CN114317575A

CN114317575A - Method for improving in vitro synthesis efficiency of protein

Info

Publication number: CN114317575A
Application number: CN202111668229.9A
Authority: CN
Inventors: 郭敏; 李克绪; 徐丽琼; 于雪
Original assignee: Kangma Healthcode Shanghai Biotech Co Ltd
Current assignee: Kangma Healthcode Shanghai Biotech Co Ltd
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2022-04-12

Abstract

In the production setting of protein in vitro synthesis, the invention limits the liquid level height of the reaction liquid of the in vitro cell-free protein synthesis system in the reaction vessel, and ensures that the reaction liquid can be spread in the reaction vessel as much as possible. The invention is applied to industrial production, can use a large container to carry out protein in vitro synthesis, and can process IVTT reaction liquid with larger volume at one time so as to realize obtaining protein with higher yield through single reaction, thereby greatly improving the efficiency of protein in vitro synthesis.

Description

Method for improving in vitro synthesis efficiency of protein

Technical Field

The invention belongs to the technical field of molecular biological experiments, and particularly relates to a method for improving in-vitro synthesis efficiency of protein.

Background

Proteins are important molecules in cells, and are involved in performing almost all functions of cells. The difference in the sequence and structure of proteins determines their function. Protein synthesis and production, which are currently performed by organelles, include a process of gene transcription (transcription) from a DNA template to mRNA and a process of linking genetic information (three base pairs) on mRNA into corresponding amino acids and then into proteins. These two steps can be either synthesized by intact cells that retain biological activity (in vivo synthesis of proteins) or in lysed cell lysates (lysates) (in vitro/cell-free synthesis of proteins), a step also called in vitro transcription/translation of protein-synthesized cells (IVTT).

Specifically, gene transcription refers to a process of synthesizing one mRNA using a template strand of DNA as a template and 4 NTPs (ATP, CTP, GTP, and UTP) as raw materials under the catalytic action of DNA-dependent RNA polymerase according to the base complementary pairing principle. For some RNA viruses, RNA may also direct the synthesis of mRNA. The translation process is specifically a process of assembling activated amino acids into protein polypeptide chains on ribosomes (also called nucleoproteins) under the action of related enzymes and cofactors by using mRNA as a template and tRNA as a carrier.

Wherein, the in vitro transcription/translation of the cell for protein synthesis specifically refers to that exogenous target mRNA or DNA is used as a template, and a target protein is synthesized by artificially controlling and supplementing substrates required by protein synthesis and substances such as protein factors related to transcription and translation. In vitro synthesis of proteins is a relatively rapid, time-saving, and convenient way of protein expression because plasmid construction, transformation, cell culture, cell collection, and disruption steps are not required, and in recent years, has been increasingly used in industrial protein production.

However, the current IVTT technology is basically performed on the basis of a small culture vessel, such as a microplate including a 96-well plate, a 48-well plate, a 24-well plate, and a 12-well plate, which are commonly used in laboratories, or a 6-cm petri dish and a 15-cm petri dish. The IVTT reaction is performed by using a smaller culture container, and the effect is relatively stable. For example, when used for expressing the Fluorescence value of EGFP (Enhanced Green Fluorescent Protein), it can be stabilized to 3000RFU (Relative Fluorescence Units) or more, and even when the activity of the lysate is high, the Fluorescence value of EGFP can reach 10000RFU or more.

When protein in vitro synthesis is applied to industrial mass production, IVTT reaction is required to be performed on a larger volume of reaction solution once to increase the amount of protein synthesized once. At this point, it is highly undesirable to use laboratory-scale small volume containers (microwell plates having only a few hundred microliters to a few milliliters per well). Since each time the reaction solution is poured, the addition of the reactant requires a precise control of each well, which is very time consuming. However, in the production practice, it has been found that when the volume of the reaction liquid for IVTT is large and a large-capacity reaction vessel such as a Erlenmeyer flask or a beaker has to be used to carry out in vitro protein synthesis, the protein expression level is generally lower than that of a small-capacity control group (the control group uses a microplate such as a 96-well plate or a 48-well plate as a reaction vessel).

Therefore, in the industrial application of protein in vitro synthesis, a method for improving the in vitro synthesis efficiency of protein in the industrialized large-volume IVTT reaction needs to be found.

