Efficient cell-free protein synthesis buffer solution as well as preparation method and application thereof

Abstract

The invention discloses an efficient cell-free protein synthesis buffer solution as well as a preparation method and application thereof, and belongs to the field of bioengineering and synthetic biology. The efficient cell-free protein synthesis buffer solution comprises 20-50 mM of HEPES, a cationic substance, an energy regeneration component, 9.5-38 mM of amino acid, an auxiliary factor, a stabilizer and 0.01%-0.03% / v of a bacteriostatic agent, the cationic substances are 8-15 mM of Mg < 2 + >, 60-150 mM of K < + > and 1-10 mM of NH4 < + >; the energy regeneration system comprises 1 to 4 mM of nucleoside triphosphate, 30 to 50 mM of phosphocreatine and 0.2 to 0.6 U / [mu] L of creatine kinase; the stabilizer comprises 1 to 3 mM of dithiothreitol; 1% to 3% w / v of PEG 8000; and a molecular chaperone GroEL / GroES with a concentration of 0.02 to 0.08 mg / mL. The activity is high after long-time normal-temperature preservation; the buffer solution is high in compatibility, low in reagent cost and suitable for industrial production.

Description

Technical Field

[0001] The present invention belongs to the fields of bioengineering and synthetic biology, and in particular relates to an efficient cell-free protein synthesis buffer solution and a preparation method and application thereof. Background Art

[0002] The cell-free protein synthesis (CFPS) system is a technology for efficiently expressing proteins in vitro. Its core lies in optimizing the reaction buffer to provide the energy, substrates and stable environment required for transcription and translation.

[0003] For example, the patent application with publication number CN 119101696 A discloses a cell-free protein synthesis system based on ribozyme regulation and its application, which includes an RNA reporter chain and a ribozyme inhibitory chain. When the pathogen target is not present, the ribozyme inhibitory chain can specifically bind to the ribozyme sequence on the RNA reporter chain and inhibit the ribozyme's cleavage activity. When the pathogen target is present, the ribozyme inhibitory chain specifically binds to the pathogen target, activates the ribozyme's cleavage activity and the RNA reporter chain's translation activity, and expresses the reporter protein, thereby achieving rapid on-site detection of pathogens. By changing the inhibitory chain sequence, different pathogens can be detected, making the cell-free protein synthesis system more convenient for detecting a variety of highly pathogenic pathogens. However, the ribozyme activity needs to be stably maintained, and strict conditions such as buffer pH and ionic strength are required, which increases the complexity of the operation. It is also not suitable for large-scale protein production. Another patent application with publication number CN 118271454A discloses a fusion protein, a nucleic acid encoding the same, and related cell-free protein synthesis to optimize the cell-free protein synthesis system and improve the efficiency of in vitro protein synthesis. The fusion protein provided by the present invention is characterized by having the following structure: the N-terminus or C-terminus of A is connected to B, wherein A is a subunit of the 20S proteasome or a variant thereof, and B is an nHis tag; there is or is no connecting peptide between A and B; n is the number of histidine residues, and n is an integer greater than or equal to 1; by optimizing the proteasome activity in the cell-free synthesis system, the correct folding and stability of the protein are improved; the buffer needs to contain specific cofactors to support the function of the fusion protein; however, the fusion protein design needs to be customized for a specific protein type, has low versatility, and is difficult to directly extend to the synthesis of other proteins.

[0004] In summary, existing cell-free systems have the following problems:

[0005] 1. Short energy supply: Traditional ATP regeneration systems (such as phosphoenolpyruvate PEP) are inefficient, with reaction times typically less than 6 hours and limited yields.

[0006] 2. Low protein folding efficiency: Complex proteins (such as antibodies containing disulfide bonds) are prone to aggregation and inactivation.

[0007] 3. Scale-up bottleneck: high reagent costs, such as purified enzymes and PEP, and activity loss of >50% after freeze-dried storage.

[0008] 4. Limited application scenarios: Incompatible with the efficient expression of toxic proteins or unnatural amino acids. Summary of the Invention

[0009] The present invention aims to provide an efficient cell-free protein synthesis buffer and a preparation method and application thereof, so as to improve the efficiency of CFPS protein synthesis.

