A cell-free synthesis system for synthesizing GLP-1 related single / double / triple target receptor agonist polypeptide or fusion protein and a preparation method thereof

Abstract

The application provides a cell-free synthesis system, a kit and a preparation method thereof for synthesizing a GLP-1 related single / dual / triple target receptor agonist polypeptide or fusion protein. By using a cell-free protein expression system, GLP-1 related variants or fusion proteins and the like can be successfully expressed, and the protein yield and biological activity are high, the production process of the GLP-1 related polypeptide or protein is shortened, and the cost is reduced.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to the field of synthetic biotechnology, and more specifically, to a cell-free synthesis system, kit, and preparation method for synthesizing GLP-1-related single / double / triple receptor agonist peptides or fusion proteins. Background Technology

[0002] Cell-free protein synthesis (CFPS) is an in vitro recombinant protein expression technique that involves synthesizing proteins in vitro using cell lysates containing essential components for protein synthesis, such as ribosomes, transfer RNA, initiation / elongation / termination factors, guanosine triphosphate, adenosine triphosphate, cytidine triphosphate, uridine triphosphate, PO4+, Mg2+, and K+. CFPS is suitable for preparing various types of proteins, including difficult-to-express proteins, toxic proteins, and complex proteins. It has gained widespread attention and application in drug research, biomanufacturing, and life sciences, encompassing research, development, and commercial applications. Currently, CFPS is primarily used in drug development, widely applied to the production of various peptides, proteins, or fusion proteins, such as in antibody preparation and biopharmaceutical production.

[0003] Glucagon-like peptide-1 (GLP-1) is an intestinal hypoglycemic agent derived from the transcription product of the proglucagon gene. It is an endogenous hormone primarily secreted by L cells in the intestine. It stimulates insulin release and reduces glucagon production, thereby lowering blood glucose levels. Studies have also unexpectedly discovered that GLP-1 has significant fat-reducing effects. These significant effects make GLP-1 an important molecule for treating diabetes and weight loss. GIP (Glucose-dependent insulinotropic polypeptide, also called gastric inhibitory peptide) is a linear peptide composed of 43 amino acids, belonging to the secretin and glucagon families. Incretin-stimulated insulin secretion, under the synergistic effect of elevated blood glucose and GIP, increases intracellular cyclic adenosine monophosphate (cAMP) levels in β cells, promoting insulin secretion in a glucose-dependent manner. Therefore, GIP receptor agonists are an important supplement to hypoglycemic therapy. GCG (glucagon) is a hormone secreted by pancreatic α cells that inhibits insulin secretion and promotes glycogenolysis, thereby raising blood glucose levels. The glycemic effect caused by GCG receptor activation can be counteracted by the glucose concentration-dependent insulinotropic effect and glucagon-inhibiting effect caused by GLP-1 receptor activation; conversely, the energy-decomposing effect of GCG receptor activation, combined with the food-reducing effect of GLP-1 receptor activation, can synergistically reduce weight. Due to the high amino acid sequence homology between GCG and GLP-1, the development of dual-receptor agonists is highly feasible.

[0004] Based on the characteristics of GLP-1, GIP, and GCG in lowering blood sugar, many pharmaceutical companies have developed dual-target receptor agonists composed of GLP-1 and GIP or GCG, and there are even successful cases of combining all three as triple-target receptor agonists. Furthermore, they can be fused with other bioactive proteins to prepare fusion proteins with multiple biological functions, which is of great significance in the biopharmaceutical industry.

[0005] However, currently used GLP-1-related receptor agonists or fusion proteins are all produced through cell expression (fermentation) or artificial synthesis. Protein products prepared by fermentation methods contain many impurities, have long production cycles, and require sophisticated purification processes. Artificial synthesis methods also suffer from high costs and low yields. In short, current synthesis methods have many shortcomings and are difficult to promote and use.

[0006] Therefore, there is an urgent need to provide a method that can effectively reduce the production cost of GLP-1-related single / double / triple receptor agonist peptides or fusion proteins. Summary of the Invention

[0007] The purpose of this invention is to overcome the problems existing in the prior art and provide a cell-free synthesis system and preparation method for synthesizing GLP-1 related single / double / triple receptor agonist peptides or fusion proteins, which can achieve efficient expression and preparation of biologically active target proteins, while shortening the production cycle and reducing costs.

[0008] The first aspect of this invention provides a cell-free synthesis system for synthesizing GLP-1-related single / dual / triple-target receptor agonist peptides or fusion proteins, characterized in that the system comprises the following components:

[0009] (1) An mRNA or DNA template encoding a protein, wherein the mRNA or DNA template contains a nucleic acid sequence of a polypeptide or fusion protein targeting any one or more of GLP-1, GIP, or GCG;

[0010] (2) Cell extracts;

[0011] The sequence of GLP-1 is the amino acid sequence shown in SEQ ID NO:1, or contains a sequence that has at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:1;

[0012] The GIP sequence is the amino acid sequence shown in SEQ ID NO:2, or contains a sequence that has at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:2;

[0013] The GCG sequence is the amino acid sequence shown in SEQ ID NO:3, or contains a sequence that has at least 80%, 85%, 90%, 95%, 99%, or 100% similarity to SEQ ID NO:3.

[0014] In a preferred embodiment, in the cell-free synthesis system, the fusion protein is a fusion of the GLP-1-related single / double / triple target receptor agonist peptide and an active protein or peptide;

[0015] Preferably, the active protein or polypeptide is selected from one or more of the following: albumin-binding domain, human immunoglobulin IgG4 CH3, superoxide dismutase, fibroblast growth factor, human immunoglobulin IgG4 Fc or fragments thereof, wherein the sequence of the active protein is the amino acid sequence shown in SEQ ID NO: 4-9.

[0016] In a preferred embodiment, the mRNA or DNA template further comprises an amino acid sequence of a SUMO tag; and the system contains a protease capable of cleaving the SUMO tag;

[0017] The amino acid sequence of the SUMO tag is the sequence shown in SEQ ID NO:2, or contains a sequence that is at least 80%, 85%, 90%, 99%, or 100% identical to SEQ ID NO:2; preferably, the SUMO tag is linked to GLP-1, and the nucleic acid sequence of the SUMO tag is at the 5' end of GLP-1.

[0018] In a preferred embodiment, the system further includes one or more components selected from the group consisting of:

[0019] Substrates for protein synthesis, substrates for RNA synthesis, magnesium ions, potassium ions, buffers, energy regeneration systems, polyethylene glycol or its analogues, dithiothreitol, and optional solvents;

[0020] The solvent is water or an aqueous solvent.

[0021] In a preferred embodiment, the energy regeneration system is selected from the creatine phosphate / creatine phosphate enzyme system, the glycolysis pathway and its intermediate product energy system, or a combination thereof; preferably, the energy regeneration system comprises carbohydrates, which are preferably a mixture of glucose and maltodextrin.

[0022] In a preferred embodiment, the concentration of glucose in the cell-free synthesis system is 8.8–128 mmol / L; and the concentration of maltodextrin is 84–500 mmol / L.

[0023] In a preferred embodiment, in the cell-free synthesis system, the volume concentration of the cell extract is 20%-70%, preferably 30%-60%, more preferably 40%-50%, based on the total volume of the cell-free synthesis system.