Accordingly, the prior art is subject to further improvements and enhancements.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a method for improving the efficiency of in vitro protein synthesis, which can be applied to the in vitro synthesis of proteins with larger volume in industrial production.

The invention provides a method for improving the in vitro synthesis efficiency of protein, which comprises the following steps:

a. adding reaction liquid of an in-vitro cell-free protein synthesis system into a reaction container;

b. adding a nucleic acid template of the protein to be expressed;

c. incubating the reaction solution for a set time under suitable conditions to synthesize a protein to be expressed encoded by the nucleic acid template;

a and b are not in sequence;

in the step a, the liquid surface area of the reaction liquid in the reaction container is 100-10000 cm²The height of the liquid surface is not more than 5 cm.

Preferably, the liquid surface area of the reaction liquid in the reaction container is 300-9000 cm²The liquid level is no more than 3cm, preferably,the liquid surface area is 300-9000 cm2, the liquid surface height is not more than 2.8cm, and more preferably, the liquid surface area is 500-8000 cm²The liquid level is not more than 1.4 cm.

Preferably, the reaction solution comprises a cell lysate, a nucleic acid synthase, a protein synthase, a substrate for synthesizing RNA, a substrate for synthesizing protein, water or an aqueous solvent.

In another preferred embodiment, the cell lysate is derived from one or more types of cells selected from the group consisting of: prokaryotic cells and eukaryotic cells. Preferably, the cell lysate is derived from one or more types of cells selected from the group consisting of: escherichia coli, mammalian cells, plant cells, yeast cells, or a combination thereof; preferably, the yeast cell is selected from saccharomyces cerevisiae, pichia pastoris, kluyveromyces, or a combination thereof; more preferably, the kluyveromyces is kluyveromyces lactis.

The cell lysate comprises ribosome for protein translation, transfer RNA, aminoacyl tRNA synthetase, initiation factors and elongation factors required by protein synthesis, termination release factors and the like. In addition, it contains some other proteins, especially soluble proteins, which originate in the cytoplasm of yeast cells.

Preferably, the concentration (v/v) of the cell extract is 20% to 90%, preferably 30% to 80%, more preferably 50% to 80%, based on the total volume of the reaction solution. In the present invention, the content and purity of the cell extract are not particularly limited.

Further, the substrate for synthesizing RNA comprises: one of nucleoside monophosphate, nucleoside triphosphate or a combination thereof.

The substrate of the synthetic protein comprises: 20 natural amino acids and non-natural amino acids. The substrate for synthesizing DNA comprises: a deoxynucleoside monophosphate, a deoxynucleoside triphosphate, or a combination thereof.

Further, suitable conditions include a reaction temperature of 20 to 35 ℃, preferably 20 to 30 ℃, more preferably 25 ℃.

Preferably, the set time is in particular from 0.5 to 20h, preferably from 1 to 18h, more preferably from 2 to 15h, more preferably from 3 to 12 h; the reaction time can be determined manually according to specific conditions, and can also be 2-4h or 6 h.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 is a flow chart of a method of increasing the efficiency of in vitro protein synthesis according to the present invention;

FIG. 2 is a graph comparing the results of experiments with a reaction solution of the present invention having a height of 1.4cm and a 24-well plate;

FIG. 3 is a graph comparing the results of experiments with a 24-well plate having a height of 2.8cm for the reaction solution of the present invention;

FIG. 4 is a graph comparing the results of experiments with a reaction solution of the present invention having a height of 7.1cm and a 24-well plate;

FIG. 5 is a graph comparing the results of experiments with a 24-well plate and a reaction solution of the present invention having a height of 0.64 cm;

FIG. 6 is a graph comparing the results of experiments with a 24-well plate having a reaction solution height of 1.3cm according to the present invention;

FIG. 7 is a graph showing the comparison of the results of the experiments in which the ratio of the carrier cell lysate of the present invention is 50% and 80%.

