In-vitro protein synthesis system, kit and preparation method thereof

Abstract

The invention provides an in-vitro protein synthesis system, a kit and a preparation method thereof, specifically, the in-vitro protein synthesis system is prepared from (a) a cell extract; and (b) a first reaction promoter is mixed to form a cell-free in-vitro protein synthesis system, so that the synthesis efficiency of the foreign protein can be remarkably improved.

Description

[0001] The present invention is a divisional application of patent application number CN201711452033.X filed on December 28, 2017, and the invention name is "An in vitro protein synthesis system, a kit and a preparation method thereof." Technical Field

[0002] The present invention relates to the field of biotechnology, and in particular to an in vitro protein synthesis system, a kit and a preparation method thereof. Background Art

[0003] Biological reactions, or biochemical reactions, refer to chemical reactions that take place in living organisms. These reactions are catalyzed by enzymes. Enzymes and reactants must be dissolved in water in the internal environment for reactions to occur. Water provides a carrier and medium for substances in the body [1]. In the last few decades of the 20th century, biochemistry has made great achievements in explaining life processes. Now, almost all fields related to life sciences, such as botany, medicine, and genetics, are engaged in biochemical research [2].

[0004] Traditional protein expression systems refer to a multi-step cascade biochemical reaction technique that expresses foreign genes through model organisms such as bacteria, fungi, plant cells or animal cells [3-4]. With the development of science and technology, cell-free expression systems, also known as in vitro protein synthesis systems, have emerged. They use exogenous target mRNA or DNA as a template for protein synthesis, and can achieve the synthesis of target proteins by artificially controlling the addition of substrates and transcription and translation-related protein factors required for protein synthesis [5-7]. Protein expression in the in vitro translation system does not require plasmid construction, transformation, cell culture, cell collection and disruption steps, and is a fast, time-saving and convenient protein expression method [8]. With the development of science and technology, protein synthesis systems have begun to be widely used. Biochemical reactions carried out in organisms or cells can be automatically regulated by positive and negative feedback in complex network systems, and biochemical reactions carried out in organisms are reactions far from equilibrium points. They need to obtain energy from the outside world or output substances, energy and entropy to the outside world [9]. In cells, the removal of byproducts of biological reactions is mainly achieved through metabolism, maintenance of homeostasis, catalysis of biological enzymes, and redox reactions far from the equilibrium point in cells, but these biochemical reactions are difficult to achieve in vitro biotechnology

[10] . In addition, a disadvantage of the protein synthesis system is that the yield is not high and the reaction is not fast or sensitive enough. In in vitro biosynthesis systems, such as in vitro protein synthesis, in vitro transcription, kinase reaction, ATPase reaction, ion pump reaction, etc., byproducts such as free phosphate ions and pyrophosphate ions (PPi) will limit the yield of the target product.

[0005] At present, there are other methods known to remove reaction byproducts, such as controlling byproducts such as free phosphate ions by semi-continuous reaction through a semipermeable membrane that allows small molecules to diffuse passively between the reaction mixture and the dialysate. However, there are the disadvantages that the semipermeable membrane dialysis device is high in cost, the reaction system cannot be expanded, and the reaction products easily clog the semipermeable membrane pores.

[0006] Therefore, there is an urgent need in the art to develop and establish a method that can improve protein synthesis efficiency by controlling the content of free phosphate ions in a protein synthesis system through in situ removal. Summary of the invention

[0007] The object of the present invention is to provide a method for improving protein synthesis efficiency by controlling the content of free phosphate ions in a protein synthesis system by in situ removal.

[0008] The first aspect of the present invention provides an in vitro protein synthesis system, comprising:

[0009] (a) cells or cell extracts, or a combination thereof;

[0010] (b) a first reaction accelerator, wherein the first reaction accelerator is selected from the group consisting of aluminum, aluminum salts, oxyaluminum complexes, or combinations thereof.

[0011] In another preferred embodiment, the cell is selected from the group consisting of prokaryotic cells and eukaryotic cells.

[0012] In another preferred embodiment, the cell is selected from the group consisting of Escherichia coli, bacteria, mammalian cells (such as HF9, Hela, CHO, HEK293), plant cells, yeast cells, or a combination thereof.

[0013] In another preferred embodiment, the yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces, or a combination thereof; preferably, the yeast cell includes Kluyveromyces, more preferably Kluyveromyces lactis.

[0014] In another preferred embodiment, the protein synthesis system further comprises:

[0015] (b') a second reaction accelerator, wherein the second reaction accelerator is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, soluble starch, or a combination thereof;

[0016] (c) polyethylene glycol;

[0017] (d) optionally exogenous sucrose; and

[0018] (e) an optional solvent, wherein the solvent is water or an aqueous solvent.

[0019] In another preferred embodiment, the protein synthesis system comprises a yeast in vitro protein synthesis system (such as a Kluyveromyces in vitro protein synthesis system, preferably, a Kluyveromyces lactis in vitro protein synthesis system).

[0020] In another preferred example, the aluminum oxide composite comprises aluminum oxide nanoparticles.

[0021] In another preferred example, the oxygen-aluminum complex includes Al2O3.

[0022] In another preferred example, the oxyaluminum complex is in particle form or nano form.

[0023] In another preferred embodiment, the particle size of the oxyaluminum composite is 0.5-20 mm, preferably 0.8-10 mm, and more preferably 1-5 mm.

[0024] In another preferred embodiment, the average weight of the oxyaluminum complex is 1-80 mg, preferably 3-50 mg, and more preferably 4-30 mg.

[0025] In another preferred embodiment, the concentration (v / v) of the oxyaluminum complex in the first reaction accelerator is 0.5-20%, preferably 0.8-10%, more preferably 1%-10%, more preferably 1%-5%, more preferably 2%-3%, based on the total volume of the first reaction accelerator.

[0026] In another preferred embodiment, in the reaction accelerator, the content (wt%) of the oxyaluminum complex is 0.1-20%, preferably 1-10%, more preferably 2%-8%, more preferably 4%-7%, more preferably 5.5%-6.5%, based on the total weight of the first reaction accelerator.

[0027] In another preferred embodiment, in the protein synthesis system, the concentration (v / v) of the first reaction promoter is 20%-70%, preferably 30%-60%, more preferably 45%-55%, based on the total volume of the protein synthesis system.

[0028] In another preferred embodiment, in the protein synthesis system, the content (wt%) of the first reaction accelerator is 20%-70%, preferably, 30%-60%, more preferably, 45%-55%, based on the total weight of the protein synthesis system.

[0029] The second aspect of the present invention provides an in vitro cell-free protein synthesis system, comprising:

[0030] (a) Cell extracts;

[0031] (b) a first reaction accelerator, wherein the first reaction accelerator is selected from the group consisting of aluminum, aluminum salts, oxyaluminum complexes, or combinations thereof.

[0032] In another preferred embodiment, the protein synthesis system further comprises:

[0033] (b') a second reaction accelerator, wherein the second reaction accelerator is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, soluble starch, or a combination thereof;

[0034] (c) polyethylene glycol;

[0035] (d) optionally exogenous sucrose; and

[0036] (e) an optional solvent, wherein the solvent is water or an aqueous solvent.

[0037] In another preferred embodiment, the protein synthesis system comprises a yeast in vitro protein synthesis system (such as a Kluyveromyces in vitro protein synthesis system, preferably, a Kluyveromyces lactis in vitro protein synthesis system).

[0038] In another preferred example, the aluminum oxide composite comprises aluminum oxide nanoparticles.

[0039] In another preferred example, the oxygen-aluminum complex includes Al2O3.

[0040] In another preferred example, the oxyaluminum complex is in particle form or nano form.

[0041] In another preferred embodiment, the particle size of the oxyaluminum composite is 0.5-20 mm, preferably 0.8-10 mm, and more preferably 1-5 mm.

[0042] In another preferred embodiment, the average weight of the oxyaluminum complex is 1-80 mg, preferably 3-50 mg, and more preferably 4-30 mg.

[0043] In another preferred embodiment, the concentration (v / v) of the oxyaluminum complex in the first reaction accelerator is 0.5-20%, preferably 0.8-10%, more preferably 1%-10%, more preferably 1%-5%, more preferably 2%-3%, based on the total volume of the first reaction accelerator.