[0024] In a preferred embodiment, in the cell-free synthesis system, the protease capable of cleaving the SUMO tag is added exogenously or derived from the cell extract.

[0025] In a preferred embodiment, in the cell-free synthesis system, the cell extract is the supernatant obtained by centrifuging a cell lysate;

[0026] The cell extract is selected from one or any combination of bacteria, mammalian cells, human cells, plant cells, yeast cells, and insect cells, preferably from yeast cells, and more preferably from Kluyveromyces oryzae.

[0027] A second aspect of the present invention provides a kit comprising a container and components of the cell-free synthesis system described in the first aspect, located within the container.

[0028] The third aspect of the present invention provides a cell-free synthesis method for GLP-1-related single / double / triple target receptor agonist peptides or fusion proteins, characterized in that the method uses the cell-free synthesis system as described in the first aspect, or the kit as described in the second aspect.

[0029] In a preferred embodiment, the cell-free synthesis method includes the following steps:

[0030] (1) Provide a DNA template: On a nucleic acid sequence encoding a polypeptide or fusion protein that encodes a GLP-1-related single / double / triple target receptor agonist, wherein the GLP-1 sequence is the amino acid sequence shown in SEQ ID NO:1, or contains a sequence that has at least 80%, 85%, 90%, 99% or 100% identity with SEQ ID NO:1.

[0031] The GIP sequence is the amino acid sequence shown in SEQ ID NO:2, or contains a sequence that has at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:2;

[0032] The GCG sequence is the amino acid sequence shown in SEQ ID NO:3, or contains a sequence that has at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:3;

[0033] The SUMO tag is the amino acid sequence shown in SEQ ID NO:2, or contains a sequence that is at least 80%, 85%, 90%, 99%, or 100% identical to SEQ ID NO:2;

[0034] (2) Cell-free protein expression: The constructed DNA template is added to a cell-free synthesis system to express the template and obtain the product.

[0035] Preferably, the mRNA or DNA template further contains an amino acid sequence of a SUMO tag, and the cell-free synthesis system further contains a protease capable of cleaving the SUMO.

[0036] In a preferred embodiment, in the cell-free synthesis method,

[0037] The temperature at which cell-free protein expression is absent in step (2) is 20℃-40℃;

[0038] And / or, the expression time of the cell-free protein is 2-12 h;

[0039] And / or, in step (2), the volume ratio of the DNA template to the cell-free synthesis system is (1-40):100.

[0040] Compared with the prior art, the present invention has the following beneficial effects or advantages:

[0041] (1) This invention provides a cell-free synthesis system for synthesizing GLP-1-related single / double / triple receptor agonist peptides or fusion proteins. It utilizes the characteristic that DNA can be transcribed and translated extracellularly. By simulating the intracellular environment and providing sufficient substrates and energy, DNA or RNA templates can be transcribed and translated smoothly, thereby synthesizing proteins or peptides corresponding to DNA or RNA templates. Compared with intracellular synthesis, this method can produce highly active GLP-related single / double / triple receptor agonist peptides or fusion proteins more efficiently, saving time and conveniently. It breaks the limitations of microbial fermentation or artificial synthesis to prepare GLP-1 and its related proteins, greatly improves production efficiency, and provides favorable conditions for the promotion and utilization of GLP-1 and its related proteins.

[0042] (2) The cell-free synthesis system provided by the present invention inserts a SUMO tag into the RNA or DNA template. Since the SUMO protease contained in the expression system can digest the SUMO tag, the target protein can be obtained by expressing the protein and digesting the SUMO tag at the same time. It is not necessary to add exogenous SUMO protease for digestion after expression, thereby shortening the protein production cycle and reducing the production cost. Compared with the existing situation where no digestion is performed during expression, the synthesis efficiency is greatly improved.

[0043] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0044] Figure 1 The electrophoretic pattern and cell viability of GLP-1 synthesized without cell protein expression in Example 1 are shown.

[0045] Figure 2 The electrophoretic pattern and cell activity of the GLP1-ABD fusion protein synthesized in Example 2 without cell protein expression are shown.

[0046] Figure 3 The electrophoretic pattern and cell activity of the GLP1-CH3 fusion protein synthesized in Example 3 without cell protein expression are shown.

[0047] Figure 4 The electrophoretic pattern and cell activity of the GLP1-ABD-CH3 fusion protein synthesized in Example 4 using cell-free protein expression are shown.

[0048] Figure 5 The electrophoretic pattern and cell activity of the GLP1-ABD-FGF21 fusion protein synthesized in Example 5 using cell-free protein expression are shown.

[0049] Figure 6 The electrophoretic pattern and cell activity of the GLP1-SOD fusion protein synthesized in Example 6 without cell protein expression are shown.

[0050] Figure 7 The electrophoretic pattern and cell activity of the GLP1-ABD-SOD fusion protein synthesized in Example 7 using cell-free protein expression are shown.

[0051] Figure 8 The electrophoretic pattern and cell activity of the GLP1-Fc fusion protein synthesized in Example 8 without cell protein expression are shown. Figure 9 The fluorescence values ​​of SGA001b and SGA002b expressed in Example 9 are shown.

[0052] Figure 10 Electrophoretic images of SGA003b and SGA004b expression in Example 9 are shown.

[0053] Figure 11 The activity data of SGA003b and SGA004b in Example 9 are shown.

[0054] Figure 12 The electrophoresis diagram showing the expression effect of enzyme digestion comparison in Example 10 is shown; the first lane band is SGA001b, the second band is SGA001b parallel experiment, and the third band is SGA005b.

[0055] Figure 13 Cell viability data for the positive control group (commercially available GLP-1) are shown.

[0056] Figure 14 Cell viability data for the negative control group (commercially available BSA) are shown. Detailed Implementation

[0057] This invention, based on extensive and in-depth research and through numerous screenings and explorations, proposes for the first time a cell-free synthetic system and its preparation method for synthesizing GLP-1-related receptor agonists. By using a cell-free protein expression system, GLP-related single / dual / triple-target receptor agonist peptides or fusion proteins can be successfully expressed with high protein yield and bioactivity, shortening the production process of GLP-1-related peptides or proteins and reducing costs. The invention is further illustrated below with specific embodiments and examples. For the specific methods or materials used in the examples, those skilled in the art can make conventional substitutions based on the technical concept of this invention and existing technologies, and are not limited to the specific descriptions in the embodiments of this invention.

[0058] Terminology Introduction

[0059] The "GLP-1-related single / dual / triple-target receptor agonists" described in this invention refer to GLP-1 single-target receptor agonists, GLP-1 dual-target receptor agonists, and GLP-1 triple-target receptor agonists, respectively. Specifically:

[0060] "GLP-1 single-target receptor agonists" are drugs that act solely on the GLP-1 receptor, exerting their effects by mimicking or enhancing the biological activity of GLP-1. They can promote insulin secretion, inhibit glucagon release, and slow gastrointestinal motility, thereby helping to lower blood sugar levels and treat diabetes.

[0061] "GLP-1 dual-target receptor agonists" refer to drugs that act on both the GLP-1 receptor and another target simultaneously. Dual-target agonists are mainly of two types: GLP-1R / GIPR and GLP-1R / GCGR. By acting on multiple targets, they synergistically exert effects such as lowering blood sugar, lowering blood pressure, reducing lipids, and improving metabolism, resulting in a more comprehensive therapeutic effect. Different combinations exert different pharmacological effects by acting on different biological processes, playing an important role in personalized diabetes treatment.