Detailed Description

The invention provides a method for improving the in vitro synthesis efficiency of protein, and in order to make the purpose, technical scheme and effect of the invention clearer and clearer, the invention is further described in detail by referring to the attached drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Terms and noun explanations

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

In vitro cell-free protein synthesis system

In the present invention, the expressions "in vitro cell-free protein synthesis system", "in vitro protein synthesis reaction system", "cell-free protein synthesis system", and the like have the same meanings. Protein in vitro synthesis system, in vitro protein synthesis system, cell-free protein synthesis system, cell-free in vitro protein synthesis system, in vitro cell-free synthesis system, CFS system (cell-free system), CFPS system (cell-free protein synthesis system), etc. Including in vitro translation systems, in vitro transcription translation systems (IVTT systems), and the like. In the present invention, the IVTT system is preferred. We also refer to the in vitro Protein synthesis system as the "Protein Factory" (Protein Factory). In vitro protein synthesis reaction refers to a reaction for synthesizing a protein in an in vitro cell-free synthesis system, and at least comprises a translation process. Including but not limited to IVTT reactions (in vitro transcription translation reactions). In the present invention, IVTT reaction is preferred. IVTT reaction, corresponding to IVTT system, is the process of in vitro transcription and translation of DNA into Protein (Protein), therefore, we also refer to such in vitro Protein synthesis system as D2P system, D-to-P system, D _ to _ P system, DNATO-Protein system; the corresponding in vitro Protein synthesis methods are also called D2P method, D-to-P method, D _ to _ P method, DNA-to-Protein method.

Cell lysis solution

In the present invention, "cell lysate" and "cell extract" have the same meaning.

The cell lysate used in the present invention may be a commercially available product or a self-made product, and the commercially available product may be a kit of the present company or the like.

In the present invention, the method for self-preparing the cell extract or cell lysate is not limited, and a preferred preparation method comprises the following steps:

(i) providing a cell;

(ii) washing the cells to obtain washed cells;

(iii) subjecting the washed cells to cell disruption treatment, thereby obtaining a crude cell extract;

(iv) and carrying out solid-liquid separation on the cell crude extract to obtain a liquid part, namely the cell extract.

In the present invention, the solid-liquid separation method is not particularly limited, and a preferable method is centrifugation.

In the present invention, the centrifugation conditions are not particularly limited, and one preferable centrifugation condition is 5000-.

In the present invention, the centrifugation time is not particularly limited, and a preferable centrifugation time is 0.5min to 2h, preferably 20min to 50 min.

In the present invention, the temperature of the centrifugation is not particularly limited, and it is preferable that the centrifugation is performed at 1 to 10 ℃, preferably, 2 to 6 ℃.

In the present invention, the washing treatment is not particularly limited, and a preferable washing treatment is a treatment with a washing solution at a pH of 7 to 8 (preferably, 7.4), the washing solution is not particularly limited, and typically the washing solution is selected from the group consisting of: potassium 4-hydroxyethylpiperazine ethanesulfonate, potassium acetate, magnesium acetate, or a combination thereof.

In the present invention, the cell disruption treatment is not particularly limited, and a preferable cell disruption treatment includes high-pressure disruption, freeze-thawing (e.g., liquid nitrogen low-temperature disruption).

Nucleic acid synthetase

The nuclease according to the present invention is not particularly limited, and may be, for example, RNA polymerase, DNA polymerase, or the like, each independently, and may be directly added by an exogenous method, or may be provided as a reaction product or an intermediate product (e.g., an exogenous nucleic acid template encoding RNA polymerase or/and encoding DNA polymerase is added).

Sources of the RNA polymerase include, but are not limited to: a cell extract comprising an endogenously expressed RNA polymerase, an exogenous RNA polymerase, a translation product of an exogenous nucleic acid template encoding an RNA polymerase, or a combination thereof. In each of the above embodiments, it is preferable that the RNA polymerase is T7RNA polymerase. The exogenous nucleic acid template for coding RNA polymerase can be translated into RNA polymerase through the in vitro protein synthesis reaction of the system.

Sources of the DNA polymerase include, but are not limited to: a cell extract comprising an endogenously expressed DNA polymerase, an exogenous DNA polymerase, a translation product of an exogenous nucleic acid template encoding a DNA polymerase, or a combination thereof. In each of the above embodiments, the DNA polymerase is preferably phi29DNA polymerase independently. The exogenous nucleic acid template for coding the DNA polymerase can be translated into the DNA polymerase through the in vitro protein synthesis reaction of the system.