[0044] In another preferred embodiment, in the reaction accelerator, the content (wt%) of the oxyaluminum complex is 0.1-20%, preferably 1-10%, more preferably 2%-8%, more preferably 4%-7%, more preferably 5.5%-6.5%, based on the total weight of the first reaction accelerator.

[0045] In another preferred embodiment, in the protein synthesis system, the concentration (v / v) of the first reaction promoter is 20%-70%, preferably 30%-60%, more preferably 45%-55%, based on the total volume of the protein synthesis system.

[0046] In another preferred embodiment, in the protein synthesis system, the content (wt%) of the first reaction accelerator is 20%-70%, preferably, 30%-60%, more preferably, 45%-55%, based on the total weight of the protein synthesis system.

[0047] In another preferred embodiment, the cell source of the cell extract is selected from one or more types of cells in the following group: prokaryotic cells and eukaryotic cells.

[0048] In another preferred embodiment, the cell source of the cell extract is selected from one or more types of cells in the following group: Escherichia coli, bacteria, mammalian cells (such as HF9, Hela, CHO, HEK293), plant cells, yeast cells, or a combination thereof.

[0049] In another preferred embodiment, the yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces, or a combination thereof; preferably, the yeast cell includes Kluyveromyces, more preferably Kluyveromyces lactis.

[0050] In another preferred embodiment, the protein synthesis system further comprises one or more components selected from the following group:

[0051] (f1) a substrate for synthesizing RNA;

[0052] (f2) substrates for protein synthesis;

[0053] (f3) magnesium ion;

[0054] (f4) potassium ion;

[0055] (f5) buffer;

[0056] (f6) RNA polymerase;

[0057] (f7) Energy regeneration system.

[0058] In another preferred embodiment, the protein synthesis system further comprises one or more components selected from the following group:

[0059] (g8) heme;

[0060] (g9) Spermidine.

[0061] In another preferred embodiment, the cell extract comprises a yeast cell extract.

[0062] In another preferred embodiment, the yeast cell extract is an aqueous extract of yeast cells.

[0063] In another preferred embodiment, the yeast cell extract does not contain long-chain nucleic acid molecules endogenous to yeast.

[0064] In another preferred embodiment, the yeast cell extract is prepared by a method comprising the following steps:

[0065] (i) providing yeast cells;

[0066] (ii) washing the yeast cells to obtain washed yeast cells;

[0067] (iii) subjecting the washed yeast cells to cell disruption to obtain a crude yeast extract; and

[0068] (iv) subjecting the crude yeast extract to solid-liquid separation to obtain a liquid portion, namely, a yeast cell extract.

[0069] In another preferred embodiment, the solid-liquid separation includes centrifugation.

[0070] In another preferred embodiment, the centrifugation is performed in a liquid state.

[0071] In another preferred embodiment, the centrifugation condition is 5000-100000g, preferably, 8000-30000g.

[0072] In another preferred embodiment, the centrifugation time is 0.5 min-2 h, preferably, 20 min-50 min.

[0073] In another preferred embodiment, the centrifugation is carried out at 1-10°C, preferably, at 2-6°C.

[0074] In another preferred embodiment, the washing treatment is carried out using a washing liquid at a pH of 7-8 (preferably 7.4).

[0075] In another preferred embodiment, the washing liquid is selected from the following group: potassium 4-hydroxyethylpiperazineethanesulfonate, potassium acetate, magnesium acetate, or a combination thereof.

[0076] In another preferred embodiment, the cell disruption treatment includes high-pressure disruption and freeze-thaw (such as liquid nitrogen low-temperature) disruption.

[0077] In another preferred embodiment, the substrate for synthesizing RNA includes: nucleoside monophosphate, nucleoside triphosphate, or a combination thereof.

[0078] In another preferred embodiment, the substrates for synthesizing proteins include: 1-20 natural amino acids and unnatural amino acids.

[0079] In another preferred embodiment, the magnesium ions are derived from a magnesium ion source, and the magnesium ion source is selected from the following group: magnesium acetate, magnesium glutamate, or a combination thereof.

[0080] In another preferred embodiment, the potassium ions are derived from a potassium ion source, and the potassium ion source is selected from the following group: potassium acetate, potassium glutamate, or a combination thereof.

[0081] In another preferred embodiment, the energy regeneration system is selected from the following group: creatine phosphate / creatine phosphate enzyme system, glycolysis pathway and its intermediate energy system, or a combination thereof.

[0082] In another preferred embodiment, the protein synthesis system further comprises (h1) artificially synthesized tRNA.

[0083] In another preferred embodiment, the buffer is selected from the following group: 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethane, or a combination thereof.

[0084] In another preferred embodiment, the protein synthesis system further comprises (i1) an exogenous DNA molecule for directing protein synthesis.

[0085] In another preferred embodiment, the DNA molecule is linear.

[0086] In another preferred embodiment, the DNA molecule is circular.

[0087] In another preferred embodiment, the DNA molecule contains a sequence encoding a foreign protein.

[0088] In another preferred embodiment, the sequence encoding the exogenous protein includes a genomic sequence and a cDNA sequence.

[0089] In another preferred embodiment, the sequence encoding the foreign protein further contains a promoter sequence, a 5' untranslated sequence, and a 3' untranslated sequence.

[0090] In another preferred embodiment, the protein synthesis system comprises components selected from the following group: 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, nucleoside triphosphates, amino acids, creatine phosphate, dithiothreitol (DTT), creatine phosphate kinase, RNA polymerase, or a combination thereof.

[0091] In another preferred embodiment, the polyethylene glycol is selected from the following group: PEG3000, PEG8000, PEG6000, PEG3350, or a combination thereof.

[0092] In another preferred embodiment, the polyethylene glycol includes polyethylene glycol with a molecular weight (Da) of 200-10000, preferably polyethylene glycol with a molecular weight of 3000-10000.

[0093] In another preferred embodiment, in the protein synthesis system, the concentration (v / v) of component (a) is 20%-70%, preferably 30-60%, more preferably 40%-50%, based on the total volume of the protein synthesis system.

[0094] In another preferred embodiment, in the protein synthesis system, the content (wt%) of component (c) is 10%-95%, preferably, 20%-80%, more preferably, 40%-60%, based on the total weight of the protein synthesis system.

[0095] In another preferred embodiment, in the protein synthesis system, the concentration (w / v, such as g / ml) of component (d) is 0.1-8%, preferably 0.5-4%, and more preferably 1-2%.

[0096] In another preferred embodiment, in the protein synthesis system, the concentration of component (e) is 0.2-4%, preferably 0.5-4%, more preferably 0.5-1%, based on the total volume of the protein synthesis system.

[0097] In another preferred embodiment, the nucleoside triphosphate is selected from the group consisting of adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, uridine triphosphate, or a combination thereof.

[0098] In another preferred embodiment, in the protein synthesis system, the concentration of component (f1) is 0.1-5 mM, preferably 0.5-3 mM, and more preferably 1-1.5 mM.

[0099] In another preferred embodiment, the amino acid is selected from the following group: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, or a combination thereof.

[0100] In another preferred embodiment, the amino acids include D-type amino acids and / or L-type amino acids.

[0101] In another preferred embodiment, in the protein synthesis system, the concentration of the component (f2) is 0.01-0.48 mM, preferably, 0.04-0.24 mM, more preferably, 0.04-0.2 mM, and most preferably, 0.08 mM.

[0102] In another preferred embodiment, in the protein synthesis system, the concentration of the component (f3) is 1-10 mM, preferably 1-5 mM, and more preferably 2-4 mM.

[0103] In another preferred embodiment, in the protein synthesis system, the concentration of the component (f4) is 30-210 mM, preferably 30-150 mM, and more preferably 30-60 mM.

[0104] In another preferred embodiment, in the protein synthesis system, the concentration of the component (f6) is 0.01-0.3 mg / mL, preferably, 0.02-0.1 mg / mL, and more preferably, 0.027-0.054 mg / mL.

