CN109988801A - A kind of novel efficient energy regeneration system (BES), kit and preparation method for external biological reaction system - Google Patents

Abstract

The present invention provides a kind of novel efficient energy regeneration system (BES), kit and preparation methods for external biological reaction system, bioenergy regenerating system of the invention, it can be applied to the energy supply of external biological synthesis, and it is applied in cell free in vitro biological respinse system, the cost for having saved external biological synthesis improves the ability of external cell-free biosynthesis.

Description

A kind of novel efficient energy regeneration system for external biological reaction system (BES), kit and preparation method

Technical field

The present invention relates to field of biotechnology, in particular it relates to a kind of for the new of external biological reaction system Efficient energy regeneration system (BES), kit and the preparation method of type.

Background technique

Biochemical reaction, that is, biochemical reaction just refers to the chemical reaction carried out in vivo.Internal biochemical reaction is all By enzymatic, enzyme and reactant are dissolved in the water of interior environment, are reacted, water provides carrier and medium for substance in vivo.

Intracellular biochemical reaction is just named the metabolism of cell, it is the basis of cell activities.New old generation It thanks and is referred to as metabolized, be that organism shows one of important feature of its vital movement, the intracorporal metabolism of biology is not complete Spontaneous progress, but be catalyzed by biocatalyst-enzyme.Metabolism synthesizes and decomposes two aspects comprising substance.Have Machine nutrients, it is either being obtained from external environment or itself storage, it is smaller by series reaction step transitions , the process of simple substance is known as catabolism.Anabolism is also known as biosynthesis, is that organism utilizes small molecule or big The structural detail of molecule is built into the process of itself macromolecular.Being built into macromolecular by small molecule is to become molecular structure more Complexity, this process need to provide energy.The all life activity of organism requires energy.The growth of organism, hair It educates, including nucleic acid, the biosynthesis of protein, body movement, the transmitting of contraction and biomembrane including muscle, transportation function Etc., require consumption energy.During biological oxidation, the energy capture that can release glucose is stored, and is risen The molecular formula adenosine triphyosphate of capture and storing energy effect, abbreviation adenosine triphosphate (ATP).It can directly provide certainly By that can push the nucleotide dealer of a variety of chemical reactions of organism in addition to ATP, there are also GTP, UTP and CTP etc..

Protein synthesis is a part of biosynthesis, and main includes intracellular synthesis and external synthesis.External protein Synthetic method is to generate [1-4] in generation nineteen sixty, synthesizes template by protein of the mRNA of external source or DNA, is added by artificially controlling Plus the substances such as substrate, energy and transcription and translation GAP-associated protein GAP factor needed for protein synthesis, realize target protein Synthesis, be a kind of relatively rapid, time saving, convenient and fast protein expression mode.

The system (in vitro transcription and translation, IVTT) with translation coupling is transcribed in vitro Using DNA as template, and then corresponding mRNA and protein are synthesized in system, be one of protein synthesis in vitro method. In IVTT system, the transcription of RNA and the translation process of protein require NTP (ATP, GTP, CTP, UTP).ATP and GTP are also It can be used directly, as primary energy.In protein building-up process, ATP participate in amino acid connect with tRNA [4] and Protein translation process [5], and GTP then takes part in all processes of protein translation, including translation initiation, translation extend and turn over Translate termination [5].Due to being unable to automatic regeneration after ATP and GTP consumption, in IVTT system, in order to maintain lasting egg White matter synthesis and high yield, how continual and steady offer energy becomes most important challenge [11,12].

The protein synthesis in vitro system having at present using the compound for containing energy-rich phosphate bond as regeneration energy source, Transfer from from corresponding enzymatic energy-rich phosphate bond to ADP, these compounds common are phosphoenolpyruvate (phosphoenolpyruvate, PEP), creatine phosphate (creatine phosphate, CrP) and acetyl phosphate (acetyl Phosphate) etc. [6].Although these compounds can be released energy by corresponding kinase reaction and generate ATP, often only Big energy quickly can be briefly provided, and these energy-rich compounds have inhibition for cell in vitro synthesis in the incipient stage It acts on [6,7], cannot enduringly energize, and higher cost, the efficiency for being unfavorable for protein synthesis in vitro system improves With industrial application [8,9,10].

Many biosynthesis system is all using the compound for containing energy-rich phosphate bond as regenerating energy source at present, at This height, while instantaneous a large amount of ATP has inhibiting effect to external synthetic reaction.

Therefore there is an urgent need in the art to a kind of slow release ATP, low cost is capable of the new energy regeneration body of industrialization System is applied to external biological reaction system.

Summary of the invention

The purpose of the present invention is to provide a kind of slow release ATP, low cost is capable of the new energy regeneration of industrialization System is applied to external biological reaction system.

First aspect present invention provides a kind of bioenergy regenerating system, and the bioenergy regeneration architectonical includes:

(a) cell extract；

(b) polyethylene glycol；

(c) carbohydrate, the carbohydrate are selected from the group: glucose, starch, glycogen, sucrose, maltose, cyclodextrin or its group It closes；With

(d) phosphate cpd.

In another preferred example, the cell origin of the cell extract one or more types selected from the group below is thin Born of the same parents: prokaryotic cell and eukaryocyte.

In another preferred example, the cell origin of the cell extract one or more types selected from the group below is thin Born of the same parents: Escherichia coli, bacterium, mammalian cell (such as HF9, Hela, CHO, HEK293), plant cell, yeast cells or its group It closes.

In another preferred example, the yeast cells is selected from the group: saccharomyces cerevisiae, pichia yeast, kluyveromyces or its Combination；Preferably, the yeast cells includes: kluyveromyces, it is more preferably Kluyveromyces lactis.

In another preferred example, in the bioenergy regenerating system, the concentration (v/v) of component (a) is 20%-70%, Preferably, 30-60%, more preferably, 40%-50%, with the total volume meter of the albumen synthetic system.

In another preferred example, in the albumen synthetic system, the content (wt%) of component (a) is 10%-95%, compared with Goodly, 20%-80%, more preferably, 40%-60%, with the total weight of the albumen synthetic system.

In another preferred example, the polyethylene glycol is selected from the group: PEG3000, PEG8000, PEG6000, PEG3350, Or combinations thereof.

In another preferred example, the polyethylene glycol includes the polyethylene glycol that molecular weight (Da) is 200-10000, preferably Ground, molecular weight are the polyethylene glycol of 3000-10000.

In another preferred example, the phosphate cpd is selected from the group: potassium phosphate, magnesium phosphate, ammonium phosphate, phosphoric acid hydrogen two Sodium, sodium dihydrogen phosphate, or combinations thereof.

In another preferred example, the concentration (w/v, such as g/ml) of the component (b) is 0.1-10%, preferably, 0.5- 8%, more preferably, 0.8-5%, more preferably, 1-2%, with the total volume meter of bioenergy regenerating system.

In another preferred example, in the albumen synthetic system, the content (wt%) of component (b) is 10%-95%, compared with Goodly, 20%-80%, more preferably, 40%-60%, with the total weight of the albumen synthetic system.

In another preferred example, in the bioenergy regenerating system, the concentration (mmol/L) of the component (c) is 10-100mM, preferably, 30-80mM, more preferably, 40-60mM.

In another preferred example, the content (v/v) of the component (c) is 1-10%, preferably, 3-8%, more preferably, 4-6%, with the total volume meter of bioenergy regenerating system.

In another preferred example, the concentration (v/v) of the component (d) is 1-6%, preferably, 2-5%, more preferably, 2-3%, with the total volume meter of bioenergy regenerating system.

In another preferred example, in the bioenergy regenerating system, the concentration (mmol/L) of the component (d) is 10-60mM, preferably, 20-50mM, more preferably, 20-30mM.

