A sumo tag-based cell-free synthesis system and method

Abstract

The application provides a SUMO tag-based cell-free protein synthesis system and method. By inserting a SUMO tag at one end of a target protein, and using a cell-free protein expression system containing a protease capable of cutting the SUMO tag, the yield and efficiency of protein synthesis can be improved, the process 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 protein synthesis system and method based on SUMO tags. Background Technology

[0002] Cell-free protein synthesis (CFPS) is an in vitro recombinant protein expression technique that uses 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+) to synthesize proteins in vitro. CFPS is suitable for preparing various types of proteins, including difficult-to-express, toxic, and complex proteins. It has gained widespread attention and application in drug research, biomanufacturing, and life sciences, both in research, development, and commercial applications. Currently, CFPS is primarily used in drug development, such as antibody preparation and biopharmaceutical production. Although CFPS systems can perform large-scale protein expression, significant obstacles remain regarding post-expression protein modification and activity activation. For example, since protein synthesis begins with mRNA or DNA templates, these templates can be added to the N-terminus of the protein during CFPS, resulting in a synthesized protein containing an undesirable initiation terminus. By fusing a SUMO tag to the target protein and then digesting the SUMO tag after the fusion protein is expressed, the invalid initiation end of the target protein is also removed, thus solving the above problem.

[0003] In addition, the SUMO tag is a small ubiquitinated protein with a molecular weight of only about 11 kDa. It is often fused to the N-terminus of the target protein to increase the protein's expression level and solubility.

[0004] Furthermore, the SUMO tag can be recognized and cleaved by the protease Ulp1 without leaving any redundant amino acid residues in the target protein. This makes the removal of the SUMO tag more efficient and convenient than other protein tags, making it an ideal protein fusion tag. Currently, the typical method for using the SUMO tag is to first express the fusion protein sequence with the SUMO tag, and then add the SUMO protease to cleave the SUMO tag after expression, thereby obtaining the complete protein.

[0005] However, in the case of large-scale protein synthesis and production, first completing the synthesis of the protein fused with the SUMO tag, and then separately digesting it to obtain the target protein, increases the number of steps and costs in the synthesis of the target protein.

[0006] In addition, although yeast-derived cell extract systems contain the protease Ulp1, in existing technologies, since SUMO is not cleaved during expression, it is often necessary to modify the SUMO sequence. By modifying SUMO, a new SUMO tag that is not cleaved by endogenous SUMO protease can be obtained. However, at the same time, a corresponding new SUMO protease must be designed, which further increases the complexity of the process and the production cost. 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 protein synthesis system and method based on SUMO tags. The system and method of this application 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 protein synthesis system based on a SUMO tag, the system comprising the following components:

[0009] (1) An mRNA or DNA template encoding a protein, wherein the template contains a nucleic acid sequence of the target protein and a nucleic acid sequence of a SUMO tag;

[0010] (2) Cell extracts;

[0011] The system contains a protease capable of cleaving the SUMO tag.

[0012] In a preferred embodiment, in the SUMO-tagged cell-free protein synthesis system, the protease capable of cleaving the SUMO tag is added exogenously or derived from the cell extract.

[0013] In a preferred embodiment, in the SUMO-tagged cell-free protein synthesis system, the 3' or 5' end of the target protein nucleic acid sequence is attached to the nucleic acid sequence of the SUMO tag; preferably at the 5' end.

[0014] In a preferred embodiment, a nucleic acid sequence may or may not be added between the SUMO tag and the nucleic acid sequence of the target protein; preferably, no nucleic acid sequence is added.

[0015] In a preferred embodiment, the cell extract is the supernatant obtained by centrifuging a cell lysate.

[0016] In a preferred embodiment, 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.

[0017] A second aspect of the present invention provides a method for cell-free protein synthesis based on a SUMO tag, wherein the method employs a cell-free protein synthesis system based on a SUMO tag as described in any one of the first aspects.

[0018] In a preferred embodiment, the SUMO-tagged cell-free protein synthesis method includes the following steps:

[0019] (1) Constructing a DNA template: ligating a nucleic acid encoding a SUMO tag to a nucleic acid sequence containing a target protein, wherein the SUMO tag is the SUMO protein shown in SEQ ID NO:1;

[0020] (2) Cell-free protein expression: The constructed DNA template is added to a cell-free synthesis system to express the template and obtain the target protein.