[0105] In another preferred embodiment, in the protein synthesis system, the concentration of 4-hydroxyethylpiperazineethanesulfonic acid is 5-50 mM, preferably 10-50 mM, preferably 15-30 mM, and more preferably 20-25 mM.

[0106] In another preferred embodiment, in the protein synthesis system, the concentration of potassium acetate is 20-210 mM, preferably 30-210 mM, preferably 30-150 mM, more preferably 30-60 mM.

[0107] In another preferred embodiment, in the protein synthesis system, the concentration of magnesium acetate is 1-10 mM, preferably 1-5 mM, and more preferably 2-4 mM.

[0108] In another preferred embodiment, in the protein synthesis system, the concentration of creatine phosphate is 10-50 mM, preferably 20-30 mM, and more preferably 25 mM.

[0109] In another preferred embodiment, in the protein synthesis system, the concentration of heme is 0.01-0.1 mM, preferably, 0.02-0.08 mM, more preferably, 0.03-0.05 mM, and most preferably, 0.04 mM.

[0110] In another preferred embodiment, in the protein synthesis system, the concentration of spermidine is 0.05-1 mM, preferably, 0.1-0.8 mM, more preferably, more preferably, 0.2-0.5 mM, more preferably, 0.3-0.4 mM, and most preferably, 0.4 mM.

[0111] In another preferred embodiment, in the protein synthesis system, the concentration of dithiothreitol (DTT) is 0.2-15 mM, preferably 0.2-7 mM, and more preferably 1-2 mM.

[0112] In another preferred embodiment, in the protein synthesis system, the concentration of the creatine kinase phosphate is 0.1-1 mg / mL, preferably, 0.2-0.5 mg / mL, and more preferably, 0.27 mg / mL.

[0113] In another preferred embodiment, in the protein synthesis system, the concentration of the T7 RNA polymerase is 0.01-0.3 mg / mL, preferably, 0.02-0.1 mg / mL, and more preferably, 0.027-0.054 mg / mL.

[0114] In another preferred embodiment, the protein synthesis system has the following properties:

[0115] In the synthetic system, the total amount of protein synthesized reached 3ug protein / mL system.

[0116] In another preferred embodiment, the composition of the protein synthesis system includes:

[0117]

[0118] In another preferred embodiment, the composition of the protein synthesis system also includes:

[0119] Spermidine, 0.2-0.4mM 0.3-0.4mM;

[0120] Heme, 0.01-0.04mM 0.03-0.04mM.

[0121] In another preferred embodiment, the PEG is selected from PEG3350, PEG3000, and / or PEG8000.

[0122] In another preferred embodiment, the RNA polymerase is T7 RNA polymerase.

[0123] The third aspect of the present invention provides a method for producing the protein synthesis system according to the first aspect of the present invention or the second aspect of the present invention, comprising the steps of:

[0124] Component (i) and component (ii) are mixed to obtain the protein synthesis system according to the first aspect or the second aspect of the present invention, wherein the component (i) is selected from the following group: cells or cell extracts, or a combination thereof; the component (ii) is a first reaction accelerator, and the first reaction accelerator is selected from the following group: aluminum, aluminum salts, oxyaluminum complexes, or a combination thereof.

[0125] In another preferred embodiment, the component (i) is a cell extract.

[0126] In another preferred embodiment, in the protein synthesis system, the ratio (mass ratio) of component (i) to component (ii) is 0.1-10:0.1-10, preferably, 0.5-8:0.5-8, more preferably, 0.8-5:0.8-5, more preferably, 0.9-2:0.9-2.

[0127] In another preferred embodiment, in the protein synthesis system, the concentration (v / v) of component (i) is 10-80%, preferably 20-60%, based on the total volume of the protein synthesis system.

[0128] In another preferred embodiment, in the protein synthesis system, the content (wt%) of component (i) is 10-80%, preferably 20-60%, based on the total weight of the protein synthesis system.

[0129] In another preferred embodiment, in the protein synthesis system, the concentration (v / v) of component (ii) is 10-80%, preferably 20-60%, based on the total volume of the protein synthesis system.

[0130] In another preferred embodiment, in the protein synthesis system, the content (wt%) of component (ii) is 10-80%, preferably 20-60%, based on the total weight of the protein synthesis system.

[0131] A fourth aspect of the present invention provides a method for in vitro protein synthesis, comprising:

[0132] (i) providing the protein synthesis system described in the first aspect of the present invention or the second aspect of the present invention, and adding an exogenous DNA molecule for directing protein synthesis;

[0133] (ii) incubating the protein synthesis system of step (i) for a period of time T1 under suitable conditions, thereby synthesizing the protein encoded by the exogenous DNA.

[0134] In another preferred embodiment, the method further comprises: (iii) optionally separating or detecting the protein encoded by the exogenous DNA from the protein synthesis system.

[0135] In another preferred embodiment, the exogenous DNA comes from prokaryotes or eukaryotes.

[0136] In another preferred embodiment, the exogenous DNA comes from animals, plants, or pathogens.

[0137] In another preferred embodiment, the exogenous DNA comes from mammals, preferably primates, rodents, including humans, mice, and rats.

[0138] In another preferred embodiment, the coding sequence of the exogenous protein encodes an exogenous protein selected from the following group: luciferin protein, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutant, α-amylase, enterobactin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon αA, interleukin-1β, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase, or a combination thereof.

[0139] In another preferred embodiment, the exogenous protein is selected from the following group: luciferin protein, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutation, α-amylase, enterobactin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon αA, interleukin-1β, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase, or a combination thereof.

[0140] In another preferred embodiment, the exogenous DNA encodes an exogenous protein selected from the following group: luciferin protein, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutant, α-amylase, enterobactin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon αA, interleukin-1β, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase, or a combination thereof.

[0141] In another preferred embodiment, the protein encoded by the exogenous DNA is selected from the following group: luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutation, α-amylase, enterobactin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon αA, interleukin-1β, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase, or a combination thereof.

[0142] In another preferred embodiment, in step (ii), the reaction temperature is 20-37°C, preferably 20-25°C.

[0143] In another preferred embodiment, in step (ii), the reaction time is 1-6 h, preferably 2-4 h.

[0144] A fifth aspect of the present invention provides a kit comprising:

[0145] (k1) a first container, and a component (i) located in the first container, wherein the component (i) is selected from the group consisting of cells or cell extracts, or a combination thereof;

[0146] (k2) a second container, and a component (ii) in the second container, wherein the component (ii) is a first reaction accelerator, wherein the first reaction accelerator is selected from the group consisting of aluminum, aluminum salts, oxyaluminum complexes, or combinations thereof; and

[0147] (kt) Label or instruction sheet.

[0148] In another preferred embodiment, the component (i) is a cell extract.

[0149] In another preferred embodiment, the first container and the second container are the same container or different containers.

[0150] In another preferred embodiment, the kit further comprises one or more containers optionally selected from the following group:

[0151] (k3) a third container, and polyethylene glycol in the third container;

[0152] (k4) an optional fourth container, and sucrose in the fourth container;

[0153] (k5) a fifth container, and a substrate for synthesizing RNA located in the fifth container;

[0154] (k6) a sixth container, and a substrate for synthesizing a protein located in the sixth container;

[0155] (k7) a seventh container, and magnesium ions located in the seventh container;

[0156] (k8) an eighth container, and potassium ions located in the eighth container; and

[0157] (k9) a ninth container, and a buffer located in the ninth container.

[0158] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0159] Figure 1It shows that reaction byproducts represented by phosphate are released in biological reactions. For example, magnesium ions are involved in several steps of transcription and translation, and the main target is produced through reactions in solid, liquid, or other phases using substrates, energy, etc. At the same time, several reaction byproducts are produced in several stages of transcription and translation, such as free phosphate ions and pyrophosphate ions; such as kinase reactions (protein kinases, lipid kinases, sugar kinases, etc.); ATPase enzyme reactions (transmembrane transport of sodium, potassium, calcium ions, etc.), molecular motors, etc., and byproducts often react with biological reaction systems and inhibit reactions. Currently, the main methods for removing reaction byproducts include dialysis, circulation, solution replacement, etc., all of which require additional equipment, have complex systems, are difficult to operate, and are not easy to complete in small reaction systems. Therefore, the removal of reaction byproducts, especially in situ removal, is the key to improving reaction efficiency and reducing reaction complexity.

