WO2020135747A1

WO2020135747A1 - Optimized in vitro cell-free protein synthesis system and application thereof

Info

Publication number: WO2020135747A1
Application number: PCT/CN2019/129289
Authority: WO
Inventors: 郭敏; 杨宁
Original assignee: 康码（上海）生物科技有限公司
Priority date: 2018-12-28
Filing date: 2019-12-27
Publication date: 2020-07-02
Also published as: CN111378708B; CN111378708A

Abstract

Disclosed is an optimized in vitro cell-free protein synthesis system and an application thereof, comprising: a cell extract, being a yeast cell extract with an inserted T7 RNA polymerase gene; a carbohydrate, being a mixture of glucose and maltodextrin; a phosphate compound; a buffering agent, being a trishydroxymethylaminomethane buffering agent; and a DNA molecule template encoding a heterologous protein prepared by using a nucleic acid isothermal amplification method. In the DNA molecule template, a sequence shown in SEQ ID NO. 1 is inserted upstream of the coding sequence of the heterologous protein. By means of optimization, the cost of in vitro protein synthesis is reduced, and the yield of target protein is increased.

Description

An optimized in vitro cell-free protein synthesis system and its application

Technical field

The invention belongs to the technical field of protein synthesis, and in particular relates to a cell-free protein synthesis system for in vitro protein synthesis.

Background technique

Protein is an important molecule in a cell, and is involved in almost all the functions of the cell. The different sequence and structure of the protein determine its different functions (1). Within the cell, proteins can act as enzymes to catalyze various biochemical reactions, can act as signaling molecules to coordinate various activities of organisms, can support biological morphology, store energy, transport molecules, and move organisms (2). In the field of biomedicine, protein antibodies, as targeted drugs, are an important means of treating cancer and other diseases (1,2).

The traditional protein expression system refers to a molecular biological technique for expressing foreign genes through model organisms such as bacteria, fungi, plant cells or animal cells (3). With the development of science and technology, the cell-free expression system, also known as the in vitro protein synthesis system, came into being. It is the foreign target mRNA or DNA as a protein synthesis template, and the substrate and transcription required for protein synthesis are supplemented by manual control. Translation-related protein factors and other substances can achieve the synthesis of the target protein (3, 4). Expressing proteins in an in vitro translation system does not require plasmid construction, transformation, cell culture, cell collection, and disruption steps. It is a fast, time-saving, and convenient way of protein expression (5,6). In vitro protein synthesis system generally refers to the addition of mRNA or DNA template, RNA polymerase, amino acids and ATP to the lysis system of bacteria, fungi, plant cells or animal cells to complete the rapid and efficient translation of foreign proteins (5, 7).

At present, commercial in vitro protein expression systems that are frequently experimented include E. coli system (ECE), rabbit reticulocyte (RRL), wheat germ (Wheat germ extract, WGE), and insect (Insect) cell extract, ICE) and human source systems (5, 6). Compared with the traditional in vivo recombinant expression system, the protein-free cell-free synthesis system has many advantages. For example, it can express special proteins that are toxic to cells or contain unnatural amino acids (such as D-amino acids). PCR products are used as templates to synthesize multiple proteins in parallel, and to conduct high-throughput drug screening and proteomics research (7). Commercially, E. coli in vitro synthesis systems are widely used. Escherichia coli is easy to culture and ferment, has low cost, simple cell disruption, and can synthesize higher-yield protein (6). Compared with prokaryotic systems, the cultivation of eukaryotic cells is more difficult and expensive, and the preparation process of their cell extracts is cumbersome. Therefore, their translation system costs more and is only suitable for special laboratory use (1,2). Therefore, eukaryotic in vitro protein expression systems suitable for industrial large-scale (ton scale) preparation and production do not currently exist.

After a stage of research and development and preparation, a high-yield and low-cost in vitro expression system has been developed in the field-yeast extract prepared by high-pressure crushing or liquid nitrogen crushing plus magnesium acetate, potassium acetate, amino acids, ATP, DNA template, polyethylene glycol and other components. However, the in vitro protein synthesis reaction system still has the shortcomings of high cost and insufficient reaction efficiency. Therefore, there is an urgent need for a low-cost and efficient in vitro protein synthesis reaction system.