[0010] The purpose of the present invention can be achieved through the following technical solutions:

[0011] A highly efficient cell-free protein synthesis buffer comprising 20-50 mM HEPES, a cationic substance, an energy regeneration component, 9.5-38 mM amino acids, a cofactor, a stabilizer, and 0.01% to 0.03% / v of an antibacterial agent;

[0012] The cationic substance is 8-15mM Mg 2+ 、60-150mM K + and 1-10 mM NH 4+ ;

[0013] The energy regeneration system includes 1-4 mM nucleoside triphosphate, 30-50 mM creatine phosphate and 0.2-0.6 U / μL creatine kinase;

[0014] The stabilizer includes 1-3 mM dithiothreitol; 1% to 3% w / v PEG8000; and 0.02-0.08 mg / mL molecular chaperone GroEL / GroES.

[0015] Furthermore, the pH of the buffer solution is 7.6±0.2.

[0016] Furthermore, the nucleoside triphosphate is one or a combination of ATP, GTP, CTP and UTP.

[0017] Nucleoside triphosphates directly provide the energy and precursors required for biosynthesis (e.g., RNA elongation and protein translation). Creatine phosphate and creatine kinase, in combination, constitute the classic ATP regeneration system, continuously regenerating ATP through the transfer of high-energy phosphate bonds from creatine phosphate to ADP, maintaining energy homeostasis in the reaction system. Creatine phosphate serves as an energy storage molecule, and creatine kinase catalyzes its reaction with ADP to generate ATP, replenishing the consumption of NTPs. NTPs directly participate in anabolism, while their breakdown products (e.g., ADP) are converted back into ATP through the regeneration system, forming an energy cycle.

[0018] Furthermore, the amino acids include 19 L-amino acids (except glutamic acid); the addition amount of the 19 L-amino acids is 0.5-2 mM each.

[0019] Furthermore, among the 19 L-amino acids, phenylalanine can be replaced with p-azidophenylalanine, lysine can be replaced with azidolysine, methionine can be replaced with azidoalanine, tyrosine can be replaced with p-azidotyrosine, tryptophan can be replaced with azidotryptophan, and cysteine ​​can be replaced with azidoalanine; the above replacements are replacements of one or a combination of multiple replacements.

[0020] Furthermore, the cofactors include 0.1–0.5 mM coenzyme A, 0.1–1.0 mM NAD + and 0.01–0.1 mM folic acid.

[0021] Furthermore, the antibacterial agent is sodium azide.

[0022] A method for preparing an efficient cell-free protein synthesis buffer comprises the following steps:

[0023] S1. Dissolve HEPES in ultrapure water and stir until completely dissolved.

[0024] S2. Adjust the pH with 1 M KOH at 25°C, add MgCl2 and potassium glutamate in sequence; add energy regeneration components, amino acids, cofactors, and antibacterial agents; and slowly add the sterile filtered stabilizer.

[0025] S3. After stirring evenly, filter the mixture through a 0.22 μm filter membrane for sterilization, divide the mixture into sterile centrifuge tubes, and store at -20°C.

[0026] A highly efficient cell-free protein synthesis buffer for CFPS protein synthesis and freeze-dried reagent storage.

[0027] Furthermore, the CFPS protein synthesis comprises the following steps:

[0028] S1. Prepare the above-mentioned cell-free protein synthesis buffer;

[0029] S2. Add E. coli S30 extract and linear DNA template, react at a constant temperature of 25-30°C for 18-24 hours, and obtain CFPS protein after treatment.

[0030] Furthermore, the volume ratio of the buffer solution to the Escherichia coli S30 extract is (60-70): (30-40).

[0031] Furthermore, the E. coli S30 extract is prepared by lysing E. coli, centrifuging the supernatant, and dialyzing to remove small molecule metabolites.

[0032] Furthermore, the linear DNA template is a plasmid encoding GFP, and the concentration is 0.5–1 μg / μL.

[0033] Furthermore, the freeze-dried reagent is stored, comprising the following steps:

[0034] S1. Premix the buffer with the crude enzyme and add 5% v / v trehalose as a lyophilization protectant;

[0035] S2. Freeze-dry and seal the sample and store at 25°C. Reconstitute and test the activity every month.

[0036] Beneficial effects of the present invention:

[0037] (1) The present invention adopts the creatine phosphate / creatine kinase system to replace the traditional phosphoenolpyruvate energy supply system, significantly improving the ATP regeneration efficiency, maintaining the steady-state concentration of nucleoside triphosphates in the reaction solution, extending the reaction time of cell-free protein synthesis, and improving the yield of the target protein.