[0160] Figure 2 The diagram shows the effect of adding the first reaction promoter (phosphoric acid control agent) to a 30 μL in vitro protein synthesis system at 2 hours of reaction. The solid circle line is the cell-free reaction system without the addition of the first reaction promoter, the square is the cell-free reaction system with Fe2O3 added at 2 hours, the equilateral triangle is the cell-free reaction system with Fe3O4 added at 2 hours, and the inverted triangle is the cell-free reaction system with an equal mass of alumina particles added at 2 hours. The reaction conditions are 20-25℃ for 3 hours. All errors are the standard deviation of three replicates.

[0161] Figure 3 The diagram shows the effect of adding the first reaction promoter to a 120 μL cell-free reaction system at 2 h of reaction. The solid circle line is the cell-free reaction system without adding the first reaction promoter, the square is the cell-free reaction system with Fe2O3 added at 2 h, the equilateral triangle is the cell-free reaction system with the same mass of Fe3O4 added at 2 h, and the inverted triangle is the cell-free reaction system with the same mass of alumina particles added at 2 h. The reaction conditions were 20-25 ° C for 4 h and 5 h. All errors are the standard deviation of three replicates.

[0162] Figure 4 A schematic diagram showing the addition of the first reaction promoter to a 120 μL cell-free reaction system at 2 hours of reaction and the determination of the phosphate concentration in the system at 4.5 hours. The first column is the cell-free reaction system without the addition of the first reaction promoter, the second column is the cell-free reaction system with the addition of Fe2O3 at 2 hours, the third column is the cell-free reaction system with the addition of an equal mass of Fe3O4 at 2 hours, and the fourth column is the cell-free reaction system with the addition of an equal mass of alumina particles at 2 hours. The reaction conditions were 20-25°C for 4.5 hours.

[0163] Figure 5The effect of adding 30 mg of alumina particles to a 90 μL cell-free reaction system at different time periods (1 h and 2 h and without addition) is shown, and the reaction conditions are 20-25° C. All errors are standard deviations of three replicates.

[0164] Figure 6 The figure shows the phosphoric acid concentration in a 90 μL cell-free reaction system with 30 mg of alumina particles added at different time periods (1 h and 2 h, and without addition), and the reaction conditions are 20-25° C. All errors are standard deviations of three replicates.

[0165] Figure 7 The figure shows the effect of adding different masses of aluminum oxide particles to a 90 μL cell-free reaction system when the reaction was carried out for 1 hour, and the reaction conditions were 20-25° C. All errors are standard deviations of three replicates.

[0166] Figure 8 This is a schematic diagram of the phosphate control system to improve the efficacy of biological reactions. In short, the phosphate control in the system and the biochemical reaction in the system are a pair of competing reactions. Too much phosphate or pyrophosphate in the system will cause pH imbalance in the system, affect the activity of the biological enzymes involved in the biochemical reaction in the system, and reduce the yield of the target protein; at the same time, phosphate is easy to combine with the magnesium ions required in the reaction system to form magnesium sulfate that is insoluble in water, resulting in a decrease in the reaction activity of many biochemical reaction steps due to the lack of magnesium ions. The addition of phosphate control agents adjusts the balance of the biochemical reactions mentioned above by physical (adsorption) or chemical (formation of precipitation products out of the reaction system), thereby creating a relatively ideal reaction environment for the protein synthesis system. DETAILED DESCRIPTION

[0167] After extensive and in-depth research, the inventor unexpectedly discovered for the first time that an in vitro protein synthesis system formed by mixing a specific ratio of (a) cells or cell extracts, or a combination thereof; and (b) a first reaction promoter (such as aluminum, aluminum salts, oxyaluminum complexes, or a combination thereof) can significantly improve the synthesis efficiency of exogenous proteins, and its RLU can reach up to 3×109-4×109. And under the action of the first reaction promoter, compared with the yeast cell-free expression system to which the first reaction promoter is not added, the relative light unit value of the activity of the exogenous protein (such as luciferase) synthesized by the yeast cell-free expression system to which the reaction promoter is added 1 hour after the reaction starts is significantly increased by 2-3 times, thereby improving the reaction efficiency and increasing the yield of the target protein. On this basis, the present invention is completed.

[0168] In addition, the experimental results of the present invention show that a cell-free in vitro protein synthesis system to which a first reaction promoter is added within a specific time period (such as when the reaction is carried out for about 1 hour) can significantly promote the synthesis of in vitro protein in a protein synthesis system (such as a yeast in vitro protein synthesis system), and the first reaction promoter of the present invention can be added at the start of the reaction of the protein synthesis system (such as a yeast in vitro protein synthesis system), 1 hour after the start of the reaction, or 2 hours after the start of the reaction. Among them, adding the first reaction promoter 1 hour after the start of the reaction of the protein synthesis system (such as a yeast in vitro protein synthesis system) has a better promoting effect on protein synthesis.

[0169] Reaction accelerator

[0170] In the present invention, the reaction accelerator is not particularly limited, and any reaction accelerator that can significantly improve the protein synthesis efficiency of the in vitro protein synthesis system of the present invention is within the protection scope of the present invention.

[0171] In a preferred embodiment, the reaction accelerator of the present invention includes a first reaction accelerator and a second reaction accelerator.

[0172] In a preferred embodiment, the first reaction accelerator is selected from the following group: aluminum, aluminum salt, oxyaluminum complex, or a combination thereof.

[0173] In a preferred embodiment, the second reaction accelerator is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, soluble starch, or a combination thereof.

[0174] In a preferred embodiment, the second reaction accelerator is selected from one or more of the following groups:

[0175] a) Sugars / polyols: sucrose, trehalose, mannitol, lactose, glucose, maltose, etc.;

[0176] b) Polymers: PEG, dextran, albumin, etc.;

[0177] c) Anhydrous solvents: glycerol, DMSO, etc.

[0178] In a preferred embodiment, the second reaction accelerator is selected from the group consisting of sucrose, trehalose, mannitol, lactose, glucose, maltose, galactose, or a combination thereof.

[0179] In a preferred embodiment, the second reaction accelerator is selected from the group consisting of sucrose, trehalose, lactose, or a combination thereof.

[0180] Biological reactions

[0181] Biological reactions, or biochemical reactions, refer to chemical reactions that take place in living organisms. These reactions are catalyzed by enzymes. Enzymes and reactants must be dissolved in water in the internal environment for reactions to occur. Water provides carriers and media for substances in the body. In the last few decades of the 20th century, biochemistry has made great achievements in explaining life processes. Now, almost all fields related to life sciences, such as botany, medicine, and genetics, are engaged in biochemical research. Biochemical reactions in organisms or cells can be automatically regulated by positive and negative feedback in complex network systems. Biochemical reactions in cells require enzyme catalysis. Enzymes have high catalytic efficiency, mild reaction conditions, directionality, and high specificity for substrates.

[0182] Cell extracts

[0183] In a preferred embodiment, the cell source of the cell extract is one or more types of cells selected from the group consisting of prokaryotic cells and eukaryotic cells.

[0184] In a preferred embodiment, the cell source of the cell extract is selected from one or more types of cells in the following group: Escherichia coli, bacteria, mammalian cells (such as HF9, Hela, CHO, HEK293), plant cells, yeast cells, insect cells, or a combination thereof.

[0185] In a preferred embodiment, the yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces, or a combination thereof; preferably, the yeast cell comprises Kluyveromyces, more preferably Kluyveromyces lactis.

[0186] In the present invention, the cell extract includes a yeast cell extract.

[0187] In the present invention, the content and purity of the cell extract are not particularly limited.

[0188] In a preferred embodiment, in the protein synthesis system, the content (wt%) of the cell extract (such as yeast cell extract) is 10%-95%, preferably, 20%-80%, more preferably, 40%-60%, based on the total weight of the protein synthesis system.

