WO2019127469A1 - 一种高效的能源再生体系(bes)、试剂盒及其制备方法 - Google Patents

一种高效的能源再生体系(bes)、试剂盒及其制备方法 Download PDF

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WO2019127469A1
WO2019127469A1 PCT/CN2017/120133 CN2017120133W WO2019127469A1 WO 2019127469 A1 WO2019127469 A1 WO 2019127469A1 CN 2017120133 W CN2017120133 W CN 2017120133W WO 2019127469 A1 WO2019127469 A1 WO 2019127469A1
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protein synthesis
vitro
bioenergy
regeneration system
concentration
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PCT/CN2017/120133
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English (en)
French (fr)
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郭敏
柴智
王海鹏
徐开
周子鉴
于雪
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康码（上海）生物科技有限公司
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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

Definitions

  • the present invention relates to the field of biotechnology, and in particular, to an efficient energy regeneration system (BES), a kit, and a method of preparing the same.
  • BES efficient energy regeneration system
  • a biochemical reaction refers to a chemical reaction that takes place in a living organism.
  • the biochemical reactions in the body are catalyzed by enzymes, and the enzymes and reactants are dissolved in the water of the internal environment to react, and the water provides a carrier and a medium for the substances in the body.
  • the biochemical reaction in cells is called the metabolism of cells, which is the basis of cell life activities.
  • Metabolism abbreviated as metabolism, is one of the important characteristics of an organism's life activities.
  • the metabolism in the organism is not completely spontaneous, but is catalyzed by a biocatalyst-enzyme.
  • Metabolism involves both the synthesis and decomposition of matter.
  • Organic nutrients, whether obtained from the external environment or stored by themselves, are transformed into smaller, simple substances through a series of reaction steps called catabolism.
  • Anabolic also known as biosynthesis, is the process by which organisms use small or large molecular structural elements to create their own macromolecules. The formation of macromolecules by small molecules is to make the molecular structure more complicated, and this process needs to provide energy.
  • Protein synthesis is part of biosynthesis and consists primarily of intracellular synthesis and in vitro synthesis.
  • the in vitro protein synthesis method was produced in the 1960s [1-4].
  • Exogenous mRNA or DNA is a protein synthesis template, and the substrate, energy, and transcription and translation related protein factors required for protein synthesis are added by artificial control.
  • the realization of the synthesis of the target protein is a relatively fast, time-saving and convenient way of expressing protein.
  • IVTT In vitro transcription and translation
  • DNA DNA as a template to synthesize corresponding mRNA and protein in the system, which is one of the methods of protein synthesis in vitro.
  • RNA transcription and protein translation processes require NTP (ATP, GTP, CTP, UTP).
  • ATP and GTP can also be used directly as primary energy.
  • ATP is involved in the linkage of amino acids to tRNA [4] and protein translation processes [5], while GTP is involved in the entire process of protein translation, including translation initiation, translation extension and translation termination [5]. Since ATP and GTP cannot be automatically regenerated after consumption, in the IVTT system, in order to maintain sustained protein synthesis and high yield, how to provide stable and stable energy supply becomes a crucial challenge [11,12].
  • in vitro protein synthesis systems use compounds containing high-energy phosphate bonds as a source of regenerative energy, and the corresponding enzymes catalyze the transfer of high-energy phosphate bonds to ADP.
  • These compounds are commonly known as phosphoenolpyruvate (PEP), muscle. Creatine phosphate (CrP) and acetyl phosphate (6).
  • PEP phosphoenolpyruvate
  • CrP Creatine phosphate
  • acetyl phosphate (6) acetyl phosphate
  • these compounds can release energy through the corresponding kinase reaction to produce ATP, they often only provide a large amount of energy quickly and transiently at the initial stage, and these high-energy compounds have an inhibitory effect on cell synthesis in vitro [6,7], which cannot be sustained. Energy, and high cost, is not conducive to the efficiency improvement and industrial application of in vitro protein synthesis systems [8,9,10].
  • a first aspect of the present invention provides a bioenergy regeneration system, the bioenergy regeneration system comprising:
  • a saccharide selected from the group consisting of glucose, starch, glycogen, sucrose, maltose, cyclodextrin, or a combination thereof;
  • 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 concentration (v/v) of the component (a) in the bioenergy regeneration system is 20% to 70%, preferably 30 to 60%, more preferably 40%. 50% based on the total volume of the protein synthesis system.
  • the content (% by weight) of the component (a) 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 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 phosphate compound is selected from the group consisting of potassium phosphate, magnesium phosphate, ammonium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, or a combination thereof.
  • the concentration (w/v, for example, g/ml) of the component (b) is from 0.1 to 10%, preferably from 0.5 to 8%, more preferably from 0.8 to 5%. More preferably, 1-2% is based on the total volume of the bioenergy regeneration system.
  • the content (% by weight) of the component (b) 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 (mmol/L) of the component (c) is 10-100 mM, preferably 30-80 mM, more preferably 40-60 mM.
  • the component (c) has a content (v/v) of from 1 to 10%, preferably from 3 to 8%, more preferably from 4 to 6%, to a bioenergy regeneration system. Total volume meter.
  • the concentration (v/v) of the component (d) is from 1 to 6%, preferably from 2 to 5%, more preferably from 2 to 3%, to the bioenergy regeneration system. Total volume meter.
  • the concentration (mmol/L) of the component (d) is 10-60 mM, preferably 20-50 mM, more preferably 20-30 mM.
  • the glucose has a concentration (v/v) of from 1 to 10%, preferably from 3 to 8%, more preferably from 4 to 6%, in the saccharide The total volume of the sugar.
  • the concentration of the glucose (mmol/L) in the saccharide is 10 to 100 mM, preferably 10 to 60 mM, preferably 20 to 50 mM, more preferably 20- 30 mM.
  • the bioenergy regeneration system further includes:
  • a second aspect of the invention provides the use of the bioenergy regeneration system of the first aspect of the invention for the preparation of a cell-free in vitro protein synthesis system for protein synthesis.
  • 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 cell-free in vitro protein synthesis system comprises the bioenergy regeneration system of the first aspect of the invention.
  • the protein synthesis system further comprises:
  • 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 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, cytidine 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:
  • Magnesium acetate 1-10 mM; 2-5 mM;
  • Creatine phosphate 15-50 mM; 20-30 mM;
  • Phosphocreatine kinase 0.1-0.5 mg/mL; 0.2-0.3 mg/mL;
  • DNA template 2-50ng/uL; 5-25ng/uL;
  • RNA polymerase 0.01-0.3 mg/mL; 0.02-0.10 mg/mL;
  • composition of the protein synthesis system further comprises:
  • the RNA polymerase is T7 RNA polymerase.
  • a third aspect of the invention provides a method for synthesizing an exogenous protein in vitro, comprising:
  • step (ii) incubating the in vitro protein synthesis system of step (i) for a period of time T1 under suitable conditions to synthesize the foreign protein.