[0038] (2) The present invention uses the crowding effect of PEG-8000 molecules and the synergistic effect of endogenous molecular chaperones to simulate the high-density environment within the cell, effectively promote the assembly of membrane protein transmembrane domains and the correct folding of antibodies containing multiple pairs of disulfide bonds, and improve the soluble expression rate of membrane proteins.

[0039] (3) The buffer provided by the present invention is used to prepare freeze-dried reagents, and the activity retention is greater than 90% after being stored at room temperature for 6 months, which is suitable for use in cold chain-free scenarios such as the field and battlefield. On the other hand, the buffer has strong compatibility and supports the efficient expression of linear DNA templates, non-natural amino acid insertions, and toxic proteins. In addition, the present invention uses crude enzymes instead of purified enzymes and creatine phosphate instead of PEP, which reduces the reagent cost by 70%, making it suitable for industrial production. DETAILED DESCRIPTION

[0040] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0041] Example 1

[0042] This embodiment provides an efficient cell-free protein synthesis buffer and its preparation method and application, comprising the following steps:

[0043] S1. Dissolve HEPES in ultrapure water and stir until completely dissolved.

[0044] S2. Adjust the pH to 7.6 with 1 M KOH at 25°C, add the cationic substance in sequence; add the energy regeneration system, 20 mM amino acid, cofactor and antibacterial agent; slowly add the sterile filtered stabilizer;

[0045] S3. After stirring evenly, filter and sterilize using a 0.22 μm filter membrane to obtain a cell-free protein synthesis buffer solution; dispense into sterile centrifuge tubes and store at -20°C to obtain a cell-free protein synthesis buffer solution;

[0046] The cationic substance is 12 mM Mg 2+ , 100mM K + and 5 mM NH 4+ ;

[0047] The energy regeneration system includes 3 mM nucleoside triphosphates (ATP, GTP, CTP, and UTP mixed in a mass ratio of 1:1:1:1), 40 mM creatine phosphate, and 0.5 U / μL creatine kinase;

[0048] The stabilizer includes 2mM dithiothreitol; 2% w / v polyethylene glycol 8000; 0.05mg / mL molecular chaperone GroEL / GroES;

[0049] The cofactors include 0.3mM coenzyme A, 0.5mM NAD + and 0.05 mM folic acid;

[0050] S4. Remove the S30 cell extract from -80°C and thaw it; mix the cell extract and reaction buffer at a volume ratio of 30:60, add a DNA template, and set the DNA template at 15 ng / uL; set the negative control group without adding DNA template; place the reaction on a constant temperature oscillator with the following parameters: temperature 25°C, rotation speed 200 rpm, and reaction time 24 hours; obtain CFPS protein after treatment.

[0051] Example 2

[0052] Compared with Example 1, this embodiment differs in that the amount of energy regeneration system is increased. The specific implementation steps are as follows:

[0053] S1. Dissolve HEPES in ultrapure water and stir until completely dissolved.

[0054] S2. Adjust the pH to 7.6 with 1 M KOH at 25°C, add the cationic substance in sequence; add the energy regeneration system, 20 mM amino acid, cofactor and antibacterial agent; slowly add the sterile filtered stabilizer;

[0055] S3. After stirring evenly, filter and sterilize using a 0.22 μm filter membrane to obtain a cell-free protein synthesis buffer solution; dispense into sterile centrifuge tubes and store at -20°C to obtain a cell-free protein synthesis buffer solution;

[0056] The cationic substance is 12 mM Mg 2+ , 100mM K + and 5 mM NH4+ ;

[0057] The energy regeneration system includes 4 mM nucleoside triphosphates (ATP, GTP, CTP, and UTP mixed in a mass ratio of 1:1:1:1), 50 mM creatine phosphate, and 0.6 U / μL creatine kinase;

[0058] The stabilizer includes 2mM dithiothreitol; 2% w / v polyethylene glycol 8000; 0.05mg / mL molecular chaperone GroEL / GroES;

[0059] The cofactors include 0.3mM coenzyme A, 0.5mM NAD + and 0.05 mM folic acid;

[0060] S4. Remove the S30 cell extract from -80°C and thaw it; mix the cell extract and reaction buffer at a volume ratio of 30:60, add a DNA template, and set the DNA template at 15 ng / uL; set the negative control group without adding DNA template; place the reaction on a constant temperature oscillator with the following parameters: temperature 25°C, rotation speed 200 rpm, and reaction time 24 hours; obtain CFPS protein after treatment.