1. Garcia RA, Riley MR. Applied biochemistry and biotechnology. Humana Press, 1981.263-264p.

2.Fromm HJ, Hargrove M. Essentials of Biochemistry. 2012;

3.

S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, et al. Protein production and purification. Nat Methods. 2008; 5(2):135–46.

4.Assenberg R,Wan PT,Geisse S,Mayr LM.Advances inrecombinant protein expression for pharmaceutical research.CurrOpinStructBiol[Internet].2013;23(3):393–402.Availablefrom from:http:/ /dx.doi.org/10.1016/j.sbi.2013.03.008

5. Katzen F, Chang G, Kudlicki W. The past, present and future of cell-free protein synthesis.

Trends Biotechnol. 2005; 23(3): 150–6.

6.Lu”Y.Cell-free synthetic theology:Engineering in the open world.Synth Syst Biotechnol[Internet].2017; 2(1):23–7.Available from from:

http://linkinghub.elsevier.com/retrieve/pii/S2405805X1730008X

7.SpirinAS,SwartzJR.Chapter1.Cell-FreeProteinSynthesisSystems:HistoricalLandmarks, Classification,andGeneralMethods.Wiley-VCHVerlagGmbH&Co.KGaA; 2008.1-34p.

Summary of the invention

The object of the present invention is to provide a low-cost and efficient in vitro protein synthesis reaction system. It mainly solves the technical problems of excessively high cost of protein synthesis system and insufficient reaction efficiency in the prior art.

The technical solutions adopted by the present invention to solve the above technical problems are as follows:

An in vitro cell-free protein synthesis system. The cell-free protein synthesis system includes:

(a) Cell extracts, which are yeast cell extracts inserted with the T7 RNA polymerase gene;

(b) carbohydrates, which are mixtures of glucose and maltodextrin;

(c) Phosphoric acid compounds;

(d) a buffering agent, the buffering agent is a trishydroxymethylaminomethane buffering agent;

(e) A DNA molecule template encoding a foreign protein, the DNA molecule template is prepared using a nucleic acid isothermal amplification method, and the DNA molecule template is inserted with SEQ ID NO. 1 upstream of the encoding sequence of the foreign protein The sequence shown.

Preferably, the protein synthesis system further includes one or more components of the following group:

(f1) polyethylene glycol;

(f2) Synthetic RNA substrate;

(f3) Amino acid mixture;

(f4) magnesium ions;

(f5) potassium ions;

(f6) Dithiothreitol (DTT);

(f7) Active enzymes, which can catalyze the metabolism of carbohydrates to produce ATP;

(f8) Optional water or aqueous solvent.

Further preferably, the active enzyme is selected from amylase, phosphorylase, galactosidase, glucose phosphate mutase, or a combination thereof. Further preferably, the amylase is alpha amylase.

Preferably, the phosphoric acid compound is selected from orthophosphate, dihydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, or a combination thereof.

Preferably, the cell source of the cell extract is one or more types of cells selected from the group consisting of E. coli, bacteria, 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.

Preferably, the glucose concentration is 8.8-128 mmol/L.

Preferably, the concentration of the maltodextrin is 84-500 mmol/L.

Preferably, the concentration (v/v) of the cell extract is 20%-70%, preferably 30-60%, more preferably 40%-50%, based on the total volume of the protein synthesis system meter.

The present invention also provides a kit, which includes a container and the above-mentioned cell-free protein synthesis system components located in the container.

The invention also provides a method for synthesizing exogenous protein in vitro, including:

(i) Provide the above-mentioned in vitro cell-free protein synthesis system;

(ii) Carry out an incubation reaction under suitable conditions to synthesize the foreign protein.

Preferably, the method further comprises: (iii) optionally isolating or detecting the foreign protein from the in vitro cell-free protein synthesis system.

Compared with the prior art, the beneficial effects of the present invention are as follows:

1. Use glucose, maltodextrin and other low-cost substances to replace compounds containing high-energy phosphate bonds such as phosphoenolpyruvate, creatine phosphate and acetyl phosphate, as energy sources to provide ATP for in vitro reactions, reduce costs while reducing costs Release the way of providing energy, prolong the reaction time and increase the yield of target protein.

