Reaction system and method for cell-free synthesis of brazzein

Abstract

The application provides a reaction system and method for synthesizing a cell-free sweet protein, characterized by comprising: 1) a cell-free reaction system; and 2) a nucleic acid encoding the sweet protein. The system and method can be used to synthesize various sweet proteins, which indicates that the system and method disclosed by the application are universal for the synthesis of sweet proteins, and the sweet proteins purified by the system and method have a high concentration. In addition, the method is simple and the reaction conditions are mild, and is particularly suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the cell-free synthesis of sweet proteins. Background Technology

[0002] Lifestyle-related diseases, such as hypertension, hyperlipidemia, diabetes, and obesity, are serious and increasingly prevalent problems worldwide. Various efforts have been made to prevent these diseases. The introduction of low-calorie sweeteners is one promising potential approach to overcoming this problem.

[0003] Low-calorie sweeteners (sugar substitutes) are widely used in food, mainly including sucrose, aspartame, sorbitol, xylitol, mannitol, cyclamate, o-benzoyl sulfimide, and steviol glycosides. Safety challenges and debates surrounding these sweeteners continue, although extensive reviews by government agencies in many countries and regions have consistently concluded that they are safe when used as intended. In addition, these sweeteners also have drawbacks such as being unsuitable for most cooking and baking applications and having poor sensory quality.

[0004] As people place increasing emphasis on a healthy lifestyle, the market demand for low-calorie sweeteners is also growing. Sweet proteins that can be digested into amino acids are receiving increasing attention.

[0005] Sweet proteins are a class of proteins that can produce a sweet taste. Similar to sucrose, sweet proteins induce a sweet taste through T1R2-T2R3 receptors. Currently known sweet proteins mainly include thaumatin (including Thaumatin I and Thaumatin II), Monellin, Mabinlin, Brazzein, egg whitelysozyme, Curculin (Neoculin), and Miraculin.

[0006] Miracle fruit protein (also known as kiwifruit protein) is a 191-amino acid protein found in red berries native to West Africa, with an oligosaccharide linked to its amino terminus. Miracle fruit protein itself is tasteless, but it can transform sour tastes into a sweetness similar to that of curculigo acid protein. Sun et al. obtained an active recombinant protein in transgenic lettuce, with a yield of 33.7–43.5 μg / kg fresh lettuce.

[0007] Curculin, a sweet protein isolated from C. latifolia in western Malaysia, is a homodimer formed by two 114-amino acid residues of NBS (Basic Subunit), and its protein structure is still unknown. In recent years, researchers have discovered a new gene in C. latifolia and named it Neoculin Acidic Subunit (NAS), a 113-amino acid polypeptide with an amino-terminal glycosylated structure. The heterodimer protein obtained by fusing the NAS and NBS genes and expressing it in A. oryzae is called Neoculin, which exhibits strong sweetness-inducing activity and a sweet taste.

[0008] Egg white lysozyme is a 129-amino acid single-chain protein, classified into four types (C, G, V, and λ) based on its amino acid sequence and substrate specificity. Type C has a sweet taste, with a sweetness threshold of 10 μM.

[0009] Areca catechu protein is a sweet-tasting protein extracted from the seeds of *Capparis masaikai* Levi, a plant in the Brassicaceae family, from high-altitude provinces such as Yunnan, my country. It exists in at least four types (I-1 / II / III / IV), with type II showing no significant decrease in sweetness at 80℃ for 48 hours. Amino acid sequencing revealed that areca catechu protein II is a heterodimer formed by a 33-amino acid A subunit and a 72-amino acid B subunit.

[0010] Thaumatine is derived from the West African plant *Thaumatococcus daniellii*. Thaumatine I and Thaumatine II are the two most important sweet proteins mentioned above. At the same molar concentration, its sweetness is 100,000 times that of sucrose, with a sweetness threshold below 50 nM. Thaumatine itself is a high-calorie sweetener, but the amount used is negligible. Thaumatine is a single-chain protein with 207 amino acids. It is stable during high-temperature processes such as pasteurization and baking at pH < 5.5, but aggregates and loses its sweetness at 70°C when pH > 7. Thaumatine can be expressed by bacteria such as *E. coli*, fungi such as yeast and filamentous fungi, and transgenic plants.

[0011] Brazil nuts, a single-chain protein first isolated from *P. brazzeana* by Ming and Hellekant in 1994, contain 54 amino acids and are currently the smallest known sweet protein. Local indigenous peoples have used Brazil nuts for centuries. At the same molar concentration, its sweetness is 9500 times that of sucrose. Brazil nuts contain four disulfide bonds, resulting in high protein stability; they retain their sweetness even after heating at 98°C for 2 hours or 80°C for 4.5 hours under pH conditions of 2.5–8.0. Both the amino acid termini and carboxyl termini are important for the sweetness of Brazil nuts. They are generally expressed using *E. coli* or yeast, or synthesized in solid phase using the Fmoc strategy.

