WO2019127259A1 - 一种体外蛋白合成体系、试剂盒及其制备方法 - Google Patents

一种体外蛋白合成体系、试剂盒及其制备方法 Download PDF

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WO2019127259A1
WO2019127259A1 PCT/CN2017/119540 CN2017119540W WO2019127259A1 WO 2019127259 A1 WO2019127259 A1 WO 2019127259A1 CN 2017119540 W CN2017119540 W CN 2017119540W WO 2019127259 A1 WO2019127259 A1 WO 2019127259A1
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protein synthesis
synthesis system
reaction
cell
another preferred
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PCT/CN2017/119540
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English (en)
French (fr)
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郭敏
徐开
陈鉴冰
周子鉴
柴智
章小铃
王海鹏
于雪
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康码（上海）生物科技有限公司
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Priority to PCT/CN2017/119540 priority Critical patent/WO2019127259A1/zh
Priority to EP17935873.4A priority patent/EP3733861A4/en
Priority to KR1020207022011A priority patent/KR102560378B1/ko
Publication of WO2019127259A1 publication Critical patent/WO2019127259A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M45/00—Means for pre-treatment of biological substances
    • C12M45/05—Means for pre-treatment of biological substances by centrifugation

Definitions

  • the invention relates to the field of biotechnology, in particular to an in vitro protein synthesis system, a kit and a preparation method thereof.
  • Bio reactions that is, biochemical reactions, refer to chemical reactions carried out in living organisms. These reactions are catalyzed by enzymes. The enzymes and reactants are dissolved in the water of the internal environment to react. Water provides carriers and media for the body's substances [1] .
  • biochemistry has made great achievements in explaining life processes.
  • almost all fields related to life sciences such as botany, medicine, and genetics are engaged in biochemistry research [2].
  • the biochemical reactions carried out in organisms or cells can be automatically regulated by positive and negative feedback in complex network systems, and the biochemical reactions carried out in organisms are reactions away from equilibrium points. It is necessary to extract energy from the outside world or to output matter, energy and entropy to the outside world [9].
  • the removal of by-products of biological reactions is mainly achieved by the body's metabolism, the maintenance of the body's steady state, the catalytic action of biological enzymes, and the redox reaction in the cells away from the equilibrium point, but these biochemical reactions are difficult to achieve in vitro biotechnology. [10].
  • a disadvantage of protein synthesis systems is that the yield is not high, the reaction is not fast and sensitive, and in vitro biosynthesis systems, such as in vitro protein synthesis, in vitro transcription, kinase reaction, ATPase reaction, ion pump reaction, etc., such as free phosphoric acid Root ions, pyrophosphate ions (PPi), limit the yield of the target product.
  • in vitro biosynthesis systems such as in vitro protein synthesis, in vitro transcription, kinase reaction, ATPase reaction, ion pump reaction, etc., such as free phosphoric acid Root ions, pyrophosphate ions (PPi), limit the yield of the target product.
  • a first aspect of the invention provides an in vitro protein synthesis system comprising:
  • a first reaction promoter selected from the group consisting of aluminum, an aluminum salt, an aluminum oxide complex, or a combination thereof.
  • the cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell.
  • 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.
  • mammalian cells such as HF9, Hela, CHO, HEK293
  • plant cells such as HF9, Hela, CHO, HEK293
  • yeast cells or a combination thereof.
  • 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, preferably The ground is Kluyveromyces lactis.
  • the protein synthesis system further comprises:
  • a second reaction promoter selected from the group consisting of a monosaccharide, a disaccharide, an oligosaccharide, a soluble starch, or a combination thereof;
  • the protein synthesis system comprises a yeast in vitro protein synthesis system (eg, Kluyveromyces in vitro protein synthesis system, preferably, Kluyveromyces lactis in vitro protein synthesis system).
  • a yeast in vitro protein synthesis system eg, Kluyveromyces in vitro protein synthesis system, preferably, Kluyveromyces lactis in vitro protein synthesis system.
  • the oxyaluminum complex comprises oxyaluminum nanoparticles.
  • the oxyaluminum composite comprises Al 2 O 3 .
  • the oxyaluminum complex is in the form of particles or nano.
  • the oxyaluminum composite has a particle diameter of from 0.5 to 20 mm, preferably from 0.8 to 10 mm, more preferably from 1 to 5 mm.
  • the oxyaluminum complex has an average weight of from 1 to 80 mg, preferably from 3 to 50 mg, more preferably from 4 to 30 mg.
  • the concentration (v/v) of the oxy-aluminum complex in the first reaction accelerator is 0.5-20%, preferably 0.8-10%, more preferably, 1%. -10%, more preferably, 1% to 5%, more preferably 2% to 3%, based on the total volume of the first reaction accelerator.
  • the content of the oxyaluminum complex (wt%) in the reaction accelerator is 0.1-20%, preferably 1-10%, more preferably 2%-8%. More preferably, 4% to 7%, more preferably 5.5% to 6.5%, based on the total weight of the first reaction accelerator.
  • the concentration (v/v) of the first reaction accelerator is from 20% to 70%, preferably from 30% to 60%, more preferably, 45. %-55%, based on the total volume of the protein synthesis system.
  • the content of the first reaction accelerator (wt%) in the protein synthesis system is 20% to 70%, preferably 30% to 60%, more preferably 45%. -55% based on the total weight of the protein synthesis system.
  • a second aspect of the invention provides an in vitro cell-free protein synthesis system comprising:
  • a first reaction promoter selected from the group consisting of aluminum, an aluminum salt, an aluminum oxide complex, or a combination thereof.
  • the protein synthesis system further comprises:
  • a second reaction promoter selected from the group consisting of a monosaccharide, a disaccharide, an oligosaccharide, a soluble starch, or a combination thereof;
  • the protein synthesis system comprises a yeast in vitro protein synthesis system (eg, Kluyveromyces in vitro protein synthesis system, preferably, Kluyveromyces lactis in vitro protein synthesis system).
  • a yeast in vitro protein synthesis system eg, Kluyveromyces in vitro protein synthesis system, preferably, Kluyveromyces lactis in vitro protein synthesis system.
  • the oxyaluminum complex comprises oxyaluminum nanoparticles.
  • the oxyaluminum composite comprises Al 2 O 3 .
  • the oxyaluminum complex is in the form of particles or nano.
  • the oxyaluminum composite has a particle diameter of from 0.5 to 20 mm, preferably from 0.8 to 10 mm, more preferably from 1 to 5 mm.
  • the oxyaluminum complex has an average weight of from 1 to 80 mg, preferably from 3 to 50 mg, more preferably from 4 to 30 mg.
  • the concentration (v/v) of the oxy-aluminum complex in the first reaction accelerator is 0.5-20%, preferably 0.8-10%, more preferably, 1%. -10%, more preferably, 1% to 5%, more preferably 2% to 3%, based on the total volume of the first reaction accelerator.
  • the content of the oxyaluminum complex (wt%) in the reaction accelerator is 0.1-20%, preferably 1-10%, more preferably 2%-8%. More preferably, 4% to 7%, more preferably 5.5% to 6.5%, based on the total weight of the first reaction accelerator.
  • the concentration (v/v) of the first reaction accelerator is from 20% to 70%, preferably from 30% to 60%, more preferably, 45. %-55%, based on the total volume of the protein synthesis system.
  • the content of the first reaction accelerator (wt%) in the protein synthesis system is 20% to 70%, preferably 30% to 60%, more preferably 45%. -55% based on the total weight of the protein synthesis system.
  • the cell extract of the cell extract is selected from the group consisting of one or more types of cells: prokaryotic cells and eukaryotic cells.
  • the cell extract is obtained from a cell source selected from the group consisting of one or more types of cells: Escherichia coli, bacteria, mammalian cells (eg, HF9, Hela, CHO, HEK293), plant cells , yeast cells, or a combination thereof.
  • a cell source selected from the group consisting of one or more types of cells: Escherichia coli, bacteria, mammalian cells (eg, HF9, Hela, CHO, HEK293), plant cells , yeast cells, or a combination thereof.
  • 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, preferably The ground is Kluyveromyces lactis.
  • the protein synthesis system further comprises one or more components selected from the group consisting of:
  • the protein synthesis system further comprises one or more components selected from the group consisting of:
  • the cell extract comprises a yeast cell extract.
  • the yeast cell extract is an aqueous extract of yeast cells.
  • the yeast cell extract is free of yeast endogenous long chain nucleic acid molecules.
  • the yeast cell extract is prepared by a method comprising the steps of:
  • the solid-liquid separation comprises centrifugation.
  • centrifugation is carried out in a liquid state.
  • the centrifugation conditions are from 5,000 to 100,000 g, preferably from 8,000 to 30,000 g.
  • the centrifugation time is from 0.5 min to 2 h, preferably from 20 min to 50 min.
  • the centrifugation is carried out at 1-10 ° C, preferably at 2-6 ° C.
  • the washing treatment is carried out using a washing liquid at a pH of 7-8 (preferably, 7.4).
  • the washing liquid is selected from the group consisting of potassium 4-hydroxyethylpiperazine ethanesulfonate, potassium acetate, magnesium acetate, or a combination thereof.
  • the cell disruption treatment comprises high pressure disruption, freeze-thaw (eg, liquid nitrogen cryolysis) disruption.
  • the substrate for the synthetic RNA comprises: a nucleoside monophosphate, a nucleoside triphosphate, or a combination thereof.
  • the substrate of the synthetic protein comprises: 1-20 natural amino acids, and unnatural amino acids.
  • the magnesium ion is derived from a source of magnesium ions selected from the group consisting of magnesium acetate, magnesium glutamate, or a combination thereof.
  • the potassium ion is derived from a source of potassium ions selected from the group consisting of potassium acetate, potassium glutamate, or a combination thereof.
  • the energy regeneration system is selected from the group consisting of a phosphocreatine/phosphocreatase system, a glycolysis pathway and its intermediate energy system, or a combination thereof.
  • the protein synthesis system further comprises (h1) a synthetic tRNA.
  • the buffering agent is selected from the group consisting of 4-hydroxyethylpiperazineethanesulfonic acid, trishydroxymethylaminomethane, or a combination thereof.
  • the protein synthesis system further comprises (i1) an exogenous DNA molecule for directing protein synthesis.
  • the DNA molecule is linear.
  • the DNA molecule is cyclic.
  • the DNA molecule contains a sequence encoding a foreign protein.
  • the sequence encoding the foreign protein comprises a genomic sequence, a cDNA sequence.
  • sequence encoding the foreign protein further comprises a promoter sequence, a 5' untranslated sequence, and a 3' untranslated sequence.
  • the protein synthesis system comprises a component selected from the group consisting of 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, nucleoside triphosphate, amino acid, creatine phosphate, disulfide Threitol (DTT), phosphocreatine kinase, RNA polymerase, or a combination thereof.
  • the polyethylene glycol is selected from the group consisting of PEG3000, PEG 8000, PEG 6000, PEG 3350, or a combination thereof.
  • the polyethylene glycol comprises polyethylene glycol having a molecular weight (Da) of from 200 to 10,000, preferably polyethylene glycol having a molecular weight of from 3,000 to 10,000.
  • the concentration (v/v) of the component (a) in the protein synthesis system is from 20% to 70%, preferably from 30% to 60%, more preferably from 40% to 50%. %, based on the total volume of the protein synthesis system.
  • the content (% by weight) of the component (c) in the protein synthesis system is from 10% to 95%, preferably from 20% to 80%, more preferably from 40% to 60%. %, based on the total weight of the protein synthesis system.
  • the concentration (w/v, for example, g/ml) of the component (d) in the protein synthesis system is from 0.1 to 8%, preferably from 0.5 to 4%, more preferably, 1-2%.
  • the concentration of the component (e) in the protein synthesis system is 0.2 to 4%, preferably 0.5 to 4%, more preferably 0.5 to 1%, to synthesize the protein.
  • the total volume of the system is 0.2 to 4%, preferably 0.5 to 4%, more preferably 0.5 to 1%, to synthesize the protein.
  • the nucleoside triphosphate is selected from the group consisting of adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, uridine nucleoside triphosphate, or a combination thereof.
  • the concentration of the component (f1) in the protein synthesis system is from 0.1 to 5 mM, preferably from 0.5 to 3 mM, more preferably from 1 to 1.5 mM.
  • the amino acid is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, serine, Tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, or a combination thereof.
  • the amino acid comprises a D-form amino acid and/or an L-form amino acid.
  • the concentration of the component (f2) in the protein synthesis system is 0.01 to 0.48 mM, preferably 0.04 to 0.24 mM, more preferably 0.04 to 0.2 mM, optimally , 0.08 mM.
  • the concentration of the component (f3) in the protein synthesis system is 1-10 mM, preferably 1-5 mM, more preferably 2-4 mM.
  • the concentration of the component (f4) in the protein synthesis system is 30-210 mM, preferably 30-150 mM, more preferably 30-60 mM.
  • the concentration of the component (f6) in the protein synthesis system is 0.01 to 0.3 mg/mL, preferably 0.02 to 0.1 mg/mL, more preferably 0.027 to 0.054 mg. /mL.
  • the concentration of 4-hydroxyethylpiperazineethanesulfonic acid in the protein synthesis system is 5 to 50 mM, preferably 10 to 50 mM, preferably 15 to 30 mM, more preferably , 20-25 mM.