[0021] In a preferred embodiment, the temperature at which cell protein expression is absent in step (2) is 20°C-40°C; preferably 25°C-37°C, more preferably 30°C;

[0022] And / or, the expression time of the cell-free protein is 2-12 h; preferably 2-6 h, more preferably 3-5 h.

[0023] In a preferred embodiment, the volume ratio of DNA template to cell-free synthesis system in step (2) is (1-40):100; preferably (1-10):100, more preferably 1:30.

[0024] Compared with the prior art, the present invention has the following beneficial effects or advantages:

[0025] (1) The cell-free protein synthesis system and method based on SUMO tag provided by the present invention can digest the SUMO tag by the SUMO protease contained in the expression system. Therefore, the target protein can be obtained by expressing the protein and digesting the SUMO tag at the same time in the whole reaction system. 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 in the expression process, the synthesis efficiency is greatly improved.

[0026] (2) Since the SUMO tag of the present invention does not need to remain fused to the target protein after expression, it can provide protein expression or achieve the first expression of certain amino acids. Therefore, for yeast-derived systems, compared with the prior art, there is no need to specifically modify the SUMO tag to prevent it from being digested by enzymes, and there is no need to add exogenous SUMO protease for digestion after expression. The method is simple and easy to operate, makes full use of the advantages of yeast cells in SUMO digestion, and reasonably saves the process and cost of protein synthesis. It is suitable for cell-free synthesis of various proteins and has a wide range of application prospects.

[0027] 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

[0028] Figure 1 The electrophoretic pattern of the expression of the toxin protein in Example 1 is shown.

[0029] Figure 2 The fluorescence values ​​of each group in the toxin protein expression experiment in Example 1 are shown.

[0030] Figure 3 The concentration of the toxin protein after purification in Example 1 is shown.

[0031] Figure 4 The fluorescence values ​​of SGA001b and SGA002b expressed in Example 2 are shown.

[0032] Figure 5 Electrophoretic images of SGA003b and SGA004b expression in Example 2 are shown.

[0033] Figure 6 The activity data of SGA003b and SGA004b in Example 2 are shown.

[0034] Figure 7 The electrophoresis diagrams showing the expression effects of enzyme digestion comparison in Example 3 are displayed; the first lane band is SGA001b, the second band is SGA001b in parallel experiments, and the third band is SGA005b. Detailed Implementation

[0035] This invention, based on extensive and in-depth research and through numerous screenings and experiments, proposes for the first time a method and corresponding synthetic system for improving cell-free protein expression efficiency by adding a SUMO tag. This method involves inserting a SUMO tag into a DNA template containing the target protein. During the expression of the target protein in a cell-free protein system containing SUMO protease, the presence of the SUMO tag significantly increases the expression level of the target protein, while also ensuring the expression of the target protein at its first amino acid, thereby enhancing the biological activity of the target protein.

[0036] The present invention will be further illustrated below with reference to 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 the present invention and existing technologies, and are not limited to the specific descriptions in the embodiments of the present invention.

[0037] Terminology Introduction

[0038] 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.

[0039] In this invention, "cell-free" or "cell-free system" refers to a method of 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 protein synthesis system of this invention allows 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 that provided by cell extracts) is also within the scope of protection of this invention.

[0040] In this invention, one specific operation method of the cell-free protein synthesis system includes, but is not limited to, the cell-free protein synthesis system based on *E. coli* described in WO2016005982A1. Other cited references, and their direct and indirect citations, describing in vitro cell-free protein 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 the "2.1 Systems and Advantages" section (pages 27-28) of the document "Lu, Y. Advances in Cell-Free Biosynthetic Technology. Current Developments in Biotechnology and Bioengineering, 2019, Chapter 2, 23-45," including but not limited to those cited in the references cited in the "2.1 Systems and Advantages" section, 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, CN109423 The in vitro cell-free protein synthesis system and the DNA template construction and amplification methods described in CN1099A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN110093284A, CN110408635A, etc., and the referenced literature, can all be used as the in vitro protein synthesis system and the DNA template construction and amplification methods of the present invention.

[0041] In this invention, "protein" and "protein protein" have the same meaning and are both translated as protein, and can be used interchangeably.

[0042] In this invention, "system" and "structure" are both translated as "system" and can be used interchangeably.