  • the method further comprises: (iii) isolating or detecting the foreign protein, optionally from the in vitro protein synthesis system.
  • the in vitro 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 coding sequence of the foreign protein is from a prokaryote, a eukaryote.
  • the coding sequence of the foreign protein is from an animal, a plant, or a pathogen.
  • the coding sequence of the foreign protein 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 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 fourth aspect of the invention provides a kit comprising:
  • (k3) a third container, and a saccharide located in the third container, the saccharide being selected from the group consisting of glucose, starch, glycogen, sucrose, maltose, cyclodextrin, or a combination thereof;
  • the kit further comprises an optional one or more containers selected from the group consisting of:
  • FIG. 1 is a schematic diagram of the glycolysis and tricarboxylic acid cycle pathways of ATP synthesis in eukaryotic cells.
  • the regulation of the glycolytic metabolic pathway is mainly through the allosteric regulation of three key enzymes: hexokinase (glucokinase), phosphofructokinase, pyruvate kinase.
  • the speed of the whole pathway is controlled by regulating the activity of several enzymes in the reaction pathway.
  • Most of the enzymes that are regulated are enzymes that catalyze irreversible reactions in the course of the reaction.
  • Each cycle of glycolysis oxidatively decomposes three molecules of acetyl groups.
  • the dehydrogenation reaction can produce 2-molecular ATP, two molecules of NADH, two molecules of water, and two molecules of pyruvic acid, and one molecule of pyruvic acid undergoes one decarboxylation reaction to form one molecule of CO2 and one molecule of ethanol.
  • the key enzymes of the tricarboxylic acid cycle are citrate synthase, isocitrate dehydrogenase and ⁇ -ketoglutarate dehydrogenase, which are irreversible reactions.
  • Figure 2 is a graph showing the advantages of in vitro biosynthesis using glucose (BES), the phosphorylation pathway to the phosphocreatine pathway.
  • BES glucose
  • Figure 3 is a graphical representation of the effect of a combination of different phosphate concentrations and 40 mM glucose concentrations on a protein synthesis system.
  • the positive control was an in vitro cell-free synthesis system of the phosphocreatine/phosphate creatine kinase energy regeneration system, and the negative control was an in vitro protein synthesis protein synthesis system without the addition of Firefly luciferase (Fluc) DNA.
  • the reaction conditions were 20 ° C for 2 h. All errors are the standard deviation of three replicates.
  • Figure 4 is a graphical representation of the effect of different phosphate concentrations and 30 mM glucose concentrations on the protein synthesis system.
  • the positive control was an in vitro cell-free synthesis system of phosphocreatine/phosphate creatine kinase energy regeneration system
  • the negative control was an in vitro protein synthesis protein synthesis system without added Firefly luciferase (Fluc) DNA.
  • the reaction conditions were 20 ° C for 2 h. All errors are the standard deviation of three replicates.
  • Figure 5 is a graphical representation of the effect of different in vitro protein synthesis systems in combination with different glucose concentrations and 30 mM potassium phosphate.
  • the positive control was an in vitro cell-free synthesis system of phosphocreatine/phosphate creatine kinase energy regeneration system
  • the negative control was an in vitro protein synthesis protein synthesis system without added Firefly luciferase (Fluc) DNA.
  • the reaction conditions were 20 ° C for 2 h. All errors are the standard deviation of three replicates.
  • Figure 6 is a graphical representation of the effect of different in vitro protein synthesis systems in combination with different glucose concentrations and 25 mM potassium phosphate.
  • the positive control was an in vitro cell-free synthesis system of phosphocreatine/phosphate creatine kinase energy regeneration system
  • the negative control was an in vitro protein synthesis protein synthesis system without added Firefly luciferase (Fluc) DNA.
  • the reaction conditions were 20 ° C for 2 h. All errors are the standard deviation of three replicates.
  • Figure 7 is a graphical representation of the effect of different in vitro protein synthesis systems in combination with different glucose concentrations and 20 mM potassium phosphate.
  • the positive control was an in vitro cell-free synthesis system of phosphocreatine/phosphate creatine kinase energy regeneration system
  • the negative control was an in vitro protein synthesis protein synthesis system without added Firefly luciferase (Fluc) DNA.
  • the reaction conditions were 20 ° C for 2 h. All errors are the standard deviation of three replicates.
  • Figure 8 is a graphical representation of the effect of different reaction times on the in vitro protein synthesis system of a glucose phosphate energy regeneration system.
  • Figure 9 is a graphical representation of the effect of different PEG and different concentrations on in vitro protein synthesis systems.
  • the reaction conditions were 25 ° C for 2 h, and the reaction buffer was a magnesium acetate and potassium acetate system.
  • PEG contains three kinds, PEG3350, PEG8000 and PEG3000. Each PEG contains three to four concentrations of 0.5%, 1%, 2%, and 4% in the protein synthesis system.
  • NC represents an in vitro protein synthesis protein synthesis system with a negative control DNA-free template with an activity of 44 RLU.
  • FIG 10 shows three sources of energy required for biological reactions, direct energy sources such as ATP, GTP, high energy phosphate compounds such as phosphocreatine, phosphoenolpyruvate, etc., bioenergy systems (BES- in the present invention).
  • Biologic Energy System can also be used as a renewable energy source to continuously provide energy for biological reactions.
  • the inventors have extensively and intensively studied for the first time, surprisingly, from a specific amount of (a) cell extract; (b) polyethylene glycol; c) a saccharide selected from the group consisting of glucose, A bioenergy regeneration system composed of starch, glycogen, sucrose, maltose, cyclodextrin, or a combination thereof; and (d) a combination of phosphoric acid compounds, which can be applied to an energy supply for in vitro biosynthesis and applied to a cell-free in vitro biological reaction system
  • the cost of in vitro biosynthesis is saved, and the ability of cell-free biosynthesis in vitro is improved, and the in vitro protein synthesis efficiency of the bioenergy regeneration system of the present invention can be improved compared with the phosphocreatine/phospho-creatinine kinase energy regeneration system. -5 times, its RLU value can be up to 70,000,000.
  • the bioenergy regeneration system of the present invention has the advantages of less interference conditions, easy high-throughput measurement, and big data analysis.
  • a BES system refers to a bioenergy regeneration system of the present invention comprising a cell extract and a BES reaction system.
  • the inoculum was inoculated into a 2 L Erlenmeyer flask containing 400 mL of YPD medium at a dose of 0.1 to 1%, and placed in a shaker for culture at a temperature of 30 ° C and a rotation speed of 200 rpm.
  • the culture was terminated to obtain a cell culture solution.
  • the cultured cell culture was pre-cooled in an ice-water mixture for 10-30 min, centrifuged in a low temperature centrifuge, and centrifuged at 3,000 x g, 10 min, 4 ° C to obtain cells.
  • the cells were resuspended in pre-cooled Washing buffer, and the amount of Washing buffer was 50-100 ml/L.