[0061] The remaining raw materials and preparation process remain the same as in Example 1.

[0062] Example 3

[0063] Compared with Example 1, this embodiment differs in that the amount of energy regeneration system used is reduced. The specific implementation steps are as follows:

[0064] S1. Dissolve HEPES in ultrapure water and stir until completely dissolved.

[0065] S2. Adjust the pH to 7.6 with 1 M KOH at 25°C, add the cationic substance in sequence; add the energy regeneration system, 20 mM amino acid, cofactor and antibacterial agent; slowly add the sterile filtered stabilizer;

[0066] S3. After stirring evenly, filter and sterilize using a 0.22 μm filter membrane to obtain a cell-free protein synthesis buffer solution; dispense into sterile centrifuge tubes and store at -20°C to obtain a cell-free protein synthesis buffer solution;

[0067] The cationic substance is 12 mM Mg 2+ , 100mM K + and 5 mM NH 4+ ;

[0068] The energy regeneration system includes 1 mM nucleoside triphosphate (ATP, GTP, CTP and UTP mixed in a mass ratio of 1:1:1:1), 30 mM creatine phosphate and 0.2 U / μL creatine kinase;

[0069] The stabilizer includes 2mM dithiothreitol; 2% w / v polyethylene glycol 8000; 0.05mg / mL molecular chaperone GroEL / GroES;

[0070] The cofactors include 0.3mM coenzyme A, 0.5mM NAD + and 0.05 mM folic acid;

[0071] S4. Remove the S30 cell extract from -80°C and thaw it; mix the cell extract and reaction buffer at a volume ratio of 30:60, add a DNA template, and set the DNA template at 15 ng / uL; set the negative control group without adding DNA template; place the reaction on a constant temperature oscillator with the following parameters: temperature 25°C, rotation speed 200 rpm, and reaction time 24 hours; obtain CFPS protein after treatment.

[0072] The remaining raw materials and preparation process remain the same as in Example 1.

[0073] Example 4

[0074] Compared with Example 1, the difference between this embodiment and Example 1 is that, in the L-amino acid, phenylalanine can be replaced by p-azidophenylalanine, and lysine can be replaced by azidolysine;

[0075] The remaining raw materials and preparation process remain the same as in Example 1.

[0076] Example 5

[0077] Compared with Example 1, this embodiment differs in that the dosage of the auxiliary factor is increased. The specific implementation steps are as follows:

[0078] S1. Dissolve HEPES in ultrapure water and stir until completely dissolved.

[0079] S2. Adjust the pH to 7.6 with 1 M KOH at 25°C, add the cationic substance in sequence; add the energy regeneration system, 20 mM amino acid, cofactor and antibacterial agent; slowly add the sterile filtered stabilizer;

[0080] S3. After stirring evenly, filter and sterilize using a 0.22 μm filter membrane to obtain a cell-free protein synthesis buffer solution; dispense into sterile centrifuge tubes and store at -20°C to obtain a cell-free protein synthesis buffer solution;

[0081] The cationic substance is 12 mM Mg 2+ , 100mM K + and 5 mM NH 4+ ;

[0082] The energy regeneration system includes 3 mM nucleoside triphosphates (ATP, GTP, CTP, and UTP mixed in a mass ratio of 1:1:1:1), 40 mM creatine phosphate, and 0.5 U / μL creatine kinase;

[0083] The stabilizer includes 2mM dithiothreitol; 2% w / v polyethylene glycol 8000; 0.05mg / mL molecular chaperone GroEL / GroES;

[0084] The cofactors include 0.5 mM coenzyme A, 1.0 mM NAD + and 0.1 mM folic acid;

[0085] S4. Remove the S30 cell extract from -80°C and thaw it; mix the cell extract and reaction buffer at a volume ratio of 30:60, add a DNA template, and set the DNA template at 15 ng / uL; set the negative control group without adding DNA template; place the reaction on a constant temperature oscillator with the following parameters: temperature 25°C, rotation speed 200 rpm, and reaction time 24 hours; obtain CFPS protein after treatment.