Protein synthases

The protein synthase of the present invention is not particularly limited, and may be a protein synthase that promotes any link of protein biosynthesis: activating amino acid; secondly, the transfer of the amino acid is activated; ③ condensation of activated amino acids on the ribosome. Such as aminoacyl tRNA synthetases, and the like.

Nucleic acid templates for proteins to be expressed

The "nucleic acid template for protein to be expressed" in the present invention refers to a nucleic acid template for directing protein synthesis, which may be mRNA, DNA or a combination thereof, preferably a DNA template. The plasmid may be linear or circular, and one of the preferred plasmids is a circular plasmid.

When performing an in vitro protein synthesis reaction, the promoter in the template for promoting the synthesis of the target protein may be selected from any one of the following groups: AOD1, MOX, AUG1, AOX1, GAP, FLD1, PEX8, YPT1, LAC4, PGK, ADH4, AMY1, GAM1, XYL1, XPR2, TEF, RPS7, T7, and any suitable combination thereof. One of the preferred is the T7 promoter.

The term "DNA template" is used interchangeably with "exogenous template DNA" and "template DNA" and refers to a foreign DNA molecule used to direct protein synthesis. Examples include (but are not limited to): genome sequence and cDNA sequence. The sequence for encoding the foreign protein also comprises a promoter sequence, a 5 'untranslated sequence and a 3' untranslated sequence.

In the present invention, the selection of the foreign DNA is not particularly limited, and in general, the foreign DNA is selected from the group consisting of: exogenous DNA encoding a luciferin protein, or luciferase (e.g., firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyltrna synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of an antibody, DNA of a luciferase mutant, or a combination thereof.

The foreign DNA may also be selected from the group consisting of: exogenous DNA encoding alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single-chain antibody fragments (scFV), transthyretin, tyrosinase, xylanase, or a combination thereof.

In a preferred embodiment, the exogenous D na encodes a protein selected from the group consisting of: green fluorescent protein (e GFP), Yellow Fluorescent Protein (YFP), escherichia coli β -galactosidase (lactasise, LacZ), human Lysine-tRNA synthetase (Lysine-tRNA synthetase), human Leucine-tRNA synthetase (Leucine-tRNA synthetase), arabidopsis thaliana Glyceraldehyde 3-phosphate dehydrogenase (Glyceraldehyde-3-phosphate dehydrogenase), murine Catalase (Catalase), or combinations thereof, with green fluorescent protein (e GFP) being particularly preferred.

Method for improving in vitro synthesis efficiency of protein in large-scale production

When the volume of the IVTT reaction solution is large and protein synthesis in vitro has to be carried out using a large-volume reaction vessel such as a flask or a beaker, the protein expression level is generally lower than that of a small-volume control group (the control group uses a microplate such as a 96-well plate or a 48-well plate as a reaction vessel). The present inventors have made extensive and intensive studies and, as a result of extensive screening and investigation, have unexpectedly found that the reaction activity can be improved by limiting the liquid level of the reaction solution in the reaction vessel of the in vitro cell-free protein synthesis system in the production setting for the in vitro protein synthesis, and by "spreading" the reaction solution as much as possible in the reaction vessel.

Specifically, the invention provides a method for improving the in vitro synthesis efficiency of protein, which comprises the following steps:

d. adding reaction liquid of an in-vitro cell-free protein synthesis system into a reaction container;

e. adding a nucleic acid template of the protein to be expressed;

f. incubating the reaction solution for a set time under suitable conditions to synthesize a protein to be expressed encoded by the nucleic acid template;

a and b are not in sequence;

Preferably, the liquid surface area of the reaction liquid in the reaction container is 300-9000 cm²The height of the liquid surface is not more than 3cm, preferably, the area of the liquid surface is 300-9000 cm²The height of the liquid surface is not more than 2.8cm, and the area of the liquid surface is preferably 500-8000 cm²The liquid level is not more than 1.4 cm.

Further, the reaction solution includes a cell lysate, a nucleic acid synthase, a protein synthase, a substrate for synthesizing RNA, a substrate for synthesizing protein, water, or an aqueous solvent.