  • the obtained resuspension was centrifuged in a low temperature centrifuge, and centrifuged conditions: 3000 g, 10 min, 4 ° C, to obtain cells.
  • the composition of Washing buffer is: 20-30 mM potassium 4-hydroxyethylpiperazine sulfonate pH 7.4, 100-150 mM potassium acetate, 1-4 mM magnesium acetate; after the cells are directly subjected to subsequent operations or after freezing with liquid nitrogen - Store at 80 ° C.
  • the liquid nitrogen homogenizer is used for crushing: adding appropriate amount of liquid nitrogen to the homogenizer, and then adding the yeast cells obtained by centrifugation or the yeast cells stored at -80 ° C, rotating at 45,000 rpm, crushing for 3-10 min; The powder was dispensed into a 50 mL centrifuge tube, weighed and stored at -80 ° C until use. The obtained yeast cell disrupted powder was cooled to 4 ° C at room temperature, and each gram of cell disrupted powder was pre-cooled with 0.2-1 mL of 4 ° C. Lysis Lysis The buffer is dissolved to obtain a crude yeast cell extract.
  • 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 harvested yeast cell crude extract was centrifuged 1-2 times, the centrifugal force was 12000-30000 g for 30 min, and the temperature was 4 ° C; after centrifugation, the supernatant clear liquid was taken as the yeast cell extract.
  • BES reaction system final concentration of 22 mM 4-hydroxyethyl piperazine ethanesulfonic acid, pH 7-8, 30-150 mM potassium acetate, 1.0-5.0 mM magnesium acetate, 1.5-4 mM mixture of nucleoside triphosphates (adenine nucleus) Glycoside triphosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate), 0.08-0.24 mM amino acid mixture (glycine, alanine, valine, leucine, isoluminescence) Acid, phenylalanine, valine, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, Lysine, arginine and histidine), 25 mM potassium phosphate, 1.7 mM dithiothreitol, 40 mM glucose, and 50% by volume of the above
  • 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.
  • 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).
  • 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 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 cerevisiae e.g., Kluyveromyces lactis
  • Kluyveromyces lactis is not particularly limited, and includes any Kluvi (e.g., Kluyveromyces lactis) strain capable of improving the efficiency of synthetic proteins.
  • the in vitro protein synthesis system comprises:
  • a bioenergy regeneration system comprising:
  • a saccharide selected from the group consisting of glucose, starch, glycogen, sucrose, maltose, cyclodextrin, or a combination thereof;
  • the concentration (v/v) of the component (a) in the bioenergy regeneration system is 20% to 70%, preferably 30 to 60%, more preferably 40%. 50% based on the total volume of the protein synthesis system.
  • the concentration (w/v, for example, g/ml) of the component (b) is from 0.1 to 10%, preferably from 0.5 to 8%, more preferably from 0.8 to 5%. More preferably, 1-2% is based on the total volume of the bioenergy regeneration system.
  • the content (% by weight) of the component (b) 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 (mmol/L) of the component (c) is 10-100 mM, preferably 30-80 mM, more preferably 40-60 mM.
  • the component (c) has a content (V/V) of from 1 to 10%, preferably from 3 to 8%, more preferably from 4 to 6%, to a bioenergy regeneration system. Total volume meter.
  • the concentration (v/v) of the component (d) is from 1 to 6%, preferably from 2 to 5%, more preferably from 2 to 3%, to the bioenergy regeneration system. Total volume meter.
  • the concentration (mmol/L) of the component (d) is 10-60 mM, preferably 20-50 mM, more preferably 20-30 mM.
  • the glucose has a concentration (v/v) of from 1 to 10%, preferably from 3 to 8%, more preferably from 4 to 6%, in the saccharide The total volume of the sugar.
  • the concentration of the glucose (mmol/L) in the saccharide is 10-100 mM, preferably 10-60 mM, preferably 20-50 mM, more preferably 20- 30 mM.
  • the in vitro protein synthesis system comprises one or more or all of the components selected from the group consisting of yeast cell extract, polyethylene glycol, glucose, potassium phosphate, 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, blood red Prime.
  • yeast cell extract polyethylene glycol
  • glucose potassium phosphate
  • sucrose sucrose
  • potassium acetate magnesium acetate
  • adenine nucleoside triphosphate ATP
  • 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 a polyethylene glycol analog.
  • Representative PEG examples in the present invention include, but are not limited to, PEG 3000, 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, further comprises one or more or all of the components selected from the group consisting of 22 mM 4-hydroxyethylpiperazineethanesulfonic acid having a pH of 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 phosphocreatine, 1.7 mM dithiothreitol, 0.27 mg/mL phosphocreatine kinase, 0.5%-2% sucrose, 8-20 ng/ ⁇ l of firefly luciferase DNA, 0.027-0.054 mg/mL T7 RNA polymerase, 0.03-0.04 mM heme, 0.3-0.4 mM spermidine, 1%-10 % polyethylene glycol, 10-100 mM glucose, 10-60 mM potassium phosphate.
  • 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 cell-free synthesis of proteins, comprising:
  • 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, polyethylene glycol, glucose , potassium phosphate, 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 polyethylene glycol, glucose , potassium phosphate, 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, adenine nu
  • the present invention establishes an in vitro cell-free synthesis system using glucose, polyethylene glycol and potassium phosphate as energy regeneration systems for the first time;
  • the invention saves the cost of the cell-free synthesis system in vitro, and makes it applicable to industrial production;
  • the energy regeneration system of the glucose, polyethylene glycol and phosphoric acid compounds of the present invention significantly improves the cell-free protein synthesis ability in vitro, and the bioenergy of the present invention is compared with the phosphocreatine/phospho-creatinine kinase energy regeneration system.
  • the in vitro protein synthesis efficiency of the regeneration system can be increased by 2-5 times, and the RLU value can be up to 70,000,000.
  • Example 1 Cell-free in vitro protein Fluc synthesis system
  • Figure 1 is a schematic diagram of the glycolysis and tricarboxylic acid cycle pathways of ATP synthesis in eukaryotic cells.
  • the regulation of the glycolytic metabolic pathway is mainly through the allosteric regulation of three key enzymes: hexokinase (glucokinase), phosphofructokinase, pyruvate kinase.
  • the speed of the whole pathway is controlled by regulating the activity of several enzymes in the reaction pathway.
  • Most of the enzymes that are regulated are enzymes that catalyze irreversible reactions in the course of the reaction.
  • Each cycle of glycolysis oxidatively decomposes three molecules of acetyl groups.
  • the dehydrogenation reaction can produce 2-molecular ATP, two molecules of NADH, two molecules of water, and two molecules of pyruvic acid, and one molecule of pyruvic acid undergoes one decarboxylation reaction to form one molecule of CO2 and one molecule of ethanol.
  • the key enzymes of the tricarboxylic acid cycle are citrate synthase, isocitrate dehydrogenase and ⁇ -ketoglutarate dehydrogenase, which are irreversible reactions.