In another preferred example, in the carbohydrate, the concentration (v/v) of the glucose is 1-10%, preferably, 3- 8%, more preferably, 4-6%, with the total volume meter of the carbohydrate.

In another preferred example, in the carbohydrate, the concentration (mmol/L) of the glucose is 10-100mM, preferably, 10-60mM, preferably, 20-50mM, more preferably, 20-30mM.

In another preferred example, the bioenergy regenerating system further include:

(e) optional Exogenous Sucrose；With

(f) optional solvent, the solvent are water or aqueous solvent.

Second aspect of the present invention provides a kind of purposes of bioenergy regenerating system described in first aspect present invention, uses In cell-free external albumen synthetic system of the preparation for albumen synthesis.

In another preferred example, the albumen synthetic system includes yeast outer protein synthesis system (such as Crewe dimension ferment The outer albumen synthetic system of parent, preferably, the external albumen synthetic system of Kluyveromyces lactis).

In another preferred example, the cell-free external albumen synthetic system includes life described in first aspect present invention Object energy regeneration system.

In another preferred example, the albumen synthetic system further include:

(e) optional Exogenous Sucrose；With

(f) optional solvent, the solvent are water or aqueous solvent.

In another preferred example, the albumen synthetic system further includes one or more components selected from the group below:

(f1) for synthesizing the substrate of RNA；

(f2) it is used for the substrate of synthetic proteins；

(f3) magnesium ion；

(f4) potassium ion；

(f5) buffer；

(f6) RNA polymerase；

(f7) energy-regenerating system.

In another preferred example, the albumen synthetic system further includes one or more components selected from the group below:

(g8) ferroheme；

(g9) spermidine.

In another preferred example, the cell extract includes yeast cell extract.

In another preferred example, the yeast cell extract is the aqueous extract to yeast cells.

In another preferred example, the yeast cell extract is free of the long nucleic acid molecule of yeast entogenous.

In another preferred example, the yeast cell extract is prepared with method comprising the following steps:

(i) yeast cells is provided；

(ii) carrying out washing treatment is carried out to yeast cells, obtains washed yeast cells；

(iii) broken cell processing is carried out to washed yeast cells, to obtain yeast crude extract；With

(iv) the yeast crude extract is separated by solid-liquid separation, obtains liquid portion, as yeast cell extract.

In another preferred example, the separation of solid and liquid includes centrifugation.

In another preferred example, it is centrifuged in the liquid state.

In another preferred example, the centrifugal condition is 5000-100000g, preferably, 8000-30000g.

In another preferred example, the centrifugation time is 0.5min-2h, preferably, 20min-50min.

In another preferred example, the centrifugation carries out at 1-10 DEG C, preferably, carrying out at 2-6 DEG C.

In another preferred example, the carrying out washing treatment uses cleaning solution in pH under 7-8 (preferably, 7.4) Reason.

In another preferred example, the cleaning solution is selected from the group: 4- hydroxyethyl piperazineethanesulfonic acid potassium, potassium acetate, magnesium acetate, Or combinations thereof.

In another preferred example, broken cell processing includes that high pressure is broken, freeze thawing (such as liquid nitrogen cryogenics) is broken.

In another preferred example, the substrate of the synthesis RNA includes: Nucleotide monophosphates, ribonucleoside triphosphote or its group It closes.

In another preferred example, the substrate of the synthetic proteins includes: 1-20 kind natural amino acid and non-natural ammonia Base acid.

In another preferred example, the magnesium ion derives from magnesium ion source, and the magnesium ion source is selected from the group: magnesium acetate, Psicosoma, or combinations thereof.

In another preferred example, the source of potassium ions is selected from the group in potassium ion source, the potassium ion source: potassium acetate, Potassium glutamate, or combinations thereof.

In another preferred example, the energy-regenerating system is selected from the group: phosphocreatine/phosphocreatine enzyme system, sugared ferment Solution approach and its intermediate product energy system, or combinations thereof.

In another preferred example, the albumen synthetic system further includes (h1) artificial synthesized tRNA.

In another preferred example, the buffer is selected from the group: 4- hydroxyethyl piperazineethanesulfonic acid, trihydroxy methyl amino first Alkane, or combinations thereof.

In another preferred example, the albumen synthetic system further includes (i1) external source for instructing protein to synthesize DNA molecular.

In another preferred example, the DNA molecular is linear.

In another preferred example, the DNA molecular is cricoid.

In another preferred example, the DNA molecular contains the sequence of encoding foreign proteins.

In another preferred example, the sequence of the encoding foreign proteins includes genome sequence, cDNA sequence.

In another preferred example, the sequence of the encoding foreign proteins also contain promoter sequence, 5' non-translated sequence, 3' non-translated sequence.

In another preferred example, the albumen synthetic system includes ingredient selected from the group below: 4- hydroxyethyl piperazineethanesulfonic acid, Potassium acetate, magnesium acetate, ribonucleoside triphosphote, amino acid, phosphocreatine, dithiothreitol (DTT) (DTT), creatine phosphokinase, RNA polymerization Enzyme, or combinations thereof.

In another preferred example, in the albumen synthetic system, the concentration of component (e) is 0.2-4%, preferably, 0.5- 4%, more preferably, 0.5-1%, with the total volume meter of the albumen synthetic system.

In another preferred example, the ribonucleoside triphosphote is selected from the group: adenosine triphyosphate, three phosphorus of guanosine Acid, cytidine triphosphate, uridine diphosphate guanosine triphosphate, or combinations thereof.

In another preferred example, in the albumen synthetic system, the concentration of component (f1) is 0.1-5mM, preferably, 0.5- 3mM, more preferably, 1-1.5mM.

In another preferred example, the amino acid is to be selected from the group: glycine, alanine, valine, leucine, different bright Propylhomoserin, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, Threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, or combinations thereof.

In another preferred example, the amino acid includes D type amino acid and/or L-type amino acid.

In another preferred example, in the albumen synthetic system, the concentration of the component (f2) is 0.01-0.48 mM, compared with Goodly, 0.04-0.24mM, more preferably, 0.04-0.2mM, most preferably, 0.08mM.

In another preferred example, in the albumen synthetic system, the concentration of the component (f3) is 1-10mM, preferably, 1-5mM, more preferably, 2-4mM.

In another preferred example, in the albumen synthetic system, the concentration of the component (f4) is 30-210mM, preferably Ground, 30-150mM, more preferably, 30-60mM.

In another preferred example, in the albumen synthetic system, the concentration of the component (f6) is 0.01-0.3 mg/mL, Preferably, 0.02-0.1mg/mL, more preferably, 0.027-0.054mg/mL.

In another preferred example, in the albumen synthetic system, the concentration of 4- hydroxyethyl piperazineethanesulfonic acid is 5-50 mM, Preferably, 10-50mM, preferably, 15-30mM, more preferably, 20-25mM.

In another preferred example, in the albumen synthetic system, the concentration of the potassium acetate is 20-210mM, preferably, 30-210mM, preferably, 30-150mM, more preferably, 30-60mM.

In another preferred example, in the albumen synthetic system, the concentration of the magnesium acetate is 1-10mM, preferably, 1- 5mM, more preferably, 2-4mM.

In another preferred example, in the albumen synthetic system, the concentration of the phosphocreatine is 10-50mM, preferably, 20-30mM, more preferably, 25mM.

In another preferred example, in the albumen synthetic system, the concentration of the ferroheme is 0.01-0.1mM, preferably Ground, 0.02-0.08mM, more preferably, 0.03-0.05mM, most preferably, 0.04mM.