  • the concentration of the potassium acetate in the protein synthesis system is 20-210 mM, preferably 30-210 mM, preferably 30-150 mM, more preferably 30-60 mM.
  • the magnesium acetate has a concentration of 1-10 mM, preferably 1-5 mM, more preferably 2-4 mM.
  • the concentration of creatine phosphate is 10-50 mM, preferably 20-30 mM, more preferably 25 mM.
  • the concentration of the heme in the protein synthesis system is 0.01 to 0.1 mM, preferably 0.02 to 0.08 mM, more preferably 0.03 to 0.05 mM, most preferably 0.04 mM. .
  • the spermidine concentration in the protein synthesis system is 0.05-1 mM, preferably 0.1-0.8 mM, more preferably, more preferably 0.2-0.5 mM, more preferably Ground, 0.3-0.4 mM, optimally, 0.4 mM.
  • the concentration of the dithiothreitol (DTT) in the protein synthesis system is from 0.2 to 15 mM, preferably from 0.2 to 7 mM, more preferably from 1 to 2 mM.
  • the concentration of the phosphocreatine kinase in the protein synthesis system is 0.1 to 1 mg/mL, preferably 0.2 to 0.5 mg/mL, more preferably 0.27 mg/mL.
  • the concentration of the T7 RNA polymerase in the protein synthesis system is 0.01-0.3 mg/mL, preferably 0.02-0.1 mg/mL, more preferably 0.027-0.054 mg/mL. .
  • the protein synthesis system has the following properties:
  • composition of the protein synthesis system comprises:
  • composition of the protein synthesis system further comprises:
  • the PEG is selected from the group consisting of PEG 3350, PEG 3000, and/or PEG 8000.
  • the RNA polymerase is T7 RNA polymerase.
  • a third aspect of the invention provides a method for producing a protein synthesis system according to the first aspect of the invention or the second aspect of the invention, comprising the steps of:
  • the component (i) and the component (ii) are mixed to obtain the protein synthesis system according to the first or second aspect of the invention, wherein the component (i) is selected from the group consisting of: cell or cell extraction And a combination thereof; the component (ii) is a first reaction accelerator, and the first reaction promoter is selected from the group consisting of aluminum, an aluminum salt, an aluminum oxide composite, or a combination thereof.
  • the component (i) is a cell extract.
  • the ratio (mass ratio) of component (i) to component (ii) in the protein synthesis system is from 0.1 to 10:0.1 to 10, preferably from 0.5 to 8:0.5. 8, more preferably, 0.8-5: 0.8-5, more preferably, 0.9-2: 0.9-2.
  • the concentration (v/v) of component (i) in the protein synthesis system is 10-80%, preferably 20-60%, based on the total volume of the protein synthesis system. meter.
  • the content (% by weight) of the component (i) in the protein synthesis system is 10-80%, preferably 20-60%, based on the total weight of the protein synthesis system. .
  • the concentration (v/v) of component (ii) in the protein synthesis system is 10-80%, preferably 20-60%, based on the total volume of the protein synthesis system. meter.
  • the content (wt%) of the component (ii) in the protein synthesis system is 10-80%, preferably 20-60%, based on the total weight of the protein synthesis system. .
  • a fourth aspect of the invention provides a method of in vitro protein synthesis comprising:
  • step (ii) incubating the protein synthesis system of step (i) for a period of time T1 under suitable conditions to synthesize the protein encoded by the exogenous DNA.
  • the method further comprises: (iii) isolating or detecting the protein encoded by the exogenous DNA, optionally from the protein synthesis system.
  • the exogenous DNA is from a prokaryote, a eukaryote.
  • the exogenous DNA is from an animal, a plant, or a pathogen.
  • the exogenous DNA is from a mammal, preferably a primate, a rodent, including a human, a mouse, a rat.
  • the coding sequence of the foreign protein encodes a foreign protein selected from the group consisting of 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 mutant, alpha-amylase, enterobacterin A, C Hepatitis B virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylan Enzyme, or a combination thereof.
  • luciferin or luciferase (such as firefly luciferase)
  • green fluorescent protein yellow fluorescent protein
  • Aminoacyl tRNA synthetase
  • the exogenous protein is selected from the group consisting of 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, alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin precursor Interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase, or a combination thereof.
  • luciferin or luciferase (such as firefly luciferase)
  • green fluorescent protein yellow fluorescent protein
  • aminoacyl tRNA synthetase aminoacyl tRNA synthetase
  • the exogenous DNA encodes a foreign protein selected from the group consisting of 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 mutant, alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase, or Its combination.
  • luciferin or luciferase (such as firefly luciferase)
  • green fluorescent protein yellow fluorescent protein
  • aminoacyl tRNA Synthetase glyceraldehyde
  • the protein encoded by the exogenous DNA is selected from the group consisting of 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, alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, Insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase, or a combination thereof.
  • luciferin or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, Glyceraldehyde-3-
  • the reaction temperature is 20 to 37 ° C, preferably 20 to 25 ° C.
  • the reaction time is from 1 to 6 h, preferably from 2 to 4 h.
  • a fifth aspect of the invention provides a kit comprising:
  • (k1) a first container, and a component (i) located in the first container, the component (i) being selected from the group consisting of a cell or a cell extract, or a combination thereof;
  • component (k2) a second container, and component (ii) located in the second container, said component (ii) being a first reaction promoter, said first reaction promoter being selected from the group consisting of aluminum and aluminum salts , an aluminum oxide composite, or a combination thereof; and
  • the component (i) is a cell extract.
  • first container and the second container are the same container or different containers.
  • the kit further comprises an optional one or more containers selected from the group consisting of:
  • Figure 1 shows the release of reaction by-products represented by phosphate in a biological reaction.
  • reaction by-products such as free phosphate ions, pyrophosphate ions; such as kinase reaction (protein kinase, lipid kinase, glycokinase, etc.); ATPase enzyme reaction (sodium, potassium, calcium, etc.)
  • kinase reaction protein kinase, lipid kinase, glycokinase, etc.
  • ATPase enzyme reaction sodium, potassium, calcium, etc.
  • reaction by-products mainly include dialysis method, circulation method, solution replacement, etc., all of which require additional equipment, are complicated in system, are not easy to operate, and are not easily completed in a small reaction system. Therefore, the removal of reaction by-products, especially in situ removal, is the key to improving the efficiency of the reaction and reducing the complexity of the reaction.
  • Figure 2 is a graph showing the effect of adding 30 ⁇ L of the in vitro protein synthesis system to the first reaction promoter (phosphoric acid control preparation) at 2 h of reaction.
  • the solid circle line is a cell-free reaction system without the addition of the first reaction promoter.
  • the cell-free reaction system of Fe 2 O 3 is added when the square is 2 hours, and the cell-free reaction system of the mass Fe 3 O 4 is added when the equilateral triangle is 2 hours.
  • a cell-free reaction system of equal mass alumina particles was added when the triangle was 2 h.
  • the reaction conditions were 20-25 ° C for 3 h. All errors are the standard deviation of three replicates.
  • Figure 3 shows a schematic diagram of the effect of adding a first reaction promoter to a 120 ⁇ L cell-free reaction system at 2 h of reaction.
  • the solid circle line is a cell-free reaction system without adding the first reaction accelerator, the cell-free reaction system of Fe 2 O 3 is added when the square is 2 h, and the cell-free reaction system of equal mass of Fe 3 O 4 is added when the equilateral triangle is 2 h.
  • a cell-free reaction system of equal mass alumina particles was added when the inverted triangle was 2 h.
  • the reaction conditions were 20-25 ° C for 4 h and 5 h. All errors are the standard deviation of three replicates.
  • Figure 4 is a graph showing the concentration of phosphoric acid in the system at a reaction time of 2 hours when a 120 ⁇ L cell-free reaction system was added to the first reaction promoter at 2 h.
  • 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 Fe 2 O 3 added at 2 h
  • the third column is the cell-free system with equal mass of Fe 3 O 4 at 2 h.
  • the fourth column is a cell-free reaction system in which equal-mass alumina particles are added at 2 hours.
  • the reaction conditions were 20-25 ° C for 4.5 h.
  • Figure 5 shows the effect of the addition of 30 mg of alumina particles in a 90 ⁇ L cell-free reaction system at different time periods (1 h and 2 h and without), with reaction conditions of 20-25 °C. All errors are the standard deviation of three replicates.
  • Figure 6 is a graph showing the concentration of phosphoric acid in a system in which 90 ⁇ L of the cell-free reaction system was added to 30 mg of alumina particles at different time periods (1 h and 2 h, and without), and the reaction conditions were 20-25 °C. All errors are the standard deviation of three replicates.
  • Fig. 7 is a view showing the effect of adding 90 ⁇ L of the cell-free reaction system to different degrees of alumina particles in the system at the reaction for 1 hour, and the reaction conditions were 20-25 °C. All errors are the standard deviation of three replicates.
  • FIG 8 is a schematic illustration of the efficacy of a phosphoric acid control system to enhance biological response.
  • the phosphoric acid control in the system and the biochemical reactions in the system are a pair of competing reactions. Excessive phosphate or pyrophosphate in the system will lead to pH imbalance in the system, affecting the activity of the bio-enzyme involved in the biochemical reaction in the system, and reducing the yield of the target protein.
  • the phosphate is easily associated with the magnesium ion required in the reaction system.
  • the combination of the formation of water-insoluble magnesium sulfate results in numerous biochemical reaction steps resulting in a decrease in reactivity due to the lack of magnesium ions.
  • the addition of the phosphoric acid control formulation adjusts the balance of the biochemical reaction as described above by physical (adsorption) or chemistry (formation of the precipitated product from the reaction system), thereby creating a relatively ideal reaction environment for the protein synthesis system.
  • the inventors have extensively and intensively studied for the first time, surprisingly, by a specific ratio of (a) cells or cell extracts, or a combination thereof; and (b) a first reaction promoter (such as aluminum, aluminum salts, aluminum oxide).
  • a first reaction promoter such as aluminum, aluminum salts, aluminum oxide.
  • the in vitro protein synthesis system formed by the combination of the complex or a combination thereof can significantly improve the synthesis efficiency of the foreign protein, and the RLU can be up to 3 ⁇ 10 9 -4 ⁇ 10 9 .
  • the foreign protein synthesized by the yeast cell-free expression system added to the reaction promoter 1 h after the start of the reaction for example
  • the relative light unit value of luciferase activity is significantly increased by 2-3 times, the reaction efficiency is increased, and the yield of the target protein is increased.
  • the experimental results of the present invention indicate that the cell-free in vitro protein synthesis system in which the first reaction promoter is added during a specific period of time (for example, when the reaction is carried out for about 1 h) can significantly promote the protein synthesis system (such as the yeast in vitro protein synthesis system).
  • the protein synthesis system such as yeast in vitro protein synthesis system
  • the first reaction promoter of the present invention is added, wherein, in the protein synthesis system (such as Yeast in vitro protein synthesis system)
  • the addition of the first reaction promoter after 1 h of the reaction started to promote the protein synthesis.
  • the reaction accelerator is not particularly limited as long as a reaction accelerator capable of remarkably improving the protein synthesis efficiency of the in vitro protein synthesis system of the present invention is within the scope of the present invention.
  • the reaction accelerator of the present invention comprises a first reaction accelerator and a second reaction accelerator.
  • the first reaction promoter is selected from the group consisting of aluminum, aluminum salts, aluminum oxide complexes, or combinations thereof.
  • the second reaction promoter is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, soluble starches, or combinations thereof.
  • the second reaction promoter is selected from one or more of the group consisting of:
  • sugars/polyols sucrose, trehalose, mannitol, lactose, glucose, maltose, etc.;
  • polymer PEG, dextran, albumin, etc.
  • Anhydrous solvent glycerin, DMSO, and the like.
  • the second reaction promoter is selected from the group consisting of sucrose, trehalose, mannitol, lactose, glucose, maltose, galactose, or a combination thereof.
  • the second reaction promoter is selected from the group consisting of sucrose, trehalose, lactose, or a combination thereof.
  • a biological reaction that is, a biochemical reaction, refers to a chemical reaction carried out in a living body. These reactions are catalyzed by an enzyme, and the enzyme and the reactant are dissolved in the water of the internal environment to react, and the water provides a carrier and a medium for the body substance.
  • biochemistry has made great achievements in explaining life processes, and now almost all areas related to life sciences such as botany, medicine, and genetics are engaged in biochemistry research.
  • the biochemical reactions carried out in organisms or cells can be automatically regulated by positive and negative feedback in complex network systems.
  • the biochemical reactions carried out in cells require enzyme catalysis.
  • the enzyme has high catalytic efficiency, mild reaction conditions, directionality, and high specificity for the substrate.
  • the cell extract of the cell extract is selected from the group consisting of one or more types of cells: prokaryotic cells and eukaryotic cells.
  • the cell extract of the cell extract is selected from the group consisting of one or more types of cells: Escherichia coli, bacteria, mammalian cells (eg, HF9, Hela, CHO, HEK293), plant cells , yeast cells, insect cells, or a combination thereof.
  • 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, preferably The ground is Kluyveromyces lactis.
  • the cell extract comprises a yeast cell extract.