[0189] In vitro expression system

[0190] Yeast has the advantages of simple cultivation, efficient protein folding, and post-translational modification. Saccharomyces cerevisiae and Pichia pastoris are model organisms for expressing complex eukaryotic proteins and membrane proteins. Yeast can also be used as a raw material for preparing in vitro translation systems.

[0191] Kluyveromyces is an ascospore yeast, of which Kluyveromyces marxianus and Kluyveromyces lactis are widely used in industry. Compared with other yeasts, Kluyveromyces lactis has many advantages, such as strong secretion ability, better large-scale fermentation characteristics, food safety level, and the ability to modify proteins post-translationally.

[0192] In the present invention, the yeast in vitro expression system is not particularly limited. A preferred yeast in vitro expression system is a Kluyveromyces expression system (more preferably, a Kluyveromyces lactis expression system).

[0193] Controlling the free phosphate content in protein synthesis systems

[0194] In the present invention, the “controlling the content of free phosphate in the protein synthesis system” refers to controlling the free phosphate as a reaction byproduct in the protein synthesis system, regulating the effect on the protein synthesis system, and thus improving the protein synthesis efficiency in the protein synthesis system.

[0195] In the present invention, the reaction accelerator of the present invention can be added to the protein synthesis system to achieve the above purpose.

[0196] In a preferred embodiment, the free phosphorus content control stage includes a pre-reaction stage before phosphoric acid control, a phosphoric acid content control stage and a post-phosphoric acid control stage.

[0197] In a preferred embodiment, there is no particular time limit for the phosphoric acid content control stage, and the amount of free phosphorus added to control the amount of free phosphorus added is determined based on the reaction time.

[0198] In a preferred embodiment, the duration of the pre-reaction phase before phosphate control is 0±2h, preferably 1h; the phosphate content control phase is 0±5h after the pre-reaction phase before phosphate control, preferably 2-5h; the duration of the post-phosphate control phase is determined according to the characteristics of each target protein and biochemical reaction. In summary, the duration of the entire phosphate control reaction system is 1-7h, preferably 2-4h.

[0199] In vitro cell-free protein synthesis system

[0200] In a preferred embodiment, the in vitro cell-free protein synthesis system of the present invention comprises a yeast in vitro protein synthesis system.

[0201] Yeast has the advantages of simple cultivation, efficient protein folding, and post-translational modification. Saccharomyces cerevisiae and Pichia pastoris are model organisms for expressing complex eukaryotic proteins and membrane proteins. Yeast can also be used as a raw material for preparing in vitro translation systems.

[0202] Kluyveromyces is an ascospore yeast, of which Kluyveromyces marxianus and Kluyveromyces lactis are widely used in industry. Compared with other yeasts, Kluyveromyces lactis has many advantages, such as super strong secretion ability, better large-scale fermentation characteristics, food safety level, and the ability to modify proteins after translation.

[0203] In the present invention, the yeast in vitro protein synthesis system is not particularly limited. A preferred yeast in vitro protein synthesis system is a Kluyveromyces expression system (more preferably, a Kluyveromyces lactis expression system).

[0204] In the present invention, the Kluyveromyces (such as Kluyveromyces lactis) is not particularly limited, and includes any Kluyveromyces (such as Kluyveromyces lactis) strain that can improve the efficiency of protein synthesis.

[0205] In the present invention, the yeast in vitro protein synthesis system comprises:

[0206] (a) cell extracts (such as yeast cell extracts);

[0207] (b) a first reaction accelerator, wherein the first reaction accelerator is selected from the group consisting of aluminum, aluminum salts, oxyaluminum complexes, or combinations thereof;

[0208] In a preferred embodiment, the yeast in vitro protein synthesis system further comprises:

[0209] (b') a second reaction accelerator, wherein the second reaction accelerator is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, soluble starch, or a combination thereof;

[0210] (c) polyethylene glycol;

[0211] (d) optionally exogenous sucrose; and

[0212] (e) an optional solvent, wherein the solvent is water or an aqueous solvent.

[0213] In a preferred embodiment, the particle size of the oxyaluminum composite is 0.5-20 mm, preferably, 0.8-10 mm, more preferably, 1-5 mm, and more preferably, 1-3 mm.

[0214] In another preferred embodiment, the average weight of the oxyaluminum complex is 1-80 mg, 3-50 mg, more preferably, 4-30 mg, more preferably, 8-15 mg.

[0215] In another preferred embodiment, in the reaction accelerator, the concentration (v / v) of the oxyaluminum complex is 0.5-20%, preferably 0.8-10%, more preferably 1%-10%, more preferably 1%-5%, more preferably 2%-3%, based on the total volume of the first reaction accelerator.

[0216] In another preferred embodiment, in the reaction accelerator, the content (wt%) of the oxyaluminum complex is 0.1-20%, preferably 1-10%, more preferably 2%-8%, more preferably 4%-7%, more preferably 5.5%-6.5%, based on the total weight of the first reaction accelerator.

[0217] In another preferred embodiment, in the protein synthesis system, the concentration (v / v) of the first reaction promoter is 20%-70%, preferably 30%-60%, more preferably 45%-55%, based on the total volume of the protein synthesis system.

[0218] In another preferred embodiment, in the protein synthesis system, the content (wt%) of the first reaction accelerator is 20%-70%, preferably 30%-60%, more preferably 45%-55%, based on the total weight of the protein synthesis system.

[0219] In another preferred embodiment, in the protein synthesis system, the ratio (mass ratio) of component (i) to component (ii) is 0.1-10:0.1-10, preferably, 0.5-8:0.5-8, more preferably, 0.8-5:0.8-5, more preferably, 0.9-2:0.9-2.

[0220] In a particularly preferred embodiment, the in vitro protein synthesis system provided by the present invention comprises one or more or all of the components selected from the following group: yeast cell extract, oxyaluminum complex (such as aluminum oxide), polyethylene glycol, sucrose, 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytosine triphosphate (CTP), thymidine triphosphate (TTP), amino acid mixture, creatine phosphate, dithiothreitol (DTT), creatine phosphate kinase, RNase inhibitor, luciferin, luciferase DNA, RNA polymerase, spermidine, and heme.

[0221] In the present invention, the RNA polymerase is not particularly limited and can be selected from one or more RNA polymerases, and a typical RNA polymerase is T7 RNA polymerase.

[0222] In the present invention, the proportion of the yeast cell extract in the in vitro protein synthesis system is not particularly limited. Generally, the content (wt%) of the yeast cell extract is 10%-95%, preferably 20%-80%, and more preferably 40%-60%, based on the total weight of the protein synthesis system.

[0223] In the present invention, the yeast cell extract does not contain intact cells. A typical yeast cell extract includes ribosomes, transfer RNA, aminoacyl tRNA synthetase, initiation factors and elongation factors required for protein synthesis, and termination release factors. In addition, the yeast extract also contains some other proteins derived from the cytoplasm of yeast cells, especially soluble proteins.

[0224] In the present invention, the protein content of the yeast cell extract is 20-100 mg / mL, preferably 50-100 mg / mL. The method for determining the protein content is the Coomassie Brilliant Blue determination method.

[0225] In the present invention, the preparation method of the yeast cell extract is not limited. A preferred preparation method comprises the following steps:

[0226] (i) providing yeast cells;

[0227] (ii) washing the yeast cells to obtain washed yeast cells;

[0228] (iii) disrupting the washed yeast cells to obtain a crude yeast extract;

[0229] (iv) subjecting the crude yeast extract to solid-liquid separation to obtain a liquid portion, namely, a yeast cell extract.

[0230] In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

[0231] In a preferred embodiment, the centrifugation is performed in a liquid state.

[0232] In the present invention, the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5000-100000 g, preferably 8000-30000 g.

[0233] In the present invention, the centrifugation time is not particularly limited, and a preferred centrifugation time is 0.5 min-2 h, preferably 20 min-50 min.

[0234] In the present invention, the centrifugation temperature is not particularly limited. Preferably, the centrifugation is performed at 1-10°C, more preferably, at 2-6°C.