[0012] Monetine was first isolated from the fruit of the tropical plant *Dioscoreophyllum cumminsii* Diels, and consists of a 44-amino acid A chain and a 50-amino acid B chain. Given the proximity of the N-terminus of the A chain and the C-terminus of the B chain, they were genetically engineered to form a single-chain protein with no intramolecular disulfide bonds. Its sweetness is 90,000 times that of sucrose at the same molar concentration. It can be expressed in *E. coli*, yeast, and transgenic plants, and can also be synthesized in a solid-phase manner.

[0013] Cell-free protein expression (IVTT) is an in vitro recombinant protein expression technology that uses cell lysates containing essential components for protein synthesis to synthesize proteins in vitro. Compared to traditional cell-based protein synthesis techniques, cell-free protein synthesis avoids many problems associated with traditional techniques because it does not require a cell system, such as low expression efficiency, difficulty in protein purification, and cytotoxicity. Its advantages are also obvious, including: 1. High efficiency: Cell-free protein expression technology can achieve large-scale protein preparation in a short time, greatly improving screening efficiency. 2. High purity: The purity of cell-free protein expression technology can reach ~90%, meeting the requirements of industrial production. 3. Flexibility: Cell-free protein expression technology allows for optimization of the expression system by adjusting reaction conditions or reactants, providing greater flexibility for scientific research and industrial applications.

[0014] Currently, the large-scale industrial production of sematrandini, monomelin, and brassinolide is still in the exploratory stage. The expression of sweet proteins is mainly achieved through intracellular expression, which suffers from problems such as low protein expression levels, complex processes, low recovery rates, long production cycles, and high costs. Furthermore, there is currently no mature process for expressing sweet proteins using cell-free synthesis methods. Summary of the Invention

[0015] To address the aforementioned shortcomings in the industrial production of sweet proteins, this invention utilizes cell-free in vitro protein synthesis technology (IVTT) to synthesize sweet proteins in large quantities in vitro, offering advantages such as simple process, short production cycle, and low cost.

[0016] The first aspect of the present invention provides a reaction system for the cell-free synthesis of sweet proteins, characterized in that it comprises: 1) a cell-free reaction system; and 2) nucleic acid encoding the sweet protein.

[0017] In another preferred embodiment, the cell-free reaction system includes at least a cell-free extract.

[0018] In another preferred embodiment, the cell extract is preferably selected from any of the following sources: Escherichia coli, yeast cells, mammalian cells, plant cells, insect cells, or a combination thereof.

[0019] In another preferred embodiment, the cell extract is more preferably selected from any of the following sources: Escherichia coli, wheat germ cells, insect cells, rabbit reticulocytes, CHO cells, COS cells, VERO cells, BHK cells, human fibrosarcoma HT1080 cells, or combinations thereof.

[0020] In another preferred embodiment, the yeast cells are selected from Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichiaminuta, Ogataeaminuta, Pichia lindneri, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichiapijperi, Pichiastiptis, Pichia methanolica, Pichiasp., and Saccharomyces cerevisiae. The yeasts include: cerevisiae, brewer's yeast, sugarcane molasses yeast, Saccharomyces sp., Hansenula polymorpha, Candida utilis, Kluyveromyces, or a combination thereof.

[0021] In another preferred embodiment, 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 yaros. One or a combination of (yarrowii); preferably, the yeast cell is a Kluyveromyces cell, more preferably a Kluyveromyces lactis cell.

[0022] In another preferred embodiment, the yeast cell extract is an aqueous extract of yeast cells.

[0023] In another preferred embodiment, the yeast cell extract does not contain yeast endogenous long-chain nucleic acid molecules.

[0024] In another preferred embodiment, the yeast cell extract is prepared using a method comprising the following steps:

[0025] (i) Provide yeast cells;

[0026] (ii) Wash the yeast cells to obtain washed yeast cells;

[0027] (iii) The washed yeast cells are subjected to cell-breaking treatment to obtain crude yeast extract; and

[0028] (iv) The crude yeast extract is subjected to solid-liquid separation to obtain the liquid fraction, which is the yeast cell extract.

[0029] In another preferred embodiment, centrifugation is performed in a liquid state.

[0030] In another preferred embodiment, the centrifugation conditions are 5000-100000g, more preferably 8000-30000g.

[0031] In another preferred embodiment, the centrifugation time is 0.5 min–2 h, more preferably 20–50 min.

[0032] In another preferred embodiment, the centrifugation is performed at 1-10°C, more preferably at 2-6°C.

[0033] In another preferred embodiment, the washing treatment is carried out using a washing solution at a pH of 7-8 (preferably 7.4).

[0034] In another preferred embodiment, the washing liquid is selected from the group consisting of potassium 4-hydroxyethylpiperazine ethanesulfonate, potassium acetate, magnesium acetate, or combinations thereof.