  • the content and purity of the cell extract are not particularly limited.
  • the content (wt%) of the cell extract (such as yeast cell extract) in the protein synthesis system is 10%-95%, preferably 20%-80%, more Preferably, 40% to 60%, based on the total weight of the protein synthesis system.
  • Yeast combines the advantages of simple, efficient protein folding, and post-translational modification. Among them, Saccharomyces cerevisiae and Pichia pastoris are model organisms that express complex eukaryotic proteins and membrane proteins. Yeast can also be used as a raw material for the preparation of in vitro translation systems.
  • Kluyveromyces is an ascomycete, in which Kluyveromyces marxianus and Kluyveromyces lactis are industrially widely used yeasts.
  • Kluyveromyces cerevisiae has many advantages over other yeasts, such as superior secretion capacity, better large-scale fermentation characteristics, food safety levels, and the ability to simultaneously modify post-translational proteins.
  • the yeast in vitro expression system is not particularly limited, and a preferred yeast in vitro expression system is the Kluyveromyces expression system (more preferably, the K. lactis expression system).
  • controlling the content of free phosphate in the protein synthesis system means controlling the free phosphate of the reaction by-product in the protein synthesis system, regulating the influence on the protein synthesis system, thereby improving protein synthesis in the protein synthesis system. effectiveness.
  • reaction accelerator of the present invention can be added to a protein synthesis system to achieve the above object.
  • the free phosphorus content control stage includes a pre-reaction phase prior to phosphoric acid control, a phosphoric acid content control phase, and a post-phosphorus control phase.
  • the phosphoric acid content control stage has no particular time limit, and the addition of the free phosphorus control dose is determined based on the time of the reaction.
  • the pre-reaction phase of the phosphoric acid control is 0 ⁇ 2h, preferably 1h; the phosphoric acid content control phase is 0 ⁇ 5h after the pre-reaction phase of the phosphoric acid control, preferably 2 -5h; the stage of phosphoric acid control is based on the target protein and biochemical reaction characteristics.
  • the entire phosphoric acid controlled reaction system has a duration of 1-7 h, preferably 2-4 h.
  • the in vitro cell-free protein synthesis system of the invention comprises a yeast in vitro protein synthesis system.
  • Yeast combines the advantages of simple, efficient protein folding, and post-translational modification. Among them, Saccharomyces cerevisiae and Pichia pastoris are model organisms that express complex eukaryotic proteins and membrane proteins. Yeast can also be used as a raw material for the preparation of in vitro translation systems.
  • Kluyveromyces is an ascomycete, in which Kluyveromyces marxianus and Kluyveromyces lactis are industrially widely used yeasts.
  • Kluyveromyces cerevisiae has many advantages over other yeasts, such as superior secretion capacity, better large-scale fermentation characteristics, food safety levels, and the ability to simultaneously modify post-translational proteins.
  • the yeast in vitro protein synthesis system is not particularly limited, and a preferred yeast in vitro protein synthesis system is the Kluyveromyces expression system (more preferably, the K. lactis expression system).
  • Kluyveromyces yeast e.g., Kluyveromyces lactis
  • the yeast in vitro protein synthesis system comprises:
  • a cell extract such as a yeast cell extract
  • a first reaction promoter selected from the group consisting of aluminum, an aluminum salt, an aluminum oxide complex, or a combination thereof;
  • the yeast in vitro protein synthesis system further comprises:
  • a second reaction promoter selected from the group consisting of a monosaccharide, a disaccharide, an oligosaccharide, a soluble starch, or a combination thereof;
  • the oxyaluminum composite has a particle size of from 0.5 to 20 mm, preferably from 0.8 to 10 mm, more preferably from 1 to 5 mm, still more preferably from 1 to 3 mm.
  • the oxyaluminum complex has an average weight of from 1 to 80 mg, from 3 to 50 mg, more preferably from 4 to 30 mg, more preferably from 8 to 15 mg.
  • the concentration (v/v) of the oxyaluminum complex in the reaction accelerator is from 0.5 to 20%, preferably from 0.8 to 10%, more preferably from 1% to 10%. %, more preferably, 1% to 5%, more preferably 2% to 3%, based on the total volume of the first reaction accelerator.
  • the content of the oxyaluminum complex (wt%) in the reaction accelerator is 0.1-20%, preferably 1-10%, more preferably 2%-8%. More preferably, 4% to 7%, more preferably 5.5% to 6.5%, based on the total weight of the first reaction accelerator.
  • the concentration (v/v) of the first reaction accelerator is from 20% to 70%, preferably from 30% to 60%, more preferably, 45. %-55%, based on the total volume of the protein synthesis system.
  • the content (wt%) of the first reaction accelerator is 20% to 70%, preferably 30% to 60%, more preferably 45%. 55% based on the total weight of the protein synthesis system.
  • the ratio (mass ratio) of component (i) to component (ii) in the protein synthesis system is from 0.1 to 10:0.1 to 10, preferably from 0.5 to 8:0.5. 8, more preferably, 0.8-5: 0.8-5, more preferably, 0.9-2: 0.9-2.
  • the in vitro protein synthesis system comprises one or more or all of the components selected from the group consisting of yeast cell extracts, aluminum oxide complexes (such as alumina), and polyethylene glycol.
  • Alcohol sucrose, 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, adenine nucleoside triphosphate (ATP), guanosine triphosphate (GTP), cytosine triphosphate (CTP) Thymidine triphosphate (TTP), amino acid mixture, creatine phosphate, dithiothreitol (DTT), phosphocreatine kinase, RNase inhibitor, fluorescein, luciferase DNA, RNA polymerase, Spermidine, heme.
  • the RNA polymerase is not particularly limited and may be selected from one or more RNA polymerases, and a typical RNA polymerase is T7 RNA polymerase.
  • the ratio of the yeast cell extract in the in vitro protein synthesis system is not particularly limited, and usually the content (wt%) of the yeast cell extract is 10% to 95%, preferably 20%. -80%, more preferably, 40%-60%, based on the total weight of the protein synthesis system.
  • the yeast cell extract does not contain intact cells, and typical yeast cell extracts include ribosomes for protein translation, transfer RNA, aminoacyl tRNA synthetase, initiation factors required for protein synthesis, and The elongation factor and the termination release factor.
  • the yeast extract contains some other proteins in the cytoplasm derived from yeast cells, especially soluble proteins.
  • the yeast cell extract contains a protein content of 20 to 100 mg/mL, preferably 50 to 100 mg/mL.
  • the method for determining protein content is a Coomassie Brilliant Blue assay.
  • the preparation method of the yeast cell extract is not limited, and a preferred preparation method comprises the following steps:
  • the solid-liquid separation method is not particularly limited, and a preferred mode is centrifugation.
  • the centrifugation is carried out in a liquid state.
  • the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5,000 to 100,000 g, preferably 8,000 to 30,000 g.
  • the centrifugation time is not particularly limited, and a preferred centrifugation time is from 0.5 min to 2 h, preferably from 20 min to 50 min.
  • the temperature of the centrifugation is not particularly limited.
  • the centrifugation is carried out at 1-10 ° C, preferably at 2-6 ° C.
  • the washing treatment method is not particularly limited, and a preferred washing treatment method is treatment with a washing liquid at a pH of 7-8 (preferably, 7.4), and the washing liquid is not particularly Typically, the wash liquor is typically selected from the group consisting of potassium 4-hydroxyethylpiperazine ethanesulfonate, potassium acetate, magnesium acetate, or combinations thereof.
  • the manner of the cell disruption treatment is not particularly limited, and a preferred cell disruption treatment includes high pressure disruption, freeze-thaw (e.g., liquid nitrogen low temperature) disruption.
  • the mixture of nucleoside triphosphates in the in vitro protein synthesis system is adenine nucleoside triphosphate, guanosine triphosphate, cytidine triphosphate, and uridine nucleoside triphosphate.
  • the concentration of each of the single nucleotides is not particularly limited, and usually the concentration of each single nucleotide is from 0.5 to 5 mM, preferably from 1.0 to 2.0 mM.
  • the mixture of amino acids in the in vitro protein synthesis system can include natural or unnatural amino acids, and can include D-form or L-form amino acids.
  • Representative amino acids include, but are not limited to, 20 natural amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, serine, Tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.
  • the concentration of each amino acid is usually from 0.01 to 0.5 mM, preferably from 0.02 to 0.2 mM, such as 0.05, 0.06, 0.07, 0.08 mM.
  • the in vitro protein synthesis system further comprises polyethylene glycol or an analog thereof.
  • concentration of polyethylene glycol or the like is not particularly limited, and usually, the concentration (w/v) of polyethylene glycol or the like is from 0.1 to 8%, preferably from 0.5 to 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, PEG 8000, PEG 6000, and PEG 3350. It should be understood that the system of the present invention may also include other various molecular weight polyethylene glycols (e.g., PEG 200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc.).
  • the in vitro protein synthesis system further comprises sucrose.
  • concentration of sucrose is not particularly limited, and usually, the concentration (w/v) of sucrose is 0.2 to 4%, preferably 0.5 to 4%, more preferably 0.5 to 1%, based on the total volume of the protein synthesis system. meter.
  • the in vitro protein synthesis system further comprises heme.
  • concentration of hemoglobin is not particularly limited, and usually, the concentration of heme is 0.01 to 0.1 mM, preferably 0.02 to 0.08 mM, more preferably 0.03 to 0.05 mM, most preferably 0.04 mM.
  • the in vitro protein synthesis system further comprises spermidine.
  • concentration of spermidine is not particularly limited, and usually, the concentration of spermidine is 0.05 to 1 mM, preferably 0.1 to 0.8 mM, more preferably, more preferably 0.2 to 0.5 mM, still more preferably 0.3 to 0.4. mM, optimally, 0.4 mM.
  • the in vitro protein synthesis system further contains a buffer, the composition of which is not particularly limited, and a preferred buffer contains 4-hydroxyethylpiperazineethanesulfonic acid, and/or Tris buffer. liquid.
  • the buffer may further contain other buffer components such as potassium acetate or magnesium acetate to form a reaction solution or a reaction buffer having a pH of 6.5 to 8.5 (preferably 7.0 to 8.0).
  • the type and content of the buffer are not particularly limited.
  • the buffer is present at a concentration of 1-200 mM or 1-100 mM, preferably 5-50 mM.
  • a particularly preferred in vitro protein synthesis system in addition to the yeast extract, the aluminum oxide complex (such as alumina), further comprises one or more or all of the components selected from the group consisting of 22 mM, pH 7.4 - hydroxyethylpiperazine ethanesulfonic acid, 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 phosphocreatine, 1.7 mM dithios Sugar alcohol, 0.27mg/mL phosphocreatine kinase, 1%-4% polyethylene glycol, 0.5%-2% sucrose, 8-20ng/ ⁇ L firefly luciferase DNA, 0.027-0.054mg/mL T7 RNA polymerization Enzyme, 0.03-0.04 mM heme, 0.3-0.4 mM spermidine.
  • the aluminum oxide complex such as alumina
  • coding sequence of a foreign protein is used interchangeably with “foreign DNA” and refers to a foreign DNA molecule for directing protein synthesis.
  • the DNA molecule is linear or circular.
  • the DNA molecule contains a sequence encoding a foreign protein.
  • examples of the sequence encoding the foreign protein include, but are not limited to, a genomic sequence, a cDNA sequence.
  • the sequence encoding the foreign protein further comprises a promoter sequence, a 5' untranslated sequence, and a 3' untranslated sequence.
  • the selection of the exogenous DNA is not particularly limited.
  • the exogenous DNA is selected from the group consisting of a luciferin protein, or a luciferase (such as firefly luciferase), a green fluorescent protein, and a yellow fluorescent protein. , aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, exogenous DNA of a variable region of an antibody, DNA of a luciferase mutant, or a combination thereof.
  • the exogenous DNA may also be selected from the group consisting of alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum white. Protein, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, exogenous DNA of xylanase, or a combination thereof.
  • alpha-amylase enteromycin A
  • hepatitis C virus E2 glycoprotein insulin precursor
  • interferon alpha A interleukin-1 beta
  • lysozyme serum white.
  • Protein single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, exogenous DNA of xylanase, or a combination thereof.
  • the exogenous DNA encodes a protein selected from the group consisting of: green fluorescent protein (eGFP), yellow fluorescent protein (YFP), and Escherichia coli beta-galactosidase ( ⁇ -galactosidase, LacZ), human lysine-tRNA synthetase, human leucine-tRNA synthetase, Arabidopsis glyceraldehyde 3-phosphate dehydrogenase (Glyceraldehyde-3-phosphate) Dehydrogenase), murine catalase (Catalase), or a combination thereof.
  • eGFP green fluorescent protein
  • YFP yellow fluorescent protein
  • Escherichia coli beta-galactosidase ⁇ -galactosidase, LacZ
  • human lysine-tRNA synthetase human leucine-tRNA synthetase
  • the invention provides a kit for in vitro protein synthesis, comprising:
  • (k1) a first container, and a component (i) located in the first container, the component (i) being selected from the group consisting of a cell or a cell extract, or a combination thereof;
  • component (k2) a second container, and component (ii) located in the second container, said component (ii) being a first reaction promoter, said first reaction promoter being selected from the group consisting of aluminum and aluminum salts , an aluminum oxide composite, or a combination thereof; and
  • first container and the second container are the same container or different containers.