[0062] "GLP-1 triple receptor agonist" refers to a drug or treatment strategy that acts simultaneously on the GLP-1 receptor, another diabetes-related receptor, and another target for treating diabetes, such as GLP-1 / GCG / GIP triple target.

[0063] In this invention, GLP-1 has a broad concept, encompassing variants of GLP-1. A variant of GLP-1 refers to a peptide having one or more amino acid sequences different from the amino acid sequence of natural glucagon-like peptide-1 (GLP-1), while exhibiting significant activity against the GLP-1 receptor, and is interchangeable with "analytes." These GLP-1 variants can be modified by substitution, addition, deletion, or modification of a subset of amino acids in natural GLP-1, or a combination thereof. In this invention, GLP-1 and its variants are often used as a whole concept, broadly representing GLP-1 and polypeptides with similar sequences and the same biological activity as GLP-1. Their sequences are the amino acid sequences shown in SEQ ID NO: 1, or closely related sequences, for example, sequences with more than 80% identity.

[0064] The "cell-free protein expression" described in this invention, also known as "cell-free protein synthesis," or "in vitro protein synthesis," or "in vitro cell-free protein synthesis," refers to the reaction that synthesizes proteins in an in vitro cell-free environment synthesis system, including at least the translation process. This includes, but is not limited to, IVT (in vitro translation reaction), IVTT (in vitro transcription-translation reaction), and IVDTT (in vitro replication-transcription-translation reaction). In this invention, the IVTT reaction is preferred. The IVTT reaction, corresponding to the IVTT system, is the process of transcribing and translating DNA into protein in vitro. Therefore, we also refer to this type of in vitro protein synthesis system as the D2P system, D-to-P system, D_to_P system, DNA-to-Protein system, IVTT system, etc.; the corresponding in vitro protein synthesis methods are also called the D2P method, D-to-P method, D_to_P method, DNA-to-Protein method, IVTT reaction, IVTT, etc.

[0065] In this invention, "cell-free" or "cell-free system" refers to in vitro protein synthesis that does not involve secretion and expression by intact cells. It should be noted that while the in vitro cell-free synthesis system of this invention allows for the addition of cellular components to promote the reaction, the added cells are not primarily intended for the secretion and expression of exogenous target proteins. Furthermore, in the D2P system constructed under the guidance of this invention, the intentional addition of a small number of intact cells (e.g., whose protein content does not exceed 30 wt% compared to the protein content provided by cell extracts) is also within the scope of protection of this invention.

[0066] In this invention, one specific operation method of the cell-free synthesis system includes, but is not limited to, the cell-free synthesis system based on *E. coli* described in WO2016005982A1. Other cited references, and their direct and indirect citations, describing in vitro cell-free synthesis systems based on wheat germ cells, rabbit reticulocytes, *Saccharomyces cerevisiae*, *Pichia pastoris*, and *Kluyveromyces martensii*, are also included as embodiments of the in vitro protein synthesis system of this invention. For example, the in vitro cell-free protein synthesis system described in the references cited in "Lu, Y. Advances in Cell-Free Biosynthetic Technology. Current Developments in Biotechnology and Bioengineering, 2019, Chapter 2, 23-45," including but not limited to the references cited on pages 27-28 of section "2.1 Systems and Advantages," can all be used as the in vitro protein synthesis system for implementing this invention. For example (unless otherwise stated, the following documents and their references are cited in whole and in all respects for all purposes): CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN10942 The in vitro cell-free synthesis system and the method for constructing and amplifying DNA templates described in references 3509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN110093284A, CN110408635A, etc., and the references cited therein, can all be used as the in vitro protein synthesis system and the method for constructing and amplifying the DNA template of the present invention.

[0067] In this invention, "transcription" refers to the process of transferring information contained in a DNA nucleic acid sequence to a complementary RNA sequence. "Translation" refers to the process of using the genetic information of messenger RNA (mRNA) to specify and guide the synthesis of polypeptide chains.

[0068] In this invention, "protein" and "protein protein" have the same meaning and are both translated as "protein," and can be used interchangeably. Since polypeptides are also combinations of amino acids, they can be synthesized using the same cell-free synthesis system as proteins. Therefore, for ease of expression, polypeptides are sometimes also classified as proteins. The protein synthesis described in this application, depending on the specific circumstances (e.g., when using a GLP-1 DNA template for translational expression, the resulting protein is a polypeptide), can also refer to polypeptide synthesis.

[0069] In this invention, "system" and "structure" are both translated as "system" and can be used interchangeably.

[0070] In this invention, "expression activity", "synthetic activity", "protein synthesis amount", "protein expression amount" and "protein expression yield" have the same meaning and can be used interchangeably. They all represent the ability of a cell-free synthetic system to synthesize proteins.

[0071] In this invention, the terms "cell extract", "cell extract", "cell extract", "cell lysate", "cell fragments" and "cell lysate" have the same meaning and can be used interchangeably. In English, they can be described as cell extract, cell lysate, etc.

[0072] "Target protein," also known as "purpose protein," "protein to be expressed," etc., refers to the protein product to be obtained through cell-free protein synthesis. Specifically, in this invention, it refers to a GLP-1 receptor agonist.

[0073] In this invention, "vector," "plasmid," "primer," "target gene," "gene," "transformation," "PCR," etc., are all conventional meanings in the prior art and will not be elaborated here.

[0074] Specifically, the present invention provides a cell-free synthesis system for synthesizing GLP-1-related single / dual / triple-target receptor agonist peptides or fusion proteins, the system comprising the following components:

[0075] (1) An mRNA or DNA template encoding a protein, wherein the mRNA or DNA template contains a nucleic acid sequence of a polypeptide or fusion protein targeting any one or more of GLP-1, GIP, or GCG;

[0076] (2) Cell extracts;

[0077] The sequence of GLP-1 is the amino acid sequence shown in SEQ ID NO:1, or contains a sequence that has at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:1;

[0078] The GIP sequence is the amino acid sequence shown in SEQ ID NO:2, or contains a sequence that has at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:2;

[0079] The GCG sequence is the amino acid sequence shown in SEQ ID NO:3, or contains a sequence that has at least 80%, 85%, 90%, 95%, 99%, or 100% similarity to SEQ ID NO:3.

[0080] The cell-free synthesis system of this invention is proposed by the applicant based on long-term research in cell-free synthesis systems. This system uses an mRNA or DNA template containing a nucleic acid sequence of a GLP-1-related single / double / triple-target receptor agonist polypeptide or fusion protein, thereby providing a gene template for transcription and translation to synthesize proteins. The cell extract can provide many factors required for cell-free protein synthesis, such as ribosomes, enzymes, tRNA, amino acids, and other essential biomolecules. These components can mimic the intracellular translation and transcription processes in an in vitro environment, thereby achieving protein synthesis. In this application, the "GLP-1 sequence" includes sequences of GLP-1 and its variants, and is not strictly limited in specific terms. Any sequence similar to GLP-1 and possessing the same biological activity can serve as a gene template for cell-free synthesis. As mentioned above, a "GLP-1 single-target receptor agonist" refers to a drug that acts only on the GLP-1 receptor, exerting its effect by mimicking or enhancing the biological activity of GLP-1.