[0086] The remaining raw materials and preparation process remain the same as in Example 1.

[0087] Example 6

[0088] Compared with Example 1, this embodiment differs in that the amount of the auxiliary factor is reduced. The specific implementation steps are as follows:

[0089] S1. Dissolve HEPES in ultrapure water and stir until completely dissolved.

[0090] S2. Adjust the pH to 7.6 with 1 M KOH at 25°C, add the cationic substance in sequence; add the energy regeneration system, 20 mM amino acid, cofactor and antibacterial agent; slowly add the sterile filtered stabilizer;

[0091] S3. After stirring evenly, filter and sterilize using a 0.22 μm filter membrane to obtain a cell-free protein synthesis buffer solution; dispense into sterile centrifuge tubes and store at -20°C to obtain a cell-free protein synthesis buffer solution;

[0092] The cationic substance is 12 mM Mg 2+ , 100mM K + and 5 mM NH 4+ ;

[0093] The energy regeneration system includes 3 mM nucleoside triphosphates (ATP, GTP, CTP, and UTP mixed in a mass ratio of 1:1:1:1), 40 mM creatine phosphate, and 0.5 U / μL creatine kinase;

[0094] The stabilizer includes 2mM dithiothreitol; 2% w / v polyethylene glycol 8000; 0.05mg / mL molecular chaperone GroEL / GroES;

[0095] The cofactors include 0.1 mM coenzyme A, 0.1 mM NAD + and 0.01 mM folic acid;

[0096] S4. Remove the S30 cell extract from -80°C and thaw it; mix the cell extract and reaction buffer at a volume ratio of 30:60, add a DNA template, and set the DNA template at 15 ng / uL; set the negative control group without adding DNA template; place the reaction on a constant temperature oscillator with the following parameters: temperature 25°C, rotation speed 200 rpm, and reaction time 24 hours; obtain CFPS protein after treatment.

[0097] The remaining raw materials and preparation process remain the same as in Example 1.

[0098] Comparative Example 1

[0099] Compared with Example 1, this comparative example differs in that creatine phosphate and creatine kinase are replaced by phosphoenolpyruvate and pyruvate kinase. The specific implementation steps are as follows:

[0100] S1. Dissolve HEPES in ultrapure water and stir until completely dissolved.

[0101] S2. Adjust the pH to 7.6 with 1 M KOH at 25°C, add the cationic substance in sequence; add the energy regeneration system, 20 mM amino acid, cofactor and antibacterial agent; slowly add the sterile filtered stabilizer;

[0102] S3. After stirring evenly, filter and sterilize using a 0.22 μm filter membrane to obtain a cell-free protein synthesis buffer solution; dispense into sterile centrifuge tubes and store at -20°C to obtain a cell-free protein synthesis buffer solution;

[0103] The cationic substance is 12 mM Mg 2+ , 100mM K + and 5 mM NH 4+ ;

[0104] The energy regeneration system includes 3 mM nucleoside triphosphates (ATP, GTP, CTP, and UTP mixed in a mass ratio of 1:1:1:1), 30 mM phosphoenolpyruvate, and 0.4 U / μL pyruvate kinase;

[0105] The stabilizer includes 2mM dithiothreitol; 2% w / v polyethylene glycol 8000; 0.05mg / mL molecular chaperone GroEL / GroES;

[0106] The cofactors include 0.3mM coenzyme A, 0.5mM NAD + and 0.05 mM folic acid;

[0107] S4. Remove the S30 cell extract from -80°C and thaw it; mix the cell extract and reaction buffer at a volume ratio of 30:60, add a DNA template, and set the DNA template at 15 ng / uL; set the negative control group without adding DNA template; place the reaction on a constant temperature oscillator with the following parameters: temperature 25°C, rotation speed 200 rpm, and reaction time 24 hours; obtain CFPS protein after treatment.

[0108] The remaining raw materials and preparation process remain the same as in Example 1.

[0109] Comparative Example 2

[0110] Compared with Example 1, this comparative example is different in that PEG8000 is not added. The specific implementation steps are as follows:

[0111] S1. Dissolve HEPES in ultrapure water and stir until completely dissolved.

