before and after administration of the different administration groups
Analysis of the data shows that GLP-1-FGF21-P4-FA is significantly better than control molecule Semaglutide in blood glucose, body weight and lipid metabolism, and has obvious dose dependence.
The foregoing has described in detail preferred embodiments of the present invention, which are described in detail. 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 should be included in the protection scope of the present invention.
Claims (14)
1. A GLP-1-FGF21 fusion protein modified with a fatty acid chain, wherein the GLP-1-FGF21 fusion protein comprises GLP-1 or an active fragment thereof, a linker, a FGF21 variant or an active fragment thereof, wherein the C-terminus of the FGF21 variant or active fragment thereof comprises a-LPXTG motif or any other motif recognizable by the Sortase-a enzyme and wherein the C-terminus comprises a fatty acid chain modification, wherein X is any amino acid, and wherein the linker is a connecting peptide.
2. The fatty acid chain modified GLP-1-FGF21 fusion protein of claim 1, wherein said FGF21 variant is mutated on the basis of the sequence of a wild-type human FGF21 protein, said wild-type human FGF21 having the sequence shown in SEQ ID No. 1.
3. The fatty acid chain modified GLP-1-FGF21 fusion protein of claim 1 or 2, wherein said mutation comprises a substitution of N at position 121 with Q and a substitution of M at position 168 with L of the sequence shown in SEQ ID No. 1, and wherein the FGF21 variant has the sequence shown in SEQ ID No. 4.
4. The fatty acid chain modified GLP-1-FGF21 fusion protein of any one of claims 1-3, wherein said FGF21 variant sequence is as set forth in any one of SEQ ID NOs 5-8; preferably, the FGF21 variant sequence is shown in SEQ ID NO. 8.
5. The fatty acid chain modified GLP-1-FGF21 fusion protein of claim 4, wherein said GLP-1 polypeptide is selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 11 and SEQ ID NO. 13.
6. The fatty acid chain modified GLP-1-FGF21 fusion protein of claim 5, wherein said fatty acid chain modification is coupling a fatty acid chain to the side chain NH 2-of amino acid K at position 180 of the sequence shown in SEQ ID No. 8.
7. The fatty acid chain modified GLP-1-FGF21 fusion protein of any one of claims 1-6, wherein said linker peptide is GGGSGGGGS（SEQ ID NO:14）、（GGGGS）n、（GGGGS）n、SGGGGSGGGG（SEQ ID NO:15）、GGGGGSGGGGSSGGGGS（SEQ ID NO:16）、GGGGSGGGGSGGGG（SEQ ID NO:17）、GGGGSGGGGSGGGSGGGGS（SEQ ID NO:18）、GSPGSSSSGS（SEQ ID NO:19）、GSGSGSGS（SEQ ID NO:20）、GSGSGNGS（SEQ ID NO:21）、GGSGSGSG（SEQ ID NO:22）、GGSGSG（SEQ ID NO:23）、GGSG（SEQ ID NO:24）、GGSGNGSG（SEQ ID NO:25）、GGNGSGSG（SEQ ID NO:26）、GGNGSG（SEQ ID NO:27） or (GGGGGS) n, which may be an integer between 1 and 5; preferably, the connecting peptide is GGGGSGGGGS (SEQ ID NO: 28) or (GGGGS) 4 (SEQ ID NO: 29).
8. The fatty acid chain modified GLP-1-FGF21 fusion protein of any one of claims 1 to 7, wherein the sequence of said GLP-1-FGF21 fusion protein is shown in SEQ ID No. 9 or SEQ ID No. 12.
9. The method for producing a fatty acid chain modified GLP-1-FGF21 fusion protein according to any one of claims 1 to 8, comprising: 1) Obtaining the GLP-1-FGF21 fusion protein, 2) obtaining a coupled polypeptide molecule modified by a fatty acid chain, and performing fatty acid chain modification on the GLP-1-FGF21 fusion protein by using a Sortase-A enzyme.
10. The method of preparing a GLP-1-FGF21 fusion protein of claim 9, wherein said step of fatty acid modification comprises: synthesizing a coupled polypeptide molecule, mixing the coupled polypeptide molecule with a fatty acid chain solution, and stirring at room temperature to obtain a coupled product A.
11. The method of preparing a GLP-1-FGF21 fusion protein of claim 10, wherein said step of fatty acid modification further comprises: mixing the coupling product A with the GLP-1-FGF21 fusion protein, adding a Sortase-A enzyme solution, and stirring at room temperature to obtain the GLP-1-FGF21 fusion protein modified by fatty acid chains; optionally, the fatty acid chain modified GLP-1-FGF21 fusion protein is further purified.
12. The method of any one of claims 10 to 11, wherein the conjugated polypeptide is as shown in SEQ ID No. 10, and the fatty acid chain modification is to conjugate a fatty acid chain to the side chain NH 2-of amino acid K at position 8 of the sequence shown in SEQ ID No. 10.
13. A pharmaceutical composition comprising a safe and effective amount of the fatty acid chain modified GLP-1-FGF21 fusion protein of any one of claims 1-8 or the fatty acid chain modified GLP-1-FGF21 fusion protein prepared by the method of any one of claims 9-12, and a pharmaceutically acceptable carrier.
14. The fatty acid chain modified GLP-1-FGF21 fusion protein of any one of claims 1-8, the method of preparation of any one of claims 9-12, for use in the preparation of a medicament for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, metabolic syndrome, and neurodegenerative diseases, in particular for use in the delay or prevention of disease progression in type 2 diabetes, the treatment of metabolic syndrome, the treatment of obesity or the prevention of overweight, for use in reducing food intake, increasing energy expenditure, reducing weight, delay progression from Impaired Glucose Tolerance (IGT) to type 2 diabetes; controlling blood sugar and blood fat; delay progression from type 2 diabetes to diabetes requiring insulin; regulating appetite; inducing satiety; preventing weight recovery after successful weight loss; treating a disease or condition associated with overweight or obesity; treating bulimia; treating binge eating; the application of the medicine for treating atherosclerosis, hypertension, IGT, dyslipidemia, coronary heart disease, fatty liver and beta-blocker poisoning in inhibiting gastrointestinal tract movement.
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