In another preferred embodiment, the cell lysate is derived from one or more types of cells selected from the group consisting of: prokaryotic cells and eukaryotic cells. Preferably, the cell extract is derived from one or more types of cells selected from the group consisting of: escherichia coli, mammalian cells, plant cells, yeast cells, or a combination thereof; preferably, the yeast cell is selected from saccharomyces cerevisiae, pichia pastoris, kluyveromyces, or a combination thereof; more preferably, the kluyveromyces is kluyveromyces lactis.

Further, suitable conditions include a reaction temperature of 20 to 35 ℃, preferably 20 to 30 ℃, more preferably 25 ℃. The setting time can be comprehensively determined according to the factors such as the consumption of raw materials (such as the amount of reaction substrate, the expected protein content and the like), the reaction efficiency and the like.

Further, the set time is specifically 0.5 to 20 hours, preferably 1 to 18 hours, more preferably 2 to 15 hours, more preferably 3 to 12 hours; the reaction time can be determined manually according to specific conditions, and can also be 2-4h or 6 h.

The nucleoside triphosphate mixture in the reaction solution is adenosine triphosphate, guanosine triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate.

The amino acid mixture of the reaction solution may include natural or unnatural amino acids, and may include D-or L-form amino acids. Representative amino acids include (but are not limited to) the 20 natural amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

A particularly preferred in vitro cell-free protein synthesis system comprises, in addition to the yeast cell extract, the following components: 22mM 4-hydroxyethylpiperazine ethanesulfonic acid with pH of 7.4, 30-150mM potassium acetate, 1.0-5.0mM magnesium acetate, 1.5-4mM nucleoside triphosphate mixture, 0.08-0.24mM amino acid mixture, 25mM phosphocreatine, 1.7mM dithiothreitol, 0.27mg/mL phosphocreatine kinase, 1% -4% polyethylene glycol, 0.5% -2% sucrose, 0.027-0.054mg/mL T7RNA polymerase.

The flow chart of the method for improving the in vitro synthesis efficiency of the protein provided by the invention is shown in figure 1.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The embodiment of the invention adopts the kit (model parameters) of the company, comprising Kluyveromyces cell lysate, nucleic acid synthetase, protein synthetase, a substrate for synthesizing RNA, a substrate for synthesizing protein, water or aqueous solvent and the like.

Other materials and reagents used in examples of the present invention are commercially available products unless otherwise specified.

Example 1

Preparing a nucleic acid template: constructing a plasmid vector for expressing EGFP, carrying out in-vitro DNA amplification, and preparing the coding foreign protein DNA template of EGFP (plasmid DNA template)

Selecting enhanced green fluorescent protein (mEGFP) as a foreign protein to serve as a target expression product, wherein the amino acid sequence of the foreign protein is shown as SEQ ID No. X.

And selecting a plasmid vector. The artificial construction plasmid vector designed aiming at the kluyveromyces lactis cell extract is adopted, and the artificial construction plasmid vector contains functional elements such as a T7 promoter, a 5 'UTR and a 3' UTR. The plasmid vector can be combined with a Kluyveromyces lactis cell extract containing endogenously expressed T7RNA polymerase to construct an in vitro cell-free protein synthesis system, and various exogenous proteins can be synthesized in vitro.

Inserting a DNA fragment containing the mEGFP coding gene into a plasmid vector by adopting a PCR amplification and homologous fragment recombination method to construct a plasmid vector for expressing the mEGFP, and marking the plasmid vector as a D2P plasmid or pD 2P. The plasmid was confirmed to be correct by gene sequencing. Wherein, the gene sequence of the code mEGFP is shown as SEQ ID No. X.

DNA amplification was performed. The amplification reaction system comprises the following components in final concentration: 1. mu.M-5. mu.M random primer (primer sequence: NNNNN), 1.14 ng/. mu.L of the above plasmid template, 0.5mM-1mM dNTP, 0.1mg/mL BSA, 0.05mg/mL-0.1mg/mL phi29DNA polymerase, 1 XPhi 29 reaction buffer (composition 200mM Tris-HCl,20mM MgCl2,10mM (NH4)2SO4,10mM KCl, pH 7.5). And mixing the reaction system uniformly, and placing the mixture in an environment at 30 ℃ for reaction for 2 hours. Obtaining a plasmid DNA template (double-stranded DNA structure), measuring the nucleic acid concentration of the plasmid DNA template by an ultraviolet spectrophotometer, and refrigerating the reaction solution for later use.