[0035] In another preferred embodiment, the cell disruption treatment includes high-pressure disruption and freeze-thaw (e.g., liquid nitrogen cryogenic) disruption.

[0036] In another preferred embodiment, the reaction system further comprises one or more components selected from the following: a buffer, potassium ions, magnesium ions, polyethylene glycol, an optional aqueous solvent, and a phosphate.

[0037] In another preferred embodiment, the buffer is selected from the group consisting of Tris-HCl, Tris base, HEPES, Tris-citric acid, citric acid-citrate, Tris-citrate, or a combination thereof.

[0038] In another preferred embodiment, the potassium ions are derived from a potassium ion source, which is not particularly limited and is selected from the group consisting of potassium acetate, potassium glutamate, potassium citrate, or a combination thereof.

[0039] In another preferred embodiment, the potassium ion concentration is 30-210 mM, more preferably 30-150 mM, and even more preferably 30-80 mM.

[0040] In another preferred embodiment, the magnesium ions are derived from a magnesium ion source, which is not particularly limited, and the magnesium ion source is selected from one or a combination of magnesium acetate, magnesium glutamate, magnesium citrate, and magnesium aspartate.

[0041] In another preferred embodiment, the polyethylene glycol is selected from the group consisting of PEG3000, PEG8000, PEG6000, PEG3350, or combinations thereof.

[0042] In another preferred embodiment, the phosphate is selected from orthophosphate, dihydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, or combinations thereof; orthophosphate is preferred.

[0043] In another preferred embodiment, the concentration v / v of the cell extract is 20-80%.

[0044] In another preferred embodiment, the concentration (w / v) of the polyethylene glycol is 0.1-8%, more preferably 0.5-4%, and even more preferably 1-2%.

[0045] In another preferred embodiment, the sweet protein includes one or more of semasin, monetin, areca nut protein, Brazil nut, egg white lysozyme, curculigo syrup protein, or miracle fruit protein.

[0046] In another preferred embodiment, the semathymidine is selected from semathymidine I and semathymidine II.

[0047] In another preferred embodiment, the sweet protein includes wild-type, mutant, recombinant, and fusion proteins.

[0048] In another preferred embodiment, the sweet protein structure includes the sequence of SEQ ID NO: 1 to 4; or includes a polypeptide having ≥85%, ≥90%, ≥95%, ≥97%, ≥98%, or ≥99% homology to the amino acid sequence shown in SEQ ID NO: 1 to 4 and having the same activity as the sequence of SEQ ID NO: 1 to 4.

[0049] A second aspect of the present invention provides a method for cell-free synthesis of a sweet protein, characterized by comprising: step 1), providing a cell-free reaction system containing the cell-free reaction system described in the first aspect of the present invention; and step 2), adding nucleic acid encoding a sweet protein into the cell-free reaction system to synthesize the sweet protein.

[0050] In another preferred embodiment, the synthesis method further includes: step 3), a purification process.

[0051] In another preferred embodiment, magnetic beads are used to purify the sweet protein.

[0052] In another preferred embodiment, the sweet protein is further purified by desalting.

[0053] In another preferred embodiment, a desalting column is used for desalting purification. Attached Figure Description

[0054] Figure 1 The image shows the electrophoresis result of the extracted plasmid (1.8% agarose), from left to right: Marker, PTMI-4, PTMII-4, PBZ-4, PMN-94.

[0055] Figure 2 This is an SDS-PAGE electrophoresis image showing the reduction of nickel affinity proteins, from left to right: Marker, PTMI-4, PTMII-4, PBZ-4, and PMN-94. Detailed Implementation

[0056] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0057] The present invention will be further described below with reference to specific embodiments and examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions should preferably be performed according to the conditions indicated in the specific embodiments described above, and then may be performed under conventional conditions or as recommended by the manufacturer.

[0058] Unless otherwise stated, percentages and parts mentioned in this invention are weight percentages and weight parts.

[0059] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available products.

[0060] Unless otherwise specified, all temperature units in this application are Celsius (°C).

[0061] Nouns and terms

[0062] The following are explanations or descriptions of the meanings of some relevant "nouns" and "terms" used in this invention to facilitate a better understanding of the invention. These explanations or descriptions apply to the entire text of this invention, both below and above. When references are made in this invention, the definitions of relevant terms, nouns, and phrases in the referenced documents are also cited; however, in case of conflict with the definitions in this invention, the definitions in this invention shall prevail. The conflict between the definitions in the referenced documents and the definitions in this invention does not affect the application of the cited components, substances, compositions, materials, systems, formulations, types, methods, equipment, etc., which shall be determined in the referenced documents.

[0063] In this invention, preferred embodiments such as “preferred,” “better,” “more preferred,” “even better,” “most preferred,” and “further preferred” do not constitute any limitation on the scope of the invention or its protection scope, and are not intended to limit the scope and implementation of the invention, but are only used to provide some embodiments as examples.