In another preferred example, in the albumen synthetic system, the concentration of the spermidine is 0.05-1mM, preferably, 0.1-0.8mM, more preferably, more preferably, 0.2-0.5mM, more preferably, 0.3-0.4mM, most preferably, 0.4mM.

In another preferred example, in the albumen synthetic system, the concentration of the dithiothreitol (DTT) (DTT) is 0.2- 15mM, preferably, 0.2-7mM, more preferably, 1-2mM.

In another preferred example, in the albumen synthetic system, the concentration of the creatine phosphokinase is 0.1-1 mg/ ML, preferably, 0.2-0.5mg/mL, more preferably, 0.27mg/mL.

In another preferred example, in the albumen synthetic system, the concentration of the t7 rna polymerase is 0. 01-0.3mg/ ML, preferably, 0.02-0.1mg/mL, more preferably, 0.027-0.054mg/mL.

In another preferred example, the albumen synthetic system has the following performance:

In synthetic system, albumen synthesis total amount reaches 3ug albumen/ml system.

In another preferred example, the composition of the albumen synthetic system includes:

In another preferred example, the composition of the albumen synthetic system further include:

Spermidine, 0.2-0.4mM 0.3-0.4mM；

Ferroheme, 0.01-0.04mM 0.03-0.04mM.

In another preferred example, the RNA polymerase is t7 rna polymerase.

Third aspect present invention provides a kind of synthetic method of external foreign protein, comprising:

(i) in vitro in the presence of albumen synthetic system, bioenergy regenerating system described in first aspect present invention is provided；

(ii) under the suitable conditions, the external albumen synthetic system of incubation step (i) T1 for a period of time, to synthesize institute State foreign protein.

In another preferred example, the method further include: (iii) optionally from the external albumen synthetic system, Foreign protein described in separation or detection.

In another preferred example, the external albumen synthetic system includes outer protein synthesis system (such as Crewe of yeast The outer albumen synthetic system of yeast is tieed up, preferably, the external albumen synthetic system of Kluyveromyces lactis).

In another preferred example, the coded sequence of the foreign protein comes from prokaryotes, eucaryote.

In another preferred example, the coded sequence of the foreign protein comes from animal, plant, pathogen.

In another preferred example, the coded sequence of the foreign protein comes from mammal, preferably Primate, grinding tooth Animal, including people, mouse, rat.

In another preferred example, the coded sequence of the foreign protein encodes foreign protein selected from the group below: fluorescein Albumen or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescence protein, aminoacyl tRNA synthetase, Glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, the Variable Area of antibody, luciferase mutant, alphalise starch Enzyme, enterocin A, hepatitis C virus E 2 glycoprotein, insulin precurosor, Interferon α A, interleukin-1 ' beta ', lysozyme element, Seralbumin, single-chain antibody section (scFV), transthyretin, tyrosinase, zytase, or combinations thereof.

In another preferred example, the foreign protein is selected from the group: (such as firefly is glimmering for fluorescent proteins or luciferase Light element enzyme), green fluorescent protein, yellow fluorescence protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, hydrogen peroxide Enzyme, actin, the Variable Area of antibody, luciferase mutation, alpha-amylase, enterocin A, hepatitis C virus E 2 sugar egg White, insulin precurosor, Interferon α A, interleukin 1 β, lysozyme element, seralbumin, single-chain antibody section (scFV), first Shape parathyrine transporter, tyrosinase, zytase, or combinations thereof.

In another preferred example, in the step (ii), reaction temperature is 20-37 DEG C, preferably, 20-25 DEG C.

In another preferred example, in the step (ii), reaction time 1-6h, preferably, 2-4h.

Fourth aspect present invention provides a kind of kit, comprising:

(k1) the first container, and the cell extract in the first container；

(k2) second container, and the polyethylene glycol in second container；

(k3) third container, and the carbohydrate in third container, the carbohydrate are selected from the group: glucose, starch, sugar Original, sucrose, maltose, cyclodextrin, or combinations thereof；

(k4) the 4th container, and the phosphate cpd in the 4th container；With

(kt) label or specification.

In another preferred example, the kit further includes optional one or more containers selected from the group below:

(k5) the 5th container, and positioned at the 5th container for synthesizing the substrate of RNA；

(k6) the 6th container, and the substrate for synthetic proteins positioned at the 6th container；

(k7) the 7th container, and the magnesium ion positioned at the 7th container；

(k8) the 8th container, and the potassium ion positioned at the 8th container；With

(k9) the 9th container, and the buffer positioned at the 9th container.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Detailed description of the invention

Fig. 1 is the glycolysis and krebs cycle pathway's schematic diagram that ATP is synthesized in eukaryocyte.Glycolysis metabolism approach It adjusts mainly by various allosteric agents to three key enzymes: hexokinase (glucokinase), phosphofructokinase, pyruvic acid Kinases carries out allosteric adjusting.The speed of entire approach is controlled by adjusting the activity of several enzymes in reaction path, is conditioned Enzyme majority is the enzyme for being catalyzed irreversible reaction in the reaction mechanism mechanism of reaction, every completion one cycle in glycolysis, and oxygenolysis falls 3 molecule second Acyl group, by 2 times, dehydrogenation reaction produces 2- molecule ATP, 2 molecule NADH, the pyruvic acid of 2 molecular waters and 2 molecules, while 1 Molecule pyruvic acid generates 1 molecule CO2 and 1 molecules of ethanol using 1 decarboxylic reaction.The key enzyme of tricarboxylic acid cycle is lemon Acid synthase, isocitric dehydrogenase and ketoglurate dehydrogenase system are irreversible reaction.Every completion one cycle, oxidation point A molecule acetyl group is taken off, producible 10 molecule ATP, and meanwhile decarboxylic reaction twice, two molecule CO2 of generation, four dehydrogenation reactions, Generate three molecule NADH and a molecule FADH2.

Fig. 2 be external biological synthesis using glucose (BES), phosphate pathway to the advantage figure of phosphocreatine approach.It utilizes Bio-energy manufacture approach is greatly repeated in BES, effectively generates ATP functional approach, has not only saved cost, realized simultaneously Slow release ATP participates in biosynthesis.

Fig. 3 is different influence signal of the combination of phosphate concn and 40mM concentration of glucose to albumen synthetic system Figure.Positive control is the external cell-free synthetic system of phosphocreatine/creatine phosphokinase energy regeneration system, and negative control is It is not added with the protein synthesis in vitro albumen compound body of firefly luciferase (Firefly luciferase, Fluc) DNA System.Reaction condition is 20 DEG C of reaction 2h.All errors are duplicate standard deviation three times.

Fig. 4 is different the influence schematic diagram of phosphate concn and the combination of 30mM concentration of glucose to albumen synthetic system. Positive control is the external cell-free synthetic system of phosphocreatine/creatine phosphokinase energy regeneration system, and negative control is not Add the protein synthesis in vitro albumen synthetic system of firefly luciferase (Firefly luciferase, Fluc) DNA.Instead Answering condition is 20 DEG C of reaction 2h.All errors are duplicate standard deviation three times.

Fig. 5 is different the influence signal of the protein synthesis in vitro system of concentration of glucose and the combination of 30mM potassium phosphate Figure.Positive control is the external cell-free synthetic system of phosphocreatine/creatine phosphokinase energy regeneration system, and negative control is It is not added with the protein synthesis in vitro albumen compound body of firefly luciferase (Firefly luciferase, Fluc) DNA System.Reaction condition is 20 DEG C of reaction 2h.All errors are duplicate standard deviation three times.