[0235] In the present invention, the washing treatment method is not particularly limited. A preferred washing treatment method is to use a washing liquid at a pH of 7-8 (preferably 7.4). The washing liquid is not particularly limited. Typically, the washing liquid is selected from the following group: potassium 4-hydroxyethylpiperazineethanesulfonate, potassium acetate, magnesium acetate, or a combination thereof.

[0236] In the present invention, the cell disruption treatment method is not particularly limited. A preferred cell disruption treatment includes high-pressure disruption and freeze-thaw (such as liquid nitrogen cryogenic) disruption.

[0237] The nucleoside triphosphate mixture in the in vitro protein synthesis system is adenosine triphosphate, guanosine triphosphate, cytosine triphosphate and uridine triphosphate. In the present invention, the concentration of various mononucleotides is not particularly limited, and usually the concentration of each mononucleotide is 0.5-5mM, preferably 1.0-2.0mM.

[0238] The amino acid mixture in the in vitro 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 generally 0.01-0.5 mM, preferably 0.02-0.2 mM, such as 0.05, 0.06, 0.07, 0.08 mM.

[0239] In a preferred embodiment, the in vitro protein synthesis system further contains polyethylene glycol or its analogs. The concentration of polyethylene glycol or its analogs is not particularly limited. Generally, the concentration (w / v) of polyethylene glycol or its analogs is 0.1-8%, preferably 0.5-4%, more preferably 1-2%, based on the total weight of the protein synthesis 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.).

[0240] In a preferred embodiment, the in vitro protein synthesis system further contains sucrose. The concentration of sucrose is not particularly limited, and generally, the concentration (w / v) of sucrose is 0.2-4%, preferably 0.5-4%, more preferably 0.5-1%, based on the total volume of the protein synthesis system.

[0241] In a preferred embodiment, the in vitro protein synthesis system further contains heme. The concentration of heme is not particularly limited, and generally, the concentration of heme is 0.01-0.1 mM, preferably 0.02-0.08 mM, more preferably 0.03-0.05 mM, and most preferably 0.04 mM.

[0242] In a preferred embodiment, the in vitro protein synthesis system further contains spermidine. The concentration of spermidine is not particularly limited, and generally, the concentration of spermidine is 0.05-1 mM, preferably, 0.1-0.8 mM, more preferably, 0.2-0.5 mM, more preferably, 0.3-0.4 mM, and most preferably, 0.4 mM.

[0243] In a preferred embodiment, the in vitro protein synthesis system further contains a buffer, and the composition of the buffer is not particularly limited. A preferred buffer contains 4-hydroxyethylpiperazineethanesulfonic acid and / or Tris buffer. In the present invention, the buffer may also contain other buffer components, such as potassium acetate and magnesium acetate, to form a reaction solution or reaction buffer with a pH of 6.5-8.5 (preferably 7.0-8.0). In the present invention, the type and content of the buffer are not particularly limited. Generally, the concentration of the buffer is 1-200mM or 1-100mM, preferably 5-50mM.

[0244] A particularly preferred in vitro protein synthesis system, in addition to yeast extract and oxygen-aluminum complex (such as aluminum oxide), also contains one or more or all of the following components: 22 mM 4-hydroxyethylpiperazineethanesulfonic acid, 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, 8-20 ng / μL firefly luciferase DNA, 0.027-0.054 mg / mL T7 RNA polymerase, 0.03-0.04 mM heme, and 0.3-0.4 mM spermidine.

[0245] Coding sequence of foreign protein (foreign DNA)

[0246] As used herein, the terms "coding sequence of exogenous protein" and "exogenous DNA" are used interchangeably, both referring to exogenous DNA molecules used to direct protein synthesis. Typically, the DNA molecules are linear or circular. The DNA molecules contain sequences encoding exogenous proteins. In the present invention, examples of sequences encoding exogenous proteins include (but are not limited to): genomic sequences, cDNA sequences. The sequences encoding exogenous proteins also contain promoter sequences, 5' non-translated sequences, and 3' non-translated sequences.

[0247] In the present invention, the selection of the exogenous DNA is not particularly limited. Typically, the exogenous DNA is selected from the following group: exogenous DNA encoding luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, exogenous DNA of the variable region of an antibody, DNA of a luciferase mutant, or a combination thereof.

[0248] The exogenous DNA can also be selected from the following group: exogenous DNA encoding α-amylase, enterobactin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon αA, interleukin-1β, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase, or a combination thereof.

[0249] In a preferred embodiment, the exogenous DNA encodes a protein selected from the following group: green fluorescent protein (enhanced GFP, eGFP), yellow fluorescent protein (YFP), Escherichia coli β-galactosidase (β-galactosidase, LacZ), human lysine-tRNA synthetase (Lysine-tRNA synthetase), human leucine-tRNA synthetase (Leucine-tRNA synthetase), Arabidopsis glyceraldehyde 3-phosphate dehydrogenase (Glyceraldehyde-3-phosphatedehydrogenase), mouse catalase (Catalase), or a combination thereof.

[0250] Reagent test kit

[0251] The present invention provides a kit for in vitro protein synthesis, comprising:

[0252] (k1) a first container, and a component (i) located in the first container, wherein the component (i) is selected from the group consisting of cells or cell extracts, or a combination thereof;

[0253] (k2) a second container, and a component (ii) in the second container, wherein the component (ii) is a first reaction accelerator, wherein the first reaction accelerator is selected from the group consisting of aluminum, aluminum salts, oxyaluminum complexes, or combinations thereof; and

[0254] (kt) Label or instruction sheet.

[0255] In a preferred embodiment, the first container and the second container are the same container or different containers.

[0256] The present invention also provides a kit for cell-free in vitro protein synthesis, comprising:

[0257] (k1) a first container, and component (i) located in the first container, wherein component (i) is a cell extract;

[0258] (k2) a second container, and a component (ii) in the second container, wherein the component (ii) is a first reaction accelerator, wherein the first reaction accelerator is selected from the group consisting of aluminum, aluminum salts, oxyaluminum complexes, or combinations thereof; and

[0259] (kt) Label or instruction sheet.

[0260] A particularly preferred kit for in vitro protein synthesis comprises an in vitro protein synthesis system, which includes one or more or all of the components selected from the following group: yeast cell extract, oxyaluminum complex (such as aluminum oxide), 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytosine triphosphate (CTP), thymidine triphosphate (TTP), amino acid mixture, creatine phosphate, dithiothreitol (DTT), creatine phosphate kinase, RNase inhibitor, luciferin, luciferase DNA, T7 RNA polymerase, spermidine, and heme.

[0261] The main advantages of the present invention include:

[0262] (1) Compared with the general in vitro protein synthesis system, the in vitro protein synthesis system containing the first reaction accelerator of the present invention has a 2-3 times higher ability to synthesize protein, which has obvious advantages.

[0263] (2) The in vitro protein synthesis system of the present invention can more conveniently and rapidly control biochemical reactions, such as enzyme reactions, biosynthesis reactions, biodegradation reactions, cell-free biological reactions, etc.

[0264] (3) The present invention first discovered that adding a reaction promoter to an in vitro protein synthesis system can control the concentration of phosphate ions in the protein synthesis system and significantly improve the protein synthesis efficiency.

[0265] (4) The present invention adopts the in situ removal method for the first time to improve the efficiency of biological reaction and reduce the complexity of reaction.

[0266] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0267] Unless otherwise specified, the materials and reagents used in the examples of the present invention are all commercially available products.

[0268] General Methods

[0269] In the present invention, after the yeast extract is obtained, sucrose and / or mannitol are added, and then freeze-dried to obtain freeze-dried yeast extract.

[0270] In the present invention, the biological reaction system is taken as an in vitro protein synthesis system, and the biological reactant is taken as an example of a yeast cell extract, but it is not limited thereto.

[0271] Example 1 Preparation of yeast cell extract by liquid nitrogen disruption method

[0272] 1.1. Primary seed culture: Inoculate the frozen bacteria at -80℃ into the shake flask medium and culture at 30℃ and 200rpm until the logarithmic growth phase.

[0273] 1.2. Secondary seed culture: Take an appropriate amount of primary seed culture solution and inoculate it into the secondary seeds, and culture at 30°C and 200rpm until the logarithmic growth phase.