[0064] In the description of this invention, terms such as "one preferred method," "one preferred embodiment," "one preferred example," "preferred example," "in a preferred embodiment," "in some preferred examples," "in some preferred methods," "preferred," "preferred," "more preferred," "more preferably," "further preferred," and "most preferred," as well as illustrative enumerations such as "one embodiment," "one method," "example," "specific example," "for instance," "as an example," "for example," "like," etc., do not constitute any limitation on the scope or protection of the invention. The specific features described in each method are included in at least one specific embodiment of this invention. In this invention, the specific features described in each method can be combined in any suitable manner in one or more specific embodiments. In this invention, the technical features or technical solutions corresponding to each preferred method can also be combined in any suitable manner.

[0065] In this invention, "any combination thereof" means "greater than 1" in quantity and "a group consisting of the following situations in terms of scope: "any one of them, or a group consisting of at least two of them".

[0066] In this invention, the descriptions of "one or more", "one or more", etc., have the same meaning as "at least one", "at least one", "a combination thereof", "or a combination thereof", "and a combination thereof", "or any combination thereof", "and any combination thereof", etc., and can be used interchangeably to indicate a quantity equal to "1" or "greater than 1".

[0067] In this invention, "or / and" or "and / or" means "either one or a combination thereof", or at least one of them.

[0068] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0069] Sequence identity (or homology) is determined by comparing two aligned sequences along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of a reference nucleotide sequence or protein) and determining the number of positions where identical residues occur. This is typically expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

[0070] The prior art methods described in this invention using terms such as "usually", "conventionally", "generally", "frequently", and "often" are also cited as references to the content of this invention. Unless otherwise specified, they can be regarded as one of the preferred embodiments of some technical features of this invention. It should be noted that this does not constitute any limitation on the scope of coverage and protection of the invention.

[0071] All documents mentioned in this invention, and those directly or indirectly cited by such documents, are incorporated herein by reference as if each document were cited individually.

[0072] 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 (including but not limited to embodiments) can be combined with each other to form new or preferred technical solutions, as long as they can be used to implement this invention. Due to space limitations, they will not be described in detail here.

[0073] The "mutation" described in this invention includes the insertion, deletion, and substitution of amino acids.

[0074] In vitro protein synthesis refers to the synthesis of proteins in a cell-free in vitro synthesis system, including at least the translation process. This includes, but is not limited to, IVT (in vitro translation), IVTT (in vitro transcription-translation), and IVDTT (in vitro replication-transcription-translation). 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 a D2P system, D-to-P system, or DNA-to-Protein system; and the corresponding in vitro protein synthesis methods are also referred to as D2P methods, D-to-P methods, or DNA-to-Protein methods.

[0075] "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 in the in vitro cell-free protein synthesis system of this invention, the addition of cellular components to promote the reaction is also permitted, but 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.

[0076] D2P, DNA-to-Protein, refers to the process of converting a DNA template into a protein product. Examples include D2P technology, D2P systems, D2P methods, and D2P kits.

[0077] The terms "expression system of the present invention," "cell-free reaction system," "in vitro expression system of the present invention," "in vitro cell-free expression system," and "in vitro cell-free expression system" are used interchangeably and all refer to the in vitro protein expression system of the present invention. Other descriptive methods may also be used, such as: in vitro protein synthesis system, in vitro protein synthesis system, cell-free system, cell-free protein synthesis system, cell-free in vitro protein synthesis system, in vitro cell-free protein synthesis system, in vitro cell-free synthesis system, CFS system (cell-free system), CFPS system (cell-free protein synthesis system), etc. Depending on the reaction mechanism, it may include an in vitro translation system (which can be abbreviated as IVT system, a type of mR2P system), an in vitro transcription-translation system (which can be abbreviated as IVTT system, a type of D2P system), an in vitro replication-transcription-translation system (which can be abbreviated as IVDTT system, a type of D2P system), etc. In this invention, the IVTT system is preferred. We also refer to the in vitro protein synthesis system as a "protein factory" (or "protein factory"). The in vitro protein synthesis system provided by this invention uses an open-ended description of its components. The cell-free protein synthesis system of the present invention uses exogenous DNA, mRNA or a combination thereof as nucleic acid templates for protein synthesis, and achieves in vitro synthesis of target proteins by artificially controlling the addition of substrates and transcription and translation-related protein factors required for protein synthesis.

[0078] Target protein: The target expression product of the in vitro protein synthesis system of this invention is not synthesized by host cell secretion, but is synthesized in vitro based on an exogenous nucleic acid template, and can also be called an exogenous protein. The exogenous protein can be a protein, a fusion protein, or a mixture containing protein molecules or fusion protein molecules; it also broadly includes polypeptides. The product obtained after the in vitro protein synthesis reaction based on the nucleic acid template encoding the target protein can be a single substance or a combination of two or more substances. "Exogenous protein," "target protein," "target protein," and "target translation product" have the same meaning and can be translated as "objective protein," "interested protein," "objective translated product," "interested protein product," etc., and can be used interchangeably in this invention.