Fig. 6 is different the influence signal of the protein synthesis in vitro system of concentration of glucose and the combination of 25mM potassium phosphate Figure.Positive control is the external cell-free synthetic system of phosphocreatine/creatine phosphokinase energy regeneration system, and negative control is It is not added with the protein synthesis in vitro albumen compound body of firefly luciferase (Firefly luciferase, Fluc) DNA System.Reaction condition is 20 DEG C of reaction 2h.All errors are duplicate standard deviation three times.

Fig. 7 is different the influence signal of the protein synthesis in vitro system of concentration of glucose and the combination of 20mM potassium phosphate Figure.Positive control is the external cell-free synthetic system of phosphocreatine/creatine phosphokinase energy regeneration system, and negative control is It is not added with the protein synthesis in vitro albumen compound body of firefly luciferase (Firefly luciferase, Fluc) DNA System.Reaction condition is 20 DEG C of reaction 2h.All errors are duplicate standard deviation three times.

Fig. 8 is different the reaction time and shows the influence of the protein synthesis in vitro system of glucose phosphate energy regeneration system It is intended to.

Fig. 9 is the influence schematic diagram of different PEG and different concentration to protein synthesis in vitro system.Reaction condition is 25 DEG C reaction 2h, reaction buffer be magnesium acetate and potassium acetate system.Wherein PEG include three kinds, PEG3350, PEG8000 and PEG3000.Every kind of PEG includes 0.5%, 1%, 2% and 4% three to four kind of concentration in albumen synthetic system.NC indicate be Protein synthesis in vitro albumen synthetic system of the negative control without DNA profiling, activity are 44RLU.

Figure 10 is that there are three types of source, direct energy source such as ATP, GTP, high energy for required energy in biological respinse Phosphoric acid key compound such as phosphocreatine, phosphoenolpyruvate etc., the present invention in bioenergy system (BES-Biologic Energy System) it also can be used as the renewable sources of energy, energy is constantly provided, biological respinse is carried out.

Specific embodiment

The present inventor after extensive and in-depth study, for the first time it was unexpectedly observed that by certain content (a) cell extraction Object；(b) polyethylene glycol；C) carbohydrate, the carbohydrate are selected from the group: glucose, starch, glycogen, sucrose, maltose, cyclodextrin or A combination thereof；(d) the bioenergy regenerating system that phosphate cpd combination is constituted, the energy that can be applied to external biological synthesis supply It answers, and is applied in cell free in vitro biological respinse system, saved the cost of external biological synthesis, improve external cell-free The ability of biosynthesis, compared with phosphocreatine/creatine phosphokinase energy regeneration system, bioenergy of the invention raw body again The external albumen combined coefficient of system can be improved 2-5 times, and RLU value reaches as high as 70,000,000.Also, biological energy source of the invention Source regenerating system also has many advantages, such as that disturbed condition is few, is easy to high pass measurement and big data analysis.On this basis, of the invention People completes the present invention.

BES system prepares (bioenergy regenerating system)

As used herein, BES system refers to bioenergy regenerating system of the invention, reacts comprising cell extract and BES System two parts.

Cell extract preparation:

It is inoculated into the 2L conical flask containing 400mL YPD culture medium by the inoculum concentration of 0.1-1%, and is placed in shaking table Middle culture, condition of culture: temperature is 30 DEG C, revolving speed 200rpm.In the middle and later periods (OD600=3.0- of yeast growth logarithmic phase 6.9), terminate culture, obtain cell culture fluid.Cultured cell culture is placed in mixture of ice and water and is pre-chilled, the time is 10-30min is centrifuged in refrigerated centrifuge, and centrifugal condition: 3,000 × g, 10min, 4 DEG C obtain cell.With pre-cooling Cell is resuspended in Washing buffer, and Washing buffer dosage is 50-100ml/L culture solution.The weight that will be obtained Suspension is centrifuged in refrigerated centrifuge, centrifugal condition: 3000g, 10min, 4 DEG C obtain cell.Washing buffer group Become: the 4- hydroxyethyl piperazineethanesulfonic acid potassium that 20-30mM pH is 7.4,100-150mM potassium acetate, 1-4mM magnesium acetate；It will be thin Born of the same parents directly carry out subsequent operation or carry out quick-frozen rear -80 DEG C of preservations using liquid nitrogen.It is crushed using liquid nitrogen homogenizer: even Appropriate liquid nitrogen is added in slurry device, adds the yeast cells of the yeast cells that centrifugation obtains or -80 DEG C of preservations, revolving speed: 45, 000rpm is crushed 3-10min；The low-temperature powder being crushed is dispensed into 50mL centrifuge tube, weigh and be stored in -80 DEG C to With obtained yeast cells is crushed powder and is cooled to 4 DEG C at room temperature, what every gram of clasmatosis powder was pre-chilled with 4 DEG C of 0.2-1mL Lysis Lysis buffer is dissolved, and yeast cells crude extract is obtained.Lysis buffer is 7.4 by 10-40mM pH 4- hydroxyethyl piperazineethanesulfonic acid potassium, 50-150mM potassium acetate, 1-4mM magnesium acetate, 2-7mM dithiothreitol (DTT), 0.5-2mM benzene first Base sulfuryl fluoride composition.The yeast cells crude extract of harvest is subjected to centrifugation 1-2 times, centrifugal force is the 12000-30000g time to be 30min, temperature are 4 DEG C；After centrifugation, taking supernatant liquid body is yeast cell extract.

The preparation of BES reaction system:

BES reaction system: 4- hydroxyethyl piperazineethanesulfonic acid of the final concentration of 22mM pH for 7-8,30-150mM potassium acetate, 1.0-5.0mM magnesium acetate, 1.5-4mM ribonucleoside triphosphote mixture (adenosine triphyosphate, guanopterin nucleoside triphosphate, born of the same parents Pyrimidine nucleoside triphosphoric acid and uridine diphosphate guanosine triphosphate), ispol (glycine, alanine, the figured silk fabrics of 0.08-0.24mM Propylhomoserin, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, day Winter amide, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine), 25mM potassium phosphate, The above-mentioned cell extract of 1.7mM dithiothreitol (DTT), 40mM glucose and 50% volume.

Biological respinse

Biological respinse, that is, biochemical reaction just refers to the chemical reaction carried out in vivo, these reactions are all by enzyme Catalysis, enzyme and reactant are dissolved in the water of interior environment, can just react, water provides carrier and medium for substance in vivo.20 In the last decades in century, biochemistry has been achieved for very big achievement in terms of explaining life process, now almost Biochemical research is engaged in botany, medicine, the relevant field of the life sciences such as science of heredity.In organism or cell institute into Capable biochemical reaction can obtain auto-control by positive-negative feedback in the complicated network system.Intracellular institute into Capable biochemical reaction requires the catalysis of enzyme.The high catalytic efficiency of enzyme, reaction condition is mild, has directionality, the bottom of to Object has high specificity.

Cell extract

In a preferred embodiment, the cell origin of the cell extract one or more types selected from the group below Cell: prokaryotic cell and eukaryocyte.

In a preferred embodiment, the cell origin of the cell extract one or more types selected from the group below Cell: Escherichia coli, bacterium, mammalian cell (such as HF9, Hela, CHO, HEK293), plant cell, yeast cells, insect Cell, or combinations thereof.

In a preferred embodiment, the yeast cells is selected from the group: saccharomyces cerevisiae, pichia yeast, kluyveromyces, Or combinations thereof；Preferably, the yeast cells includes: kluyveromyces, it is more preferably Kluyveromyces lactis.

In the present invention, the cell extract includes yeast cell extract.

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

In a preferred embodiment, in the albumen synthetic system, (such as yeast cells extracts the cell extract Object) content (wt%) be 10%-95%, preferably, 20%-80%, more preferably, 40%- 60%, with albumen conjunction The total weight of architectonical.