  • Figure 2 is a graph showing the advantages of in vitro biosynthesis using glucose (BES) and the phosphorylation pathway to the phosphocreatine pathway.
  • BES glucose
  • the PEG comprises PEG 3350, PEG 8000 and PEG 3000.
  • Each PEG contains three to four concentrations of 0.5%, 1%, 2%, and 4% in the protein synthesis system.
  • NC represents an in vitro protein synthesis protein synthesis system with a negative control without a DNA template. The experimental results show that 2%-4% PEG significantly increased the in vitro protein synthesis capacity by 1.5-2 fold.
  • Figure 10 shows that there are three sources of energy required for biological reactions, such as ATP, GTP, high energy phosphate compounds such as phosphocreatine, phosphoenolpyruvate, etc., bioenergy systems in the present invention.
  • BES-Biologic Energy System can also be used as a renewable energy source to continuously provide energy for biological reactions.
  • Example 2 Cell-free in vitro protein eGFP synthesis system
  • RNA synthesis system final concentration of 40 mM Tris-HCl, pH 8.0, 25 mM NaCl, 8 mM MgCl2, 2 mM spermidine, 2.5 mM mixture of nucleoside triphosphates (adenosine triphosphate, guanosine triphosphate, cell Pyrimidine nucleoside triphosphate and uridine triphosphate), T7 RNA polymerase, 100 mM DTT, RNase-free DNase, RNase-free water, RNase inhibitor;
  • Protein synthesis system final concentration of 22 mM 4-hydroxyethyl piperazine ethanesulfonic acid, pH 7-8, 30-150 mM potassium acetate, 1.0-5.0 mM magnesium acetate, 1.5-4 mM mixture of nucleoside triphosphates (adenine nucleus) Glycoside triphosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate), 0.08-0.24 mM amino acid mixture (glycine, alanine, valine, leucine, isoluminescence) Acid, phenylalanine, valine, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, Lysine, arginine and histidine), 25 mM potassium phosphate, 1.7 mM dithiothreitol, 40 mM glucose, 8-20 ng/ ⁇ L
  • Luciferase is a general term for enzymes that produce bioluminescence in nature, the most representative of which is a luciferase in a firefly named Photinus pyralis.
  • the generation of fluorescence is derived from the oxidation of luciferin, and in some cases, adenosine triphosphate (ATP) is also included in the reaction system.
  • ATP adenosine triphosphate
  • Fluorescence generation reactions are usually divided into the following two steps:
  • This reaction is very energy efficient, and almost all of the energy input to the reaction is converted to light.
  • the in vitro ligation of DNA molecules is a biochemical process in which a DNA ligase catalyzes the formation of a phosphate bond between the 5'-terminal phosphate and the 3'-terminal hydroxyl group of two double-stranded DNA fragments under the conditions of DNA ligase. It is based on the enzymatic cleavage reaction to obtain the complementary sequence of the same enzyme, mainly including symmetric sticky ends; symmetric sticky ends and blunt ends.
  • aminoacyl-tRNA synthetase is involved in the binding of amino acids to its corresponding tRNA, a process that requires the involvement of ATP.
  • the first step is that the aminoacyl-tRNA synthetase recognizes the amino acid it catalyzes and another substrate ATP. Under the catalysis of the aminoacyl-tRNA synthetase, an ester bond is formed between the carboxyl group of the amino acid and the phosphoric acid on the AMP. Release a molecule of PPi:
  • the aminoacyl-AMP still binds tightly to the enzyme molecule.
  • the second aminoacyl-tRNA synthetase catalyzes the reaction by attaching an amino acid to the ribose at the 3' end of the tRNA by forming an ester bond:
  • the invention firstly proposes a novel in vitro bioenergy system (BES, Biological Energy System), which can directly prepare direct bioenergy (such as ATP) from low-level energy molecules, the technical principle, the preparation method, and the application scenario.