[0079] In this invention, "protein" and "protein protein" have the same meaning and are both translated as protein, and can be used interchangeably.

[0080] In this invention, "wild-type protein" refers to a protein that is common in nature and has not undergone mutation.

[0081] In this invention, "mutant protein" is a protein encoded by a gene with a mutation. These mutations can alter the structure and function of the protein, thereby affecting its behavior and role in the cell.

[0082] In this invention, "recombinant protein" refers to a protein obtained by using recombinant DNA or recombinant RNA technology.

[0083] In this invention, a "fusion protein" refers to a protein product obtained by connecting the coding regions of two or more genes end-to-end using genetic engineering techniques, and expressing the resulting gene under the control of the same regulatory sequence. Such proteins typically consist of at least two domains encoded by separate genes, which are then linked together as a single unit and transcribed and translated to produce a polypeptide.

[0084] In this invention, "system" and "structure" are both translated as "system" and can be used interchangeably.

[0085] In this invention, "protein synthesis amount", "protein expression amount" and "protein expression yield" have the same meaning and can be used interchangeably.

[0086] In this invention, cell extract, cell extract solution, 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.

[0087] In this invention, the terms "energy system," "energy system," and "energy supply system" have equivalent meanings and can be used interchangeably. Similarly, "energy regeneration system" and "energy regeneration system" have equivalent meanings and can be used interchangeably. An energy regeneration system is a preferred embodiment or component of an energy system.

[0088] Furthermore, the present invention provides a cell-free protein synthesis system, comprising at least cell extracts or cell lysates.

[0089] More preferably, the cell-free protein synthesis system further includes one or more components selected from the group consisting of: a substrate for RNA synthesis, a substrate for protein synthesis, polyethylene glycol or an analogue thereof, magnesium ions, potassium ions, a buffer, RNA polymerase, an energy regeneration system, dithiothreitol, and optionally an aqueous solvent.

[0090] More preferably, the substrate for the synthesized RNA includes one or a combination of nucleoside monophosphate, nucleoside triphosphate, or nucleoside triphosphate.

[0091] More preferably, the substrate for the synthesized protein includes 20 natural amino acids and non-natural amino acids.

[0092] More preferably, the magnesium ions are derived from a magnesium ion source, which is selected from the group consisting of magnesium acetate, magnesium glutamate, or a combination thereof.

[0093] More preferably, the potassium ions are derived from a potassium ion source, which is selected from the group consisting of potassium acetate, potassium glutamate, or a combination thereof.

[0094] More preferably, the energy regeneration system is selected from the group consisting of: creatine phosphate / creatine phosphate enzyme system, glycolysis pathway and its intermediate product energy system, or a combination thereof.

[0095] More preferably, the energy regeneration system includes a glucose / phosphate system, wherein the phosphate is selected from the group consisting of tripotassium phosphate, triammonium phosphate, trisodium phosphate, dipotassium hydrogen phosphate, diammonium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, ammonium dihydrogen phosphate, sodium dihydrogen phosphate, or a combination thereof.

[0096] More preferably, the buffer is selected from the group consisting of 4-hydroxyethylpiperazine ethanesulfonic acid, tris(hydroxymethyl)aminomethane, or a combination thereof.

[0097] Further preferably, the in vitro protein synthesis system contains polyethylene glycol (PEG) or an analogue thereof. The concentration of PEG or an analogue thereof is not particularly limited, but typically, the concentration (w / v) of PEG or an analogue thereof is 0.1-8%, more preferably 0.5-4%, and even 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] More preferably, 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 more preferably, polyethylene glycol with a molecular weight of 3000-10000.

[0099] In this invention, the RNA polymerase is not particularly limited and can be selected from one or more RNA polymerases, with T7 RNA polymerase being a typical RNA polymerase.

[0100] An optional approach is that the in vitro protein synthesis system provided by the present invention includes: cell extract, 4-hydroxyethylpiperazine ethanesulfonic acid, potassium acetate, magnesium acetate, adenine nucleoside triphosphate (ATP), guanine nucleoside triphosphate (GTP), cytosine nucleoside triphosphate (CTP), thymidine nucleoside triphosphate (TTP), a mixture of amino acids, creatine phosphate, dithiothreitol (DTT), creatine phosphate kinase, and RNA polymerase.

[0101] In this invention, the cell extract does not contain intact cells. Typical cell extracts include ribosomes for protein translation, aminoacyl-tRNA synthetase, initiation and elongation factors required for protein synthesis, and termination release factors. Furthermore, the cell extract also contains other proteins derived from the cytoplasm of cells, especially soluble proteins.

[0102] In this invention, the proportion of the cell extract in the in vitro cell-free protein synthesis system is not particularly limited. Typically, the cell extract accounts for 20-70% of the system, preferably 30-60%, and more preferably 40-50%.