Vitro expression systems

Yeast (yeast) has both the advantage for cultivating simple efficient protein matter folding and posttranslational modification.It wherein makes wine ferment Female (Saccharomyces cerevisiae) and pichia yeast (Pichia pastoris) be express complicated eukaryotic protein with The model organism of memebrane protein, yeast also can be used as the raw material for preparing external translating system.

Kluyveromyces (Kluyveromyces) are a kind of ascospore yeast, kluyveromyces marxianus therein (Kluyveromyces marxianus) and Kluyveromyces lactis (Kluyveromyces lactis) are industrially to make extensively Yeast.Compared with other yeast, Kluyveromyces lactis is had many advantages, such as superpower secretion capacity, preferably big Scale fermentation characteristic, the rank of food safety and there is the ability modified after protein translation simultaneously etc..

In the present invention, yeast vitro expression systems are not particularly limited, and a kind of preferred yeast vitro expression systems are Kluyveromyces expression system (more preferably, Kluyveromyces lactis expression system).

External cell-free albumen synthetic system

In a preferred embodiment, external cell-free albumen synthetic system of the invention includes the outer albumen of yeast Synthetic system.

Yeast (yeast) has both the advantage for cultivating simple efficient protein matter folding and posttranslational modification.It wherein makes wine ferment Female (Saccharomyces cerevisiae) and pichia yeast (Pichia pastoris) be express complicated eukaryotic protein with The model organism of memebrane protein, yeast also can be used as the raw material for preparing external translating system.

Kluyveromyces (Kluyveromyces) are a kind of ascospore yeast, kluyveromyces marxianus therein (Kluyveromyces marxianus) and Kluyveromyces lactis (Kluyveromyces lactis) are industrially to make extensively Yeast.Compared with other yeast, Kluyveromyces lactis is had many advantages, such as superpower secretion capacity, preferably big Scale fermentation characteristic, the rank of food safety and there is the ability modified after protein translation simultaneously etc..

In the present invention, the outer protein synthesis system of yeast is not particularly limited, a kind of outer albumen of preferred yeast Matter synthetic system is kluyveromyces expression system (more preferably, Kluyveromyces lactis expression system).

In the present invention, kluyveromyces (such as Kluyveromyces lactis) are not particularly limited, including any one can The Crewe for improving synthetic proteins efficiency ties up (such as Kluyveromyces lactis) bacterial strain.

In the present invention, the protein synthesis in vitro system includes:

(i) bioenergy regenerating system, the bioenergy regeneration architectonical include:

(a) cell extract；

(b) polyethylene glycol；

(c) carbohydrate, the carbohydrate are selected from the group: glucose, starch, glycogen, sucrose, maltose, cyclodextrin or its group It closes；

(d) phosphate cpd.

In another preferred example, in the bioenergy regenerating system, the concentration (v/v) of component (a) is 20%-70%, Preferably, 30-60%, more preferably, 40%-50%, with the total volume meter of the albumen synthetic system.

In another preferred example, the concentration (w/v, such as g/ml) of the component (b) is 0.1-10%, preferably, 0.5- 8%, more preferably, 0.8-5%, more preferably, 1-2%, with the total volume meter of bioenergy regenerating system.

In another preferred example, in the albumen synthetic system, the content (wt%) of component (b) is 10%-95%, compared with Goodly, 20%-80%, more preferably, 40%-60%, with the total weight of the albumen synthetic system.

In another preferred example, in the bioenergy regenerating system, the concentration (mmol/L) of the component (c) is 10-100mM, preferably, 30-80mM, more preferably, 40-60mM.

In another preferred example, the content (V/V) of the component (c) is 1-10%, preferably, 3-8%, more preferably, 4-6%, with the total volume meter of bioenergy regenerating system.

In another preferred example, the concentration (v/v) of the component (d) is 1-6%, preferably, 2-5%, more preferably, 2-3%, with the total volume meter of bioenergy regenerating system.

In another preferred example, in the bioenergy regenerating system, the concentration (mmol/L) of the component (d) is 10-60mM, preferably, 20-50mM, more preferably, 20-30mM.

In another preferred example, in the carbohydrate, the concentration (v/v) of the glucose is 1-10%, preferably, 3- 8%, more preferably, 4-6%, with the total volume meter of the carbohydrate.

In another preferred example, in the carbohydrate, the concentration (mmol/L) of the glucose is 10-100mM, preferably, 10-60mM, preferably, 20-50mM, more preferably, 20-30mM.

In a particularly preferred embodiment, external albumen synthetic system provided by the invention includes selected from the group below one Kind or a variety of or whole components: yeast cell extract, polyethylene glycol, glucose, potassium phosphate, sucrose, 4- hydroxyethyl piperazine second Sulfonic acid, potassium acetate, magnesium acetate, adenosine triphyosphate (ATP), guanopterin nucleoside triphosphate (GTP), three phosphorus of cytidine Sour (CTP), thymidine triphosphate (TTP), ispol, phosphocreatine, dithiothreitol (DTT) (DTT), phosphoric acid flesh Acid kinase, RNase inhibitor, fluorescein, luciferin enzyme dna, RNA polymerase, spermidine, ferroheme.

In the present invention, RNA polymerase is not particularly limited, and can be selected from one or more RNA polymerases, typically RNA polymerase is t7 rna polymerase.

In the present invention, ratio of the yeast cell extract in vitro in albumen synthetic system is not particularly limited, The content (wt%) of the usual yeast cell extract is 10%-95%, preferably, 20%-80%, more preferably, 40%-60%, with the total weight of the albumen synthetic system.

In the present invention, the yeast cell extract is free of complete cell, typical yeast cell extract packet Include the initiation factor and extension of the ribosomes for protein translation, transfer RNA, aminoacyl tRNA synthetase, protein synthesis needs The factor and termination releasing factor.In addition, also containing other in some cytoplasm from yeast cells in yeast extract Albumen, especially soluble protein.

In the present invention, protein content contained by the yeast cell extract is 20-100mg/mL, preferably 50- 100mg/mL.The measurement protein content method is Coomassie brilliant blue measuring method.

In the present invention, the preparation method of the yeast cell extract is unrestricted, a kind of preferred preparation method The following steps are included:

(i) yeast cells is provided；

(ii) carrying out washing treatment is carried out to yeast cells, obtains washed yeast cells；

(iii) broken cell processing is carried out to washed yeast cells, to obtain yeast crude extract；

(iv) the yeast crude extract is separated by solid-liquid separation, obtains liquid portion, as yeast cell extract.

In the present invention, the solid-liquid separation method is not particularly limited, and a kind of preferred mode is centrifugation.

In a preferred embodiment, the centrifugation carries out in the liquid state.

In the present invention, the centrifugal condition is not particularly limited, and a kind of preferred centrifugal condition is 5000-100000g, Preferably, 8000-30000g.

In the present invention, the centrifugation time is not particularly limited, and a kind of preferred centrifugation time is 0.5min -2h, Preferably, 20min-50min.

In the present invention, the temperature of the centrifugation is not particularly limited, it is preferred that and the centrifugation carries out at 1-10 DEG C, Preferably, being carried out at 2-6 DEG C.

In the present invention, the carrying out washing treatment mode is not particularly limited, and a kind of preferred carrying out washing treatment mode is to adopt It is handled with cleaning solution in the case where pH is 7-8 (preferably, 7.4), the cleaning solution is not particularly limited, the typical washing Liquid is selected from the group: 4- hydroxyethyl piperazineethanesulfonic acid potassium, potassium acetate, magnesium acetate, or combinations thereof.

In the present invention, the mode of broken cell processing is not particularly limited, at a kind of preferred broken cell Reason includes that high pressure is broken, freeze thawing (such as liquid nitrogen cryogenics) is broken.