[0081] In one example, the fusion protein is a fusion of the GLP-1-related single / double / triple target receptor agonist peptide with an active protein or peptide;

[0082] Preferably, the active protein or polypeptide is selected from one or more of the following: albumin-binding domain, human immunoglobulin IgG4 CH3, superoxide dismutase, fibroblast growth factor, human immunoglobulin IgG4 Fc or fragments thereof, wherein the sequence of the active protein is the amino acid sequence shown in SEQ ID NO: 4-9.

[0083] In one example, the mRNA or DNA template further contains an amino acid sequence of a SUMO tag; and the system contains a protease capable of cleaving the SUMO tag.

[0084] The amino acid sequence of the SUMO tag is the sequence shown in SEQ ID NO:2, or contains a sequence that has at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:2; preferably, the SUMO tag is linked to GLP-1, and the nucleic acid sequence of the SUMO tag is at the 5' end of GLP-1.

[0085] To address the potential issue of invalid start ends in cell-free protein synthesis products, a SUMO tag is inserted. However, the SUMO tag is simultaneously cleaved by the SUMO protease in the system after expression and is not retained, ensuring that the structure of the target protein starts from the initial amino acid. This eliminates the need for subsequent cleavage processes, allowing direct acquisition of the target protein product, significantly shortening the process flow, greatly simplifying operations, and reducing costs. Furthermore, the addition of the SUMO tag can further increase protein yield, and compared to existing non-cleavage expression methods, the cleavage in this application results in a higher protein yield.

[0086] In one example, the system further includes one or more components selected from the group consisting of:

[0087] Substrates for protein synthesis, substrates for RNA synthesis, magnesium ions, potassium ions, buffers, energy regeneration systems, polyethylene glycol or its analogues, dithiothreitol, and optional solvents;

[0088] The solvent is water or an aqueous solvent.

[0089] The above-mentioned components are all important components in the cell-free synthesis system obtained by the applicant based on long-term research. Apart from the substrate used for synthesis, the other components all contribute to the smooth reaction of the system and extend the reaction time as much as possible to improve the reaction efficiency.

[0090] In one example, the energy regeneration system is selected from the group consisting of creatine phosphophosphate / creatine phosphokinase systems, glycolysis pathways and their intermediate product energy systems, or combinations thereof. Preferably, the energy regeneration system comprises carbohydrates, preferably a mixture of glucose and maltodextrin. The mixture of glucose and maltodextrin is a combination selected by the applicant that is both economical and possesses excellent ability to sustain the reaction, offering significant advantages over traditional creatine phosphophosphate / creatine phosphokinase systems, etc.

[0091] In a preferred example, the substrate for the synthesized protein comprises 1-20 natural amino acids and non-natural amino acids. The substrate for the synthesized protein is preferably a mixture of amino acids, including at least the mixture of amino acids required for the synthesis of the exogenous protein. Preferably, the amino acid mixture is a mixture of natural amino acids. Representative amino acids include (but are not limited to) 20 natural amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine. The concentration of each amino acid is typically 0.01-0.5 mM, preferably 0.02-0.2 mM, such as 0.05, 0.06, 0.07, or 0.08 mM.

[0092] In a preferred embodiment, the substrate for the synthesized RNA is preferably a mixture of nucleotides selected from: nucleoside monophosphates, nucleoside triphosphates, or combinations thereof. The nucleoside triphosphate mixture is adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate, and uracil nucleoside triphosphate. In another preferred embodiment, in the protein synthesis system, the concentration of the substrate for the synthesized RNA is 0.1-5 mM, preferably 0.5-3 mM, more preferably 1-1.5 mM. The concentration of each mononucleotide is not particularly limited, but typically the concentration of each mononucleotide is 0.5-5 mM, preferably 1.0-2.0 mM.

[0093] In a preferred example, the magnesium ion source is preferably selected from: magnesium aspartate, magnesium acetate, magnesium glutamate, magnesium chloride, magnesium phosphate, magnesium sulfate, magnesium citrate, magnesium hydrogen phosphate, magnesium iodide, magnesium lactate, magnesium nitrate, magnesium oxalate, or combinations thereof; in the protein synthesis system, the concentration of the magnesium ions is 1-10 mM, more preferably 1-5 mM, and even more preferably 2-4 mM.

[0094] In a preferred embodiment, the potassium ion source is preferably selected from: potassium acetate, potassium glutamate, potassium chloride, potassium phosphate, potassium sulfate, potassium citrate, potassium hydrogen phosphate, potassium iodide, potassium lactate, potassium nitrate, potassium oxalate, or combinations thereof. In the protein synthesis system, the concentration of the potassium ions is 30-210 mM, more preferably 30-150 mM, and even more preferably 30-60 mM.

[0095] Inorganic salt ions, including magnesium and potassium ions, are commonly used additives in in vitro protein synthesis systems. These ions play an important role in protein translation, promoting ribosome assembly, improving RNA stability, and facilitating polymerase binding. In a preferred example, the buffer is selected from the group consisting of 4-hydroxyethylpiperazine ethanesulfonic acid, tris(hydroxymethyl)aminomethane, or combinations thereof. The main functions of buffers in cell-free synthesis systems include maintaining a stable pH environment, reducing protein degradation and aggregation, and optimizing ionic strength and charge environment, thereby improving protein synthesis efficiency and purity. 4-hydroxyethylpiperazine ethanesulfonic acid and tris(hydroxymethyl)aminomethane are commonly used buffers. The concentration of the buffer is not strictly limited, typically 5-50 mM, preferably 10-50 mM, more preferably 15-30 mM, and even more preferably 20-25 mM.

[0096] In a preferred example, when the energy regeneration system uses a mixture of glucose and maltodextrin, the concentration of glucose is 8.8-128 mmol / L, preferably 15-50 mmol / L; and the concentration of maltodextrin is 84-500 mmol / L, preferably 100-350 mmol / L.

[0097] The concentration of the polyethylene glycol or its analogue is not particularly limited, but typically the concentration (w / v) of the polyethylene glycol or its analogue is 0.1-8%, preferably 0.5-4%, and more preferably 1-2%, based on the total weight of the protein synthesis system. Representative PEGs are selected from the group consisting of PEG3000, PEG3350, PEG6000, PEG8000, or combinations thereof.

[0098] Furthermore, the polyethylene glycol includes polyethylene glycol with a molecular weight (Da) of 200-10000, such as PEG200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc., and preferably, polyethylene glycol with a molecular weight of 3000-10000.

[0099] In a preferred embodiment, the concentration of dithiothreitol (DTT) in the protein synthesis system is 0.2-15 mM, more preferably 0.2-7 mM, and even more preferably 1-2 mM.

[0100] In a preferred example, the volumetric concentration of the cell extract is 20%-70%, preferably 30%-60%, more preferably 40%-50%, based on the total volume of the cell-free synthesis system.

[0101] In one example, the protease capable of cleaving the SUMO tag is added exogenously or derived from the cell extract. In this invention, the cell-free synthesis system involves simultaneous enzymatic cleavage of the SUMO tag during synthesis. Therefore, the system must contain a protease capable of cleaving the SUMO tag. The source of the protease is not strictly limited; it can be an endogenous enzyme contained in the cell extract or added exogenously.