[0043] 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 protein synthesis system to synthesize proteins.

[0044] 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.

[0045] In this invention, "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.

[0046] 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.

[0047] Specifically, the cell-free protein expression system based on the SUMO tag described in this invention comprises the following components:

[0048] (1) An mRNA or DNA template encoding a protein, wherein the template contains a nucleic acid sequence of the target protein and a nucleic acid sequence of a SUMO tag;

[0049] (2) Cell extracts;

[0050] The system contains a protease capable of cleaving the SUMO tag.

[0051] The cell-free protein synthesis system of this invention was discovered accidentally by the applicant during long-term research on cell-free protein synthesis systems. It addresses the problem of invalid starting ends in cell-free protein synthesis products by inserting a SUMO tag. 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. No subsequent cleavage process is required, and the target protein product can be obtained directly, significantly shortening the process flow, greatly simplifying the operation, 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.

[0052] In addition, since the added yeast lysate extract contains natural endogenous SUMO protease, SUMO protein cleavage can be achieved without the need for additional addition in the reaction system, which greatly reduces costs. Furthermore, compared with the existing technology that requires additional gene modification to prevent SUMO tag cleavage during the reaction, this application does not require related gene modification, further reducing the process flow and improving efficiency compared with the existing technology.

[0053] In one example, in the SUMO-tagged cell-free protein synthesis system, the protease capable of cleaving the SUMO tag is added exogenously or derived from the cell extract. The purpose of this invention is to simultaneously cleave the SUMO tag during synthesis; therefore, the system must contain a protease capable of cleaving the SUMO tag. The source of this protease is not strictly limited; it can be an endogenous enzyme contained in the cell extract or added exogenously.

[0054] In one example, in the SUMO-tagged cell-free protein synthesis system, the 3' or 5' end of the target protein is attached with the SUMO tag's nucleic acid sequence; 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.

[0055] 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.

[0056] In one example, the cell extract is the supernatant obtained by centrifuging the cell lysate. The cell lysate refers to the suspension obtained after disrupting the bacterial cells. The specific methods and parameters for cell fermentation and disruption are adjusted according to the bacterial species 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.

[0057] In one 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. More preferably, the Kluyveromyces further comprises: 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.

[0058] 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.

[0059] The SUMO-tagged cell-free protein synthesis system further includes one or more components selected from the group consisting of: a substrate for protein synthesis, a substrate for RNA synthesis, RNA polymerase, magnesium ions, potassium ions, a buffer, an energy regeneration system, polyethylene glycol (PEG) or an analogue thereof, dithiothreitol (DTT), and optionally a solvent, said solvent being water or an aqueous solvent.

[0060] Furthermore, the substrates for the synthesized RNA include: one or a combination of nucleoside monophosphate, nucleoside triphosphate, or nucleoside monophosphate.

[0061] Furthermore, the substrates for the synthesized protein include 20 natural amino acids and non-natural amino acids.

[0062] Furthermore, the magnesium ions are derived from a magnesium ion source selected from the group consisting of magnesium acetate, magnesium glutamate, or a combination thereof.

[0063] Furthermore, the potassium ions are derived from a potassium ion source selected from the group consisting of potassium acetate, potassium glutamate, or a combination thereof.

[0064] Furthermore, the energy regeneration system is selected from the group consisting of: creatine phosphate / creatine phosphate enzyme system, one of the energy systems of intermediate products in the glycolysis pathway, glucose, maltodextrin, sucrose, or combinations thereof.

[0065] Furthermore, the buffer is selected from the group consisting of 4-hydroxyethylpiperazine ethanesulfonic acid, tris(hydroxymethyl)aminomethane, or a combination thereof.

[0066] Furthermore, the cell-free protein synthesis system contains polyethylene glycol (PEG) or its analogues. The concentration of PEG or its analogues is not particularly limited, but typically, the concentration (w / v) of PEG or its analogues 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.

[0067] 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.

[0068] In one example, in the cell-free protein synthesis system provided by the present invention, the yeast cell extract accounts for 50-80% of the total volume of the cell-free in vitro protein synthesis system, or 50%-55%, 50-60%, or 50-65%.

[0069] 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), a mixture of amino acids, 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. 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.