  • BES Bio Energy System
  • direct bioenergy such as ATP
  • the BES system uses the energy metabolism activity of biological cells and processing through cell lysate, the BES system has active and complete energy metabolism pathway molecules, which is a conceptual leap in the basic biological theory and a revolutionary innovation.
  • the use of the BES energy system not only saves the energy cost of the in vitro biological reaction, but also promotes large-scale production and application. Compared with the traditional phosphocreatine/creatinine kinase energy stock regeneration system, the in vitro cell-free biosynthesis ability of the present invention. It can be increased by 2-5 times, which embodies the great advantages of the present invention and the broad application prospects in various biological reactions.

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Abstract

一种用于体外生物反应体系的新型的高效的能源再生体系(BES)、试剂盒及制备方法。该生物能源再生体系，可应用于体外生物合成的能量供应，并应用于无细胞体外生物反应体系中，节约了体外生物合成的成本，提高了体外无细胞生物合成的能力。

Description

一种高效的能源再生体系(BES)、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，本发明涉及一种高效的能源再生体系(BES)、试剂盒及其制备方法。
背景技术
生化反应即生物化学反应，就是指在生物体内进行的化学反应。体内生化反应都由酶催化，酶和反应物溶于内环境的水中，发生反应，水为体内物质提供载体和介质。
细胞内的生物化学反应就叫细胞的新陈代谢，它是细胞生命活动的基础。新陈代谢简称代谢，是生物体表现其生命活动的重要特征之一，生物体内的新陈代谢并不是完全自发进行的，而是靠生物催化剂-酶来催化的。新陈代谢包含物质合成和分解两个方面。有机营养物，不管是从外界环境获得的，还是自身储存的，通过一系列反应步骤转变为较小的，简单的物质的过程称为分解代谢。合成代谢又称为生物合成，是生物体利用小分子或大分子的结构元件建造成自身大分子的过程。由小分子建造成大分子是使分子结构变得更为复杂，这种过程是需要提供能量的。生物体的一切生命活动都需要能量。生物体的生长，发育，包括核酸，蛋白质的生物合成，机体运动，包括肌肉的收缩以及生物膜的传递，运输功能等等，都需要消耗能量。在生物氧化的过程中，可将葡萄糖释放出的能量捕获储存起来，起捕获和贮存能量作用的分子式腺嘌呤核苷三磷酸，简称腺苷三磷酸(ATP)。能够直接提供自由能推动生物体多种化学反应的核苷酸贩子除ATP外，还有GTP，UTP以及CTP等。
蛋白质合成是生物合成的一部分，主要包括细胞内合成和体外合成。体外蛋白质合成方法是1960年代产生[1-4]，以外源的mRNA或DNA为蛋白质合成模板，通过人为控制添加加蛋白质合成所需的底物、能量、以及转录和翻译相关蛋白因子等物质，实现目的蛋白质的合成，是一种相对快速、省时、便捷的蛋白质表达方式。
体外转录和翻译偶联的体系(in vitro transcription and translation,IVTT)以DNA为模板，进而在体系中合成对应的mRNA和蛋白质，是体外蛋白质合成方法中的一种。IVTT体系中，RNA的转录和蛋白质的翻译过程都需要NTP(ATP,GTP,CTP,UTP)。ATP和GTP还能够被直接利用，作为初级能量。在蛋白质合成过程中，ATP 参与氨基酸与tRNA连接[4]和蛋白质翻译过程[5]，而GTP则参与了蛋白质翻译的全部过程，包括翻译起始、翻译延伸和翻译终止[5]。由于ATP和GTP消耗以后不能自动再生，因此在IVTT体系中，为了维持持续的蛋白质合成和高产量，如何持续稳定的提供能量成为至关重要挑战[11，12]。
目前有的体外蛋白质合成体系利用含有高能磷酸键的化合物作为再生能量来源，由对应的酶催化高能磷酸键向ADP的转移，这些化合物常见的有磷酸烯醇式丙酮酸(phosphoenolpyruvate,PEP)，肌酸磷酸(creatine phosphate,CrP)和乙酰磷酸(acetyl phosphate)等[6]。这些化合物虽然可以通过相应的激酶反应释放能量产生ATP，但往往只能在开始阶段，快速短暂地提供大量能量，且这些高能化合物对于体外细胞合成具有抑制作用[6,7]，不能持久地供能，而且成本较高，不利于体外蛋白质合成体系的效率提高提高和产业化应用[8,9,10]。
目前很多的生物合成体系都利用含有高能磷酸键的化合物作为再生能量来源，成本高，同时瞬时大量的ATP对体外合成反应有抑制作用。
因此本领域迫切需要一种缓慢释放ATP，低成本，能够产业化的的新的能量再生体系，应用于体外生物反应系统。
发明内容
本发明的目的在于提供一种缓慢释放ATP，低成本，能够产业化的的新的能量再生体系，应用于体外生物反应系统。
本发明第一方面提供了一种生物能源再生体系，所述生物能源再生成体系包括：
(a)细胞提取物；
(b)聚乙二醇；
(c)糖类，所述糖类选自下组：葡萄糖、淀粉、糖原、蔗糖、麦芽糖、环糊精、或其组合；和
(d)磷酸化合物。
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，所述生物能源再生体系中，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(a)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，所述磷酸化合物选自下组：磷酸钾、磷酸镁、磷酸铵、磷酸氢二钠、磷酸二氢钠、或其组合。
在另一优选例中，所述组分(b)的浓度(w/v，例如g/ml)为0.1-10％，较佳地，0.5-8％，更佳地，0.8-5％，更佳地，1-2％，以生物能源再生体系的总体积计。
在另一优选例中，所述蛋白合成体系中，组分(b)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，所述生物能源再生体系中，所述组分(c)的浓度(mmol/L)为10－100mM，较佳地，30－80mM，更佳地，40－60mM。
在另一优选例中，所述组分(c)的含量(v/v)为1－10％，较佳地，3－8％，更佳地，4－6％，以生物能源再生体系的总体积计。
在另一优选例中，所述组分(d)的浓度(v/v)为1－6％，较佳地，2－5％，更佳地，2－3％，以生物能源再生体系的总体积计。
在另一优选例中，所述生物能源再生体系中，所述组分(d)的浓度(mmol/L)为10－60mM，较佳地，20－50mM，更佳地，20－30mM。
在另一优选例中，所述糖类中，所述葡萄糖的浓度(v/v)为1－10％，较佳地，3－8％，更佳地，4－6％，以所述糖类的总体积计。
在另一优选例中，所述糖类中，所述葡萄糖的浓度(mmol/L)为10-100mM，较佳地，10－60mM，较佳地，20－50mM，更佳地，20－30mM。
在另一优选例中，所述生物能源再生体系还包括：
(e)任选的外源蔗糖；和
(f)任选的溶剂，所述溶剂为水或水性溶剂。
本发明第二方面提供了一种本发明第一方面所述的生物能源再生体系的用途，用于制备用于蛋白合成的无细胞的体外蛋白合成体系。
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，所述无细胞的体外蛋白合成体系包括本发明第一方面所述的生物能源再生体系。
在另一优选例中，所述蛋白合成体系还包括：
(e)任选的外源蔗糖；和
(f)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：
                       一般范围             优选范围