[0103] In this invention, the protein content of the cell extract is 20-100 mg / mL, preferably 50-100 mg / mL. The method for determining the protein content is the Coomassie Brilliant Blue assay.

[0104] This invention also provides a vector or combination of vectors containing the nucleic acid constructs of this invention. Preferably, the vector is selected from bacterial plasmids, bacteriophages, yeast plasmids, animal cell vectors, and shuttle vectors; the vector is a transposon vector. Methods for preparing recombinant vectors are well known to those skilled in the art. Any plasmid and vector can be used as long as it can replicate and remain stable in the host.

[0105] Those skilled in the art can use well-known methods to construct expression vectors containing the promoter and / or target gene sequence described in this invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc.

[0106] Template DNA

[0107] Template DNA is a nucleotide sequence encoding any target protein to be synthesized. It can be a primitive sequence, an artificially synthesized sequence, or an artificially modified sequence. The corresponding RNA and / or protein can be synthesized using this template DNA.

[0108] In this invention, the preparation method of the cell extract is not limited, but a preferred preparation method is described below.

[0109] Includes the following steps:

[0110] (i) Provide cells;

[0111] (ii) The cells are washed to obtain washed cells;

[0112] (iii) The washed cells are subjected to cell-breaking treatment to obtain crude cell extract;

[0113] (iv) The crude cell extract is subjected to solid-liquid separation to obtain the liquid fraction, which is the cell extract.

[0114] In this invention, the solid-liquid separation method is not particularly limited, but centrifugation is a preferred method.

[0115] In a preferred embodiment, the centrifugation is performed in a liquid state.

[0116] In this invention, the centrifugation conditions are not particularly limited, but a preferred centrifugation condition is 5000-100000g, and more preferably, 8000-30000g.

[0117] In this invention, the centrifugation time is not particularly limited, but a preferred centrifugation time is 0.5 min to 2 h, and more preferably, 20 min to 50 min.

[0118] In this invention, the temperature of the centrifugation is not particularly limited. Preferably, the centrifugation is carried out at 1-10°C, and more preferably, at 2-6°C.

[0119] In this invention, the washing treatment method is not particularly limited. A preferred washing treatment method is to use a washing solution at a pH of 7-8 (preferably 7.4). The washing solution is not particularly limited, and a typical washing solution is selected from the group consisting of potassium 4-hydroxyethylpiperazine ethanesulfonate, potassium acetate, magnesium acetate, or a combination thereof.

[0120] In this invention, the method of cell disruption is not particularly limited, but a preferred method of cell disruption includes high-pressure disruption and freeze-thaw (e.g., liquid nitrogen cryogenic) disruption.

[0121] The nucleoside triphosphate mixture in the in vitro cell-free protein synthesis system comprises adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate, and uracil nucleoside triphosphate. In this invention, the concentration of each mononucleotide is not particularly limited; typically, the concentration of each mononucleotide is 0.5-5 mM, preferably 1.0-2.0 mM.

[0122] The amino acid mixture in the in vitro cell-free protein synthesis system may include natural or non-natural amino acids, and may include D-type or L-type 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.

[0123] In a preferred embodiment, the in vitro cell-free protein synthesis system further contains polyethylene glycol or its analogues. The concentration of polyethylene glycol or its analogues is not particularly limited, but typically, the concentration (w / v) is 0.1-8%, more preferably 0.5-4%, and even more preferably 1-2%, based on the total weight of the biosynthetic system. Representative examples of PEG include (but are not limited to): PEG3000, PEG8000, PEG6000, and PEG3350. It should be understood that the system of the present invention may also include polyethylene glycols of various other molecular weights (such as PEG200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc.).

[0124] In a preferred embodiment, the in vitro cell-free protein synthesis system further contains sucrose. The concentration of sucrose is not particularly limited, but typically it is 0.03-40 wt%, more preferably 0.08-10 wt%, and even more preferably 0.1-5 wt%, based on the total weight of the protein synthesis system.

[0125] A particularly preferred in vitro cell-free protein synthesis system, in addition to yeast cell extract, contains the following components: 22 mM 4-hydroxyethylpiperazine ethanesulfonic acid at pH 7.4, 30-150 mM potassium acetate, 1.0-5.0 mM magnesium acetate, 1.5-4 mM nucleoside triphosphate mixture, 0.08-0.24 mM amino acid mixture, 25 mM creatine phosphate, 1.7 mM dithiothreitol, 0.27 mg / mL creatine phosphate kinase, 1%-4% polyethylene glycol, 0.5%-2% sucrose, and 0.027-0.054 mg / mL T7 RNA polymerase (provided by endogenous expression or exogenous addition; for endogenous expression of T7 RNA polymerase, see patent CN109423496B).