[0102] In one example, in the cell-free synthesis system based on the SUMO tag, the 3' or 5' end of the target protein is attached with the nucleic acid sequence of the SUMO tag; preferably at the 5' end. To achieve the regulatory effect of the SUMO tag on the expression of the target protein, the SUMO tag is usually inserted at one end of the target protein's nucleic acid sequence, such as the 3' or 5' end, with the 5' end being the most common.

[0103] In one example, a linking nucleic acid sequence may or may not be added between the SUMO tag and the nucleic acid sequence of the target protein; preferably, no linking nucleic acid sequence is added. Considering that one of the purposes of inserting the SUMO tag is to express the target protein without the front-end sequence, in a preferred example, no linking amino acid is added between the SUMO tag and the nucleic acid sequence of the target protein.

[0104] In a preferred example, the cell-free protein expression system comprises: yeast cell extract, mRNA or DNA template encoding the protein, glucose, maltodextrin, tripotassium phosphate, 4-hydroxyethylpiperazine ethanesulfonic acid (Hepes-KOH), potassium acetate, adenine triphosphate (ATP), guanine triphosphate (GTP), cytosine triphosphate (CTP), thymidine triphosphate (TTP), an amino acid mixture, dithiothreitol (DTT), magnesium L-aspartate, polyethylene glycol, and trehalose; the template contains the nucleic acid sequence of the target protein and the nucleic acid sequence of the SUMO tag. The cell-free protein expression system can be self-prepared or directly using a cell-free protein synthesis kit, such as the Fast series and HighYield series products sold by our company.

[0105] In subsequent specific embodiments, the final concentrations of each component in the cell-free expression system (IVTT system) were as follows: 80% (v / v) Kluyveromyces lactis extract, 15 mM glucose, 320 mM maltodextrin (based on glucose monomer molar concentration), 24 mM tripotassium phosphate, 22 mM 4-hydroxyethylpiperazine ethanesulfonic acid at pH 7.4, 1.8 mM nucleoside triphosphate mixture (a mixture of adenine, guanine, cytosine, and uracil triphosphates, each with a final concentration of 1.8 mM), 0.7 mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine, each with a final concentration of 0.7 mM), and 5 mM... L-Aspartate magnesium, 80 mM potassium acetate, 0.44 mM dithiothreitol, 2% (w / v) polyethylene glycol 8000, 9.78 mM pH 8.0 Tris·HCl buffer, and 6% (w / v) trehalose. The Kluyveromyces lactis extract contains endogenously expressed T7 RNA polymerase. Alternatively, cell-free synthesis kits sold by Kangma (Shanghai) Biotechnology, such as the ProteinFactory series, can be used directly.

[0106] In a preferred example, the cell extract is the supernatant obtained by centrifuging a cell lysate;

[0107] The cell disruption solution refers to the suspension obtained after disrupting bacterial cells. The specific methods and parameters for cell fermentation and disruption are adjusted according to the type of bacteria and the actual needs of the metabolites. Specific methods can be obtained by referring to existing technologies; common disruption methods include liquid nitrogen pulverization and mechanical pulverization. Centrifugation mainly aims to remove larger cells or fragments; therefore, there are no strict limitations on the specific operating conditions. Cell extracts can typically be obtained under conditions ranging from 5000g to 20000g. To ensure the activity of various biological components in the cell extract, disruption and centrifugation are usually performed at relatively low temperatures.

[0108] In a preferred example, the cell extract is selected from one or any combination of bacteria, mammalian cells, human cells, plant cells, yeast cells, and insect cells, preferably from yeast cells, and more preferably from Kluyveromyces oryzae.

[0109] The Kluyveromyces further includes: Kluyveromyces lactis, K. lactis, Kluyveromyces marxianus, Kluyveromyces dobzhanskii, Kluyveromyces aestuarii, Kluyveromyces nonfermentans, Kluyveromyces wickerhamii, Kluyveromyces thermotolerans, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polysporus, Kluyveromyces siamensis, and Kluyveromyces yarrowii, or a combination thereof. The preferred choice is to use Kluyveromyces lactis or Kluyveromyces marxi.

[0110] The yeast used in this invention possesses advantages such as simple cultivation, efficient protein folding, and post-translational modification. Currently, *Saccharomyces cerevisiae* and *Pichia pastoris* are commonly used model organisms for expressing complex eukaryotic and membrane proteins. Yeast can also be used as a raw material for preparing in vitro translation systems; that is, yeast extracts can be used for in vitro protein translation expression. *Pichia pastoris* or *Saccharomyces cerevisiae* are commonly used yeasts for in vitro protein expression. *Kluyveromyces* is an ascospore-forming yeast, with *Kluyveromyces marxianus* and *Kluyveromyces lactis* being widely used industrially. Compared to other yeasts, *Kluyveromyces lactis* has many advantages, such as superior secretion capacity, better large-scale fermentation characteristics, food safety standards, and the ability to simultaneously perform post-translational modification, demonstrating significant application potential. In a preferred embodiment of this invention, an in vitro protein expression system based on *Kluyveromyces lactis* is employed.

[0111] In one example, a kit is also provided, comprising a container and components of the cell-free synthesis system described in any of the first aspects located within the container. The kit contains the aforementioned cell-free synthesis system for synthesizing GLP-1 receptor agonists, and by combining all components in the kit form according to the reaction amounts, a one-step reaction can be achieved, offering advantages of convenience and efficiency.

[0112] In one example, a cell-free protein synthesis method for a GLP-1 receptor agonist is also provided, using the cell-free synthesis system or kit described above. The method of this invention is similar to the aforementioned system, expressing GLP-1-related proteins in a cell-free synthesis system using yeast lysate extract as a raw material. It also allows for direct enzymatic removal of the SUMO tag after target synthesis, thereby obtaining a complete and biologically active target protein product, significantly improving production efficiency and reducing costs.

[0113] In a preferred example, the cell-free protein synthesis method of the GLP-1 receptor agonist specifically includes the following steps:

[0114] (1) Constructing a DNA template: Linking the nucleic acid encoding the SUMO tag to a nucleic acid sequence encoding a GLP-1 receptor agonist;

[0115] The sequence of the GLP-1 and its variants therein is the amino acid sequence shown in SEQ ID NO:1, or contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:1;

[0116] The GIP sequence is the amino acid sequence shown in SEQ ID NO:2, or contains a sequence that has at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:2;

[0117] The GCG sequence is the amino acid sequence shown in SEQ ID NO:3, or contains a sequence that has at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:3;

[0118] The SUMO tag is the amino acid sequence shown in SEQ ID NO:2, or contains a sequence that has at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:2;

[0119] (2) Cell-free protein expression: The constructed DNA template is added to a cell-free synthesis system to express the template and obtain the product;

[0120] Preferably, the mRNA or DNA template further contains an amino acid sequence of a SUMO tag, and the cell-free synthesis system further contains a protease capable of cleaving the SUMO.

[0121] By constructing an expression template containing both a SUMO tag and the target protein nucleic acid sequence, and expressing the protein in a cell-free synthesis system containing cell extracts, the expression efficiency and the activity of the target protein can be significantly improved.