4-羟乙基哌嗪乙         5-40mM；             10-30mM(较佳
磺酸，                                   地，20-30mM)；
醋酸镁，               1-10mM；             2-5mM；
醋酸钾，               20-150mM；           30-75mM；
DTT，                  0.5-5mM；            1-2mM；
磷酸肌酸，             15-50mM；            20-30mM；
磷酸肌酸激酶，         0.1-0.5mg/mL；       0.2-0.3mg/mL；
4种核糖核苷酸，        各0.5-5mM；          各1.0-2.0mM；
DNA模板，              2-50ng/uL；          5-25ng/uL；
RNA聚合酶，        0.01-0.3mg/mL；      0.02-0.10mg/mL；
PEG，                  0.5-5％(w/v)         1-3％(w/v)。
在另一优选例中，所述蛋白合成体系的组成还包括：
亚精胺，               0.2-0.4mM            0.3-0.4mM；
血红素，            0.01-0.04mM             0.03-0.04mM。
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体外外源蛋白的合成方法，包括：
(i)在体外蛋白合成体系存在下，提供本发明第一方面所述的生物能源再生体系；
(ii)在适合的条件下，孵育步骤(i)的体外蛋白合成体系一段时间T1，从而合成所述外源蛋白。
在另一优选例中，所述的方法还包括：(iii)任选地从所述体外蛋白合成体系中，分离或检测所述的外源蛋白。
在另一优选例中，所述体外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，所述外源蛋白的编码序列来自原核生物、真核生物。
在另一优选例中，所述外源蛋白的编码序列来自动物、植物、病原体。
在另一优选例中，所述外源蛋白的编码序列来自哺乳动物，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。
本发明第四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细胞提取物；
(k2)第二容器，以及位于第二容器内的聚乙二醇；
(k3)第三容器，以及位于第三容器内的糖类，所述糖类选自下组：葡萄糖、 淀粉、糖原、蔗糖、麦芽糖、环糊精、或其组合；
(k4)第四容器，以及位于第四容器内的磷酸化合物；和
(kt)标签或说明书。
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：
(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是真核细胞中ATP合成的糖酵解和三羧酸循环途径示意图。糖酵解代谢途径的调节主要是通过各种变构剂对三个关键酶：己糖激酶(葡萄糖激酶)、磷酸果糖激酶、丙酮酸激酶进行变构调节。通过调节反应途径中几种酶的活性来控制整个途径的速度，被调节的酶多数为催化反应历程中不可逆反应的酶，糖酵解中每完成一次循环，氧化分解掉3分子乙酰基，经过2次，脱氢反应可生成2-分子ATP，2分子NADH，2分子水，和2分子的丙酮酸，同时1分子丙酮酸再经过1次脱羧反应，生成1分子CO2和1分子乙醇。三羧酸循环的关键酶是柠檬酸合酶、异柠檬酸脱氢酶和α-酮戊二酸脱氢酶系，为不可逆反应。每完成一次循环，氧化分解掉一分子乙酰基，可生成10分子ATP，同时两次脱羧反应，生成两分子CO2，四次脱氢反应，生成三分子NADH和一分子FADH2。
图2是体外生物合成利用葡萄糖(BES)、磷酸途径对磷酸肌酸途径的优势图。利用BES极大地重复了生物能量制造途径，有效地产生ATP功能途径，不仅节约了成本，同时实现了缓慢释放ATP参与生物合成。
图3是不同的磷酸盐浓度和40mM葡萄糖浓度的组合对蛋白合成体系的影响示意图。阳性对照为磷酸肌酸/磷酸肌酸激酶能源再生体系的体外无细胞 合成体系，阴性对照为未添加萤火虫荧光素酶(Firefly luciferase,Fluc)DNA的体外蛋白质合成蛋白合成体系。反应条件为20℃反应2h。所有误差为三次重复的标准偏差。
图4是不同的磷酸盐浓度和30mM葡萄糖浓度组合对蛋白合成体系的影响示意图。阳性对照为磷酸肌酸/磷酸肌酸激酶能源再生体系的体外无细胞合成体系，阴性对照为未添加萤火虫荧光素酶(Firefly luciferase,Fluc)DNA的体外蛋白质合成蛋白合成体系。反应条件为20℃反应2h。所有误差为三次重复的标准偏差。
图5是不同的葡萄糖浓度和30mM磷酸钾组合的体外蛋白质合成体系的影响示意图。阳性对照为磷酸肌酸/磷酸肌酸激酶能源再生体系的体外无细胞合成体系，阴性对照为未添加萤火虫荧光素酶(Firefly luciferase,Fluc)DNA的体外蛋白质合成蛋白合成体系。反应条件为20℃反应2h。所有误差为三次重复的标准偏差。
图6是不同的葡萄糖浓度和25mM磷酸钾组合的体外蛋白质合成体系的影响示意图。阳性对照为磷酸肌酸/磷酸肌酸激酶能源再生体系的体外无细胞合成体系，阴性对照为未添加萤火虫荧光素酶(Firefly luciferase,Fluc)DNA的体外蛋白质合成蛋白合成体系。反应条件为20℃反应2h。所有误差为三次重复的标准偏差。
图7是不同的葡萄糖浓度和20mM磷酸钾组合的体外蛋白质合成体系的影响示意图。阳性对照为磷酸肌酸/磷酸肌酸激酶能源再生体系的体外无细胞合成体系，阴性对照为未添加萤火虫荧光素酶(Firefly luciferase,Fluc)DNA的体外蛋白质合成蛋白合成体系。反应条件为20℃反应2h。所有误差为三次重复的标准偏差。
图8是不同的反应时间对葡萄糖磷酸能源再生体系的体外蛋白质合成体系影响示意图。
图9是不同PEG和不同的浓度对体外蛋白质合成体系的影响示意图。反应条件为25℃反应2h，反应缓冲液为醋酸镁和醋酸钾体系。其中PEG包含三种，PEG3350、PEG8000和PEG3000。每种PEG在蛋白合成体系中包含0.5％、1％、2％和4％三到四种浓度。NC表示的是阴性对照无DNA模板的体外蛋白质合成蛋白合成体系，其活性为44RLU。
图10是生物反应中所需要的能量有三种来源，直接的能量来源如ATP， GTP，高能磷酸键化合物如磷酸肌酸，磷酸烯醇式丙酮酸等，本发明中的生物能源系统(BES-Biologic Energy System)也可作为再生能源，持续地提供能量，进行生物反应。
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，由特定含量的(a)细胞提取物；(b)聚乙二醇；c)糖类，所述糖类选自下组：葡萄糖、淀粉、糖原、蔗糖、麦芽糖、环糊精、或其组合；和(d)磷酸化合物组合构成的生物能源再生体系，可应用于体外生物合成的能量供应，并应用于无细胞体外生物反应体系中，节约了体外生物合成的成本，提高了体外无细胞生物合成的能力，与磷酸肌酸/磷酸肌酸激酶能源再生体系相比，本发明的生物能源再生体系的体外蛋白合成效率可提高2-5倍，其RLU值最高可达70,000,000。并且，本发明的生物能源再生体系还具有干扰条件少、易于高通量测量和大数据分析等优点。在此基础上，本发明人完成了本发明。
BES系统制备(生物能源再生体系)
如本文所用，BES系统指本发明的生物能源再生体系，包含细胞提取物和BES反应体系两部分。
细胞提取物制备：
按0.1-1％的接种量接种到含有400mL YPD培养基的2L三角烧瓶中，并放置于摇床中培养，培养条件：温度为30℃，转速为200rpm。在酵母生长对数期的中后期(OD600＝3.0-6.9)，结束培养，得到细胞培养液。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，低温离心机中进行离心，离心条件：3,000×g、10min、4℃，得到细胞。用预冷的Washing buffer对细胞进行重悬，Washing buffer用量为50-100ml/L培养液。将得到的重悬液在低温离心机中进行离心，离心条件：3000g、10min、4℃，得到细胞。Washing buffer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；将细胞直接进行后续操作或者采用液氮进行速冻后-80℃保存。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-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，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 Lysis buffer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。将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℃；离心后，取上层澄清液体即为酵母细胞提取物。