[0126] This invention uses Kluyveromyces lactis (K. lactis) as an example, but the same design, analysis, and experimental methods are also applicable to other yeasts, animal cells, eukaryotic cells, and prokaryotic cells.

[0127] An in vitro protein synthesis reaction mixture system, also described as an in vitro protein synthesis reaction mixture, reaction mixture system, or reaction mixture, refers to a mixture system including an in vitro protein synthesis system and a nucleic acid template encoding the target protein; it can be homogeneous or heterogeneous, and is allowed to be a liquid system such as a solution, emulsion, or suspension.

[0128] The relevant sequence names and sequence information involved in this invention are shown in Tables 1 and 2 below.

[0129] Table 1. Sequence List of Sweet Proteins

[0130]

[0131]

[0132] Table 2 Primer sequence list

[0133]

[0134] Example 1: Plasmid preparation (using PBZ-4 as an example)

[0135] (1) PCR amplification was performed using primers 1 and 2 to obtain the vector fragment, and PCR amplification was performed using primers 3 and 4 to obtain the target gene.

[0136] (2) The amplified vector fragment and the target gene were mixed at a ratio of 3:7, and one-tenth of the volume of Dpn I enzyme was added. The mixture was incubated at 37°C for 5 hours before transformation. The transformed bacterial culture was spread evenly on a plate containing ampicillin and cultured overnight. Single clones were then picked for sequencing. After confirming the strain by first-generation sequencing, the culture was expanded by shaking, and plasmids were extracted. The same steps (or methods) were performed on PMN94, PTMI-4, and PTMII-4. The plasmid concentrations were measured as shown in Table 3, and the extracted plasmids were subjected to electrophoresis (1.8% agarose) (see [reference]). Figure 1 The results showed that the size of the target plasmid was consistent with the expected size, indicating that the plasmid preparation was successful and the target plasmid was obtained.

[0137] Table 3: Plasmid Extraction Concentration Determination

[0138] Molecular name Concentration (ng / μL) <![CDATA[OD 260 / 280 ]]> <![CDATA[OD 260 / 230 ]]> PMN94 90.05 2.00 2.072 PBZ-4 110.9 1.947 2.102 PTMI-4 108.85 1.93 2.139 PTMII-4 124.1 1.932 2.196

[0139] Wherein, OD260 represents the absorbance of nucleic acids, OD280 represents the absorbance of proteins, OD230 represents the absorbance of pollutants, and OD... 260 / 280 The ratio of nucleic acid to protein, with a ratio of approximately 1.8 to 2.0, indicates high purity of the nucleic acid; OD 260 / 230 The ratio of nucleic acid to contaminants is approximately 2.0, further demonstrating the high purity of the nucleic acid.

[0140] Example 2: Expression and purification of sweet proteins (using sweet protein PBZ-4 as an example)

[0141] 1. Preparation of DNA amplification products

[0142] The extracted plasmid obtained in Example 2 was added to AMPi (produced by Kangma Company, model PROTN-APMiN10V00360) at a final concentration of 2 ng / μL, mixed well, and incubated overnight at 37°C to prepare the DNA amplification product solution.

[0143] 2. IVTT and protein purification.

[0144] The final concentrations of each component in the IVTT reaction system described in this embodiment are as follows: 80% (v / v) Kluyveromyces lactis extract, 15 mM glucose, 320 mM maltodextrin (molar concentration based on glucose monomer), 24 mM tripotassium phosphate, 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), L-aspartate magnesium, 80 mM potassium acetate, 2% (w / v) polyethylene glycol 8000, and 9.78 mM... The extract was prepared using a pH 8.0 Tris·HCl buffer and 6% (w / v) trehalose. The *Kluyveromyces lactis* extract contained endogenously expressed T7 RNA polymerase. The preparation of the *Kluyveromyces lactis* cell extract employed conventional techniques, referring to the method described in CN109593656A. The preparation steps, in general, included: providing an appropriate amount of fermented *Kluyveromyces lactis* cells; flash-freezing the cells with liquid nitrogen; breaking up the cells; centrifuging and collecting the supernatant to obtain the cell extract. The protein concentration in the obtained *Kluyveromyces lactis* cell extract was 20–40 mg / mL.

[0145] Add 20 mL of DNA amplification product solution to the IVTT reaction system, incubate at 30°C and 100 rpm on a shaker, and then purify the protein according to the following steps:

[0146] a. After 3 hours of IVTT reaction, 6 mL of nickel affinity magnetic beads were added, and incubation continued for 1 hour; magnetic attraction;

[0147] b. After magnetic attraction, add 10 mL of 20 mM imidazole to wash once; then magnetic attraction again.

[0148] c. After magnetic attraction, add 10 mL of 0.1% Triton X-114 and 0.2% Triton X-100, rinse once with 2M sodium chloride; then magnetic attraction again.