Ribonucleoside triphosphote mixture in the protein synthesis in vitro system is adenosine triphyosphate, guanosint Guanosine triphosphate, cytidine triphosphate and uridine diphosphate guanosine triphosphate.In the present invention, the concentration of various mononucleotides does not have Especially limitation, the concentration of usual every kind of mononucleotide are 0.5-5mM, preferably 1.0-2.0 mM.

Ispol in the protein synthesis in vitro system may include natural or non-natural amino acids, it may include D type or L-type amino acid.Representative amino acid includes (but being not limited to) 20 kinds of natural amino acids: glycine, alanine, figured silk fabrics Propylhomoserin, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, day Winter amide, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.The concentration of every kind of amino acid Usually 0.01-0.5mM, preferably 0.02-0.2mM, such as 0.05,0.06,0.07,0.08mM.

In a preferred embodiment, the protein synthesis in vitro system also contains polyethylene glycol similar to object.

In the present invention, representative PEG example include (but being not limited to): PEG3000, PEG8000, PEG6000 and PEG3350.It should be understood that system of the invention may also include other various molecular weight polyethylene glycol (such as PEG200,400, 1500,2000,4000,6000,8000,10000 etc.).

In preference, the protein synthesis in vitro system also contains sucrose.The concentration of sucrose is not particularly limited, and leads to Often, the concentration (w/v) of sucrose is 0.2-4%, preferably, 0.5-4%, more preferably, 0.5-1%, with the albumen synthetic system Total volume meter.

In preference, the protein synthesis in vitro system also contains ferroheme.The concentration of ferroheme does not limit especially System, in general, the concentration of ferroheme is 0.01-0.1mM, preferably, 0.02-0.08mM, more preferably, 0.03-0.05mM, most preferably Ground, 0.04mM.

In preference, the protein synthesis in vitro system also contains spermidine.The concentration of spermidine does not limit especially System, in general, the concentration of spermidine is 0.05-1mM, preferably, 0.1-0.8mM, more preferably, more preferably, 0.2-0.5mM, more preferably Ground, 0.3-0.4mM, most preferably, 0.4mM.

In preference, the protein synthesis in vitro system also contains buffer, and the ingredient of the buffer is not by spy It does not limit, a kind of preferred buffer contains 4- hydroxyethyl piperazineethanesulfonic acid, and/or Tris buffer.In the present invention, described Buffer can also contain other buffer compositions, such as potassium acetate, magnesium acetate, so that forming pH is 6.5-8.5 (preferably 7.0-8.0) Reaction solution or reaction buffer.In the present invention, the type and content of buffer are not particularly limited.In general, buffer is dense Degree is 1-200mM or 1-100 mM, preferably, 5-50mM.

A kind of particularly preferred protein synthesis in vitro system also contains one kind selected from the group below in addition to yeast extract Or a variety of or whole components: the 4- hydroxyethyl piperazineethanesulfonic acid that 22mM, pH are 7.4,30-150mM potassium acetate, 1.0-5.0mM vinegar Sour magnesium, 1.5-4mM ribonucleoside triphosphote mixture, the ispol of 0.08-0.24mM, 25mM phosphocreatine, bis- sulphur of 1.7mM Threitol, 0.27mg/mL creatine phosphokinase, 0.5%-2% sucrose, the DNA of 8-20ng/ μ l firefly luciferase, 0.027-0.054mg/mL T7 RNA polymerase, the ferroheme of 0.03-0.04mM, the spermidine of 0.3-0.4mM, 1%-10% Polyethylene glycol, 10-100mM glucose, 10-60mM potassium phosphate.

The coded sequence (exogenous DNA) of foreign protein

As used herein, term " coded sequence of foreign protein " is used interchangeably with " exogenous DNA ", refers both to the use of external source In the DNA molecular for instructing protein to synthesize.In general, the DNA molecular is linear or cricoid.The DNA molecular contains There is the sequence of encoding foreign proteins.In the present invention, the example of the sequence of the encoding foreign proteins includes (but and unlimited In): genome sequence, cDNA sequence.The sequence of the encoding foreign proteins also contains promoter sequence, 5 ' untranslated sequences Column, 3 ' non-translated sequences.

In the present invention, the selection of the exogenous DNA is not particularly limited, in general, exogenous DNA is selected from the group: coding is glimmering Light fibroin or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescence protein, aminoacyl tRNA synthesis Enzyme, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, the exogenous DNA of the Variable Area of antibody, luciferase are prominent The DNA of variant, or combinations thereof.

Exogenous DNA is also selected from the following group: coding alpha-amylase, enterocin A, hepatitis C virus E 2 glycoprotein, pancreas Island element precursor, Interferon α A, interleukin-1 ' beta ', lysozyme element, seralbumin, single-chain antibody section (scFV), thyroxine Transporter, tyrosinase, zytase exogenous DNA, or combinations thereof.

In a preferred embodiment, the exogenous DNA encodes albumen selected from the group below: green fluorescent protein (enhanced GFP, eGFP), yellow fluorescence protein (YFP), Escherichia coli beta galactosidase (β-galactosidase, LacZ), people's lysine-tRNA synzyme (Lysine-tRNA synthetase), human leucine-tRNA synzyme (Leucine-tRNA synthetase), arabidopsis glyceraldehyde 3 phosphate dehydrogenase (Glyceraldehyde-3- Phosphate dehydrogenase), mouse catalase (Catalase), or combinations thereof.

Kit

The present invention provides a kind of kits for external cell-free synthetic proteins, comprising:

(k1) the first container, and the raw body again of bioenergy described in first aspect present invention in the first container System；With

(kt) label or specification.

A kind of kit of particularly preferred protein synthesis in vitro includes an external protein synthetic proteins compound body System, the albumen synthetic system include selected from the group below one or more or whole components: yeast cell extract, polyethylene glycol, Glucose, potassium phosphate, 4- hydroxyethyl piperazineethanesulfonic acid, potassium acetate, magnesium acetate, adenosine triphyosphate (ATP), guanosint Guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymidine triphosphate (TTP), ispol, phosphoric acid Creatine, dithiothreitol (DTT) (DTT), creatine phosphokinase, RNase inhibitor, fluorescein, luciferin enzyme dna, t7 rna polymerase, Spermidine, ferroheme.

Main advantages of the present invention include:

(1) present invention establishes the external nothing using glucose, polyethylene glycol and potassium phosphate for energy regeneration system for the first time Cell synthetic system；

(2) present invention saves the cost of external cell-free synthetic system, it is made to can be applied to industrialized production；

(3) energy regenerating system of glucose of the invention, polyethylene glycol and phosphate cpd significantly improves external nothing The albumen synthesis capability of cell, with compared with phosphocreatine/creatine phosphokinase energy regeneration system, bioenergy of the invention The external albumen combined coefficient of regenerating system can be improved 2-5 times, and RLU value reaches as high as 70,000,000.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no Then percentage and number are weight percent and parts by weight.

Unless otherwise instructed, then material used in the embodiment of the present invention and reagent are commercial product.