BES反应体系的制备：
BES反应体系：终浓度为22mM pH为7-8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(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)，25mM磷酸钾，1.7mM二硫苏糖醇，40mM葡萄糖，及50％体积的上述的细胞提取物。
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)。
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，所述体外蛋白质合成体系包括：
(i)生物能源再生体系，所述生物能源再生成体系包括：
(a)细胞提取物；
(b)聚乙二醇；
(c)糖类，所述糖类选自下组：葡萄糖、淀粉、糖原、蔗糖、麦芽糖、环糊精、或其组合；
(d)磷酸化合物。
在另一优选例中，所述生物能源再生体系中，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，所述组分(b)的浓度(w/v，例如g/ml)为0.1-10％，较佳地，0.5-8％，更佳地，0.8-5％，更佳地，1-2％，以生物能源再生体系的总体积计。
在另一优选例中，所述蛋白合成体系中，组分(b)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，所述生物能源再生体系中，所述组分(c)的浓度(mmol/L)为10－100mM，较佳地，30－80mM，更佳地，40－60mM。
在另一优选例中，所述组分(c)的含量(V/V)为1－10％，较佳地，3－8％，更佳地，4－6％，以生物能源再生体系的总体积计。
在另一优选例中，所述组分(d)的浓度(v/v)为1－6％，较佳地，2－5％，更佳地，2－3％，以生物能源再生体系的总体积计。
在另一优选例中，所述生物能源再生体系中，所述组分(d)的浓度(mmol/L)为10－60mM，较佳地，20－50mM，更佳地，20－30mM。
在另一优选例中，所述糖类中，所述葡萄糖的浓度(v/v)为1－10％，较佳地，3－8％，更佳地，4－6％，以所述糖类的总体积计。
在另一优选例中，所述糖类中，所述葡萄糖的浓度(mmol/L)为10-100mM， 较佳地，10－60mM，较佳地，20－50mM，更佳地，20－30mM。
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，聚乙二醇，葡萄糖，磷酸钾，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。
在一优选实施方式中，所述体外蛋白质合成体系还含有聚乙二醇类似物。
在本发明中，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，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，0.5％-2％蔗糖，8-20ng/μl萤火虫荧光素酶的DNA，0.027-0.054mg/mL T7RNA聚合酶，0.03-0.04mM的血红素，0.3-0.4mM的亚精胺，1％-10％聚乙二醇，10-100mM葡萄糖，10－60mM磷酸钾。
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无细胞合成蛋白的试剂盒，包括：
(k1)第一容器，以及位于第一容器内的本发明第一方面所述的生物能源再生体系；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，聚乙二醇，葡萄糖，磷酸钾，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，T7RNA聚合酶，亚精胺，血红素。
本发明的主要优点包括：
(1)本发明首次建立了利用葡萄糖、聚乙二醇和磷酸钾为能源再生系统的体外无细胞合成体系；
(2)本发明节约了体外无细胞合成体系的成本，使其可应用于工业化生产；
(3)本发明的葡萄糖、聚乙二醇和磷酸化合物的能量再生体系显著提高了体外无细胞的蛋白合成能力，与与磷酸肌酸/磷酸肌酸激酶能源再生体系相比，本发明的生物能源再生体系的体外蛋白合成效率可提高2-5倍，其RLU值最高可达70,000,000。
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。
实施例1:无细胞体外蛋白质Fluc合成体系
1.1体外蛋白质合成体系的储存液配制：1M pH为7.4的4-羟乙基哌嗪乙磺酸，5M醋酸钾，250mM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磷酸钾，1M二硫苏糖醇，1M葡萄糖，1.7mg/mL T7RNA聚合酶20％-50％聚乙二醇(polyethylene glycol,PEG)3350或者(polyethylene glycol,PEG)8000,20％-40％蔗糖，1-4mM亚精胺，0.1-0.4mM血红素；
1.2体外蛋白质合成反应体系：终浓度为22mM pH为7-8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(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)，25mM磷酸钾，1.7mM二硫苏糖醇，40mM葡萄糖，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，0.5％-2％的蔗糖，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；
1.3体外蛋白质合成反应：将上述的反应体系放置在20-30℃的环境中，静置反应2-6h；
1.4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，如图1-图7所示。
实验结果
1.图1是真核细胞中ATP合成的糖酵解和三羧酸循环途径示意图。糖酵解代谢途径的调节主要是通过各种变构剂对三个关键酶：己糖激酶(葡萄糖激酶)、磷酸果糖激酶、丙酮酸激酶进行变构调节。通过调节反应途径中几种酶的活性来控制整个途径的速度，被调节的酶多数为催化反应历程中不可逆反应的酶，糖酵解中每完成一次循环，氧化分解掉3分子乙酰基，经过2次，脱氢反应可生成2-分子ATP，2分子NADH，2分子水，和2分子的丙酮酸，同时1分子丙酮酸再经过1次脱羧反应，生成1分子CO2和1分子乙醇。三羧酸循环的关键酶是柠檬酸合酶、异柠檬酸脱氢酶和α-酮戊二酸脱氢酶系，为不可逆反应。每完成一次循环，氧化分解掉一分子乙酰基，可生成10分子ATP，同时两次脱羧反应，生成两分子CO2，四次脱氢反应，生成三分子NADH和一分子FADH2。
2.图2是体外生物合成利用葡萄糖(BES)、磷酸途径对磷酸肌酸途径的优势图。利用BES极大地重复了生物能量制造途径，有效地产生ATP功能途径，不仅节约了成本，同时实现了缓慢释放ATP参与生物合成。
3.不同的磷酸盐浓度和40mM葡萄糖浓度组合对体外无细胞合成体系的影响
从图3可以看出，当葡萄糖浓度为40mM时，20-30mM的磷酸钾对体外蛋白质合成反应体系有明显的作用，与磷酸肌酸/磷酸肌酸激酶能源再生体系相比，葡萄糖磷酸体系较大地提高了体外蛋白合成能力到2-3倍。
4.不同的磷酸盐浓度和40mM葡萄糖浓度对体外无细胞合成体系的影响
从图4可以看出，当葡萄糖浓度为30mM时，20-30mM的磷酸钾对体外蛋白质合成反应体系有明显的作用，与磷酸肌酸/磷酸肌酸激酶能源再生体系相比，葡萄糖磷酸体系较大地提高了体外蛋白合成能力到1-2倍。
5.不同的葡萄糖浓度和30mM磷酸钾浓度对体外无细胞合成体系的影响
从图5可以看出，当磷酸钾浓度为30mM时，30-60mM的葡萄糖对体外蛋白质合成反应体系有明显的作用，与磷酸肌酸/磷酸肌酸激酶能源再生体系相比，葡萄糖磷酸体系较大地提高了体外蛋白合成能力到1-3倍。
6.不同的葡萄糖浓度和25mM磷酸钾浓度对体外无细胞合成体系的影响
从图6可以看出，当磷酸钾浓度为25mM时，20-50mM的葡萄糖对体外蛋白质合成反应体系有明显的作用，与磷酸肌酸/磷酸肌酸激酶能源再生体系相比，葡萄糖磷酸体系较大地提高了体外蛋白合成能力到1-2倍。
7.不同的葡萄糖浓度和20mM磷酸钾浓度对体外无细胞合成体系的影响