[0112] S2. Adjust the pH to 7.6 with 1 M KOH at 25°C, add the cationic substance in sequence; add the energy regeneration system, 20 mM amino acid, cofactor and antibacterial agent; slowly add the sterile filtered stabilizer;

[0113] S3. After stirring evenly, filter and sterilize using a 0.22 μm filter membrane to obtain a cell-free protein synthesis buffer solution; dispense into sterile centrifuge tubes and store at -20°C to obtain a cell-free protein synthesis buffer solution;

[0114] The cationic substance is 12 mM Mg 2+ , 100mM K + and 5 mM NH 4+ ;

[0115] The energy regeneration system includes 3 mM nucleoside triphosphates (ATP, GTP, CTP, and UTP mixed in a mass ratio of 1:1:1:1), 40 mM creatine phosphate, and 0.5 U / μL creatine kinase;

[0116] The stabilizer includes 2mM dithiothreitol; 0.05mg / mL molecular chaperone GroEL / GroES;

[0117] The cofactors include 0.3mM coenzyme A, 0.5mM NAD + and 0.05 mM folic acid;

[0118] S4. Remove the S30 cell extract from -80°C and thaw it; mix the cell extract and reaction buffer at a volume ratio of 30:60, add a DNA template, and set the DNA template at 15 ng / uL; set the negative control group without adding DNA template; place the reaction on a constant temperature oscillator with the following parameters: temperature 25°C, rotation speed 200 rpm, and reaction time 24 hours; obtain CFPS protein after treatment.

[0119] The remaining raw materials and preparation process remain the same as in Example 1.

[0120] Performance Testing

[0121] The performance of the CFPS proteins prepared in Examples 1 to 6 and Comparative Examples 1 to 2 was tested, and the specific steps were as follows:

[0122] GFP fluorescence quantitative detection: First, a standard curve was established using a serial dilution of purified GFP (0, 0.5, 1.0, 2.0, 4.0 mg / mL). The reaction system was centrifuged at 4°C, 12,000 × g for 5 minutes, and the supernatant was collected. The test group consisted of 10 μL of supernatant and 40 μL of PBS (pH 7.4, containing 0.05% Tween-20); the blank control consisted of 50 μL of PBS (the same volume replacing the supernatant). The absorbance was measured using a microplate reader (excitation wavelength 488 nm, emission wavelength 509 nm) and the concentration was calculated by substituting the standard curve into the absorbance. (Three replicates were collected for each group).

[0123] SDS-PAGE protein electrophoresis: take 2 μL supernatant + 18 μL PBS + 5 μL 4× Laemmli loading buffer (containing 2% SDS, 5% β-mercaptoethanol), heat in a 95°C metal bath for 5 minutes, and cool on ice for 2 minutes; prepare gel and electrophoresis buffer, load the sample, run electrophoresis at a constant voltage of 180 V for 60 minutes, stain, scan with a gel imaging system, analyze with ImageJ, and calculate the grayscale value.

[0124] GFP fluorescence activity detection: Redissolve the freeze-dried sample (with sterile water or original buffer), centrifuge and collect the supernatant. Detection parameters: excitation wavelength 488nm, emission wavelength 509nm; activity retention rate calculation: retention rate = T n Fluorescence value / T0 fluorescence value×100%.

[0125] The results are shown in Table 1:

[0126] Table 1

[0127] project Concentration (mg / mL) Gray value (AU) 6-month activity (%) 12-month activity (%) Example 1 2.5±0.4 16040±800 92.6 85.4 Example 2 2.3±0.3 14800±650 88.2 80.6 Example 3 2.4±0.2 15400±500 90.9 84.0 Example 4 2.1±0.4 13700±700 89.8 82.7 Example 5 2.3±0.3 14750±650 87.2 84.6 Example 6 2.2±0.4 13900±400 88.5 81.2 Comparative Example 1 1.2±0.3 6000±350 75.0 64.1 Comparative Example 2 1.8±0.3 11000±400 80.7 69.5

[0128] As can be seen from Table 1, the cell-free protein synthesis buffer prepared by the present invention is applied to GFP synthesis, and the GFP yield is as high as 2.5±0.4 mg / mL (Example 1), while the traditional PEP system control group (Comparative Example 1) is 1.2±0.3 mg / mL. Among them, the synthetic activity of the protein in Example 1 is retained by 92% after 16 months and by 85% after 12 months, which is a significant improvement over the 75.0% and 64.1% of Comparative Example 1.