[0149] d. After magnetic attraction, add 10 mL of 20 mM imidazole and wash 4 times; then magnetic attraction again;

[0150] e. After magnetic adsorption, add 3 mL of 250 mM imidazole (containing Tris) to elute for 3-5 min;

[0151] f. Collect the supernatant by magnetic attraction, and then collect the supernatant at 12000 rpm for 10 min.

[0152] The same steps (or methods) are applied to PMN94, PTMI-4, and PTMII-4.

[0153] The concentrations of nickel-affinity purified proteins are shown in Table 4, and protein reducing SDS-PAGE electrophoresis was performed (see Table 4). Figure 2 The results showed that the size of the target protein was consistent with the expected size (the theoretical molecular weights of PTMI4, PTMII4, PBZ-4, and PMN94 were 23447 Da, 23530 Da, 7738 Da, and 12512 Da, respectively. The theoretical value of PBZ-4 was smaller than that shown in the gel image because the amino acid of PBZ-4 was fused with the SUMO protein tag and was not removed. The theoretical molecular weight of the fusion protein was 20903 Da). This indicates that the expression was successful and the target protein was obtained.

[0154] Table 4: Concentration of proteins purified by nickel affinity

[0155]

[0156]

[0157] Wherein, OD260 represents the absorbance of nucleic acids, OD280 represents the absorbance of proteins, and OD... 260 / 280 The ratio of nucleic acid to protein is approximately 0.5 to 0.7, indicating that the target protein has high purity.

[0158] As can be seen from the above embodiments, the system and method of this application can synthesize a variety of different sweet proteins, indicating that the system and method disclosed in this application are universal for the synthesis of sweet proteins. In addition, the method of this application is simple, the reaction conditions are mild, the cycle is short, and it can be produced quickly, making it particularly suitable for large-scale production.

[0159] Based on the above-described preferred embodiments according to this application, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the technical concept of this application. The technical scope of this application is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A cell-free reaction system for synthesizing sweet proteins, characterized in that, include: 1) Cell-free reaction system; 2) The nucleic acid encoding the sweet protein.

2. The cell-free reaction system for synthesizing sweet proteins according to claim 1, characterized in that, The cell-free reaction system includes at least a cell-free extract.

3. The cell-free reaction system for synthesizing sweet proteins according to claim 2, characterized in that, The cell extract is selected from any of the following sources: Escherichia coli, yeast cells, mammalian cells, plant cells, insect cells, or a combination thereof.

4. The cell-free reaction system for synthesizing sweet proteins according to claim 3, characterized in that, The yeast cells were selected from Pichia pastoris, Pichia finlandica, Pichiatrehalophila, Pichia koclamae, Pichiamembranaefaciens, Pichia minuta, Ogataeaminuta, Pichia lindneri, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia apijperi, Pichiastiptis, Pichia methanolica, and other Pichia species. Saccharomyces cerevisiae, brewer's yeast, sugarcane molasses yeast, Saccharomyces sp., Hansenula polymorpha, Candida utilis, Kluyveromyces, or a combination thereof; preferably, the Kluyveromyces includes: Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces dobzhanskii, Kluyveromyces aestuarii, Kluyveromyces nonfermentans, Kluyveromyces wickerhamii, Kluyveromyces thermotolerans, Kluyveromyces fragilis, and Kluyveromyces hubeiense. Kluyveromyces hubeiensis, Kluyveromyces polysporus, Kluyveromyces siamensis, Kluyveromyces yarrowii, or a combination thereof.

5. The cell-free reaction system for synthesizing sweet proteins according to any one of claims 1-4, characterized in that, The reaction system further includes one or more components selected from buffer, potassium ions, magnesium ions, polyethylene glycol, optional aqueous solvent, and phosphate.

6. The cell-free reaction system for synthesizing sweet proteins according to any one of claims 1-5, characterized in that, The sweet protein is selected from one or more of the following: semaphore, monetin, areca nut protein, Brazil sweetener, egg white lysozyme, curculigo sweet protein, or miracle fruit protein.

7. The cell-free reaction system for synthesizing sweet proteins according to any one of claims 1-6, characterized in that, The sweet protein mentioned includes wild-type, mutant, recombinant, or fusion proteins.

8. The cell-free reaction system for synthesizing sweet proteins according to any one of claims 1-7, characterized in that, The structure of the sweet protein includes sequences containing SEQ ID NO: 1 to 4; or includes polypeptides having ≥85%, ≥90%, ≥95%, ≥97%, ≥98%, or ≥99% homology to the amino acid sequences shown in SEQ ID NO: 1 to 4 and having the same activity as the sequences in SEQ ID NO: 1 to 4.

9. A method for cell-free synthesis of sweet proteins, characterized in that, include: Step 1), providing the cell-free reaction system according to any one of claims 1-9; Step 2), adding nucleic acid encoding the sweet protein to the cell-free reaction system to synthesize the sweet protein.

10. A method for cell-free synthesis of sweet proteins according to claim 9, characterized in that, Further includes: Step 3), purification process.

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