[0070] In one example, the present invention also provides a method for cell-free protein expression based on a SUMO tag, wherein the method employs a cell-free protein synthesis system based on a SUMO tag as described in any of the preceding claims. The method of the present invention is similar to the aforementioned system, both introducing a SUMO tag into a cell-free protein synthesis system using yeast lysate extract as a raw material, thereby directly removing the SUMO tag by enzymatic cleavage after synthesis, thus obtaining a complete and biologically active target protein product, significantly improving production efficiency and reducing costs.

[0071] In a preferred example, the SUMO-tagged cell-free protein synthesis method includes the following steps:

[0072] (1) Constructing a DNA template: ligating a nucleic acid encoding a SUMO tag to a nucleic acid sequence containing a target protein, wherein the SUMO tag is the SUMO protein shown in SEQ ID NO:1;

[0073] (2) Cell-free protein expression: The constructed DNA template is added to a cell-free synthesis system to express the template and obtain the target protein.

[0074] 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 protein synthesis system containing cell extracts, the expression efficiency and the activity of the target protein can be significantly improved.

[0075] In one example, the temperature at which cell expression is absent in step (2) is 20°C-40°C; preferably 25°C-37°C, more preferably 30°C;

[0076] And / or, the time for cell-free expression is 2-12 h; preferably 2-6 h, more preferably 3-5 h.

[0077] As mentioned above, the cell-free protein synthesis method of the present invention has no special requirements and adopts conventional operating methods and parameters.

[0078] In a preferred example, the volume ratio of DNA template to cell-free synthesis system in step (2) is (1-40):100; preferably (1-10):100, more preferably 1:30. 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, and is generally in the range of (1-40):100; for example, (1-30):100; or (1-10):100; or (1-5):100; or 1:30.

[0079] 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.

[0080] Example 1 Immunotoxin (DT) Expression Experiment

[0081] 1.1 Experimental Design:

[0082] Different DNA templates were prepared according to the design schemes shown in Table 1 below. The sequences of each protein component are shown in Table 2.

[0083] Table 1 Immunotoxin DNA Template Design Scheme

[0084]

[0085]

[0086] Table 2. Sequence details of the fusion protein in Example 1

[0087]

[0088]

[0089] 1.2 Experimental Methods

[0090] 1.2.1 Expression of immunotoxins using an in vitro protein synthesis system (30 mL as an example reaction system)

[0091] First, prepare the DNA template for the expression plasmid, i.e., the AMPi amplification system (AMPi amplification system is a product sold on Kangma: product number PROTN_AMPiN10V03500). Take 100uL of AMPi reaction solution and add it to 900uL of ultrapure water. Add approximately 0.5uL of AMPiase enzyme, and then add the different plasmids mentioned above as templates, with a final concentration of 6ng / uL. Incubate overnight at 37℃.

[0092] On the second day, nucleic acid electrophoresis analysis was performed on the AMPi amplification system to confirm that the expression plasmid had been amplified. Then, 1 mL of the AMPi amplification system was added to 30 mL of D2P in vitro protein synthesis system (IVTT) at a ratio of 1:30. The mixture was incubated in a shaker at 30°C for 3-4 hours. 10 μL was then taken for eGFP fluorescence reading (RFU). Two to three replicate experiments were set up for each group.

[0093] 1.2.2 Purification of Immunotoxins

[0094] Take out 0.2% (v / v) of the IVTT volume of Ni magnetic beads and wash them 2-3 times with ultrapure water for later use.

[0095] After the protein synthesis system was incubated for 3 hours, magnetic beads were added to the system, and incubation continued for another hour. The magnetic beads were then removed from the reaction system using a magnet, and eluted once and four times sequentially with 1 mL of wash buffer 1 (500 mM NaCl, 0.4% Tritium-100, 50 mM Tris-HCl, pH 8.0, 5 mM imidazole) and wash buffer 2 (500 mM NaCl, 50 mM Tris-HCl, pH 8.0, 5 mM imidazole). Finally, elution was performed with 0.8 mL of elution buffer (500 mM NaCl, 50 mM Tris-HCl, pH 8.0, 250 mM imidazole).

[0096] The washing buffer and elution buffer were subjected to SDS-PAGE purity analysis, and the corresponding protein concentrations were calculated using the eGFP fluorescence readings.