2. By changing the type, concentration and pH of the reaction buffer to provide a stable reaction environment for the in vitro protein synthesis reaction system to improve the reaction efficiency. By modifying the yeast genome, no additional expensive biological enzymes (such as RNA polymerase) are needed in the reaction system. By using rNTP powder and buffer to prepare a nucleoside triphosphate mixture to replace the commercially available mixture of rNTP, the reaction cost is greatly reduced without reducing the activity of the in vitro protein synthesis reaction system; through the optimization of the DNA template Increase the target output per unit time; use nucleic acid isothermal amplification technology to prepare DNA molecular templates encoding foreign proteins, and use a very small amount (nack-microgram) of DNA template to complete efficient, high-throughput, and extremely simple protein synthesis, etc. .

3. The invention can greatly reduce the cost of in vitro protein synthesis reaction, and at the same time increase the in vitro protein synthesis capacity by more than 30 times.

BRIEF DESCRIPTION

FIG. 1 is a graph of data results of RFU values of fluorescent proteins synthesized by two protein synthesis systems in Example 2 of the present invention; wherein System 1 is the optimized protein synthesis system of Example 2, and System 2 is the original protein synthesis system of Example 2, The negative control is that system 1 does not add DNA template, and the detection time is 3 hours and 20 hours, respectively.

2 is a graph showing the results of the RFU value of the fluorescent protein synthesized by the maltodextrin + glucose protein synthesis system in Example 1 of the present invention; wherein the glucose concentration is 20 mM, the maltodextrin concentration is 0-500 mM, and the detection time is 3 hours, respectively And 20 hours. NC is a protein synthesis system without adding DNA template.

3 is a graph showing the data results of the RFU value of the fluorescent protein synthesized in the maltodextrin + glucose protein synthesis system in Example 1 of the present invention; wherein the glucose concentration is 0-200 mM, the maltodextrin concentration is 320 mM, and the detection time is 20 hours.

detailed description

The expressions "in vitro cell-free protein synthesis system", "in vitro expression system", "in vitro protein synthesis system", "in vitro protein synthesis reaction system", "in vitro protein synthesis system" and the like in the present invention have the same meaning.

After extensive and in-depth research, the inventors found that in the in vitro protein synthesis system, phosphocreatine and phosphocreatine kinase are used as energy sources to provide ATP for in vitro reactions. Although energy can be produced by corresponding kinase reactions to release ATP, it is often It can only provide a large amount of energy in the initial stage, and these high-energy compounds have an inhibitory effect on in vitro cell synthesis, can not provide energy for a long time, and the cost is high, which is not conducive to the efficiency of the in vitro protein synthesis system and industrial application.

Using glucose, maltodextrin and phosphoric acid compounds (potassium phosphate) as energy sources for in vitro biological reactions can slowly release ATP and reduce costs. It is a new energy regeneration system that can be industrialized. Optimized, the reaction system containing glucose + maltodextrin has an RFU value increased by more than 30 times compared to the phosphocreatine + phosphocreatine kinase system; the RFU value has increased compared to the reaction system with glucose as the energy source More than 5 times. Based on this, the present invention optimizes the in vitro cell-free protein synthesis system from various aspects, and provides an efficient and low-cost protein synthesis system.

In vitro cell-free protein synthesis system:

In the present invention, the in vitro cell-free protein synthesis system is not particularly limited, and a preferred cell-free protein synthesis system is yeast in vitro protein synthesis system, preferably Kluyveromyces in vitro protein synthesis system (more preferably, lactic acid Kluyveromyces in vitro protein synthesis system).

Yeast has the advantages of simple culture, efficient protein folding, and post-translational modification. Among them, Saccharomyces cerevisiae and Pichia pastoris are model organisms that express complex eukaryotic and membrane proteins. Yeast can also be used as a raw material for the preparation of in vitro translation systems.

Kluyveromyces (Kluyveromyces) is an ascomycete yeast, Kluyveromyces marxianus and Kluyveromyces lactis are yeasts widely used in industry. Compared with other yeasts, Kluyveromyces lactis has many advantages, such as superb secretion ability, better large-scale fermentation characteristics, food safety level, and the ability to post-translational modification of proteins.