Embodiment 1: cell free in vitro protein Fluc synthetic system

The storing liquid of 1.1 protein synthesis in vitro systems is prepared: the 4- hydroxyethyl piperazineethanesulfonic acid that 1M pH is 7.4,5M vinegar Sour potassium, 250mM magnesium acetate, the mixture of tetra- kinds of ribonucleoside triphosphotes of 25mM, including adenosine triphyosphate, guanosine three Phosphoric acid, cytidine triphosphate and uridine diphosphate guanosine triphosphate, the mixture of 20 kinds of amino acid of 1mM: glycine, the third ammonia Acid, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, egg ammonia Acid, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine, 20 kinds of amino The concentration of acid is 1.0mM, 1M potassium phosphate, 1M dithiothreitol (DTT), 1M glucose, 1.7mg/mL T7 RNA polymerase 20%- 50% polyethylene glycol (polyethylene glycol, PEG) 3350 or (polyethylene glycol, PEG) 8000, 20%-40% sucrose, 1-4mM spermidine, 0.1-0.4 mM ferroheme；

1.2 protein synthesis in vitro reaction systems: final concentration of 22mM pH is the 4- hydroxyethyl piperazineethanesulfonic acid of 7-8, 30-150mM potassium acetate, 1.0-5.0mM magnesium acetate, (adenosine triphyosphate, bird are fast for 1.5-4mM ribonucleoside triphosphote mixture Purine ribonucleoside triphosphote, cytidine triphosphate and uridine diphosphate guanosine triphosphate), the ispol of 0.08-0.24mM is (sweet Propylhomoserin, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, half Guang Propylhomoserin, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine), 25mM potassium phosphate, 1.7mM dithiothreitol (DTT), 40mM glucose, 8-20ng/ μ L firefly luciferase DNA, 0.027- 0.054mg/mL t7 rna polymerase, the polyethylene glycol of 1%-4%, the sucrose of 0.5%-2%, 0.03-0.04mM ferroheme, 0.3-0.4mM spermidine is eventually adding the yeast cell extract of 50% volume；

The reaction of 1.3 protein synthesis in vitro: above-mentioned reaction system is placed in 20-30 DEG C of environment, stands reaction 2-6h；

The measurement of 1.4 luciferase activities: after reaction, isometric bottom is added in 96 hole blanks or 384 hole blanks Object fluorescein (luciferine) is placed in 2120 multi-function microplate reader of Envision (Perkin Elmer) immediately, reading, Firefly luciferase activity is detected, relative light unit value (RLU) is used as active unit, as shown in Fig. 1-Fig. 7.

Experimental result

1. Fig. 1 is the glycolysis and krebs cycle pathway's schematic diagram that ATP is synthesized in eukaryocyte.Glycolysis metabolism approach Adjusting mainly pass through various allosteric agents to three key enzymes: hexokinase (glucokinase), phosphofructokinase, acetone Acid kinase carries out allosteric adjusting.The speed of entire approach is controlled by adjusting the activity of several enzymes in reaction path, is conditioned Enzyme majority be the enzyme for being catalyzed irreversible reaction in the reaction mechanism mechanism of reaction, every completion one cycle in glycolysis, oxygenolysis falls 3 molecules Acetyl group, by 2 times, dehydrogenation reaction produces 2- molecule ATP, 2 molecule NADH, the pyruvic acid of 2 molecular waters and 2 molecules, simultaneously 1 molecule pyruvic acid generates 1 molecule CO2 and 1 molecules of ethanol using 1 decarboxylic reaction.The key enzyme of tricarboxylic acid cycle is lemon Acid synthase, isocitric dehydrogenase and ketoglurate dehydrogenase system are irreversible reaction.Every completion one cycle, oxidation point A molecule acetyl group is taken off, producible 10 molecule ATP, and meanwhile decarboxylic reaction twice, two molecule CO2 of generation, four dehydrogenation reactions, Generate three molecule NADH and a molecule FADH2.

2. Fig. 2 be external biological synthesis using glucose (BES), phosphate pathway to the advantage figure of phosphocreatine approach.Benefit Bio-energy is greatly repeated with BES and manufactures approach, effectively generates ATP functional approach, has not only saved cost, while real Show slow release ATP and participates in biosynthesis.

3. the influence of different phosphate concns and the combination of 40mM concentration of glucose to external cell-free synthetic system

From figure 3, it can be seen that the potassium phosphate of 20-30mM is anti-to protein synthesis in vitro when concentration of glucose is 40mM System is answered to have apparent effect, compared with phosphocreatine/creatine phosphokinase energy regeneration system, glucose phosphate system is larger Ground improves external albumen synthesis capability to 2-3 times.

4. the influence of different phosphate concn and 40mM concentration of glucose to external cell-free synthetic system

From fig. 4, it can be seen that the potassium phosphate of 20-30mM is anti-to protein synthesis in vitro when concentration of glucose is 30mM System is answered to have apparent effect, compared with phosphocreatine/creatine phosphokinase energy regeneration system, glucose phosphate system is larger Ground improves external albumen synthesis capability to 1-2 times.

5. the influence of different concentration of glucose and 30mM potassium phosphate concentration to external cell-free synthetic system

From fig. 5, it can be seen that the glucose of 30-60mM is anti-to protein synthesis in vitro when potassium phosphate concentration is 30mM System is answered to have apparent effect, compared with phosphocreatine/creatine phosphokinase energy regeneration system, glucose phosphate system is larger Ground improves external albumen synthesis capability to 1-3 times.

6. the influence of different concentration of glucose and 25mM potassium phosphate concentration to external cell-free synthetic system

From fig. 6, it can be seen that the glucose of 20-50mM is anti-to protein synthesis in vitro when potassium phosphate concentration is 25mM System is answered to have apparent effect, compared with phosphocreatine/creatine phosphokinase energy regeneration system, glucose phosphate system is larger Ground improves external albumen synthesis capability to 1-2 times.

7. the influence of different concentration of glucose and 20mM potassium phosphate concentration to external cell-free synthetic system

From figure 7 it can be seen that the glucose of 20-40mM is anti-to protein synthesis in vitro when potassium phosphate concentration is 20mM System is answered to have apparent effect, compared with phosphocreatine/creatine phosphokinase energy regeneration system, glucose phosphate system is larger Ground improves external albumen synthesis capability to 1-2 times.

8. in the different reaction time, influence of the glucose phosphate system to external cell-free synthetic system.

From figure 8, it is seen that when potassium phosphate concentration is 25mM, when glucose is 40mM, at just incipient 30 minutes, There is no synthetic proteins, with extending to 1 hour for time, glucose is hydrolyzed, the ATP of generation, the outer synthetic system of donor Carry out albumen synthesis.It is 1-4 hours between when reacted, the ability of external synthetic proteins is not significantly improved.

9. the influence schematic diagram of difference PEG and different concentration to protein synthesis in vitro system.

From fig. 9, it can be seen that wherein PEG includes PEG3350, PEG8000 and PEG3000.Every kind of PEG is synthesized in albumen It include 0.5%, 1%, 2% and 4% three to four kind of concentration in system.What NC was indicated is external egg of the negative control without DNA profiling White matter synthetic proteins synthetic system.The experimental results showed that external albumen synthesis capability is significantly improved 1.5- by 2%-4% PEG 2 times.

10. Figure 10 show in biological respinse required energy there are three types of source, direct energy source such as ATP, GTP, energy-rich phosphate key compound such as phosphocreatine, phosphoenolpyruvate etc., the present invention in bioenergy system (BES- Biologic Energy System) it also can be used as the renewable sources of energy, energy is constantly provided, biological respinse is carried out.