Example 2

Influence of the degree of the reaction solution 'spreading' on the efficiency of in vitro protein synthesis

Each experiment includes an experimental group and a control group, and it should be noted that, because the kits of the in vitro protein expression system are not produced in the same batch and have different activities in different groups, the expression effects cannot be compared in the horizontal direction among the groups. However, each group of experiments and the control experiment of the same group adopt the kit of the in vitro synthesis system of the same batch, and the expression efficiency is the same. The present invention is therefore described and analyzed with only an intra-group comparison between the experimental and control groups of each experiment.

General experimental conditions: all experiments were carried out at reaction temperatures of 20-37 deg.C, especially at room temperature of 20-25 deg.C. In the experiment, the concentration of reaction solution (requiring standard specific kit trade name, batch) is all 80%, namely the ratio of carrier cell lysate to buffer solution is 4:1, an in vitro protein expression system of the Kluyveromyces lactis is formed, and the standing incubation time is 2-8 h. After the reaction, 10. mu.L of the reaction system solution was added to a 96-well blackboard or a 384-well blackboard, and immediately placed in a Tecan 2000 microplate reader, read, and the activity of green fluorescent protein was detected with RFU as an activity reference, with the results shown in FIGS. 2 to 6.

Experimental setup of first, control group

The control group used a conventional 24-well plate as a reaction vessel, and 300. mu.l of the reaction solution (standard kit trade name, lot) was added to each well, and the bottom area of each well of the 24-well plate was 2cm²Therefore, a reaction solution having a height of 1.5mm was added to each well, and 1/30 was added by volume to the DNA product prepared in example 1.

Second, experimental setup of experimental groups

The experimental group adopts reaction vessels with different sizes, wherein, the first experiment adopts a reaction vessel with the diameter of 30cm, and reaction liquids with different amounts are respectively added (the specific kit trade name and the batch are required to be standardized): 1l (a), 2l (b), and 5l (b); experiment two, a reaction vessel with a diameter of 100cm is adopted, and 5L of the reaction solution (a) and 10L of the reaction solution (b) are respectively added. After the reaction solution was added, the DNA product prepared in example 1 was added in an amount of 1/30 vol%, and the reaction was incubated for X hours. The dimensions of the reaction vessel and the results of the experiment are shown in table 1:

table 1: settings and results for each experimental group.

The results of each set of experiments are described below with reference to the accompanying drawings.

Experiment one, group a: the height of the reaction solution in the experimental group was 1.4 cm. As can be seen from FIG. 2, the protein expression effect of the experimental group was significantly higher than that of the control group after 4 hours of reaction, except that the initial stage was lower than that of the control group. Especially from the fourth hour of the reaction, the protein expression in the 24-well plate of the control group tended to stagnate at 7000RFU, while the protein expression of the experimental group continued to increase approximately linearly until it gradually declined after 6 hours exceeded 11000 RFU.

Experiment one, group b: the height of the reaction solution in the experimental group was 2.8cm, and the experimental results are shown in FIG. 3. from the second hour, the RFU value of the experimental group exceeded that of the control group, wherein the RFU values in the 2-hour experimental group and the control group were 1830 and 1330, respectively, and the RFU values in the 3-hour experimental group and the control group were 2670 and 1550, respectively.

Experiment one, group c: the height of the reaction solution in the experimental group was 7.1cm, the experimental results are shown in FIG. 4, and the activity of the reaction solution in the experimental group was substantially equal to that of the control group, and was slightly lower than that of the control group even before the reaction was carried out for 5 hours.

Experiment two, group a: more reaction liquid can be contained because of the replacement with a reaction vessel having a larger diameter. Wherein 5L of the reaction solution was added to the group a, the height was only 0.64cm, and the results of the experiment are shown in FIG. 5, and the activity of the reaction solution in the experimental group was better than that in the control group throughout, wherein the RFU values at 2 hours (1050:1000), 3 hours (1450: 1130), 4 hours (2080: 1350), 5 hours (2380: 1490), and 6 hours (2600: 1550) remained almost linearly increased throughout, particularly until the late stage of the reaction, for example, the RFU value at 6 hours was almost twice as high as that in the control group.