The in vitro cell-free protein synthesis system of the present invention includes: (a) cell extracts, which are yeast cell extracts inserted with the T7 RNA polymerase gene; (b) carbohydrates, which are glucose Mixture with maltodextrin; (c) phosphate compound; (d) buffer, the buffer is trishydroxymethylaminomethane buffer; (e) DNA molecule template encoding foreign protein, the DNA molecule template It is prepared by the nucleic acid isothermal amplification method, and the sequence shown in SEQ ID NO. 1 is inserted in the DNA molecule template upstream of the coding sequence of the foreign protein.

Preferably, the protein synthesis system further includes one or more components of the following group: (f1) polyethylene glycol; (f2) substrate for synthesizing RNA; (f3) amino acid mixture; (f4) magnesium ion; (f5) potassium ions; (f6) dithiothreitol (DTT); (f7) active enzymes capable of catalyzing the metabolism of carbohydrates to produce ATP; (f8) optional water or aqueous solvents.

In the present invention, the content and purity of the cell extract are not particularly limited. Preferably, the concentration (v/v) of the cell extract is 20%-70%, preferably 30-60%, more preferably 40%-50%, based on the total volume of the protein synthesis system meter.

Further, the cell extract is an aqueous extract of yeast cells.

Further, the cell extract does not contain yeast endogenous long-chain nucleic acid molecules.

Further, the substrate for synthesizing RNA includes one or a combination of nucleoside monophosphate and nucleoside triphosphate.

Further, the amino acid mixture includes: 20 kinds of natural amino acids and unnatural amino acids.

Further, the magnesium ion is derived from a magnesium ion source, and the magnesium ion source is selected from the group consisting of one of magnesium acetate and magnesium glutamate or a combination thereof.

Further, the potassium ion is derived from a potassium ion source, and the potassium ion source is selected from the group consisting of one of potassium acetate and potassium glutamate or a combination thereof.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. Wherein, the concentration of the nucleoside triphosphate mixture and the amino acid mixture described in each embodiment refers to the concentration of a single substance in the mixture, not the total substance concentration of the mixture.

Comparative Example 1 In vitro cell-free protein synthesis system containing phosphocreatine + phosphocreatine kinase

In vitro protein synthesis reaction system: 4-hydroxyethylpiperazineethanesulfonic acid (Hepes-KOH) with a final concentration of 22 mM and pH 7.4, 120 mM potassium acetate, 5.0 mM magnesium acetate, 1.5 mM nucleoside triphosphate mixture (adenine nucleus) Glycoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate, each nucleoside triphosphate concentration is 1.5mM), 0.1mM amino acid mixture (glycine, alanine , Valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine Acid, aspartic acid, glutamic acid, lysine, arginine, and histidine, each at a concentration of 0.1 mM), 25 mM creatine phosphate, 1.7 mM dithiothreitol, 0.27 mg/mL creatine phosphate Acid kinase, 0.03mg/mL T7 RNA polymerase, 2% polyethylene glycol, 50% volume yeast cell extract, 15ng/μL enhanced green fluorescent protein DNA.

In vitro protein synthesis reaction: The above reaction system is mixed and placed in an environment of 20-30°C for reaction.

Fluorescent protein activity measurement: Immediately after the reaction, it was placed in the Envision 2120 multifunctional microplate reader (Perkin Elmer) to detect the intensity of the fluorescent signal, and the relative fluorescence unit value (Relative Fluorescence Unit, RFU) was used as the active unit.

In the case where the reaction conditions were 20°C for 3 hours, the relative light unit value RFU of the cell-free protein synthesis system in vitro was 60. The yield of enhanced green fluorescent protein was 1.50 μg/mL. After 3 hours of reaction, the RFU value began to decrease slowly.