  • the invention also provides a kit for cell-free in vitro protein synthesis, comprising:
  • component (k2) a second container, and component (ii) located in the second container, said component (ii) being a first reaction promoter, said first reaction promoter being selected from the group consisting of aluminum and aluminum salts , an aluminum oxide composite, or a combination thereof; and
  • a particularly preferred kit for in vitro protein synthesis comprises an in vitro protein synthesis protein synthesis system comprising one or more or all of the components selected from the group consisting of yeast cell extracts, oxyaluminum complexes (eg Alumina), 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, adenine nucleoside triphosphate (ATP), guanosine triphosphate (GTP), cytosine triphosphate (CTP) , Thymidine triphosphate (TTP), amino acid mixture, phosphocreatine, dithiothreitol (DTT), phosphocreatine kinase, RNase inhibitor, fluorescein, luciferase DNA, T7 RNA polymerase , spermidine, heme.
  • yeast cell extracts eg Alumina
  • oxyaluminum complexes eg Alumina
  • 4-hydroxyethylpiperazineethanesulfonic acid potassium
  • the in vitro protein synthesis system containing the first reaction promoter of the present invention has a 2-3 fold improvement in the ability to synthesize proteins, and has a distinct advantage.
  • the in vitro protein synthesis system of the present invention can more conveniently and quickly control biochemical reactions, such as enzymatic reactions, biosynthesis reactions, biodegradation reactions, cell-free biological reactions, and the like.
  • the present inventors have found for the first time that the addition of a reaction promoter to an in vitro protein synthesis system can control the concentration of phosphate ions in a protein synthesis system, which can significantly improve the protein synthesis efficiency.
  • the present invention adopts an in situ removal method to improve bioreaction efficiency and reduce reaction complexity.
  • sucrose and/or mannitol are added, followed by freeze-drying to obtain a lyophilized yeast extract.
  • the biological reaction system is exemplified by an in vitro protein synthesis system, and the biological reactant is exemplified by a yeast cell extract, but is not limited thereto.
  • Primary seed culture Inoculated in a shake flask medium with a -80 ° C cryopreserved strain, and cultured at 30 ° C, 200 rpm to logarithmic growth phase.
  • Secondary seed culture an appropriate amount of the first-stage seed broth is inoculated to the secondary seed, and cultured at 30 ° C, 200 rpm to logarithmic growth phase.
  • Batch culture stage The second-stage seed bacteria liquid is inoculated into the fermenter, the temperature is controlled to be cultured at 30 ° C for 10-12 h, and the feed culture stage is entered. When the OD600 value is 50-55, the cell culture is collected.
  • the cultured cell culture was pre-cooled in an ice-water mixture for 10-30 min.
  • the pre-cooled cell culture in 1.4 was centrifuged in a cryogenic centrifuge, and centrifuged conditions: 3,000 g, 10 min, 4 ° C, to obtain yeast cells.
  • yeast cells were resuspended in a washing buffer (washing solution) with a pre-cooled Washing buffer, and the resulting suspension was centrifuged, and centrifuged conditions: 3000 g, 10 min, 4 ° C, to obtain yeast cells.
  • Washing buffer composition 20-30 mM potassium 4-hydroxyethylpiperazine sulfonate pH 7.4, 100-150 mM potassium acetate, 1-4 mM magnesium acetate;
  • step 1.8 The yeast cells obtained in step 1.7 are directly subjected to subsequent operations, or are frozen at -80 ° C after rapid freezing using liquid nitrogen.
  • Fragmentation using a liquid nitrogen homogenizer add appropriate amount of liquid nitrogen to the homogenizer, and then add yeast cells obtained by centrifugation or yeast cells stored at 1.8-80 ° C, rotating at 45,000 rpm, and crushing for 3-10 min; The crushed low temperature powder was dispensed into a 50 mL centrifuge tube, weighed and stored at -80 ° C until use.
  • Lysis buffer consists of 10-40 mM potassium 4-hydroxyethylpiperazine sulfonate pH 7.4, 50-150 mM potassium acetate, 1-4 mM magnesium acetate, 2-7 mM dithiothreitol, 0.5-2 mM phenylmethyl sulfonate. Acetyl fluoride composition.
  • the crude yeast cell extract obtained in step 1.10 is centrifuged 1-2 times, the centrifugal force is 12000-30000 g for 30 min, and the temperature is 4 ° C;
  • the prepared yeast cell extract was dispensed and snap frozen in liquid nitrogen and stored at -80 °C.
  • the glass bottle and glass plate containing the cell extract in 2.4 were pre-frozen at -80 ° C, and the pre-freezing time was 2-4 h;
  • luciferase activity After the reaction is completed, add an equal volume of substrate luciferine to a 96-well white plate or a 384-well white plate, and immediately place it on the Envision 2120 Multiplate Reader (Perkin Elmer) and read. Firefly luciferase activity was measured and the relative light unit value (RLU) was used as the unit of activity.
  • RLU relative light unit value
  • reaction by-products mainly include dialysis method, circulation method, solution replacement, etc., all of which require additional equipment, are complicated in system, are not easy to operate, and are not easily completed in a small reaction system. Therefore, the removal of reaction by-products, especially in situ removal, is the key to improving the efficiency of the reaction and reducing the complexity of the reaction.
  • the phosphoric acid control in the system and the biochemical reactions in the system are a pair of competing reactions. Excessive phosphate or pyrophosphate in the system will lead to pH imbalance in the system, affecting the activity of the bio-enzyme involved in the biochemical reaction in the system, and reducing the yield of the target protein. At the same time, the phosphate is easily associated with the magnesium ion required in the reaction system. The combination of the formation of water-insoluble magnesium sulfate results in numerous biochemical reaction steps resulting in a decrease in reactivity due to the lack of magnesium ions. The addition of the phosphoric acid control formulation adjusts the balance of the biochemical reaction as described above by physical (adsorption) or chemistry (formation of the precipitated product from the reaction system), thereby creating a relatively ideal reaction environment for the protein synthesis system.