Experiment two, group b: the height was 1.3cm by adding 10L of the reaction solution, and the experimental results are shown in FIG. 6, in the whole reaction process, the RFU value increased first and then gradually, and the initial stage was almost 2 times or more as high as that of the control group, for example, the RFU value (1180) at 2 hours was 3 times as high as that of the control group (380), the RFU value (1480) at 3 hours was nearly 2 times as high as that of the control group (770), and the RFU value at 4 hours (1860:1380) was nearly 1.5 times as high, but became gentle after 5 hours, and the RFU values (1920:1780) of the experimental group and the control group were not much different.

Example 3

Effect of cell lysate concentration on protein in vitro Synthesis efficiency

The experiments were carried out at reaction temperatures of 20-37 deg.C, especially at room temperature of 20-25 deg.C. In the experiment, in-vitro protein expression systems of the Kluyveromyces lactis with the concentrations of 80% and 50% of reaction liquid (standard specific kit trade name and batch) are respectively adopted,

the reaction solution of X L (b) was charged into an XX cm diameter reaction vessel. After the reaction solution was added, 1/30 volume ratio of the DNA product prepared in example 1 was added. The standing incubation time is 2-8 h. After the reaction, 10. mu.L of the reaction system solution was added to a 96-well blackboard or a 384-well blackboard, and immediately placed in a Tecan 2000 microplate reader, and read to detect the activity of green fluorescent protein with RFU as an activity reference, the results are shown in FIG. 7.

FIG. 7 is a graph showing a comparison of the results of experiments in which the ratios of the carrier cell lysate of the present invention were 50% and 80%, and it can be seen that the RFU values of the experimental group were higher than those of the control group at the same ratio in both 50% and 80% of the reaction solution after the reaction time reached 4 hours. Furthermore, the expression effect was better in 80% of the reaction solution.

In summary, in the production setting of in vitro protein synthesis, the invention limits the height of the reaction solution of the in vitro cell-free protein synthesis system in the reaction vessel, and ensures that the reaction solution can be spread in the reaction vessel as much as possible. The invention is applied to industrial production, can use a large container to carry out protein in vitro synthesis, and can process IVTT reaction liquid with larger volume at one time so as to obtain protein with higher yield through one-time reaction, thereby greatly improving the efficiency of protein in vitro synthesis.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims

1. A method for improving the in vitro synthesis efficiency of protein comprises the following steps:

b. adding a nucleic acid template of the protein to be expressed;

a and b are not in sequence;

characterized in that in the step a, the liquid surface area of the reaction liquid in the reaction container is 100-10000 cm²The height of the liquid surface is not more than 5 cm.

2. The method according to claim 1, wherein the liquid surface area of the reaction solution in the reaction vessel is 300 to 9000cm²The height of the liquid surface is not more than 3cm, preferably, the area of the liquid surface is 300-9000 cm²The height of the liquid surface is not more than 2.8cm, and the area of the liquid surface is preferably 500-8000 cm²The liquid level is not more than 1.4 cm.

3. The method according to claim 1 or 2, wherein the reaction solution comprises a cell lysate, a nucleic acid synthase, a protein synthase, a substrate for synthesizing RNA, a substrate for synthesizing protein, water or an aqueous solvent.

4. The method according to claim 3, wherein the concentration (v/v) of the cell extract is 20% to 90%, preferably 30% to 80%, more preferably 50% to 80%, based on the total volume of the reaction solution.

5. The method of claim 3, wherein the cell lysate is derived from one or more cell types selected from the group consisting of: escherichia coli, mammalian cells, plant cells, yeast cells, or a combination thereof; preferably, the yeast cell is selected from saccharomyces cerevisiae, pichia pastoris, kluyveromyces, or a combination thereof; more preferably, the kluyveromyces is kluyveromyces lactis.

6. The method of claim 3, wherein said substrate for RNA synthesis comprises: one of nucleoside monophosphate, nucleoside triphosphate or a combination thereof.

7. The method of claim 3, wherein the substrate for the synthetic protein comprises: 20 natural amino acids and non-natural amino acids.

8. A process according to claim 1 or 2, characterized in that suitable conditions include a reaction temperature of 20-35 ℃, preferably 20-30 ℃, more preferably 25 ℃.

9. The method according to claim 6, wherein the set time is in particular 0.5-20h, preferably 1-18h, more preferably 2-15h, more preferably 3-12 h.