Example 1 In vitro cell-free protein synthesis system containing glucose + maltodextrin

In vitro protein synthesis reaction system: 4-hydroxyethylpiperazineethanesulfonic acid (Hepes-KOH) with a final concentration of 22 mM and pH 7.4, 120 mM potassium acetate, 5.0 mM magnesium acetate, 1.5 mM nucleoside triphosphate mixture (adenine nucleus) Glycoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate), 0.1 mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine , Phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine Acid, arginine and histidine), 1.7mM dithiothreitol, 20mM glucose, 20mM tripotassium phosphate, 0-500mM maltodextrin (measured as glucose monomer), 0.002mg/mL alpha starch Enzyme, 0.03mg/mL T7 RNA polymerase, 2% polyethylene glycol, 50% volume yeast cell extract, 15ng/μL enhanced green fluorescent protein DNA.

2 is a graph of the data result of the RFU value of the fluorescent protein synthesized in the maltodextrin + glucose protein synthesis system in this embodiment; wherein the glucose concentration is 20 mM, the maltodextrin concentration is 0-500 mM, and the detection time is 3 hours and At 20 hours, NC refers to a protein synthesis system without adding a DNA template. It can be seen from Figure 2 that when the maltodextrin concentration is in the range of 256-400 mM, the fluorescent protein yield is the highest. When 320 mM maltodextrin was added and reacted at 20° C. for 20 h, the relative light unit value RFU of the in vitro protein synthesis reaction system was about 1900. The yield of enhanced green fluorescent protein was 121.10 μg/mL.

The addition of alpha amylase is conducive to the hydrolysis of maltodextrin. In the case of lack of alpha amylase in this example, the relative light unit value RFU of the in vitro protein synthesis reaction system is about 1750, and the yield of enhanced green fluorescent protein is 111.06 μg /mL.

Refer to FIG. 3 for a fixed 320 mM maltodextrin concentration, test the effect of different concentrations of glucose, and obtain a graph of data results of fluorescent protein RFU values; wherein the glucose concentration is 0-200 mM, the maltodextrin concentration is 320 mM, and the detection time is 20 hours. It can be seen from Figure 3 that when the glucose concentration is around 20 mM, the fluorescent protein RFU value is the highest.

Example 2 Optimization of in vitro cell-free protein synthesis system

2.1 The original in vitro cell-free protein synthesis system (System 2): 4-hydroxyethylpiperazineethanesulfonic acid (Hepes-KOH) with a final concentration of 22 mM and a pH of 7.4, 120 mM potassium acetate, 5 mM magnesium acetate, and 1.5 mM commercialized Nucleoside triphosphate mixture liquid (adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate), 0.1 mM amino acid mixture (glycine, alanine, valine Acid, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, day Aspartic acid, glutamic acid, lysine, arginine and histidine), 25mM phosphocreatine, 1.7mM dithiothreitol, 0.27mg/mL phosphocreatine kinase, 0.03mg/mL T7 RNA polymerization Enzyme, 2% polyethylene glycol, 50% volume yeast cell extract, 15ng/μL enhanced green fluorescent protein DNA (this DNA is prepared by traditional PCR, the sequence encoding green fluorescent protein in the DNA molecule template is inserted into SEQ upstream ID No. 2 shows the sequence). Yeast cells used in yeast cell extracts in this synthetic system have not been genetically modified to express RNA polymerase endogenously, so T7 RNA polymerase needs to be added exogenously.

In vitro protein synthesis reaction: mix in the above system and place in 20-30°C environment for reaction.

2.2 Optimized in vitro cell-free protein synthesis system (System 1): Tris-HCl with a final concentration of 22 mM and a pH of 8.0, 120 mM potassium acetate, 5 mM magnesium acetate, 1.5 mM four nucleoside triphosphates (Mixed with four hydroxymethylaminomethane buffers at pH 8.0 and four nucleoside triphosphate powders), 0.1 mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine , Phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine Acid, arginine and histidine), 1.7 mM dithiothreitol, 20 mM tripotassium phosphate, 20 mM glucose, 320 mM maltodextrin (measured as glucose monomer), 0.002 mg/mL alpha amylase, 2 % Polyethylene glycol, 50% volume yeast cell extract, 15ng/μL enhanced green fluorescent protein DNA (this DNA is prepared by nucleic acid isothermal amplification, and the upstream of the coding sequence of the fluorescent protein in the DNA molecule template is inserted into SEQ ID No. 1 shows the sequence). The yeast cells used in the yeast cell extracts in this synthetic system have the T7 RNA polymerase gene inserted, so they can express T7 RNA polymerase endogenously, without the need to add T7 RNA polymerase exogenously.