[0129] The above disclosures are only a few specific embodiments of the present invention. However, the embodiments of the present invention are not limited thereto. Any changes that can be conceived by those skilled in the art should fall within the scope of protection of the present invention.

Claims

1. An efficient cell-free protein synthesis buffer, characterized in that including 20-50 mM HEPES, cationic substances, energy regeneration components, 9.5-38 mM amino acids, cofactors, stabilizers, and 0.01% to 0.03% / v antibacterial agents; The cationic substance is 8-15mM Mg 2+ 、60-150mM K + and 1-10 mM NH 4+ ; The energy regeneration system includes 1-4 mM nucleoside triphosphate, 30-50 mM creatine phosphate and 0.2-0.6 U / μL creatine kinase; The stabilizer includes 1-3 mM dithiothreitol; 1% to 3% w / v PEG8000; and 0.02-0.08 mg / mL molecular chaperone GroEL / GroES.

2. An efficient cell-free protein synthesis buffer according to claim 1, characterized in that The pH of the buffer solution was 7.6±0.

2.

3. An efficient cell-free protein synthesis buffer according to claim 1, characterized in that The nucleoside triphosphate is one or a combination of ATP, GTP, CTP and UTP.

4. An efficient cell-free protein synthesis buffer according to claim 1, characterized in that The amino acids include 19 L-amino acids (except glutamic acid); the addition amount of the 19 L-amino acids is 0.5-2 mM each.

5. An efficient cell-free protein synthesis buffer according to claim 4, characterized in that, Among the 19 L-amino acids, phenylalanine can be replaced with p-azidophenylalanine, lysine can be replaced with azidolysine, methionine can be replaced with azidoalanine, tyrosine can be replaced with p-azidotyrosine, tryptophan can be replaced with azidotryptophan, and cysteine ​​can be replaced with azidoalanine; the above replacements can be replaced by one or a combination of multiple replacements.

6. An efficient cell-free protein synthesis buffer according to claim 1, characterized in that, The cofactors include 0.1–0.5 mM coenzyme A, 0.1–1.0 mM NAD + and 0.01–0.1 mM folic acid; the antibacterial agent is sodium azide.

7. A method for preparing an efficient cell-free protein synthesis buffer, for preparing the efficient cell-free protein synthesis buffer according to any one of claims 1 to 6, characterized in that: The steps include: S1. Dissolve HEPES in ultrapure water and stir until completely dissolved. S2. Adjust the pH with 1 M KOH at 25°C, add MgCl2 and potassium glutamate in sequence; add energy regeneration components, amino acids, cofactors, and antibacterial agents; and slowly add the sterile filtered stabilizer. S3. After stirring evenly, filter the mixture through a 0.22 μm filter membrane for sterilization, divide the mixture into sterile centrifuge tubes, and store at -20°C.

8. Application of an efficient cell-free protein synthesis buffer in CFPS protein synthesis and freeze-dried reagent storage, characterized in that: The efficient cell-free protein synthesis The buffer solution is prepared according to the method for preparing an efficient cell-free protein synthesis buffer solution according to claim 7.

9. Use of an efficient cell-free protein synthesis buffer according to claim 8 in CFPS protein synthesis and freeze-dried reagent storage, characterized in that: The CFPS protein synthesis comprises the following steps: S1. Prepare the above-mentioned cell-free protein synthesis buffer; S2, add E. coli S30 extract and linear DNA template, react at 25-30°C for 18-24 hours, and obtain CFPS protein after treatment; The volume ratio of the buffer solution to the Escherichia coli S30 extract is (60-70): (30-40); The E. coli S30 extract is prepared by lysing E. coli, centrifuging and taking the supernatant, and dialyzing to remove small molecule metabolites; Furthermore, the linear DNA template is a plasmid encoding GFP, and the concentration is 0.5–1 μg / μL.

10. Use of the efficient cell-free protein synthesis buffer according to claim 8 in CFPS protein synthesis and freeze-dried reagent storage, characterized in that: The freeze-dried reagent is stored, comprising the following steps: S1. Premix the buffer with the crude enzyme and add 5% v / v trehalose as a lyophilization protectant; S2. Freeze-dry and seal the sample and store at 25°C. Reconstitute and test the activity every month.