[0097] 1.3 Experimental Results

[0098] from Figure 1 The electrophoresis results showed that the target protein—immunotoxin—was expressed in all groups, but the expression levels varied. Lanes 4, 5, and 6 showed significantly higher brightness, indicating that the expression levels were significantly higher than the first three groups. In terms of band size, the protein obtained from the groups with the added SUMO tag was similar in size to the molecular weight of the protein after the SUMO tag was removed, indicating that the SUMO tag was effectively removed by enzyme cleavage.

[0099] Table 3 shows the fluorescence data of the proteins expressed by the cell-free protein synthesis system, and Table 4 compares the fold changes of the data in Table 3. Figure 2 This displays the fluorescence values ​​for each group. (See Tables 3 and 4.) Figure 2 A comparison of fluorescence values ​​for protein expression in IVTT showed that all molecules with the SUMO tag had significantly higher expression levels than their corresponding untagged molecules. This demonstrates that adding the SUMO tag can increase the expression level of the target protein in IVTT.

[0100] Table 3 IVTT expression data (GFP fluorescence data)

[0101] Group PDT-18 PDT-25 PDT-27 PDT-69 PDT-76 PDT-78 1 424 306 261 484 475 407 2 465 365 310 540 534 408 average value 444.5 335.5 285.5 512 504.5 407.5

[0102] Table 4 IVTT expression data (fluorescence numerical increase fold)

[0103] Group PDT-69 / PDT-18 PDT-76 / PDT-25 PDT-78 / PDT-27 growth multiple 1.15 1.50 1.43

[0104] Note: This increase factor is the increase in fluorescence value with the SUMO tag added compared to without the SUMO tag added.

[0105] Table 5 shows the concentrations of immunotoxin proteins purified from Ni magnetic beads, while Table 6 further compares the data by fold. Figure 3 The concentration data of the purified toxin protein are shown. According to... Figure 3 As shown in Tables 5 and 6, compared with the protein without the SUMO tag, the protein amount of the molecule with the SUMO tag increased significantly after purification, indicating that the addition of the SUMO tag has a significant effect on increasing the expression level of recombinant protein.

[0106] Table 5. Ni purification data (µM)

[0107] Group PDT-18 PDT-25 PDT-27 PDT-69 PDT-76 PDT-78 Concentration (µM) 4.648 2.333 1.713 9.623 8.189 5.960

[0108] Table 6. Ni purification data (protein quantity increase after purification)

[0109] Group PDT-69 / PDT-18 PDT-76 / PDT-25 PDT-78 / PDT-27 growth multiple 2.07 2.81 3.48

[0110] Note: This increase factor is the increase in fluorescence value with the SUMO tag added compared to without the SUMO tag added.

[0111] Example 2: Glucagon-like peptide (GLP1) expression experiment

[0112] 2.1 Construction of fusion protein expression plasmid

[0113] Different expression vectors were constructed according to the design scheme described in Table 7. The nucleic acid sequences encoding the SUMO tag and the nucleic acid sequences encoding the GLP-1 fusion protein were inserted into the expression vectors to serve as DNA templates. Specifically, the gene sequences of the SUMO tag and GLP-1 or its fusion protein were sequentially inserted into the pD2P 1.08t plasmid (also known as the 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) via PCR. The SUMO tag sequence is the amino acid sequence shown in SEQ ID NO: 2, where M represents the start codon (methionine). For ease of separation and detection, a His tag with 10D was added to the end of each fusion protein. The specific sequences of other parts are shown in Table 8.

[0114] Table 7 GLP1 Expression Design Scheme

[0115]

[0116] Table 8 Sequence Details

[0117]

[0118] 2.2 Comparison of cell-free protein synthesis expression levels of SGA001b and SGA002b

[0119] 2.2.1 Experimental Methods:

[0120] The constructed DNA plasmid vector was amplified using the AMPI amplification system (AMPi amplification system is a product sold on Kangma: product number PROTN_AMPiN10V03500). 100 μL of AMPI reaction solution was added to 900 μL of ultrapure water, along with approximately 0.5 μL of AMPiase enzyme, and the expression plasmid constructed in the previous step was added as a template, resulting in a final concentration of 2 ng / μL. The mixture was incubated overnight at 37°C.

[0121] 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 D2P in vitro protein synthesis system (IVTT) at a volume ratio of 1:30. The mixture was incubated in a shaker at 30°C for 3-4 hours. The RFU values ​​of SGA001b and SGA002b were then measured.