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Abstract

一种体外蛋白合成体系、试剂盒及其制备方法，体外蛋白合成体系由特定比例的(a)细胞提取物，和(b)第一反应促进剂混合形成，可提高外源蛋白的合成效率。

Description

一种体外蛋白合成体系、试剂盒及其制备方法 技术领域
本发明涉及生物技术领域，具体地，本发明涉及一种体外蛋白合成体系、试剂盒及其制备方法。
背景技术
生物反应即生物化学反应，就是指在生物体内进行的化学反应，这些反应都由酶催化，酶和反应物溶于内环境的水中，才能发生反应，水为体内物质提供载体和介质[1]。在20世纪的最后几十年里，生物化学在解释生命过程方面已经取得了非常大的成就，现在几乎植物学，医学，遗传学等生命科学相关的领域都有从事生物化学研究[2]。
传统的蛋白表达系统是指通过模式生物细菌、真菌、植物细胞或动物细胞等表达外源基因的一种多步级联生物化学反应术[3-4]。随着科学技术的发展，无细胞表达体系也称为体外蛋白合成系统应运而生，其是以外源目的mRNA或DNA为蛋白质合成模板，通过人工控制补加蛋白质合成所需的底物和转录、翻译相关蛋白因子等物质，能实现目的蛋白质的合成[5-7]。体外翻译系统中表达蛋白质无需进行质粒构建、转化、细胞培养、细胞收集和破碎步骤，是一种快速、省时、便捷的蛋白质表达方式[8]。随着科学技术的发展，蛋白合成体系已经开始得到广泛应用。
在生物体或细胞中所进行的生物化学反应，在复杂的网络体系中都可以通过正、负反馈得到自动调控，并且在生物体中所进行的生物化学反应都是远离平衡点的反应，它需要从外界获取能量或向外界输出物质、能量和熵[9]。在细胞内，生物反应的副产物去除主要是靠机体新陈代谢、机体稳态的维持、生物酶的催化作用以及细胞内远离平衡点的氧化还原反应达到，但这些生化反应在体外生物技术是难以实现的[10]。另外，蛋白合成体系存在的一个缺点是产量不高，反应不够快速、灵敏，体外生物合成体系中，比如体外蛋白质合成，体外转录，激酶反应，ATPase反应，离子泵反应等的副产物如自由磷酸根离子、焦磷酸离子(PPi),都会限制目标产物的产量。
目前已知有其他去除反应副产物的方法如通过半透膜允许小分子在反应混合物和透析液之间被动扩散的方法进行半连续反应来控制副产物如自由磷 酸根离子。但存在着半透膜透析装置成本高，反应体系不能进行扩大化，反应产物容易堵塞半透膜孔的缺陷。
因此，本领域迫切需要开发并建立一种能够在蛋白合成体系中，通过原位去除(in situ removal)的方式在蛋白合成体系中控制自由磷酸根离子含量来提高蛋白合成效率的方法。
发明内容
本发明的目的在于提供一种能够在蛋白合成体系中，通过原位去除(in situ removal)的方式在蛋白合成体系中控制自由磷酸根离子含量来提高蛋白合成效率的方法。
本发明第一方面提供了一种体外蛋白合成体系，包括：
(a)细胞或细胞提取物、或其组合；
(b)第一反应促进剂，所述第一反应促进剂选自下组：铝、铝盐、氧铝复合物、或其组合。
在另一优选例中，所述细胞选自下组：原核细胞和真核细胞。
在另一优选例中，所述细胞选自下组：大肠杆菌、细菌、哺乳动物细胞(如HF9、Hela、CHO、HEK293)、植物细胞、酵母细胞、或其组合。
在另一优选例中，所述酵母细胞选自下组：酿酒酵母、毕氏酵母、克鲁维酵母、或其组合；较佳地，所述的酵母细胞包括：克鲁维酵母，更佳地为乳酸克鲁维酵母。
在另一优选例中，所述蛋白合成体系还包括：
(b’)第二反应促进剂，所述第二反应促进剂选自下组：单糖、二糖、寡糖、可溶性淀粉、或其组合；
(c)聚乙二醇；
(d)任选的外源蔗糖；和
(e)任选的溶剂，所述溶剂为水或水性溶剂。
在另一优选例中，所述蛋白合成体系包括酵母体外蛋白质合成体系(如克鲁维酵母体外蛋白合成体系，较佳地，乳酸克鲁维酵母体外蛋白合成体系)。
在另一优选例中，所述氧铝复合物包括氧铝纳米颗粒。
在另一优选例中，所述氧铝复合物包括Al 2O 3。
在另一优选例中，所述氧铝复合物为颗粒形式或纳米形式。
在另一优选例中，所述氧铝复合物的粒径为0.5-20mm，较佳地，0.8-10mm，更佳地，1-5mm。
在另一优选例中，所述氧铝复合物的平均重量为1-80mg，较佳地，3-50mg，更佳地，4-30mg。
在另一优选例中，所述第一反应促进剂中，所述氧铝复合物的浓度(v/v)为0.5-20％，较佳地，0.8-10％，更佳地，1％－10％，更佳地，1％－5％，更佳地，2％－3％，以所述第一反应促进剂的总体积计。
在另一优选例中，所述反应促进剂中，所述氧铝复合物的含量(wt％)为0.1-20％，较佳地，1-10％，更佳地，2％－8％，更佳地，4％－7％，更佳地，5.5％－6.5％，以所述第一反应促进剂的总重量计。
在另一优选例中，所述蛋白合成体系中，所述第一反应促进剂的浓度(v/v)为20％－70％，较佳地，30％－60％，更佳地，45％－55％，以蛋白合成体系的总体积计。
在另一优选例中，所述蛋白合成体系中，所述第一反应促进剂的含量(wt％)为20％－70％，较佳地，30％－60％，更佳地，45％－55％，以蛋白合成体系的总重量计。
本发明第二方面提供了一种体外的无细胞的蛋白合成体系，包括：
(a)细胞提取物；
(b)第一反应促进剂，所述第一反应促进剂选自下组：铝、铝盐、氧铝复合物、或其组合。
在另一优选例中，所述蛋白合成体系还包括：
(b’)第二反应促进剂，所述第二反应促进剂选自下组：单糖、二糖、寡糖、可溶性淀粉、或其组合；
(c)聚乙二醇；
(d)任选的外源蔗糖；和
(e)任选的溶剂，所述溶剂为水或水性溶剂。
在另一优选例中，所述蛋白合成体系包括酵母体外蛋白质合成体系(如克鲁维酵母体外蛋白合成体系，较佳地，乳酸克鲁维酵母体外蛋白合成体系)。
在另一优选例中，所述氧铝复合物包括氧铝纳米颗粒。
在另一优选例中，所述氧铝复合物包括Al 2O 3。
在另一优选例中，所述氧铝复合物为颗粒形式或纳米形式。
在另一优选例中，所述氧铝复合物的粒径为0.5-20mm，较佳地，0.8-10mm，更佳地，1-5mm。
在另一优选例中，所述氧铝复合物的平均重量为1-80mg，较佳地，3-50mg，更佳地，4-30mg。
在另一优选例中，所述第一反应促进剂中，所述氧铝复合物的浓度(v/v)为0.5-20％，较佳地，0.8-10％，更佳地，1％－10％，更佳地，1％－5％，更佳地，2％－3％，以所述第一反应促进剂的总体积计。
在另一优选例中，所述反应促进剂中，所述氧铝复合物的含量(wt％)为0.1-20％，较佳地，1-10％，更佳地，2％－8％，更佳地，4％－7％，更佳地，5.5％－6.5％，以所述第一反应促进剂的总重量计。
在另一优选例中，所述蛋白合成体系中，所述第一反应促进剂的浓度(v/v)为20％－70％，较佳地，30％－60％，更佳地，45％－55％，以蛋白合成体系的总体积计。
在另一优选例中，所述蛋白合成体系中，所述第一反应促进剂的含量(wt％)为20％－70％，较佳地，30％－60％，更佳地，45％－55％，以蛋白合成体系的总重量计。
在另一优选例中，所述细胞提取物的细胞来源选自下组的一种或多种类型的细胞：原核细胞和真核细胞。
在另一优选例中，所述细胞提取物的细胞来源选自下组的一种或多种类型的细胞：大肠杆菌、细菌、哺乳动物细胞(如HF9、Hela、CHO、HEK293)、植物细胞、酵母细胞、或其组合。
在另一优选例中，所述酵母细胞选自下组：酿酒酵母、毕氏酵母、克鲁维酵母、或其组合；较佳地，所述的酵母细胞包括：克鲁维酵母，更佳地为乳酸克鲁维酵母。
在另一优选例中，所述蛋白合成体系还包括选自下组的一种或多种组分：
(f1)用于合成RNA的底物；
(f2)用于合成蛋白的底物；
(f3)镁离子；
(f4)钾离子；
(f5)缓冲剂；
(f6)RNA聚合酶；
(f7)能量再生系统。
在另一优选例中，所述蛋白合成体系还包括选自下组的一种或多种组分：
(g8)血红素；
(g9)亚精胺。
在另一优选例中，所述细胞提取物包括酵母细胞提取物。
在另一优选例中，所述的酵母细胞提取物为对酵母细胞的水性提取物。
在另一优选例中，所述酵母细胞提取物不含酵母内源性的长链核酸分子。
在另一优选例中，所述的酵母细胞提取物是用包括以下步骤的方法制备：
(i)提供酵母细胞；
(ii)对酵母细胞进行洗涤处理，获得经洗涤的酵母细胞；
(iii)对经洗涤的酵母细胞进行破细胞处理，从而获得酵母粗提物；和
(iv)对所述酵母粗提物进行固液分离，获得液体部分，即为酵母细胞提取物。
在另一优选例中，所述的固液分离包括离心。
在另一优选例中，在液态下进行离心。
在另一优选例中，所述离心条件为5000-100000g，较佳地，8000-30000g。
在另一优选例中，所述离心时间为0.5min-2h，较佳地，20min-50min。
在另一优选例中，所述离心在1-10℃下进行，较佳地，在2-6℃下进行。
在另一优选例中，所述的洗涤处理采用洗涤液在pH为7-8(较佳地，7.4)下进行处理。
在另一优选例中，所述洗涤液选自下组：4-羟乙基哌嗪乙磺酸钾、醋酸钾、醋酸镁、或其组合。
在另一优选例中，所述的破细胞处理包括高压破碎、冻融(如液氮低温)破碎。
在另一优选例中，所述的合成RNA的底物包括：核苷单磷酸、核苷三磷酸、或其组合。
在另一优选例中，所述的合成蛋白的底物包括：1-20种天然氨基酸、以及非天然氨基酸。
在另一优选例中，所述镁离子来源于镁离子源，所述镁离子源选自下组：醋酸镁、谷氨酸镁、或其组合。
在另一优选例中，所述钾离子来源于钾离子源，所述钾离子源选自下组：醋酸钾、谷氨酸钾、或其组合。
在另一优选例中，所述能量再生系统选自下组：磷酸肌酸/磷酸肌酸酶系统、糖酵解途径及其中间产物能量系统、或其组合。
在另一优选例中，所述蛋白合成体系还包括(h1)人工合成的tRNA。
在另一优选例中，所述缓冲剂选自下组：4-羟乙基哌嗪乙磺酸、三羟甲基氨基甲烷、或其组合。
在另一优选例中，所述蛋白合成体系还包括(i1)外源的用于指导蛋白质合成的DNA分子。
在另一优选例中，所述的DNA分子为线性的。
在另一优选例中，所述的DNA分子为环状的。
在另一优选例中，所述的DNA分子含有编码外源蛋白的序列。
在另一优选例中，所述的编码外源蛋白的序列包括基因组序列、cDNA序列。
在另一优选例中，所述的编码外源蛋白的序列还含有启动子序列、5'非翻译序列、3'非翻译序列。
在另一优选例中，所述蛋白合成体系包括选自下组的成分：4-羟乙基哌嗪乙磺酸、醋酸钾、醋酸镁、核苷三磷酸、氨基酸、磷酸肌酸，二硫苏糖醇(DTT)、磷酸肌酸激酶、RNA聚合酶、或其组合。
在另一优选例中，所述聚乙二醇选自下组：PEG3000、PEG8000、PEG6000、PEG3350、或其组合。
在另一优选例中，所述聚乙二醇包括分子量(Da)为200-10000的聚乙二醇，较佳地，分子量为3000-10000的聚乙二醇。
在另一优选例中，所述蛋白合成体系中，组分(a)的浓度(v/v)为20％-70％，较佳地，30-60％，更佳地，40％-50％，以所述蛋白合成体系的总体积计。
在另一优选例中，所述蛋白合成体系中，组分(c)的含量(wt％)为10％－95％，较佳地，20％－80％，更佳地，40％－60％，以所述蛋白合成体系的总重量计。
在另一优选例中，所述蛋白合成体系中，组分(d)的浓度(w/v，例如g/ml)为0.1-8％，较佳地，0.5-4％，更佳地，1-2％。
在另一优选例中，所述蛋白合成体系中，组分(e)的浓度为0.2-4％，较佳地，0.5-4％，更佳地，0.5-1％，以所述蛋白合成体系的总体积计。
在另一优选例中，所述核苷三磷酸选自下组：腺嘌呤核苷三磷酸、鸟嘌呤核 苷三磷酸、胞嘧啶核苷三磷酸、尿嘧啶核苷三磷酸、或其组合。
在另一优选例中，所述蛋白合成体系中，组分(f1)的浓度为0.1-5mM，较佳地，0.5-3mM，更佳地，1-1.5mM。
在另一优选例中，所述氨基酸为选自下组：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、组氨酸、或其组合。
在另一优选例中，所述氨基酸包括D型氨基酸和/或L型氨基酸。
在另一优选例中，所述蛋白合成体系中，所述组分(f2)的浓度为0.01-0.48mM，较佳地，0.04-0.24mM，更佳地，0.04-0.2mM，最佳地，0.08mM。
在另一优选例中，所述蛋白合成体系中，所述组分(f3)的浓度为1-10mM，较佳地，1-5mM，更佳地，2-4mM。
在另一优选例中，所述蛋白合成体系中，所述组分(f4)的浓度为30-210mM，较佳地，30-150mM，更佳地，30-60mM。
在另一优选例中，所述蛋白合成体系中，所述组分(f6)的浓度为0.01-0.3mg/mL，较佳地，0.02-0.1mg/mL，更佳地，0.027-0.054mg/mL。
在另一优选例中，所述蛋白合成体系中，4-羟乙基哌嗪乙磺酸的浓度为5-50mM，较佳地，10-50mM，较佳地，15-30mM，更佳地，20-25mM。
在另一优选例中，所述蛋白合成体系中，所述醋酸钾的浓度为20-210mM，较佳地，30-210mM，较佳地，30-150mM，更佳地，30-60mM。
在另一优选例中，所述蛋白合成体系中，所述醋酸镁的浓度为1-10mM，较佳地，1-5mM，更佳地，2-4mM。
在另一优选例中，所述蛋白合成体系中，所述磷酸肌酸的浓度为10-50mM，较佳地，20-30mM，更佳地，25mM。
在另一优选例中，所述蛋白合成体系中，所述血红素的浓度为0.01-0.1mM，较佳地，0.02-0.08mM，更佳地，0.03-0.05mM，最佳地，0.04mM。
在另一优选例中，所述蛋白合成体系中，所述亚精胺的浓度为0.05-1mM，较佳地，0.1-0.8mM，更佳地，更佳地，0.2-0.5mM，更佳地，0.3-0.4mM，最佳地，0.4mM。
在另一优选例中，所述蛋白合成体系中，所述二硫苏糖醇(DTT)的浓度为0.2-15mM，较佳地，0.2-7mM，更佳地，1-2mM。
在另一优选例中，所述蛋白合成体系中，所述磷酸肌酸激酶的浓度为0.1-1mg/mL，较佳地，0.2-0.5mg/mL，更佳地，0.27mg/mL。
在另一优选例中，所述蛋白合成体系中，所述T7RNA聚合酶的浓度为0.01-0.3mg/mL，较佳地，0.02-0.1mg/mL，更佳地，0.027-0.054mg/mL。