[0122] In a preferred example, in the cell-free protein synthesis method,

[0123] The temperature at which cell-free protein expression is absent in step (2) is 20℃-40℃; preferably 25℃-37℃, more preferably 30℃;

[0124] And / or, the expression time of the cell-free protein is 2-12 h; preferably 2-6 h, more preferably 3-5 h.

[0125] And / or, in step (2), the volume ratio of DNA template to cell-free synthesis system is (1-40):100; preferably (1-10):100, more preferably 1:30.

[0126] As mentioned above, the cell-free protein synthesis method of the present invention has no special requirements and adopts conventional operating methods and parameters. The volume ratio of DNA template to cell-free expression system is mainly adjusted according to the concentration of DNA template and other substances in the reaction system, generally in the range of (1-40):100; for example, (1-30):100; or (1-10):100; or (1-5):100; or 1:30.

[0127] Unless otherwise specified, experimental procedures in the following examples are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), and Cell-Free Protein Synthesis Manual (Edited by Alexander S. Spirin and James R. Swartz, Cell-free protein ynthesis: methods and protocols [M]. 2008), or as recommended by the manufacturer, or as per the specific implementation guidelines described above. Unless otherwise stated, percentages and parts mentioned in this invention are weight percentages and parts by weight.

[0128] Unless otherwise specified, the reagents and materials used in the embodiments of this invention are all commercially available products.

[0129] In the following examples, no additional SUMO protease was added to digest SUMO; instead, the SUMO protease naturally present in yeast cell extracts was used.

[0130] Examples 1-8: Expression experiments of glucagon-like peptide-1 (GLP-1) and its fusion protein

[0131] (1) DNA template construction: The nucleic acid sequences encoding the SUMO tag and the nucleic acid sequences encoding GLP-1 or its fusion protein were inserted into the expression vector to construct the DNA template. Specifically, the gene sequences of the SUMO tag and GLP-1 or its fusion protein were inserted sequentially into the pD2P 1.08t plasmid (also known as pD2P_1.08t plasmid, or simply 1.08t, developed by Kangma (Shanghai) Biotechnology Co., Ltd., specifically for cell-free protein synthesis using the ProteinFactory kit) by PCR. The constructed DNA plasmid vector was amplified using the AMPI amplification system (AMPi amplification system is a Kangma product: product number PROTN_AMPiN10V03500). The experimental designs for each embodiment are shown in Table 1, where the SUMO tag sequence is the amino acid sequence shown in SEQ ID NO: 2, M represents the start codon (methionine), and the amino acid sequences of other parts are shown in Table 7.

[0132] Table 1 Experimental Design Schemes for Each Embodiment

[0133]

[0134]

[0135] (2) Cell-free protein expression and purification:

[0136] Amplified DNA plasmids were added to a cell-free synthesis system (IVTT system) to conduct an in vitro protein synthesis reaction. The reaction solution was placed in an environment of 30°C. After 3 hours, nickel magnetic beads (at a ratio of 1:100) were added to the reaction solution, and the reaction solution and nickel magnetic beads were allowed to bind for 1 hour.

[0137] After the protein binds to the magnetic beads, pour the reaction solution into a 50mL centrifuge tube and use a magnetic suction cup to pick up the beads. Pour the supernatant into a waste container. Continue this process until all the magnetic beads are adsorbed into the centrifuge tube.

[0138] Wash the magnetic beads five times with 30 mL of washing buffer (1 x PBS, 20 mM imidazole), vortexing for 5 minutes each time.

[0139] Add elution buffer (containing 1x PBS and 250mM imidazole) to the magnetic beads and elute the target protein for about 5 minutes to obtain a certain volume of eluent. Centrifuge the eluent and place it in a dialysis bag for overnight dialysis for 16 hours. Concentrate the protein dialysis solution to 1 mL using an ultrafiltration tube.

[0140] Aspirate the concentrated protein solution, centrifuge at 12,000 rpm for 8 minutes, collect the supernatant, and determine the protein concentration using Nanodrop.

[0141] (3) Cell viability detection

[0142] One day in advance, the H_GLP1R Reporter HEK-293T cells to be infected were seeded into 96-well cell culture plates at a seeding density of 1.5 × 10⁴ cells per well; the cell density was approximately 30% the day before the experiment.

[0143] The following day, the original concentration of the test protein was diluted 10-fold with Assay buffer, and then continuously diluted with Assay buffer in 10-fold increments, for a total of nine dilutions and ten gradients.

[0144] Remove the 96-well plate containing H_GLP1R Reporter HEK-293T cells from the incubator. After confirming the cell density and condition, aspirate the supernatant. Add 100 μL of protein dilution to each well along the well wall, being careful not to disturb the cells. Incubate the 96-well plate in the incubator for 7 hours.

[0145] After 7 hours, aspirate the culture medium and carefully add 100 μL of 1x PBS to each well to rinse the cells, avoiding washing away the cells. Carefully aspirate the PBS and add 35 μL of 1x cell lysis buffer to each well, then incubate at room temperature for 15–20 minutes. After 15–20 minutes, immediately test the luciferase activity, or store at -20°C.

[0146] Mix 5 μL of cell lysate with 5 μL of luciferase substrate (Promega E1501 luciferase detection system), add the mixture to a 384-well plate, and immediately detect luciferase activity using a Perkin Elmer EnVision 2102 multi-plate reader.

[0147] Similarly, commercially available pure GLP-1 was used as a positive control group to detect cell viability; in addition, commercially available bovine serum albumin (BSA) was used as a negative control group to verify the effectiveness of the experimental group and the positive control group.

[0148] Experimental results:

[0149] The concentrations of peptides or proteins obtained in each embodiment and their cell activity data are shown in Table 2.

[0150] Table 2 Experimental results of each embodiment

[0151]

[0152]

[0153] (1) According to the test results, the commercially available GLP-1 control group ( Figure 13 The IC50 value of the cell viability of the ) was 0.05607 nM, while that of the negative control group BSA ( Figure 14 The cell viability of the cells was zero.

[0154] (2) Figure 1 Protein electrophoresis results showed a protein band of approximately 10 kDa. Given that the molecular weight of GLP-1 is 6.5 kDa, and considering the small band size and potential error in electrophoresis, the band was identified as GLP-1. The purified protein concentration was 0.26 mg / mL, and the IC50 value for cell viability was 0.02567 nM. This demonstrates the validity of the results from both the experimental and positive control groups, indicating that GLP-1 prepared using a cell-free protein synthesis method exhibits significant cell viability.

[0155] (3) Figure 2 The protein electrophoresis results showed a protein band of approximately 10 kDa. Given that the molecular weight of GLP1-ABD is 11.8 kDa, and considering the small band size and potential error in electrophoresis, the band can be identified as GLP1-ABD. The purified protein concentration was measured to be 2.896 mg / mL, and the IC50 value for cell viability was 0.1115 nM. Therefore, cell-active GLP1-ABD was successfully prepared using a cell-free protein synthesis method.

[0156] (4) Figure 3 The protein electrophoresis results showed a protein band of approximately 20 kDa. Given that the molecular weight of GLP1-CH3 is 19.6 kDa, the band was identified as GLP1-CH3. The purified protein concentration was 2.664 mg / mL, and the IC50 value for cell activity was 0.01333 nM. Therefore, GLP1-CH3 with cellular activity was successfully prepared using a cell-free protein synthesis method.