[0274] 1.3. Batch culture stage: The secondary seed culture liquid is inoculated into the fermenter, the temperature is controlled at 30°C for 10-12 hours, and then enters the fed-batch culture stage. When the OD600 value is 50-55, the cell culture is collected.

[0275] 1.4. Precool the cultured cells in an ice-water mixture for 10-30 minutes.

[0276] 1.5. Centrifuge the pre-cooled cell culture in 1.4 in a low-temperature centrifuge at 3,000 g, 10 min, 4°C to obtain yeast cells.

[0277] 1.6. Resuspend the yeast cells in 1.5 with pre-cooled washing buffer, and centrifuge the resuspended solution at 3000g, 10min, 4°C to obtain yeast cells. The composition of washing buffer is: 20-30mM potassium 4-hydroxyethylpiperazineethanesulfonate with a pH of 7.4, 100-150mM potassium acetate, and 1-4mM magnesium acetate;

[0278] 1.7. Repeat step 1.6 2-3 times.

[0279] 1.8. The yeast cells obtained in step 1.7 can be directly used for subsequent operations, or quickly frozen with liquid nitrogen and stored at -80°C.

[0280] 1.9. Use liquid nitrogen homogenizer for crushing: add appropriate amount of liquid nitrogen to the homogenizer, then add yeast cells obtained by centrifugation or yeast cells stored at -80℃ in 1.8, speed: 45,000rpm, crush for 3-10min; pack the crushed cryogenic powder into 50mL centrifuge tubes, weigh and store at -80℃ for later use.

[0281] 1.10. Cool the yeast cell powder obtained in 1.9 to 4°C at room temperature, and dissolve each gram of cell powder with 0.2-1 mL of 4°C pre-cooled Lysis buffer to obtain a crude yeast cell extract. Lysis buffer is composed of 10-40 mM potassium 4-hydroxyethylpiperazineethanesulfonate with a pH of 7.4, 50-150 mM potassium acetate, 1-4 mM magnesium acetate, 2-7 mM dithiothreitol, and 0.5-2 mM phenylmethylsulfonyl fluoride.

[0282] 1.11. Centrifuge the crude yeast cell extract harvested in step 1.10 1-2 times at a centrifugal force of 12000-30000g for 30 min at a temperature of 4°C;

[0283] 1.12. After centrifugation, take the upper clear liquid as the yeast cell extract.

[0284] 1.13. Aliquot the prepared yeast cell extract, freeze it in liquid nitrogen and store it at -80°C.

[0285] Example 2 Freeze-drying of cell extracts

[0286] 2.1 Thaw the yeast extract stored at -80℃ at room temperature;

[0287] 2.2 Weigh each glass bottle or glass plate and keep records;

[0288] 2.3 Dispense the thawed yeast extract into glass bottles or glass plates, with about 500 μl of cell extract in each glass bottle and about 5 ml of cell extract in each glass plate.

[0289] 2.4 Weigh the glass bottles and glass plates containing cell extracts in 2.3 again and keep records;

[0290] 2.5 Prefreeze the glass bottles and glass plates containing cell extracts in 2.4 at -80°C for 2-4 hours;

[0291] 2.6 Set the freeze-drying program: pre-freezing stage: -55℃, 4h; first freeze-drying stage: -30℃, 4-10h; second freeze-drying stage: -30℃, 10h; final freeze-drying stage: -20℃, 10-20h.

[0292] 2.7 Place the pre-frozen cell extract glass bottle and glass plate in 2.5 on the plate of the freeze dryer and perform the freeze drying process with a vacuum degree of about 0.1Mpa.

[0293] 2.8 Store the freeze-dried cell extract at -80°C, or directly add water of equal mass to that before freeze-drying and mix well for subsequent protein in vitro synthesis activity determination.

[0294] Example 3 In vitro biological reaction system

[0295] 3.1 Preparation of storage solution for in vitro biological reaction system: 1M Tris-HCl with pH 8.0, 5M potassium acetate, 1M magnesium acetate, 25mM mixture of four nucleoside triphosphates, including adenine triphosphate, guanine triphosphate, cytosine triphosphate and uridine triphosphate, 1mM mixture of twenty kinds of 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 twenty kinds of amino acids is 1.0mM, 1M glucose, 1M dithiothreitol, 1M potassium phosphate, 2.4mg / mL T7 RNA polymerase, 20%-50% polyethylene glycol (PEG) 3350 or (polyethylene glycol) glycol, PEG) 8000, 1-4 mM spermidine, 0.1-0.4 mM heme;

[0296] 3.2 In vitro biological reaction system: final concentration of 22 mM Tris-HCl with pH 7-9, 30-150 mM potassium acetate, 1.0-5.0 mM magnesium acetate, 1.5-4 mM nucleoside triphosphate mixture (adenosine triphosphate, guanosine triphosphate, cytosine triphosphate and uridine triphosphate), 0.08-0.24 mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, aspartate amide, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine), 5-80mM glucose, 1.7mM dithiothreitol, 5-40mM potassium phosphate, 8-20ng / μL firefly luciferase DNA, 0.027-0.054mg / mL T7RNA polymerase, 1%-4% polyethylene glycol, 0.03-0.04mM heme, 0.3-0.4mM spermidine, and finally add 50% volume of yeast cell extract;

[0297] 3.3 In vitro biological reaction: Place the above reaction system in an environment of 20-25°C and let it stand or react with slight shaking for 1-7 hours;

[0298] 3.4 Luciferase activity assay: After the reaction, add an equal volume of substrate luciferin to a 96-well or 384-well white plate and immediately place it on an Envision 2120 multifunctional microplate reader (Perkin Elmer) to read and detect the firefly luciferase activity. The relative light unit (RLU) is used as the activity unit.

[0299] Experimental Results

[0300] 1. Examples of biological reactions producing phosphate ions

[0301] from Figure 1It can be seen that several steps and mechanisms in biological reactions release reaction byproducts represented by phosphate. For example: in several steps of transcription and translation, magnesium ions are involved, using substrates, energy, etc., in solid, liquid, or other phases, the main target is produced through reactions; at the same time, in several stages of transcription and translation, several reaction byproducts are produced, such as free phosphate ions and pyrophosphate ions; such as kinase reactions (protein kinases, lipid kinases, sugar kinases, etc.); ATPase enzyme reactions (transmembrane transport of sodium, potassium, calcium ions, etc.), molecular motors, etc., byproducts often react with biological reaction systems and inhibit reactions. At present, the existing methods for removing reaction byproducts mainly include dialysis, circulation, solution replacement, etc., all of which require additional equipment, have complex systems, are not easy to operate, and are not easy to complete in small reaction systems. Therefore, the removal of reaction byproducts, especially in situ removal, is the key to improving reaction efficiency and reducing reaction complexity.

[0302] 2. Effects of different free phosphorus control agents on the protein synthesis system of 30 μL cell extract

[0303] from Figure 2 It can be seen that for the 30 μL cell extract protein synthesis system, adding too much reaction promoter can inhibit the in vitro protein synthesis ability of the cell extract, and different reaction promoters have different degrees of inhibition on the in vitro protein synthesis ability. When the reaction is carried out for 3 hours, the protein synthesis relative light unit value (RLU) of the protein synthesis system in which aluminum oxide is added for 2 hours is many (several) times higher than the protein synthesis relative light unit value (RLU) of the protein synthesis system in which ferric oxide or ferric oxide is added at the same time (the RLU value of adding ferric oxide or ferric oxide is very low, almost zero). Moreover, relatively speaking, the reaction activity of aluminum oxide is relatively less reduced, so the effect of an appropriate amount of aluminum oxide as a reaction promoter for in vitro biological reactions is more advantageous than ferric oxide and ferric oxide.

[0304] 3. Effects of different free phosphorus control agents on the protein synthesis system of 120 μL cell extract

[0305] from Figure 3 It can be seen that for the 120 μL cell extract protein synthesis system, adding an appropriate amount of reaction promoter can not greatly affect the in vitro protein synthesis ability of the cell extract and can even slightly improve it. Moreover, after 2 hours of reaction, the relative light unit (RLU) of protein synthesis in the protein synthesis system with the addition of aluminum oxide is 1.4 times higher than that in the protein synthesis system with the addition of ferric oxide or ferric oxide.