在另一优选例中，所述的蛋白合成体系具有以下性能：
在合成体系里，蛋白合成总量达到3ug蛋白/mL体系。
在另一优选例中，所述蛋白合成体系的组成包括：
Figure PCTCN2017119540-appb-000001
在另一优选例中，所述蛋白合成体系的组成还包括：
Figure PCTCN2017119540-appb-000002
在另一优选例中，所述的PEG选自PEG3350、PEG3000、和/或PEG8000。
在另一优选例中，所述RNA聚合酶为T7RNA聚合酶。
本发明第三方面提供了一种本发明第一方面或本发明第二方面所述的蛋白合成体系的生产方法，包括步骤：
将组分(i)和组分(ii)混合，从而获得本发明第一方面或第二方面所述的蛋白合成体系，其中，所述组分(i)选自下组：细胞或细胞提取物、或其组合；所述组分(ii)为第一反应促进剂，所述第一反应促进剂选自下组：铝、铝盐、氧铝复合 物、或其组合。
在另一优选例中，所述组分(i)为细胞提取物。
在另一优选例中，所述蛋白合成体系中，组分(i)与组分(ii)的比例(质量比)为0.1-10：0.1-10，较佳地，0.5-8:0.5-8，更佳地，0.8-5：0.8-5，更佳地，0.9-2：0.9-2。
在另一优选例中，所述蛋白合成体系中，组分(i)的浓度(v/v)为10-80％，较佳地，20-60％，以所述蛋白合成体系的总体积计。
在另一优选例中，所述蛋白合成体系中，组分(i)的含量(wt％)为10-80％，较佳地，20-60％，以所述蛋白合成体系的总重量计。
在另一优选例中，所述蛋白合成体系中，组分(ii)的浓度(v/v)为10-80％，较佳地，20-60％，以所述蛋白合成体系的总体积计。
在另一优选例中，所述蛋白合成体系中，组分(ii)的含量(wt％)为10-80％，较佳地，20-60％，以所述蛋白合成体系的总重量计。
本发明第四方面提供了一种体外蛋白合成的方法，包括：
(i)提供本发明第一方面或本发明第二方面所述的蛋白合成体系，并加入外源的用于指导蛋白质合成的DNA分子；
(ii)在适合的条件下，孵育步骤(i)的蛋白合成体系一段时间T1，从而合成由所述外源DNA编码的蛋白质。
在另一优选例中，所述的方法还包括：(iii)任选地从所述蛋白合成体系中，分离或检测所述的由外源DNA编码的蛋白质。
在另一优选例中，所述外源DNA来自原核生物、真核生物。
在另一优选例中，所述外源DNA来自动物、植物、病原体。
在另一优选例中，所述外源DNA来自哺乳动物，较佳地灵长动物，啮齿动物，包括人、小鼠、大鼠。
在另一优选例中，所述的外源蛋白的编码序列编码选自下组的外源蛋白：荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域、萤光素酶突变体、α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶、或其组合。
在另一优选例中，所述外源蛋白选自下组：荧光素蛋白、或荧光素酶(如萤火 虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域、萤光素酶突变、α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶、或其组合。
在另一优选例中，所述的外源DNA编码选自下组的外源蛋白：荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域、萤光素酶突变体、α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶、或其组合。
在另一优选例中，所述外源DNA编码的蛋白质选自下组：荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域、萤光素酶突变、α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶、或其组合。
在另一优选例中，所述步骤(ii)中，反应温度为20-37℃，较佳地，20-25℃。
在另一优选例中，所述步骤(ii)中，反应时间为1-6h，较佳地，2-4h。
本发明第五方面提供了一种试剂盒，包括：
(k1)第一容器，以及位于第一容器内的组分(i)，所述组分(i)选自下组：细胞或细胞提取物、或其组合；
(k2)第二容器，以及位于第二容器内的组分(ii)，所述组分(ii)为第一反应促进剂，所述第一反应促进剂选自下组：铝、铝盐、氧铝复合物、或其组合；和
(kt)标签或说明书。
在另一优选例中，所述组分(i)为细胞提取物。
在另一优选例中，所述的第一容器、第二容器是同一容器或不同容器。
在另一优选例中，所述试剂盒还包括任选的选自下组的一个或多个容器：
(k3)第三容器，以及位于第三容器内的聚乙二醇；
(k4)任选的第四容器，以及位于第四容器的蔗糖；
(k5)第五容器，以及位于第五容器的用于合成RNA的底物；
(k6)第六容器，以及位于第六容器的用于合成蛋白的底物；
(k7)第七容器，以及位于第七容器的镁离子；
(k8)第八容器，以及位于第八容器的钾离子；和
(k9)第九容器，以及位于第九容器的缓冲剂。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了生物反应中释放以磷酸根为代表的反应副产物。例如：在转录和翻译的若干步骤中，都有镁离子的参与，利用底物，能量等，在固态，液态，或其它相态下，通过反应产生出主要目标物；同时，在转录和翻译的若干阶段，有若干反应副产物的产生如游离磷酸根离子，焦磷酸根离子；如激酶反应(蛋白激酶，脂质激酶，糖激酶等)；ATPase酶反应(钠、钾、钙离子等跨膜转运)，分子马达等，副产物常常会反作用与生物反应系统，抑制反应。目前已有去除反应副产物的方法主要有透析法，循环法，溶液置换等方法，都需要额外设备，体系复杂，不易操作，并且不易在小反应体系内完成。因此对反应副产物的去除，特别是原位去除(in situ removal)是提高反应效率，降低反应复杂性的关键。
图2显示了30μL的体外蛋白合成体系在反应2h时加入第一反应促进剂(磷酸控制制剂)的影响示意图。实心圆圈线为未添加第一反应促进剂的无细胞反应体系，正方形为2h时加入Fe 2O 3的无细胞反应体系，正三角形为2h时加入质量Fe 3O 4的无细胞反应体系，倒三角形为2h时加入等质量氧化铝颗粒的无细胞反应体系。反应条件为20-25℃反应3h。所有误差为三次重复的标准偏差。
图3显示了120μL的无细胞反应体系在反应2h时加入第一反应促进剂的影响示意图。实心圆圈线为未添加第一反应促进剂的无细胞反应体系，正方形为2h时加入Fe 2O 3的无细胞反应体系，正三角形为2h时加入等质量Fe 3O 4的无细胞反应体系，倒三角形为2h时加入等质量氧化铝颗粒的无细胞反应体系。反应条件为20-25℃反应4h和5h。所有误差为三次重复的标准偏差。
图4显示了120μL的无细胞反应体系在反应2h时加入第一反应促进剂并在4.5h时测定体系中的磷酸浓度的示意图。第一列为未添加第一反应促进剂的无细胞反应体系，第二列为2h时加入Fe 2O 3的无细胞反应体系，第三列为2h时加入等质量Fe 3O 4的无细胞反应体系，第四列为2h时加入等质量氧化铝颗粒的无细胞反应体系。反应条件为20-25℃反应4.5h。
图5显示了90μL的无细胞反应体系在不同时间段(1h和2h和不加)加入30mg氧化铝颗粒的影响示意图，反应条件为20-25℃。所有误差为三次重复的标准偏差。
图6显示了90μL的无细胞反应体系在不同时间段(1h和2h，和不加)加入30mg氧化铝颗粒的体系中磷酸浓度示意图，反应条件为20-25℃。所有误差为三次重复的标准偏差。
图7显示了90μL的无细胞反应体系在反应进行1小时时在体系中加入不同质量的氧化铝颗粒的影响示意图，反应条件为20-25℃。所有误差为三次重复的标准偏差。
图8是磷酸控制体系提升生物反应功效的示意图。总而言之，体系中磷酸控制与体系中的生化反应是一对互相竞争的反应。体系中磷酸根或焦磷酸根过多会导致体系中pH失调，影响体系中参与生化反应的生物酶的活性，降低目标蛋白的产量；同时，磷酸根容易和反应体系中所必须的镁离子相结合形成难溶于水的硫酸镁，导致众多生化反应步骤因为镁离子的缺乏导致反应活性的降低。而磷酸控制制剂的加入通过物理(吸附作用)或化学(形成沉淀产物脱离反应体系)的方式调节了如上所述的生化反应的平衡，从而为蛋白合成体系创造一个相对理想的反应环境。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地发现，由特定比例的(a)细胞或细胞提取物、或其组合；和(b)第一反应促进剂(如铝、铝盐、氧铝复合物、或其组合)混合所形成体外蛋白合成体系可显著提高外源蛋白的合成效率，其RLU最高可达3×10 9-4×10 9。并且在第一反应促进剂的作用下，与未加入第一反应促进剂的酵母无细胞表达体系相比，反应开始1h后加入反应促进剂的酵母无细胞表达体系所合成的外源蛋白(如荧光素酶)活性的相对光单位值显著提高了2-3倍,提高了反应效率，增加了目标蛋白的产量。在此基础上，完成 了本发明。
此外，本发明的实验结果表明，在特定的时间段(如反应进行大约1h时)加入第一反应促进剂的无细胞体外蛋白合成体系可显著促进蛋白合成体系(如酵母体外蛋白合成体系)中体外蛋白的合成，并且可以在蛋白合成体系(如酵母体外蛋白合成体系)反应开始时，反应开始1h后，反应开始2h后加入本发明的第一反应促进剂，其中，在蛋白合成体系(如酵母体外蛋白合成体系)反应开始1h后加入第一反应促进剂对蛋白合成的促进作用更好。
反应促进剂
在本发明中，所述反应促进剂没有特别限制，只要能够显著提高本发明的体外蛋白合成体系的蛋白合成效率的反应促进剂均在本发明的保护范围内。
在一优选实施方式中，本发明的反应促进剂包括第一反应促进剂、第二反应促进剂。
在一优选实施方式中，所述第一反应促进剂选自下组：铝、铝盐、氧铝复合物、或其组合。
在一优选实施方式中，所述第二反应促进剂选自下组：单糖、二糖、寡糖、可溶性淀粉、或其组合。
在一优选实施方式中，所述第二反应促进剂选自下组的一种或多种：
a)糖类/多元醇：蔗糖、海藻糖、甘露醇、乳糖、葡萄糖、麦芽糖等；
b)聚合物：PEG、葡聚糖、白蛋白等；
c)无水溶剂：甘油、DMSO等。
在一优选实施方式中，所述第二反应促进剂选自下组：蔗糖、海藻糖、甘露醇、乳糖、葡萄糖、麦芽糖、半乳糖、或其组合。
在一优选实施方式中，所述第二反应促进剂选自下组：蔗糖、海藻糖、乳糖、或其组合。
生物反应
生物反应即生物化学反应，就是指在生物体内进行的化学反应，这些反应都由酶催化，酶和反应物溶于内环境的水中，才能发生反应，水为体内物质提供载体和介质。在20世纪的最后几十年里，生物化学在解释生命过程方面已经取得了非常大的成就，现在几乎植物学，医学，遗传学等生命科学相关的领 域都有从事生物化学研究。生物体或细胞中所进行的生物化学反应，在复杂的网络体系中都可以通过正、负反馈得到自动调控。细胞内所进行的生物化学反应都需要有酶的催化。酶的催化效率高，反应条件温和，具有方向性，对底物有高度专一性。
细胞提取物
在一优选实施方式中，所述细胞提取物的细胞来源选自下组的一种或多种类型的细胞：原核细胞和真核细胞。
在一优选实施方式中，所述细胞提取物的细胞来源选自下组的一种或多种类型的细胞：大肠杆菌、细菌、哺乳动物细胞(如HF9、Hela、CHO、HEK293)、植物细胞、酵母细胞、昆虫细胞、或其组合。
在一优选实施方式中，所述酵母细胞选自下组：酿酒酵母、毕氏酵母、克鲁维酵母、或其组合；较佳地，所述的酵母细胞包括：克鲁维酵母，更佳地为乳酸克鲁维酵母。
在本发明中，所述细胞提取物包括酵母细胞提取物。
在本发明中，所述细胞提取物的含量和纯度没有特别限制。
在一优选实施方式中，所述蛋白合成体系中，所述细胞提取物(如酵母细胞提取物)的含量(wt％)为10％－95％，较佳地，20％－80％，更佳地，40％－60％，以所述蛋白合成体系的总重量计。
体外表达系统
酵母(yeast)兼具培养简单、高效蛋白质折叠、和翻译后修饰的优势。其中酿酒酵母(Saccharomyces cerevisiae)和毕氏酵母(Pichia pastoris)是表达复杂真核蛋白质和膜蛋白的模式生物，酵母也可作为制备体外翻译系统的原料。
克鲁维酵母(Kluyveromyces)是一种子囊孢子酵母，其中的马克斯克鲁维酵母(Kluyveromyces marxianus)和乳酸克鲁维酵母(Kluyveromyces lactis)是工业上广泛使用的酵母。与其他酵母相比,乳酸克鲁维酵母具有许多优点,如超强的分泌能力,更好的大规模发酵特性、食品安全的级别、以及同时具有蛋白翻译后修饰的能力等。
在本发明中，酵母体外表达系统不受特别限制，一种优选的酵母体外表达系统为克鲁维酵母表达系统(更佳地，乳酸克鲁维酵母表达系统)。
控制蛋白合成体系中游离磷酸根的含量
在本发明中，所述“控制蛋白合成体系中游离磷酸根的含量”指控制蛋白合成体系中的反应副产物游离磷酸根，调节对蛋白合成体系的影响，从而提高蛋白合成体系中的蛋白合成效率。
在本发明中，可以在蛋白合成体系中加入本发明的反应促进剂来达到以上目的。
在一优选实施方式中，游离磷含量控制阶段包括磷酸控制前预反应阶段，磷酸含量控制阶段和磷酸控制后阶段。
在一优选实施方式中，所述的磷酸含量控制阶段没有特别的时间限制，根据反应进行的时间来确定加入游离磷控制剂量。
在一优选实施方式中，所述的磷酸控制前预反应阶段的时间为0±2h，较佳地，1h；磷酸含量控制阶段为磷酸控制前预反应阶段后0±5h，较佳地，2-5h；磷酸控制后阶段根据各个目标蛋白以及生化反应特点制定本阶段时长。综上，整个磷酸控制反应体系时长为1-7h，较佳地，2-4h。
体外的无细胞的蛋白合成体系
在一优选实施方式中，本发明的体外的无细胞的蛋白合成体系包括酵母体外蛋白合成体系。
酵母(yeast)兼具培养简单、高效蛋白质折叠、和翻译后修饰的优势。其中酿酒酵母(Saccharomyces cerevisiae)和毕氏酵母(Pichia pastoris)是表达复杂真核蛋白质和膜蛋白的模式生物，酵母也可作为制备体外翻译系统的原料。
克鲁维酵母(Kluyveromyces)是一种子囊孢子酵母，其中的马克斯克鲁维酵母(Kluyveromyces marxianus)和乳酸克鲁维酵母(Kluyveromyces lactis)是工业上广泛使用的酵母。与其他酵母相比，乳酸克鲁维酵母具有许多优点,如超强的分泌能力,更好的大规模发酵特性、食品安全的级别、以及同时具有蛋白翻译后修饰的能力等。