Embodiment 2: cell free in vitro protein eGFP synthetic system

Cell free in vitro protein eGFP synthetic system: final concentration of 22mM pH is the 4- hydroxyethyl piperazine second sulphur of 7-8 Acid, 30-150mM potassium acetate, 1.0-5.0mM magnesium acetate, 1.5-4mM ribonucleoside triphosphote mixture (adenosine triphyosphate, bird Purine nucleosides triphosphoric acid, cytidine triphosphate and uridine diphosphate guanosine triphosphate), the ispol of 0.08-0.24mM (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, half Cystine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and group ammonia Acid), 25mM potassium phosphate, 1.7mM dithiothreitol (DTT), 40mM glucose, 8-20ng/ μ LeGFP DNA, 0.027-0.054mg/mL T7 rna polymerase, the polyethylene glycol of 1%-4% are eventually adding the yeast cell extract of 50% volume；

Embodiment 3: external RNA synthetic system

External RNA synthetic system: final concentration of 40mM Tris-HCl, pH 8.0,25mM NaCl, 8mM MgCl2,2mM Spermidine, 2.5mM ribonucleoside triphosphote mixture (adenosine triphyosphate, guanopterin nucleoside triphosphate, three phosphorus of cytidine Acid and uridine diphosphate guanosine triphosphate), t7 rna polymerase, 100mM DTT, the DNA enzymatic of no RNA enzyme, the water of no RNA enzyme, RNA enzyme Inhibitor；

Embodiment 4: the synthetic system that polyethylene glycol participates in

Protein synthesis system: final concentration of 22mM pH is the 4- hydroxyethyl piperazineethanesulfonic acid of 7-8,30-150mM acetic acid Potassium, 1.0-5.0mM magnesium acetate, 1.5-4mM ribonucleoside triphosphote mixture (adenosine triphyosphate, guanopterin nucleoside triphosphate, Cytidine triphosphate and uridine diphosphate guanosine triphosphate), the ispol of 0.08-0.24mM (glycine, alanine, Valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, Asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine), 25 mM potassium phosphates, 1.7mM dithiothreitol (DTT), 40mM glucose, 8-20ng/ μ L eGFP DNA, 0.027-0.054mg/mL t7 rna polymerase, It is eventually adding the yeast cell extract of 50% volume；The polyethylene glycol of 1%-4% is wherein added, improves protein significantly Synthesis rate.

Embodiment 5: external uciferase activity measurement

Luciferase (Luciferase) is the general designation that the enzyme of bioluminescence can be generated in nature, wherein most there is representative Property be a kind of intracorporal luciferase of firefly that scientific name is Photinus pyralis.In corresponding chemical reaction, fluorescence Generation come from the oxidation of luciferin, also include in reaction system in some cases atriphos (ATP).

Fluorescent generates reaction and is generally divided into following two step:

Luciferin+ATP → luciferin adenylate (luciferyl adenylate)+PPi

Luciferin adenylate+O2 → oxygen luciferin+AMP+ light

Energy is saved in this reaction very much, and the energy of nearly all input reaction is all converted into light.

Embodiment 6: external DNA fragmentation linked system

The Ligation in vitro of DNA molecular is exactly under certain condition, it is adjacent to be catalyzed two double-stranded DNA segment groups by DNA ligase 5 ' end phosphoric acid and 3 ' terminal hydroxy groups between formation phosphoric acid rouge key Biochemical processes, the connection of DNA molecule is anti-in digestion It should obtain and carry out on the basis of enzyme complementary series of the same race, mainly include symmetrical cohesive end；Symmetry cohesive end and flush end connect It connects.

External DNA linked system: final concentration of 20mM Tris-HCl, pH 7.6,5mM MgCl2,5mM DTT, T4 DNA ligase, 5mM ATP, connection reaction overnight at 12 DEG C.

Embodiment 7: external aminoacyl-tRNA synthetase system

Amino acid is integrated on its corresponding tRNA by aminoacyl-tRNA synthetase participation, this process needs ATP It participates in.

The synthesis that aminoacyl-tRNA synthetase participates in is carried out in two steps.

The first step is that aminoacyl-tRNA synthetase identifies the amino acid that it is catalyzed and another substrate A TP, in aminoacyl- Under the catalysis of tRNA synzyme, an ester bond is formed between the phosphoric acid on the carboxyl and AMP of amino acid, while releasing a molecule PPi:

Amino acid+ATP --- aminoacyl-AMP+PPi

At this moment aminoacyl-AMP is still closely in conjunction with enzyme molecule.

The reaction of second aminoacyl-tRNA synthetase catalysis is that amino acid is connected to tRNA 3' by forming ester bond On the ribose at end:

Aminoacyl-AMP+tRNA --- aminoacyl-tRNA+AMP

Conclusion

Present invention firstly provides one kind directly to prepare direct bio-energy (such as ATP) by rudimentary energy molecule Novel external biological energy system (BES, Biological Energy System), technical principle, preparation method, Yi Jiying Use scene.Using energy metabolic activity in biological cell, by cell pyrolysis liquid working process, the BES system prepared has Active complete energetic supersession pathway molecule is conceptual leap and revolutionary innovation in underlying biological theory.

Using BES energy system, the cost of energy of external biological reaction has not only been saved, push large-scale production and has been answered With, compared with traditional phosphocreatine/creatine phosphokinase Energy Resources Unit regenerating system, external cell-free biosynthesis of the invention Ability can be improved 2-5 times, embody great advantage and the wide application prospect in various biological respinses of the invention.

Bibliography

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All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Claims (11)

1. a kind of bioenergy regenerating system, which is characterized in that the bioenergy regenerates architectonical and includes:

(a) cell extract；

(b) polyethylene glycol；

(c) carbohydrate, the carbohydrate are selected from the group: glucose, starch, glycogen, sucrose, maltose, cyclodextrin, or combinations thereof；With

(d) phosphate cpd.

2. bioenergy regenerating system as described in claim 1, which is characterized in that the cell origin of the cell extract is selected From the cell of one or more types of the following group: Escherichia coli, bacterium, mammalian cell (such as HF9, Hela, CHO, HEK293), plant cell, yeast cells, or combinations thereof.

3. bioenergy regenerating system as described in claim 1, which is characterized in that in the bioenergy regenerating system, group The concentration (v/v) for dividing (a) is 20%-70%, preferably, 30-60%, more preferably, 40%-50%, with the total of the albumen synthetic system Stereometer.

4. bioenergy regenerating system as described in claim 1, which is characterized in that the concentration of the component (b) (w/v, such as It g/mL) is 0.1-10%, preferably, 0.5-8%, more preferably, 0.8-5%, more preferably, 1-2%, with bioenergy regenerating system Total volume meter.

5. bioenergy regenerating system as described in claim 1, which is characterized in that in the bioenergy regenerating system, institute The concentration (mmol/L) for stating component (c) is 10-100 mM, preferably, 30-80 mM, more preferably, 40-60 mM.

6. bioenergy regenerating system as described in claim 1, which is characterized in that the concentration (mmol/L) of the component (d) For 10-60 mM, preferably, 20-50 mM, more preferably, 20-30 mM.

7. bioenergy regenerating system as described in claim 1, which is characterized in that in the carbohydrate, the glucose it is dense Spending (mmol/L) is 10-100 mM, preferably, 10-60 mM, preferably, 20-50 mM, more preferably, 20-30 mM.

8. a kind of purposes of bioenergy regenerating system described in claim 1, which is characterized in that be used to prepare and closed for albumen At cell-free external albumen synthetic system.

9. a kind of synthetic method of external foreign protein characterized by comprising

(i) in vitro in the presence of albumen synthetic system, bioenergy regenerating system described in claim 1 is provided；

(ii) under the suitable conditions, the external albumen synthetic system of incubation step (i) T1 for a period of time, thus described in synthesis Foreign protein.

10. method as claimed in claim 9, which is characterized in that the method further include: (iii) is optionally from the body In outer albumen synthetic system, the foreign protein is separated or detected.

11. a kind of kit characterized by comprising

(k1) the first container, and the cell extract in the first container；

(k2) second container, and the polyethylene glycol in second container；

(k3) third container, and the carbohydrate in third container, the carbohydrate are selected from the group: glucose, starch, glycogen, Sucrose, maltose, cyclodextrin, or combinations thereof；

(k4) the 4th container, and the phosphate cpd in the 4th container；With

(kt) label or specification.