[0306] 4. Comparison of the dephosphorylation effects of different dephosphorylation agents in a 120 μL cell extract protein synthesis system

[0307] from Figure 4 It can be seen that when equal amounts of different reaction promoters were added after 2 hours of reaction, the reaction promoters in the system decreased to varying degrees after 4.5 hours; relatively speaking, alumina had the best control effect on free phosphorus in the in vitro biological reaction system.

[0308] 5. Effect of adding aluminum oxide to the cell extract protein synthesis system at different time periods

[0309] from Figure 5 It can be seen that in the 90 μL cell-free reaction system, after 30 mg of alumina was used to control the free phosphorus content in the system at 1 hour, the RLU value between 2-3 hours of reaction was 2-3 times that of the control group (without alumina). Therefore, in the 90 μL cell-free reaction system, controlling the free phosphorus content after 1 hour of reaction significantly improved the reaction activity of the system, and when the reaction was carried out for 3 hours, the RLU value of adding alumina for 1 hour of reaction was significantly higher than the RLU value of adding alumina for 2 hours of reaction, and the former was 2-3 times the latter.

[0310] 6. Effect of adding alumina to the cell extract protein synthesis system at different time periods on the free phosphorus concentration of the system

[0311] from Figure 6 It can be seen that in the 90 μL cell-free reaction system, the free phosphorus concentration in the system decreased significantly after 1 hour of reaction. After adding alumina at different time periods (1 hour and 2 hours), the free phosphorus concentration in the system did not increase significantly compared with the control group, indicating that alumina can significantly control the free phosphorus content in the system. Moreover, when the reaction was carried out for 3 hours, the degree of decrease in the free phosphorus concentration in the system was comparable between adding alumina for 1 hour and adding alumina for 2 hours.

[0312] 7. Effect of adding different amounts of aluminum oxide to the cell extract protein synthesis system after 1 h of reaction

[0313] from Figure 7 It can be seen that after adding different masses of aluminum oxide to the 90μL cell-free reaction system at 1h of reaction, the RLU value was significantly improved compared with the control group, and the RLU value could reach 2.5×107. After 6-7 hours of reaction, adding 10mg or 20mg of aluminum oxide significantly improved the reaction activity, and the RLU value could reach 3.0×107.

[0314] 8. Phosphoric acid control system improves biological reaction efficiency

[0315] In summary, the phosphate control in the system and the biochemical reaction in the system are a pair of competing reactions. Too much phosphate or pyrophosphate in the system will lead to pH imbalance in the system, affect the activity of the biological enzymes involved in the biochemical reaction in the system, and reduce the yield of the target protein; at the same time, phosphate is easy to combine with the magnesium ions required in the reaction system to form magnesium sulfate that is insoluble in water, resulting in the reduction of reaction activity in many biochemical reaction steps due to the lack of magnesium ions. The addition of phosphate control agents adjusts the balance of the biochemical reactions mentioned above by physical (adsorption) or chemical (formation of precipitation products out of the reaction system), thereby creating a relatively ideal reaction environment for the protein synthesis system.

[0316] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0317] References:

[0318] 1.Garcia,RA,&Riley,MR(1981).Applied biochemistryandbiotechnology.Humana Press,.

[0319] 2. Waddington, CH (1961). Molecular biology or ultrastructural biology? Nature,190(4771),184.

[0320] 3. Assenberg, R., Wan, PT, Geisse, S., & Mayr, LM (2013). Advances in recombinant protein expression for use in pharmaceutical research. Current Opinion in Structural Biology, 23(3), 393–402.

[0321] 4. S.,Nordlund,P.,Weigelt,J.,Hallberg,B.M.,Bray,J.,Gileadi,O.,…Gunsalus,K.C.(2008).Protein production and purification.Nature Methods,5(2),135–146.

[0322] 5.Katzen,F.,Chang,G.,&Kudlicki,W.(2005).The past,present and futureofcell-free protein synthesis.Trends in Biotechnology,23(3),150–156.

[0323] 6.Mcmahon,T.,Zijl,P.C.M.Van,&Gilad,A.A.(2015).NIH Public Access,27(3),320–331.

[0324] 7.Quast,R.B.,Ballion,B.,Stech,M.,Sonnabend,A.,Varga,B.R.,Wüstenhagen,D.A.,…Kubick,S.(2016).Cell-free synthesis of functional humanepidermalgrowth factor receptor:Investigation of ligand-independentdimerization inSf21 microsomal membranes using non-canonical aminoacids.Scientific Reports,6(March),1–13.

[0325] 8.Lu,Y.(2017).Cell-free synthetic biology:Engineering in anopenworld.Synthetic and Systems Biotechnology,2(1),23–27.

[0326] 9.Robertis,D.(1987).Cell and molecular biology.8th Edition.

[0327] 10.Fromm,H.J.,&Hargrove,M.(2012).Essentials of Biochemistry.

Claims

1. An in vitro protein synthesis system, characterized in that: include: (a) yeast cell extract; (b) a first reaction accelerator, wherein the first reaction accelerator is an oxyaluminum complex; The average weight of the oxyaluminum complex is 1-80 mg, preferably 3-50 mg, and more preferably 4-30 mg.

2. The in vitro protein synthesis system according to claim 1, characterized in that: The in vitro protein synthesis system also includes: exogenous DNA.

3. The protein synthesis system according to claim 1 or 2, characterized in that: The protein synthesis system also includes: (b') a second reaction accelerator, wherein the second reaction accelerator is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, soluble starch, or a combination thereof.

4. The protein synthesis system according to claim 1 or 2, characterized in that: The aluminum oxide composite includes aluminum oxide nanoparticles.

5. The protein synthesis system according to claim 1 or 2, characterized in that: The oxyaluminum complex includes Al2O3.

6. The protein synthesis system according to claim 1 or 2, characterized in that: The particle size of the oxyaluminum composite is 0.5-20 mm, preferably 0.8-10 mm, and more preferably 1-5 mm.

7. The protein synthesis system according to claim 1 or 2, characterized in that: In the first reaction accelerator, the content (wt%) of the oxyaluminum complex is 0.1-20%, preferably 1-10%, more preferably 2%-8%, more preferably 4%-7%, more preferably 5.5%-6.5%, based on the total weight of the first reaction accelerator.

8. The protein synthesis system according to claim 1 or 2, characterized in that: In the protein synthesis system, the content (wt%) of the first reaction accelerator is 20%-70%, preferably 30%-60%, more preferably 45%-55%, based on the total weight of the protein synthesis system.

9. A method for producing a protein synthesis system according to any one of claims 1 to 8, characterized in that: The method comprises the steps of: mixing component (i) and component (ii) to obtain the protein synthesis system according to claim 1 or claim 2, wherein the component (i) is selected from the following group: yeast cell extract; the component (ii) is a first reaction accelerator, and the first reaction accelerator is an oxyaluminum complex; the average weight of the oxyaluminum complex is 1-80 mg, preferably 3-50 mg, and more preferably 4-30 mg.

10. The production method according to claim 9, characterized in that In the protein synthesis system, the mass ratio of component (i) to component (ii) is 0.1-10:0.1-10, preferably, 0.5-8:0.5-8, more preferably, 0.8-5:0.8-5, more preferably, 0.9-2:0.9-2.

11. A method for in vitro protein synthesis, characterized in that: include: (i) providing the protein synthesis system according to claim 1 or claim 2, and adding an exogenous DNA molecule for directing protein synthesis; (ii) incubating the protein synthesis system of step (i) for a period of time T1 under suitable conditions, thereby synthesizing the protein encoded by the exogenous DNA.

12. A kit, characterized in that: include: (k1) a first container, and component (i) in the first container, wherein component (i) is: a yeast cell extract; (k2) a second container, and a component (ii) in the second container, wherein the component (ii) is a first reaction accelerator, and the first reaction accelerator is an oxyaluminum complex; the average weight of the oxyaluminum complex is 1-80 mg, preferably 3-50 mg, and more preferably 4-30 mg; and (kt) labels or instructions.

Images