在本发明中，酵母体外蛋白质合成体系不受特别限制，一种优选的酵母体外蛋白质合成体系为克鲁维酵母表达系统(更佳地，乳酸克鲁维酵母表达系统)。
在本发明中，克鲁维酵母(如乳酸克鲁维酵母)不受特别限制，包括任何一 种能够提高合成蛋白效率的克鲁维(如乳酸克鲁维酵母)菌株。
在本发明中，所述酵母体外蛋白质合成体系包括：
(a)细胞提取物(如酵母细胞提取物)；
(b)第一反应促进剂，所述第一反应促进剂选自下组：铝、铝盐、氧铝复合物、或其组合；
在一优选实施方式中，所述酵母体外蛋白质合成体系还包括：
(b’)第二反应促进剂，所述第二反应促进剂选自下组：单糖、二糖、寡糖、可溶性淀粉、或其组合；
(c)聚乙二醇；
(d)任选的外源蔗糖；和
(e)任选的溶剂，所述溶剂为水或水性溶剂。
在一优选的实施方式中，所述氧铝复合物的粒径为0.5-20mm，较佳地，0.8-10mm，更佳地，1-5mm，更佳地，1－3mm。
在另一优选例中，所述氧铝复合物的平均重量为1-80mg，3-50mg，更佳地，4-30mg，更佳地，8-15mg。
在另一优选例中，所述反应促进剂中，所述氧铝复合物的浓度(v/v)为0.5-20％，较佳地，0.8-10％，更佳地，1％－10％，更佳地，1％－5％，更佳地，2％－3％，以所述第一反应促进剂的总体积计。
在另一优选例中，所述反应促进剂中，所述氧铝复合物的含量(wt％)为0.1-20％，较佳地，1-10％，更佳地，2％－8％，更佳地，4％－7％，更佳地，5.5％－6.5％，以所述第一反应促进剂的总重量计。
在另一优选例中，所述蛋白合成体系中，所述第一反应促进剂的浓度(v/v)为20％－70％，较佳地，30％－60％，更佳地，45％－55％，以蛋白合成体系的总体积计。
在另一优选例中，所述蛋白合成体系中，所述第一反应促进剂的含量(wt％)为20％－70％，较佳地，30％－60％，更佳地45％－55％，以蛋白合成体系的总重量计。
在另一优选例中，所述蛋白合成体系中，组分(i)与组分(ii)的比例(质量比)为0.1-10：0.1-10，较佳地，0.5-8:0.5-8，更佳地，0.8-5：0.8-5，更佳地，0.9-2：0.9-2。
在一特别优选的实施方式中，本发明提供的体外蛋白合成体系包括选自下组的一种或多种或全部成分：酵母细胞提取物，氧铝复合物(如氧化铝)，聚乙二醇， 蔗糖，4-羟乙基哌嗪乙磺酸，醋酸钾，醋酸镁，腺嘌呤核苷三磷酸(ATP)，鸟嘌呤核苷三磷酸(GTP)，胞嘧啶核苷三磷酸(CTP)，胸腺嘧啶核苷三磷酸(TTP)，氨基酸混合物，磷酸肌酸，二硫苏糖醇(DTT)，磷酸肌酸激酶，RNA酶抑制剂，荧光素，萤光素酶DNA，RNA聚合酶，亚精胺，血红素。
在本发明中，RNA聚合酶没有特别限制，可以选自一种或多种RNA聚合酶，典型的RNA聚合酶为T7RNA聚合酶。
在本发明中，所述酵母细胞提取物在体外蛋白合成体系中的比例不受特别限制，通常所述酵母细胞提取物的含量(wt％)为10％－95％，较佳地，20％－80％，更佳地，40％－60％，以所述蛋白合成体系的总重量计。
在本发明中，所述的酵母细胞提取物不含完整的细胞，典型的酵母细胞提取物包括用于蛋白翻译的核糖体、转运RNA、氨酰tRNA合成酶、蛋白质合成需要的起始因子和延伸因子以及终止释放因子。此外，酵母提取物中还含有一些源自酵母细胞的细胞质中的其他蛋白，尤其是可溶性蛋白。
在本发明中，所述的酵母细胞提取物所含蛋白含量为20-100mg/mL,较佳为50-100mg/mL。所述的测定蛋白含量方法为考马斯亮蓝测定方法。
在本发明中，所述的酵母细胞提取物的制备方法不受限制，一种优选的制备方法包括以下步骤：
(i)提供酵母细胞；
(ii)对酵母细胞进行洗涤处理，获得经洗涤的酵母细胞；
(iii)对经洗涤的酵母细胞进行破细胞处理，从而获得酵母粗提物；
(iv)对所述酵母粗提物进行固液分离，获得液体部分，即为酵母细胞提取物。
在本发明中，所述的固液分离方式不受特别限制，一种优选的方式为离心。
在一优选实施方式中，所述离心在液态下进行。
在本发明中，所述离心条件不受特别限制，一种优选的离心条件为5000-100000g，较佳地，8000-30000g。
在本发明中，所述离心时间不受特别限制，一种优选的离心时间为0.5min-2h，较佳地，20min-50min。
在本发明中，所述离心的温度不受特别限制，优选的，所述离心在1-10℃下进行，较佳地，在2-6℃下进行。
在本发明中，所述的洗涤处理方式不受特别限制，一种优选的洗涤处理方式 为采用洗涤液在pH为7-8(较佳地，7.4)下进行处理，所述洗涤液没有特别限制，典型的所述洗涤液选自下组：4-羟乙基哌嗪乙磺酸钾、醋酸钾、醋酸镁、或其组合。
在本发明中，所述破细胞处理的方式不受特别限制，一种优选的所述的破细胞处理包括高压破碎、冻融(如液氮低温)破碎。
所述体外蛋白质合成体系中的核苷三磷酸混合物为腺嘌呤核苷三磷酸、鸟嘌呤核苷三磷酸、胞嘧啶核苷三磷酸和尿嘧啶核苷三磷酸。在本发明中，各种单核苷酸的浓度没有特别限制，通常每种单核苷酸的浓度为0.5-5mM，较佳地为1.0-2.0mM。
所述体外蛋白质合成体系中的氨基酸混合物可包括天然或非天然氨基酸，可包括D型或L型氨基酸。代表性的氨基酸包括(但并不限于)20种天然氨基酸：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸。每种氨基酸的浓度通常为0.01-0.5mM，较佳地0.02-0.2mM，如0.05、0.06、0.07、0.08mM。
在优选例中，所述体外蛋白质合成体系还含有聚乙二醇或其类似物。聚乙二醇或其类似物的浓度没有特别限制，通常，聚乙二醇或其类似物的浓度(w/v)为0.1-8％，较佳地，0.5-4％，更佳地，1-2％，以所述蛋白合成体系的总重量计。代表性的PEG例子包括(但并不限于)：PEG3000，PEG8000，PEG6000和PEG3350。应理解，本发明的体系还可包括其他各种分子量的聚乙二醇(如PEG200、400、1500、2000、4000、6000、8000、10000等)。
在优选例中，所述体外蛋白质合成体系还含有蔗糖。蔗糖的浓度没有特别限制，通常，蔗糖的浓度(w/v)为0.2-4％，较佳地，0.5-4％，更佳地，0.5-1％，以所述蛋白合成体系的总体积计。
在优选例中，所述体外蛋白质合成体系还含有血红素。血红素的浓度没有特别限制，通常，血红素的浓度为0.01-0.1mM，较佳地，0.02-0.08mM，更佳地，0.03-0.05mM，最佳地，0.04mM。
在优选例中，所述体外蛋白质合成体系还含有亚精胺。亚精胺的浓度没有特别限制，通常，亚精胺的浓度为0.05-1mM，较佳地，0.1-0.8mM，更佳地，更佳地，0.2-0.5mM，更佳地，0.3-0.4mM，最佳地，0.4mM。
在优选例中，所述体外蛋白质合成体系还含有缓冲剂，所述缓冲剂的成分 不受特别限制，一种优选的缓冲剂含有4-羟乙基哌嗪乙磺酸、和/或Tris缓冲液。在本发明中，所述缓冲剂还可含有其他缓冲成分，如醋酸钾、醋酸镁，从而形成pH为6.5-8.5(优选7.0-8.0)的反应液或反应缓冲液。在本发明中，缓冲剂的类型和含量不受特别限制。通常，缓冲剂的浓度为1-200mM或1-100mM，较佳地，5-50mM。
一种特别优选的体外蛋白质合成体系，除了酵母提取物、氧铝复合物(如氧化铝)之外，还含有选自下组的一种或多种或全部成分：22mM，pH为7.4的4-羟乙基哌嗪乙磺酸，30-150mM醋酸钾，1.0-5.0mM醋酸镁，1.5-4mM核苷三磷酸混合物，0.08-0.24mM的氨基酸混合物，25mM磷酸肌酸，1.7mM二硫苏糖醇，0.27mg/mL磷酸肌酸激酶，1％-4％聚乙二醇，0.5％-2％蔗糖，8-20ng/μL萤火虫荧光素酶的DNA，0.027-0.054mg/mL T7 RNA聚合酶，0.03-0.04mM的血红素，0.3-0.4mM的亚精胺。
外源蛋白的编码序列(外源DNA)
如本文所用，术语“外源蛋白的编码序列”与“外源DNA”可互换使用，均指外源的用于指导蛋白质合成的DNA分子。通常，所述的DNA分子为线性的或环状的。所述的DNA分子含有编码外源蛋白的序列。在本发明中，所述的编码外源蛋白的序列的例子包括(但并不限于)：基因组序列、cDNA序列。所述的编码外源蛋白的序列还含有启动子序列、5’非翻译序列、3’非翻译序列。
在本发明中，所述外源DNA的选择没有特别限制，通常，外源DNA选自下组：编码荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域的外源DNA、萤光素酶突变体的DNA、或其组合。
外源DNA还可以选自下组：编码α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶的外源DNA、或其组合。
在一优选实施方式中，所述外源DNA编码选自下组的蛋白：绿色荧光蛋白(enhanced GFP,eGFP)、黄色荧光蛋白(YFP)、大肠杆菌β-半乳糖苷酶(β-galactosidase,LacZ)、人赖氨酸-tRNA合成酶(Lysine-tRNA synthetase)、人亮氨酸-tRNA合成酶(Leucine-tRNA synthetase)、拟南芥甘油醛3-磷酸脱氢酶 (Glyceraldehyde-3-phosphate dehydrogenase)、鼠过氧化氢酶(Catalase)、或其组合。
试剂盒
本发明提供了一种用于体外蛋白合成的试剂盒，包括：
(k1)第一容器，以及位于第一容器内的组分(i)，所述组分(i)选自下组：细胞或细胞提取物、或其组合；
(k2)第二容器，以及位于第二容器内的组分(ii)，所述组分(ii)为第一反应促进剂，所述第一反应促进剂选自下组：铝、铝盐、氧铝复合物、或其组合；和
(kt)标签或说明书。
在一优选实施方式中，所述的第一容器、第二容器是同一容器或不同容器。
本发明还提供了一种用于无细胞的体外蛋白合成的试剂盒，包括：
(k1)第一容器，以及位于第一容器内的组分(i)，所述组分(i)为细胞提取物；
(k2)第二容器，以及位于第二容器内的组分(ii)，所述组分(ii)为第一反应促进剂，所述第一反应促进剂选自下组：铝、铝盐、氧铝复合物、或其组合；和
(kt)标签或说明书。
一种特别优选的体外蛋白质合成的试剂盒包含一个体外蛋白质合成蛋白合成体系，该蛋白合成体系包括选自下组的一种或多种或全部成分：酵母细胞提取物，氧铝复合物(如氧化铝)，4-羟乙基哌嗪乙磺酸，醋酸钾，醋酸镁，腺嘌呤核苷三磷酸(ATP)，鸟嘌呤核苷三磷酸(GTP)，胞嘧啶核苷三磷酸(CTP)，胸腺嘧啶核苷三磷酸(TTP)，氨基酸混合物，磷酸肌酸，二硫苏糖醇(DTT)，磷酸肌酸激酶，RNA酶抑制剂，荧光素，萤光素酶DNA，T7 RNA聚合酶，亚精胺，血红素。
本发明的主要优点包括：
(1)与一般的体外蛋白质合成系统相比，本发明的含有第一反应促进剂的体外蛋白合成体系的合成蛋白的能力提高2-3倍，具有明显的优势。
(2)本发明的体外蛋白合成体系可以更加方便快速的控制生化反应，如酶反应，生物合成反应，生物分解反应，无细胞生物反应等。
(3)本发明首次发现，将反应促进剂加入体外蛋白合成体系中可控制蛋白合成体系中的磷酸根离子的浓度，可显著提高蛋白合成效率。
(4)本发明首次采用原位去除(in situ removal)的方法提高生物反 应效率，降低反应复杂性。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
如无特别说明，则本发明实施例中所用的材料和试剂均为市售产品。
通用方法
在本发明中，在获得酵母提取物后，加入蔗糖和/或甘露醇，再进行冷冻干燥，获得冻干的酵母提取物。
在本发明中，所述生物反应体系以体外蛋白合成体系为例，生物反应物以酵母细胞提取物为例，但不以此为限。
实施例1液氮破碎法制备酵母细胞提取物
1.1.一级种子培养：用-80℃冻存菌种接种于摇瓶培养基中，30℃，200rpm培养至对数生长期。
1.2.二级种子培养：取适量一级种子菌液接种到二级种子，30℃，200rpm培养至对数生长期。
1.3.批培养阶段：将二级种子菌液接种到发酵罐中，温度控制在30℃培养10-12h,进入补料培养阶段，当OD600值为50-55时，收集细胞培养物。
1.4.将培养好的细胞培养物放在冰水混合物中预冷，时间为10-30min。
1.5.将1.4中预冷好的细胞培养物在低温离心机中进行离心，离心条件：3,000g、10min、4℃，得到酵母细胞。
1.6.用预冷的Washing buffer(洗涤液)对1.5酵母细胞用washing buffer(洗涤液)进行重悬，将得到的重悬液进行离心，离心条件：3000g、10min、4℃，得到酵母细胞。Washing buffer(洗涤液)组成为：20-30mM pH为7.4的4-羟乙基哌嗪乙磺酸钾,100-150mM醋酸钾，1-4mM醋酸镁；
1.7.重复1.6步2-3次。
1.8.将1.7步中获得的酵母细胞直接进行后续操作，或者采用液氮进行 速冻后-80℃保存。
1.9.采用液氮匀浆器进行破碎：在匀浆器中加入适量液氮，再加入离心得到的酵母细胞或1.8中-80℃保存的酵母细胞，转速：45,000rpm，破碎3-10min；将破碎好的低温粉末分装到50mL离心管中，称重并储存于-80℃待用。
1.10.将1.9中得到的酵母细胞破碎粉在室温下降温至4℃，每克细胞破碎粉用0.2-1mL 4℃预冷的Lysis buffer(裂解缓冲液)进行溶解，得到酵母细胞粗提物。Lysis buffer由10-40mM pH为7.4的4-羟乙基哌嗪乙磺酸钾,50-150mM醋酸钾，1-4mM醋酸镁，2-7mM二硫苏糖醇，0.5-2mM苯甲基磺酰氟组成。
1.11.把步骤1.10中收获的酵母细胞粗提物进行离心1-2次，离心力为12000-30000g时间为30min，温度为4℃；
1.12.离心后，取上层澄清液体即为酵母细胞提取物。
1.13.将制备好的酵母细胞提取物分装，并在液氮中速冻后于-80℃保存。
实施例2细胞提取物的冷冻干燥
2.1将-80℃保存的酵母提取物放置于室温，解冻；
2.2将每个玻璃瓶或玻璃平板进行称重，并做好记录；
2.3将解冻的酵母提取物分装于玻璃瓶或玻璃平板内，每个玻璃瓶约500微升细胞提取物，每个玻璃平板约5毫升细胞提取物。
2.4将2.3中的装有细胞提取物的玻璃瓶和玻璃平板，进行再次称量，并做好记录；
2.5将2.4中的装有细胞提取物的玻璃瓶和玻璃平板，预冻于-80℃，预冻时间为2-4h；
2.6设置冷冻干燥程序:预冻阶段：-55℃，4h；冷冻干燥第一阶段：-30℃，4-10h；冷冻干燥第二阶段：-30℃，10h；最终冻干阶段：-20℃，10-20h。
2.7将2.5中预冻好的细胞提取物玻璃瓶和玻璃平板，放置于冻干机的平板上，进行冻干程序，真空度约为0.1Mpa.