[0122] Conclusion: From Figure 4 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.

[0123] (10D). This indicates that the SUMO tag can increase the expression level of proteins in cell-free protein synthesis systems.

[0124] 2.3 Comparison of the expression of SGA003b and SGA004b

[0125] The AMPi amplification systems for the corresponding plasmids SGA003b and SGA004b were prepared according to the method in section 2.2. Then, 1 mL of the amplified AMPi system was added to 30 mL of D2P in vitro protein synthesis system (IVTT) at a volume ratio of 1:30. The mixture was incubated in a shaker at 30°C for 3-4 hours. After the reaction, Ni magnetic beads (volume ratio 1:100) were added, and the reaction was continued for another 1 hour.

[0126] After the magnetic beads have bound, wash them five times with 30 mL of washing buffer (20 mM imidazole).

[0127] 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.

[0128] 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.

[0129] Aspirate the concentrated protein solution, centrifuge at 12000 rpm for 8 min, collect the supernatant, and determine the protein concentration.

[0130] from Figure 5The 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.

[0131] 2.4 Agonist activation experiment of SGA003b and SGA004b

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] From Table 9 and Figure 6 The activity results showed that the GLP-1R cell activity of the SGA003b fusion protein was higher than that of SGA004b. This indicates that the original transcription template contained a SUMO tag, which enabled the acquisition of GLP1 with the first amino acid as the starting point, thus ensuring the biological activity of the fusion protein.

[0138] Table 9 GLP-1R cell viability

[0139]

[0140] Example 3: Enzyme digestion comparison verification

[0141] An amino acid (P-proline) was inserted between the SUMO tag and GLP1 to link them, preventing SUMO protease cleavage, thus verifying the effect of SUMO tag cleavage on cell-free expression. The specific experimental methods and procedures, as well as the corresponding sequences for each part, were the same as in Example 2.

[0142] Table 10 Experimental Design Scheme

[0143]

[0144]

[0145] Table 11 Protein expression data

[0146]

[0147] Experimental results: From the electrophoresis diagram ( Figure 7 The expression data (Table 11) 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.

[0148] It is evident that by using the cell-free protein synthesis system and method of this application, cleaving the SUMO tag during expression not only does not affect protein expression, but also increases the protein expression level compared to the traditional non-enzymatic cleavage system.

[0149] The above are only some embodiments of the present invention, and the present invention is not limited to the contents of the above embodiments.

[0150] 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 protein synthesis system based on SUMO tags, characterized in that, The system comprises the following components: (1) An mRNA or DNA template encoding a protein, wherein the template contains a nucleic acid sequence of the target protein and a nucleic acid sequence of a SUMO tag; (2) Cell extracts; The system contains a protease capable of cleaving the SUMO tag.

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

3. The cell-free protein synthesis system based on SUMO tags according to claim 1, characterized in that, The target protein nucleic acid sequence has the SUMO tag attached to its 3' or 5' end; preferably at the 5' end.

4. The cell-free protein synthesis system based on SUMO tags according to claim 1 or 2, characterized in that, The SUMO tag may or may not have a linked nucleic acid sequence added between it and the nucleic acid sequence of the target protein; preferably, no linked nucleic acid sequence is added.

5. The cell-free protein synthesis system based on SUMO tags according to any one of claims 1-3, characterized in that, The cell extract is the supernatant obtained by centrifuging the cell lysate.

6. The cell-free protein synthesis system based on SUMO tags according to any one of claims 1-3, characterized in that, 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.

7. A cell-free protein synthesis method based on SUMO tags, characterized in that, The method employs the cell-free protein synthesis system based on the SUMO tag as described in any one of claims 1-5.

8. The method for cell-free protein synthesis based on SUMO tags according to claim 6, characterized in that, Includes the following steps: (1) Constructing a DNA template: ligating a nucleic acid encoding a SUMO tag to a nucleic acid sequence containing a target protein, wherein the SUMO tag is the SUMO protein shown in SEQ ID NO:1; (2) Cell-free protein expression: The constructed DNA template is added to a cell-free synthesis system to express the template and obtain the target protein.

9. The method for cell-free protein synthesis based on SUMO tags according to claim 6, 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.

10. The method for cell-free protein expression based on SUMO tags according to claim 6, characterized in that, In step (2), the volume ratio of DNA template to cell-free synthesis system is (1-40):100.

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