[0157] (5) Figure 4The protein electrophoresis results showed a protein band of approximately 25 kDa. Given that the molecular weight of GLP1-ABD-CH3 is 24.9 kDa, the band was identified as GLP1-ABD-CH3. The purified protein concentration was 0.2484 mg / mL, and the IC50 value for cell viability was 0.3304 nM. Therefore, cell-active GLP1-ABD-CH3 was successfully prepared using a cell-free protein synthesis method.

[0158] (6) Figure 5 The protein electrophoresis results showed a protein band of approximately 33 kDa. Given that the molecular weight of GLP1-ABD-FGF21 is 33.4 kDa, the band was identified as GLP1-ABD-FGF21. The purified protein concentration was 2.885 mg / mL, and the IC50 value for cell viability was 1.325 nM. Therefore, cell-active GLP1-ABD-FGF21 was successfully prepared using a cell-free protein synthesis method.

[0159] (7) Figure 6 The protein electrophoresis results showed a protein band of approximately 20 kDa. Given that the molecular weight of GLP1-SOD is 21.4 kDa, the band was identified as GLP1-SOD. The purified protein concentration was 1.34 mg / mL, and the IC50 value for cell activity was 0.115 nM. This demonstrates that a high concentration of GLP1-SOD with good cell activity can be prepared using a cell-free protein synthesis method.

[0160] (8) Figure 7 The protein electrophoresis results showed a protein band of approximately 25 kDa. Given that the molecular weight of GLP1-ABD-SOD is 26.6 kDa, the band was identified as GLP1-ABD-SOD. The purified protein concentration was 1.45 mg / mL, and the IC50 value for cell viability was 0.1555 nM. Therefore, a high concentration of GLP1-ABD-SOD fusion protein with cellular viability was successfully prepared using a cell-free protein synthesis method.

[0161] (9) Figure 8 The protein electrophoresis results showed a protein band of approximately 33 kDa. Given that the molecular weight of GLP1-Fc is 33.1 kDa, the band can be identified as GLP1-Fc. The purified protein concentration was measured to be 3.524 mg / mL, with an IC50 value of 1.62 nM. This demonstrates that a cell-free protein synthesis method can be used to prepare a high concentration of GLP1-Fc fusion protein with good cellular activity.

[0162] Example 9: Verification of the Function of the SUMO Tag

[0163] (1) Construction of fusion protein expression plasmid

[0164] Different expression vectors were constructed according to the design scheme described in Table 3 to serve as DNA templates. In all experimental groups, the SUMO tag sequence is the amino acid sequence shown in SEQ ID NO:2, where M represents the start codon (methionine). The specific sequences of other parts are shown in Table 7. For convenient separation and detection, a His tag with 10D was added to the end of each fusion protein. The specific construction method is as described in Example 1.

[0165] Table 3. Example 9GLP-1 fusion protein expression design scheme

[0166]

[0167] (2) Comparison of cell-free protein synthesis expression levels of SGA001b and SGA002b

[0168] Experimental methods:

[0169] First, prepare the AMPi amplification system for the expression plasmid (AMPi amplification system is a product sold on Kangma: product number PROTN_AMPiN10V03500). Take 100 μL of AMPi reaction solution and add it to 900 μL of ultrapure water. Add approximately 0.5 μL of AMPiase enzyme, and then add the expression plasmid constructed in the previous step as a template, with a final concentration of 2 ng / μL. Incubate overnight at 37°C.

[0170] DNA gel analysis was performed on the AMPi amplification results. After the expression plasmid was amplified, 1 mL of the amplified AMPi amplification system was added to 30 mL of cell-free protein synthesis system (IVTT system) at a volume ratio of 1:30. The mixture was incubated in a shaker at 30℃ for 3-4 h. The RFU values ​​of SGA001b and SGA002b were then measured.

[0171] Conclusion: From Figure 8 The expression levels showed that the protein expression level of SGA001b (M-SUMO-GLP1-ABD035-EGFP-8H-10D) containing the SUMO tag was higher than that of SGA002b (M-GLP1-ABD035-EGFP-8H-10D) without the SUMO tag.

[0172] (10D). This indicates that the SUMO tag can increase the expression level of proteins in cell-free synthesis systems.

[0173] (3) Comparison of the expression of SGA003b and SGA004b

[0174] Prepare the AMPi amplification system for the corresponding plasmids SGA003b and SGA004b according to the method in step (2). Then, add 1 mL of the amplified AMPi system to 30 mL of D2P in vitro protein synthesis system (IVTT) at a volume ratio of 1:30. Incubate in a shaker at 30℃ for 3-4 h. After the reaction, add Ni magnetic beads (volume ratio of 1:100) and continue the reaction for 1 h.

[0175] After the magnetic beads have bound, wash them five times with 30 mL of washing buffer (20 mM imidazole).

[0176] Elution solvent (containing 1x PBS, 250mM imidazole) was added to the magnetic beads to elute the protein. The elution time was 5 min. After centrifugation, the eluent was placed in a dialysis bag and dialyzed overnight for 16 h.

[0177] Take ultrafiltration centrifuge tubes with different molecular cutoff values, add 5 mL of 0.2 M sodium hydroxide, soak for 5 min, clean the inner wall of the ultrafiltration tube by blowing and rinsing, rinse several times with ultrapure water, centrifuge at 4000 rpm for 5 min, remove the water inside the ultrafiltration tube, and concentrate the protein dialysate to 1 mL.

[0178] Aspirate the concentrated protein solution, centrifuge at 12000 rpm for 8 min, collect the supernatant, and determine the protein concentration.

[0179] from Figure 9 The Cobb staining gel images of SGA003b and SGA004b show that the bands of both SGA003b and SGA004b correspond to the fusion protein, indicating that both groups expressed the constructed protein. Furthermore, the band size also shows that the SGA003b group successfully removed the front-end sequence, including the SUMO tag, through enzyme digestion during the reaction.

[0180] (4) Agonist activation experiment of SGA003b and SGA004b

[0181] One day in advance, the H_GLP1R Reporter HEK-293T cells to be infected were seeded into 96-well cell culture plates at a seeding density of 1.5 × 10⁴ cells per well; the cell density was approximately 30% the day before the experiment.

[0182] The following day, the original concentrations of the test proteins (SGA003b and SGA004b) were diluted 10-fold with test buffer, and then continuously diluted with test buffer in 10-fold gradients, for a total of nine dilutions and ten gradients.

[0183] Remove the 96-well plate containing H_GLP1R Reporter HEK-293T cells from the incubator. After confirming the cell density and condition, aspirate the supernatant. Add 100 μL of protein dilution to each well along the well wall, being careful not to disturb the cells. Incubate the 96-well plate in the incubator for 7 hours.

[0184] After 7 hours, aspirate the culture medium and carefully add 100 μL of 1×PBS to each well to rinse the cells, avoiding washing away the cells. Carefully aspirate the PBS and add 35 μL of 1× cell lysis buffer to each well, then incubate at room temperature for 15–20 minutes. After 15–20 minutes, immediately test the luciferase activity, or store at -20°C.

[0185] Mix 5 μL of cell lysate with 5 μL of luciferase substrate (Promega E1501 luciferase detection system), add the mixture to a 384-well plate, and immediately detect luciferase activity using a Perkin Elmer EnVision 2102 multi-plate reader.