In vitro protein synthesis reaction: The above systems are mixed and placed in an environment of 20-30°C for reaction.

The above optimized in vitro cell-free protein synthesis system (System 1), the optimization means are as follows:

1. Establishment of efficient and stable buffer system

System buffer: Because trishydroxymethylaminomethane is one of the commonly used buffer reagents in biology, it is widely used as a solvent for nucleic acids and proteins. After optimization, Tris-HCl buffer at pH 8.0 was used instead of 4-Hydroxyethylpiperazine ethanesulfonic acid (Hepes-KOH) buffer at pH 7.4.

2. Substitution of nucleoside triphosphate powder and optimization of buffer components (high efficiency + low cost)

System 2: 25mM mixture of four nucleoside triphosphates, including adenine nucleoside triphosphate (ATP), guanine nucleoside triphosphate (GTP), cytosine nucleoside triphosphate (CTP) and uracil nucleoside triphosphate (UTP) are commercial nucleoside triphosphate mixtures, and are the highest unit cost of all reaction components.

Optimized, a mixture of nucleoside triphosphate was prepared using trishydroxymethylaminomethane at pH 8.0 and four nucleoside triphosphate powders, instead of the commercially purchased nucleoside triphosphate mixture, in an in vitro protein synthesis system Shows a higher RFU value.

3. Establishment of energy regeneration system

Phosphocreatine and phosphocreatine kinase are used as energy sources to provide ATP for in vitro reactions. Although ATP can be released through the corresponding kinase reaction to produce ATP, they often only provide a large amount of energy at the beginning stage, and these high-energy compounds are In vitro cell synthesis has an inhibitory effect, can not provide energy for a long time, and the cost is higher, which is not conducive to the improvement of the efficiency of in vitro protein synthesis system and industrial application.

Using glucose, maltodextrin and phosphate compounds as energy sources for in vitro biological reactions can slowly release ATP and reduce costs. It is a new energy regeneration system that can be industrialized. After optimization, using the final concentration of 20 mM glucose, 320 mM maltodextrin and 20 mM potassium phosphate as the energy source of the in vitro protein synthesis system showed a higher RFU value in the in vitro protein synthesis system.

4. Optimization of yeast genome transformation

System 2 requires the addition of RNA polymerase. The commercialized and laboratory-made RNA polymerase has the problems of high cost and low purity. By modifying the yeast genome and using yeast cell extracts inserted with the T7 RNA polymerase gene (for the insertion method of the T7 RNA polymerase gene, please refer to the patent content of the application number 2017107685501), so that no additional expensive biological enzymes are needed in the reaction system ( Such as RNA polymerase), without significant changes in RFU value, reducing the cost of protein synthesis in vitro.

5. Cell-free protein synthesis integration of DNA replication, transcription and translation coupling

Amplify a large number of target protein DNA templates by PCR. However, PCR technology is dependent on temperature cycling, and often requires a higher temperature to denature the DNA template and amplify and extend the newly synthesized DNA molecule, and high temperature can cause denaturation and inactivation of protein factors in the in vitro synthesis system, so it is not suitable for use. In vitro synthesis system. Compared with PCR technology, the characteristic of isothermal nucleic acid amplification is to achieve nucleic acid amplification under specific and relatively mild temperature conditions, so that DNA replication, mRNA transcription and protein synthesis can be coupled in vitro. At the same time, DNA polymerases for isothermal nucleic acid amplification, including phi29 DNA polymerase, T7 DNA polymerase, etc. have greater advantages in temperature and amplification efficiency, so that there is no need to prepare a large number of DNA molecules in advance, only a small amount The DNA template can achieve in vitro protein synthesis. After optimization, the DNA replication, transcription, and translation coupling systems listed in Table 1 have achieved RFU values in the in vitro protein synthesis system that have reached the original system’s DNA template using PCR to amplify a large number of target protein DNA templates. Preparation method.

Table 1

6. DNA template modification and optimization

In system 2: No IRES in the 5'untranslated region (KLNCE102), no GAA sequence, no fusion protein fragments and tags after the translation initiation site (ATG), the translation initiation efficiency is relatively low, and it is not fast and efficient 1. The purpose of high-throughput protein synthesis in vitro.