2.8将冻干的细胞提取物储存于-80℃，或直接加入与冻干前的相等质量的水，混匀进行后续的蛋白质体外合成活性测定。
实施例3体外生物反应体系
3.1体外生物反应体系的储存液配制：1M pH为8.0的Tris-HCl，5M醋酸钾，1M醋酸镁，25mM四种核苷三磷酸的混合物，包括腺嘌呤核苷三磷酸、鸟嘌呤核苷三磷酸、胞嘧啶核苷三磷酸和尿嘧啶核苷三磷酸，1mM二十种氨基酸的混合物：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸，二十种氨基酸的浓度均为1.0mM，1M葡萄糖，1M二硫苏糖醇，1M磷酸钾，2.4mg/mL T7 RNA聚合酶，20％-50％聚乙二醇(polyethylene glycol,PEG)3350或者(polyethylene glycol,PEG)8000,1-4mM亚精胺，0.1-0.4mM血红素；
3.2体外生物反应体系：终浓度为22mM pH为7-9的Tris-HCl，30-150mM醋酸钾，1.0-5.0mM醋酸镁，1.5-4mM核苷三磷酸混合物(腺嘌呤核苷三磷酸、鸟嘌呤核苷三磷酸、胞嘧啶核苷三磷酸和尿嘧啶核苷三磷酸)，0.08-0.24mM的氨基酸混合物(甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸)，5-80mM葡萄糖，1.7mM二硫苏糖醇，5-40mM磷酸钾，8-20ng/μL萤火虫荧光素酶DNA，0.027-0.054mg/mL T7 RNA聚合酶，1％-4％的聚乙二醇，0.03-0.04mM血红素，0.3-0.4mM亚精胺，最后加入50％体积的酵母细胞提取物；
3.3体外生物反应：将上述的反应体系放置在20-25℃的环境中，静置或者轻微震荡反应1-7h；
3.4萤光素酶活性测定：反应结束后，在96孔白板或384孔白板中加入等体积的底物荧光素(luciferine)，立即放置于Envision 2120多功能酶标仪(Perkin Elmer),读数，检测萤火虫荧光素酶活性，相对光单位值(RLU)作为活性单位。
实验结果
1.生物反应产生磷酸根离子的实例
从图1可以看出，生物反应中的若干步骤和机制都释放以磷酸根为代表的反应副产物。例如：在转录和翻译的若干步骤中，都有镁离子的参与，利用底物，能量等，在固态，液态，或其它相态下，通过反应产生出主要目标物； 同时，在转录和翻译的若干阶段，有若干反应副产物的产生如游离磷酸根离子，焦磷酸根离子；如激酶反应(蛋白激酶，脂质激酶，糖激酶等)；ATPase酶反应(钠、钾、钙离子等跨膜转运)，分子马达等，副产物常常会反作用与生物反应系统，抑制反应。目前已有去除反应副产物的方法主要有透析法，循环法，溶液置换等方法，都需要额外设备，体系复杂，不易操作，并且不易在小反应体系内完成。因此对反应副产物的去除，特别是原位去除(in situ removal)是提高反应效率，降低反应复杂性的关键。
2.不同的游离磷控制制剂对30μL细胞提取物蛋白合成体系的影响
从图2可以看出，对于30μL的细胞提取物蛋白合成体系，添加过多的反应促进剂能够使细胞提取物的体外蛋白合成能力被抑制，同时不同的反应促进剂对体外蛋白合成能力的抑制程度是不同的。在反应进行3h时，反应2h加入氧化铝的蛋白合成体系中的蛋白合成相对光单位值(RLU)比同时加入三氧化二铁或四氧化三铁的蛋白合成体系中的蛋白合成相对光单位值(RLU)高很多(若干)倍(加入三氧化二铁或四氧化三铁的RLU值非常低，几乎为零)。并且，相对而言，氧化铝反应活性降低的相对较少，因此，适量的氧化铝作为体外生物反应的反应促进剂的效果比三氧化二铁、四氧化三铁更具优势。
3.不同的游离磷控制制剂对120μL细胞提取物蛋白合成体系的影响
从图3可以看出，对于120μL的细胞提取物蛋白合成体系，添加适量的反应促进剂能够使细胞提取物的体外蛋白合成能力不受太大的影响甚至能略有提升，并且，在反应2h时，加入氧化铝的蛋白合成体系中的蛋白合成相对光单位值(RLU)比同时加入三氧化二铁或四氧化三铁的蛋白合成体系中的蛋白合成相对光单位值(RLU)高1.4倍。
4.不同的去磷制剂在120μL细胞提取物蛋白合成体系的去磷效果的比较
从图4可以看出，在反应2h加入等量的不同反应促进剂时，在4.5h的时候体系中反应促进剂都有不同程度的下降；相对而言，其中，氧化铝对体外生物反应体系的游离磷的控制效果最好。
5.在不同的时间段在细胞提取物蛋白合成体系中加入氧化铝的影响
从图5可以看出，在90μL的无细胞反应体系中，在1h时用30mg氧化铝进行体系中游离磷含量控制后，在反应进行2-3h之间的RLU值是对照组(无氧化铝)的2-3倍。因此，在90μL的无细胞反应体系中反应进行1h后进行游离磷含量控制对体系的反应活性有明显的提高，并且在反应进行3h时，反 应1h加入氧化铝的RLU值显著高于反应2h加入氧化铝的RLU值，前者为后者的2-3倍。
6.在不同的时间段在细胞提取物蛋白合成体系中加入氧化铝对体系游离磷浓度的影响
从图6可以看出，在90μL的无细胞反应体系中，在反应进行1h后体系中游离磷浓度有明显的下降，在不同时间段(1h和2h)加入氧化铝后，体系中游离磷浓度和对照组相比并没有有明显的回升，说明氧化铝在体系中可显著控制游离磷含量，并且在反应进行3h时，反应1h加入氧化铝和反应2h加入氧化铝对体系中的游离磷浓度的下降程度相当。
7.在细胞提取物蛋白合成体系反应1h后加入不同量的氧化铝的影响
从图7可以看出，在90μL的无细胞反应体系在反应进行1h时在体系中加入不同质量的氧化铝后，RLU值和对照组相比都有明显的提高，其中RLU值可达2.5×10 7。在反应进行了6-7小时后，加入10mg或者20mg氧化铝对反应活性有较为明显的提高，RLU值可达3.0×10 7。
8.磷酸控制体系提升生物反应功效
总而言之，体系中磷酸控制与体系中的生化反应是一对互相竞争的反应。体系中磷酸根或焦磷酸根过多会导致体系中pH失调，影响体系中参与生化反应的生物酶的活性，降低目标蛋白的产量；同时，磷酸根容易和反应体系中所必须的镁离子相结合形成难溶于水的硫酸镁，导致众多生化反应步骤因为镁离子的缺乏导致反应活性的降低。而磷酸控制制剂的加入通过物理(吸附作用)或化学(形成沉淀产物脱离反应体系)的方式调节了如上所述的生化反应的平衡，从而为蛋白合成体系创造一个相对理想的反应环境。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
参考文献：
1.Garcia,R.A.,&Riley,M.R.(1981).Applied biochemistry and biotechnology.Humana Press,.
2.Waddington,C.H.(1961).Molecular biology or ultrastructural biology？Nature,190(4771),184.
3.Assenberg,R.,Wan,P.T.,Geisse,S.,&Mayr,L.M.(2013).Advances in recombinant protein expression for use in pharmaceutical research.Current Opinion in Structural Biology,23(3),393–402.
4.
Figure PCTCN2017119540-appb-000003
S.,Nordlund,P.,Weigelt,J.,Hallberg,B.M.,Bray,J.,Gileadi,O.,…Gunsalus,K.C.(2008).Protein production and purification.NatureMethods,5(2),135–146.
5.Katzen,F.,Chang,G.,&Kudlicki,W.(2005).The past,present and future of cell-free protein synthesis.Trends in Biotechnology,23(3),150–156.
6.Mcmahon,T.,Zijl,P.C.M.Van,&Gilad,A.A.(2015).NIH Public Access,27(3),320–331.
7.Quast,R.B.,Ballion,B.,Stech,M.,Sonnabend,A.,Varga,B.R.,Wüstenhagen,D.A.,…Kubick,S.(2016).Cell-free synthesis of functional human epidermal growth factor receptor:Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids.Scientific Reports,6(March),1–13.
8.Lu,Y.(2017).Cell-free synthetic biology:Engineering in an open world.Synthetic and Systems Biotechnology,2(1),23–27.
9.Robertis,D.(1987).Cell and molecular biology.8th Edition.
10.Fromm,H.J.,&Hargrove,M.(2012).Essentials of Biochemistry.

Claims (14)

  1. 一种体外蛋白合成体系，其特征在于，包括：
    (a)细胞或细胞提取物、或其组合；
    (b)第一反应促进剂，所述第一反应促进剂选自下组：铝、铝盐、氧铝复合物、或其组合。
  2. 如权利要求1所述的蛋白合成体系，其特征在于，所述细胞选自下组：大肠杆菌、细菌、哺乳动物细胞(如HF9、Hela、CHO、HEK293)、植物细胞、酵母细胞、或其组合。
  3. 一种体外的无细胞的蛋白合成体系，其特征在于，包括：
    (a)细胞提取物；和
    (b)第一反应促进剂，所述第一反应促进剂选自下组：铝、铝盐、氧铝复合物、或其组合。
  4. 如权利要求1或3所述的蛋白合成体系，其特征在于，所述氧铝复合物包括氧铝纳米颗粒。
  5. 如权利要求1或3所述的蛋白合成体系，其特征在于，所述氧铝复合物包括Al2O3。
  6. 如权利要求1或3所述的蛋白合成体系，其特征在于，所述氧铝复合物的粒径为0.5-20mm，较佳地，0.8-10mm，更佳地，1-5mm。
  7. 如权利要求1或3所述的蛋白合成体系，其特征在于，所述氧铝复合物的平均重量为1-80mg，较佳地，3-50mg，更佳地，4-30mg。
  8. 如权利要求1或3所述的蛋白合成体系，其特征在于，所述第一反应促进剂中，所述氧铝复合物的含量(wt％)为0.1-20％，较佳地，1-10％，更佳地，2％－8％，更佳地，4％－7％，更佳地，5.5％－6.5％，以所述第一反应促进剂的总重量计。
  9. 如权利要求1或3所述的蛋白合成体系，其特征在于，所述蛋白合成体系中，所述第一反应促进剂的含量(wt％)为20％－70％，较佳地，30％－60％，更佳地，45％－55％，以蛋白合成体系的总重量计。
  10. 一种权利要求1或权利要求3所述的蛋白合成体系的生产方法，其特征在于，包括步骤：
    将组分(i)和组分(ii)混合，从而获得权利要求1或权利要求3所述的蛋白合成体系，其中，所述组分(i)选自下组：细胞或细胞提取物、或其组合；所述组 分(ii)为第一反应促进剂，所述第一反应促进剂选自下组：铝、铝盐、氧铝复合物、或其组合。
  11. 如权利要求10所述的生产方法，其特征在于，所述蛋白合成体系中，组分(i)与组分(ii)的质量比为0.1-10：0.1-10，较佳地，0.5-8:0.5-8，更佳地，0.8-5：0.8-5，更佳地，0.9-2：0.9-2。
  12. 一种体外蛋白合成的方法，其特征在于，包括：
    (i)提供权利要求1或权利要求3所述的蛋白合成体系，并加入外源的用于指导蛋白质合成的DNA分子；
    (ii)在适合的条件下，孵育步骤(i)的蛋白合成体系一段时间T1，从而合成由所述外源DNA编码的蛋白质。
  13. 如权利要求12所述的方法，其特征在于，所述的方法还包括：(iii)任选地从所述蛋白合成体系中，分离或检测所述的由外源DNA编码的蛋白质。
  14. 一种试剂盒，其特征在于，包括：
    (k1)第一容器，以及位于第一容器内的组分(i)，所述组分(i)选自下组：细胞或细胞提取物、或其组合；
    (k2)第二容器，以及位于第二容器内的组分(ii)，所述组分(ii)为第一反应促进剂，所述第一反应促进剂选自下组：铝、铝盐、氧铝复合物、或其组合；和
    (kt)标签或说明书。
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