[0186] From Table 4 and Figure 10 The activity results showed that the GLP-1R cell activity of the SGA003b fusion protein was higher than that of the SGA004b fusion protein. This indicates that the original transcription template contained a SUMO tag, which enabled the production of a GLP1 fusion protein with the first amino acid as the starting point, thus ensuring the biological activity of the fusion protein.

[0187] Table 4 GLP-1R cell viability

[0188]

[0189] Example 10 Enzyme digestion comparison verification

[0190] An amino acid (P-proline) was inserted between the SUMO tag and GLP1 to prevent SUMO protease cleavage, thus verifying the effect of SUMO tag cleavage on cell-free expression. For ease of isolation and detection, a 10D His tag was added to the end of each fusion protein, and the specific experimental methods and procedures were the same as in Example 9. The sequences of each part of the protein molecule were identical to the corresponding parts in Example 9.

[0191] Table 5 Experimental Design Scheme of Example 10

[0192]

[0193]

[0194] Table 6 Protein expression data

[0195]

[0196] Experimental results: From the electrophoresis diagram ( Figure 11 The expression data (Table 6) show that the SUMO tag was removed by enzyme digestion after expression of the SGA001b protein, while the SGA005b group, due to the insertion of proline (P) between the SUMO tag and the target protein sequence, could not be digested by SUMO enzyme during expression, resulting in the target protein containing the SUMO tag. The protein expression level in the SGA005b group was reduced, and the concentration of the purified protein was also significantly lower than that in the SGA001b group.

[0197] It is evident that by using the cell-free synthesis system and method of this application, cleaving the SUMO tag during the expression process can assist in the expression of the first amino acid. This not only does not affect protein expression, but also increases the protein expression level compared to the traditional non-enzymatic cleavage system.

[0198] Table 7 Summary of sequences involved in this paper

[0199]

[0200]

[0201] The above are only some embodiments of the present invention, and the present invention is not limited to the contents of the above embodiments.

[0202] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing description of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A cell-free synthesis system for synthesizing GLP-1-related single / dual / triple receptor agonist peptides or fusion proteins, characterized in that, The system comprises the following components: (1) An mRNA or DNA template encoding a protein, wherein the mRNA or DNA template contains a nucleic acid sequence of a polypeptide or fusion protein targeting any one or more of GLP-1, GIP, or GCG; (2) Cell extracts; The sequence of GLP-1 is the amino acid sequence shown in SEQ ID NO:1, or contains a sequence that has at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:1; The GIP sequence is the amino acid sequence shown in SEQ ID NO:2, or contains a sequence that has at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:2; The GCG sequence is the amino acid sequence shown in SEQ ID NO:3, or contains a sequence that has at least 80%, 85%, 90%, 95%, 99%, or 100% similarity to SEQ ID NO:

3.

2. The cell-free synthesis system according to claim 1, characterized in that: in, The fusion protein is a fusion of the GLP-1-related single / double / triple target receptor agonist peptide with an active protein or peptide; Preferably, the active protein or polypeptide is selected from one or more of the following: albumin-binding domain, human immunoglobulin IgG4 CH3, superoxide dismutase, fibroblast growth factor, human immunoglobulin IgG4 Fc or fragments thereof, wherein the sequence of the active protein is the amino acid sequence shown in SEQ ID NO: 4-9.

3. The cell-free synthesis system according to claim 1 or 2, characterized in that, The mRNA or DNA template further contains an amino acid sequence of a SUMO tag; and the system contains a protease capable of cleaving the SUMO tag; the amino acid sequence of the SUMO tag is the sequence shown in SEQ ID NO:2, or contains a sequence that has at least 80%, 85%, 90%, 99%, or 100% identity with SEQ ID NO:2; Preferably, the SUMO tag is linked to GLP-1, and the nucleic acid sequence of the SUMO tag is located at the 5' end of GLP-1.

4. The cell-free synthesis system according to any one of claims 1-3, characterized in that, The system also includes one or more components selected from the group consisting of: Substrates for protein synthesis, substrates for RNA synthesis, magnesium ions, potassium ions, buffers, energy regeneration systems, polyethylene glycol or its analogues, dithiothreitol, and optional solvents; The solvent is water or an aqueous solvent.

5. The cell-free synthesis system according to claim 4, characterized in that, The energy regeneration system is selected from the phosphocreatine / phosphocreatine enzyme system, the glycolysis pathway and its intermediate product energy system, or a combination thereof; preferably, the energy regeneration system contains carbohydrates, and the carbohydrates are preferably a mixture of glucose and maltodextrin.

6. The cell-free synthesis system according to claim 5, characterized in that, The concentration of glucose is 8.8–128 mmol / L; the concentration of maltodextrin is 84–500 mmol / L.

7. The cell-free synthesis system according to claim 1 or 2, characterized in that, The volumetric concentration of the cell extract is 20%-70%, preferably 30%-60%, more preferably 40%-50%, based on the total volume of the cell-free synthesis system.

8. The cell-free synthesis system according to claim 1 or 2, characterized in that, in, The protease capable of cleaving the SUMO tag is added exogenously or derived from the cell extract.

9. The cell-free synthesis system according to any one of claims 1-3, characterized in that, The cell extract is the supernatant obtained by centrifuging the cell lysate. The cell extract is selected from one or any combination of bacteria, mammalian cells, human cells, plant cells, yeast cells, and insect cells, preferably from yeast cells, and more preferably from Kluyveromyces oryzae.

10. A reagent kit, characterized in that, The kit includes a container and components of the cell-free synthesis system according to any one of claims 1-8 located within the container.

11. A cell-free method for synthesizing GLP-1-related single / dual / triple-target receptor agonist peptides or fusion proteins, characterized in that, The method employs the cell-free synthesis system as described in any one of claims 1-8, or the kit as described in claim 9.

12. The cell-free synthesis method according to claim 11, characterized in that, Includes the following steps: (1) Provide a DNA template: a nucleic acid sequence encoding a polypeptide or fusion protein containing a single / double / triple target receptor agonist peptide encoding GLP-1, wherein the GLP-1 sequence is the amino acid sequence shown in SEQ ID NO:1, or contains a sequence that has at least 80%, 85%, 90%, 99% or 100% identity with SEQ ID NO:

1. The GIP sequence is the amino acid sequence shown in SEQ ID NO:2, or contains a sequence that has at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:2; The GCG sequence is the amino acid sequence shown in SEQ ID NO:3, or contains a sequence that has at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:3; The SUMO tag is the amino acid sequence shown in SEQ ID NO:2, or contains a sequence that is at least 80%, 85%, 90%, 99%, or 100% identical to SEQ ID NO:2; (2) Cell-free protein expression: The constructed DNA template is added to a cell-free synthesis system to express the template and obtain the product; Preferably, the mRNA or DNA template further contains an amino acid sequence of a SUMO tag, and the cell-free synthesis system further contains a protease capable of cleaving the SUMO.

13. The cell-free synthesis method according to claim 11 or 12, characterized in that, The temperature at which cell-free protein expression is absent in step (2) is 20℃-40℃; And / or, the expression time of the cell-free protein is 2-12 h; And / or, in step (2), the volume ratio of the DNA template to the cell-free synthesis system is (1-40):100.

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