After modification of the DNA template in System 1, the GAA sequence and IRES (KLNCE102) sequence were inserted in sequence from the 5'end to the 3'end before the T7 promoter and the omega sequence of the 5'untranslated region. After the translation initiation site (ATG), the target protein is inserted with a leader peptide sequence and a His tag sequence in sequence from the 5'end to the 3'end. After optimization, the modified DNA template sequence is inserted into the sequence shown in SEQ ID NO.1 upstream of the coding sequence of the foreign protein.

The optimized protein synthesis system (System 1) and the original protein synthesis system (System 2) were placed in an environment of 20-30°C to react.

Fluorescent protein activity measurement: different fluorescence can be observed during the reaction, and the color gradually becomes darker within a certain period of time. Immediately after the reaction, place it in the Envision 2120 Multifunctional Microplate Reader (Perkin Elmer), read it, select different filters, detect the intensity of each fluorescent signal, and take the relative fluorescence unit value (Relative Fluorescence Unit, RFU) as the active unit.

See Figure 1 for the RFU values of the fluorescent proteins synthesized by the two protein synthesis systems; system 1 is the optimized protein synthesis system, system 2 is the original protein synthesis system, and the negative control is that system 1 does not add a DNA template.

It can be seen from the data results in Figure 1 that the RFU value of the fluorescent protein synthesized by the optimized protein synthesis system is about 40 times that of the original protein synthesis system.

The above are only some preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the technical solution of the present invention, and any changes and modifications made are within the protection scope of the present invention.

SEQ ID NO.1

SEQ ID NO.2

Claims

An in vitro cell-free protein synthesis system, characterized in that the cell-free protein synthesis system includes:

(a) Cell extracts, which are yeast cell extracts inserted with the T7 RNA polymerase gene;

(b) carbohydrates, which are mixtures of glucose and maltodextrin;

(c) Phosphoric acid compounds;

(d) a buffering agent, the buffering agent is a trishydroxymethylaminomethane buffering agent;

(e) A DNA molecule template encoding a foreign protein, the DNA molecule template is prepared using a nucleic acid isothermal amplification method, and the DNA molecule template is inserted with SEQ ID NO. 1 upstream of the encoding sequence of the foreign protein The sequence shown.
The cell-free protein synthesis system according to claim 1, wherein the protein synthesis system further comprises one or more components of the following group:

(f1) polyethylene glycol;

(f2) Synthetic RNA substrate;

(f3) Amino acid mixture;

(f4) magnesium ions;

(f5) potassium ions;

(f6) Dithiothreitol (DTT);

(f7) Active enzymes, which can catalyze the metabolism of carbohydrates to produce ATP;

(f8) Optional water or aqueous solvent.
The cell-free protein synthesis system according to claim 1, wherein the phosphate compound is selected from orthophosphate, dihydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, or a combination thereof.
The cell-free protein synthesis system according to claim 1, wherein the cell source of the cell extract is one or more types of cells selected from the group consisting of E. coli, mammalian cells, plant cells, yeast Preferably, the yeast cells are selected from Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces, or a combination thereof; more preferably, the Kluyveromyces is Kluyveromyces lactis.
The cell-free protein synthesis system according to claim 1, wherein the glucose concentration is 8.8-128 mmol/L.
The cell-free protein synthesis system according to claim 1, wherein the concentration of the maltodextrin is 84-500 mmol/L.
The cell-free protein synthesis system according to claim 1, wherein the concentration (v/v) of the cell extract is 20%-70%, preferably 30-60%, more preferably 40 %-50%, based on the total volume of the protein synthesis system.
A kit, characterized in that the kit includes a container and the components in the cell-free protein synthesis system according to any one of claims 1-7 located in the container.
A method for synthesizing exogenous protein in vitro, characterized in that it includes:

(i) providing the in vitro cell-free protein synthesis system of claims 1-7;

(ii) Carry out an incubation reaction under suitable conditions to synthesize the foreign protein.
The method of claim 9, wherein the method further comprises: (iii) optionally isolating or detecting the foreign protein from the in vitro cell-free protein synthesis system.