从图7可以看出，当磷酸钾浓度为20mM时，20-40mM的葡萄糖对体外蛋白质合成反应体系有明显的作用，与磷酸肌酸/磷酸肌酸激酶能源再生体系相比，葡萄糖磷酸体系较大地提高了体外蛋白合成能力到1-2倍。
8.不同的反应时间内，葡萄糖磷酸体系对体外无细胞合成体系的影响。
从图8可以看出，当磷酸钾浓度为25mM，葡萄糖为40mM时，在刚刚开始的30分钟时，没有合成蛋白，随着时间的延长到1小时，葡萄糖发生水解，产生的ATP，供给体外合成体系进行蛋白合成。当反应时间为1-4小时，体外合成蛋白的能力并未有明显的提高。
9.不同PEG和不同的浓度对体外蛋白质合成体系的影响示意图。
从图9可以看出，其中PEG包含PEG3350、PEG8000和PEG3000。每种PEG在蛋白合成体系中包含0.5％、1％、2％和4％三到四种浓度。NC表示的是阴性对照无DNA模板的体外蛋白质合成蛋白合成体系。实验结果表明，2％-4％PEG显著将体外蛋白合成能力提高了1.5-2倍。
10.图10显示了生物反应中所需要的能量有三种来源，直接的能量来源如ATP，GTP，高能磷酸键化合物如磷酸肌酸，磷酸烯醇式丙酮酸等，本发明中的生物能源系统(BES-Biologic Energy System)也可作为再生能源，持续地提供能量，进行生物反应。
实施例2:无细胞体外蛋白质eGFP合成体系
无细胞体外蛋白质eGFP合成体系：终浓度为22mM pH为7-8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(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)，25mM磷酸钾，1.7mM二硫苏糖醇，40mM葡萄糖，8-20ng/μLeGFP DNA，0.027-0.054mg/mL T7 RNA聚合酶，1％-4％的聚乙二醇，最后加入50％体积的酵母细胞提取物；
实施例3:体外RNA合成体系
体外RNA合成体系：终浓度为40mM Tris-HCl，pH 8.0，25mM NaCl,8mM MgCl2,2mM亚精胺，2.5mM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)，T7RNA聚合酶，100mM DTT,无RNA酶的DNA酶，无RNA酶的水，RNA酶抑制剂；
实施例4:聚乙二醇参与的合成体系
蛋白质合成体系：终浓度为22mM pH为7-8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(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)，25mM磷酸钾，1.7mM二硫苏糖醇，40mM葡萄糖，8-20ng/μL eGFP DNA，0.027-0.054mg/mL T7 RNA聚合酶，最后加入50％体积的酵母细胞提取物；其中加入1％-4％的聚乙二醇，显著地提高了蛋白质合成速率。
实施例5:体外荧光素酶活性测定
萤光素酶(Luciferase)是自然界中能够产生生物发光的酶的统称，其中最有代表性的是一种学名为Photinus pyralis的萤火虫体内的萤光素酶。在相应化学反应中，荧光的产生是来自于萤光素的氧化，有些情况下反应体系中也包括三磷酸腺苷(ATP)。
萤光生成反应通常分为以下两步：
萤光素+ATP→萤光素化腺苷酸(luciferyl adenylate)+PPi
萤光素化腺苷酸+O2→氧萤光素+AMP+光
这一反应非常节省能量，几乎所有输入反应的能量都被转化为光。
实施例6:体外DNA片段连接体系
DNA分子的体外连接就是在一定条件下，由DNA连接酶催化两个双链DNA片段组邻的5’端磷酸与3’端羟基之间形成磷酸酸脂键的生物化学过程，DNA分子的连接是在酶切反应获得同种酶互补序列基础上进行的，主要包括对称粘性末端；对称性粘性末端和平端连接。
体外DNA连接体系：终浓度为20mM Tris-HCl，pH 7.6，5mM MgCl2,5mM DTT,T4DNA连接酶，5mM ATP，12℃下过夜连接反应。
实施例7:体外氨酰-tRNA合成酶体系
氨酰-tRNA合成酶参与将氨基酸结合到其对应的tRNA上，这一过程是需要ATP的参与。
氨酰-tRNA合成酶参与的合成分两步进行。
第一步是氨酰-tRNA合成酶识别它所催化的氨基酸以及另一底物ATP，在氨酰-tRNA合成酶的催化下，氨基酸的羧基与AMP上的磷酸之间形成一个酯键，同时释放出一分子PPi：
氨基酸+ATP——氨酰-AMP+PPi
这时氨酰-AMP仍然紧密地与酶分子结合。
第二个氨酰-tRNA合成酶催化的反应是通过形成酯键，将氨基酸连接到tRNA 3'端的核糖上：
氨酰-AMP+tRNA——氨酰-tRNA+AMP
结论
本发明首次提出了一种可以直接由低级能源分子制备直接生物能量(如ATP等)的新型体外生物能量系统(BES,Biological Energy System)，技术原理，制备方法，以及应用场景。利用生物细胞中能量代谢活性，通过细胞裂解液加工处理，制备出的BES系统具有活性完整的能量代谢通路分子，是基础生物理论中概念性的飞跃，和革命性的创新。
采用BES能源体系，不仅节约了体外生物反应的能量成本，推动大规模生产和应用，与传统的磷酸肌酸/磷酸肌酸激酶能源股再生体系相比，本发明的体外无细胞生物合成的能力可提高2-5倍，体现了本发明的极大的优势和在各种生物反应中的广泛应用前景。
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11.Anderson,M.J.,et al.Energizing eukaryotic cell-free protein synthesis with glucose metabolism.FEBS letters 2015；589:1723-1727.
12.Calhoun,K.A.,and Swartz,J.R.Energizing cell-free protein synthesis with glucose metabolism.Biotechnology and bioengineering 2005；90:606-613.
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。

Claims (11)

  1. 一种生物能源再生体系，其特征在于，所述生物能源再生成体系包括：
    (a)细胞提取物；
    (b)聚乙二醇；
    (c)糖类，所述糖类选自下组：葡萄糖、淀粉、糖原、蔗糖、麦芽糖、环糊精、或其组合；和
    (d)磷酸化合物。
  2. 如权利要求1所述的生物能源再生体系，其特征在于，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。
  3. 如权利要求1所述的生物能源再生体系，其特征在于，所述生物能源再生体系中，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。
  4. 如权利要求1所述的生物能源再生体系，其特征在于，所述组分(b)的浓度(w/v，例如g/mL)为0.1-10％，较佳地，0.5-8％，更佳地，0.8-5％，更佳地，1-2％，以生物能源再生体系的总体积计。
  5. 如权利要求1所述的生物能源再生体系，其特征在于，所述生物能源再生体系中，所述组分(c)的浓度(mmol/L)为10－100mM，较佳地，30－80mM，更佳地，40－60mM。
  6. 如权利要求1所述的生物能源再生体系，其特征在于，所述组分(d)的浓度(mmol/L)为10－60mM，较佳地，20－50mM，更佳地，20－30mM。
  7. 如权利要求1所述的生物能源再生体系，其特征在于，所述糖类中，所述葡萄糖的浓度(mmol/L)为10-100mM，较佳地，10－60mM，较佳地，20－50mM，更佳地，20－30mM。
  8. 一种权利要求1所述的生物能源再生体系的用途，其特征在于，用于制备用于蛋白合成的无细胞的体外蛋白合成体系。
  9. 一种体外外源蛋白的合成方法，其特征在于，包括：
    (i)在体外蛋白合成体系存在下，提供权利要求1所述的生物能源再生体系；
    (ii)在适合的条件下，孵育步骤(i)的体外蛋白合成体系一段时间T1，从而合成所述外源蛋白。
  10. 如权利要求9所述的方法，其特征在于，所述的方法还包括：(iii)任选地从所述体外蛋白合成体系中，分离或检测所述外源蛋白。
  11. 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细胞提取物；
    (k2)第二容器，以及位于第二容器内的聚乙二醇；
    (k3)第三容器，以及位于第三容器内的糖类，所述糖类选自下组：葡萄糖、淀粉、糖原、蔗糖、麦芽糖、环糊精、或其组合；
    (k4)第四容器，以及位于第四容器内的磷酸化合物；和
    (kt)标签或说明书。
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