Reaction composition for cell-free protein synthesis and cell-free protein synthesis method using the same

Abstract

The present invention relates to a cell-free protein synthesis reaction composition and a cell-free protein synthesis method using the same. It has been confirmed that the cell-free protein synthesis reaction composition and the cell-free protein synthesis method according to the present invention increase the protein expression rate and reduce manufacturing costs compared to conventional methods. Accordingly, the present invention has the effect of obtaining protein at a high yield relative to cost in cell-free protein synthesis.

Description

Technology Field

[0001] The present invention relates to a reaction composition for cell-free protein synthesis and a method for cell-free protein synthesis using the same. Background Technology

[0003] Traditional protein expression systems refer to molecular biological techniques that express exogenous genes using model organisms such as bacteria, fungi, plant cells, or animal cells. With the advancement of science and technology, cell-free protein synthesis systems, also known as in vitro protein synthesis systems, have emerged. These systems utilize exogenous target mRNA or DNA as a template for protein synthesis and achieve the goal of synthesizing proteins by passively controlling and replenishing substances such as substrates required for protein synthesis and protein factors involved in transcription and translation.

[0004] Furthermore, cell-free protein synthesis involves extracting only the intracellular machinery and factors involved in protein production from cells and artificially repeating the protein synthesis process outside the cell in a state where the cell's physiological regulatory mechanisms are excluded. This allows for the mass production of target proteins in a short period, thereby enabling high-speed protein synthesis without undergoing cell culture processes. Moreover, as a completely open system without physical barriers—unlike cell culture processes where protein expression takes place within the space of the cell membrane and cell wall—it offers the advantage of allowing the conditions for protein synthesis to be freely modified for application in various research.

[0005] However, conventional cell-free protein synthesis methods suffer from the problem of high costs due to expensive reagents, so there is a need for a high-yield, low-budget cell-free protein synthesis method.

[0006] Accordingly, the inventors intended to develop an optimized cell-free protein synthesis method and composition capable of obtaining protein in high yield and reducing costs. Prior art literature

[0008] Korean Registered Patent Publication No. 10-0733712 (June 23, 2007) The problem to be solved

[0009] The inventors have identified a cell-free protein synthesis reaction composition and a cell-free protein synthesis method that can obtain protein in high yield and reduce costs in cell-free protein synthesis, and have completed the present invention based on this.

[0010] Accordingly, the object of the present invention is to provide a cell-free protein synthesis method comprising the following steps.

[0011] (a) A step of culturing and crushing bacteria in a medium;

[0012] (b) centrifuging the ruptured bacterial cell solution at 15,000xg to 35,000xg to obtain the supernatant and produce a bacterial cell extract; and

[0013] (c) A step of adding the cell extract to a cell-free protein synthesis reaction composition containing a protein synthesis energy source containing maltodextrin at a concentration of 20 mM to 50 mM, and shaking and culturing.

[0014] Another object of the present invention is to provide a cell-free protein synthesis kit used in the method described above, comprising a bacterial cell extract, plasmid DNA, an amino acid mixture, a protein synthesis energy source comprising maltodextrin at a concentration of 20 mM to 50 mM, sodium hexametaphosphate (SHMP), and a salt.

[0016] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below. means of solving the problem

[0018] To achieve the above objectives, the present invention provides a cell-free protein synthesis method comprising the following steps.

[0019] (a) A step of culturing and crushing bacteria in a medium;

[0020] (b) centrifuging the ruptured bacterial cell solution at 15,000xg to 35,000xg to obtain the supernatant and produce a bacterial cell extract; and

[0021] (c) A step of adding the cell extract to a cell-free protein synthesis reaction composition containing a protein synthesis energy source containing maltodextrin at a concentration of 20 mM to 50 mM, and shaking and culturing.

[0022] As one embodiment of the present invention, the bacteria E. coli It may be, but is not limited to.

[0023] In another embodiment of the present invention, the maltodextrin may be at a concentration of 35 mM, but is not limited thereto.

[0024] As another embodiment of the present invention, the protein synthesis energy source may additionally include one or more selected from the group consisting of HEPES, NTP, tRNA, coenzyme A, NAD, cAMP, polynic acid, and spermidine, but is not limited thereto.

[0025] As another embodiment of the present invention, the composition may additionally include one or more selected from the group consisting of plasmid DNA, amino acid mixture, sodium hexametaphosphate (SHMP), potassium glutamate, and magnesium glutamate, but is not limited thereto.

[0026] As another embodiment of the present invention, the amino acid mixture may be one or more mixtures selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, cysteine, methionine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, and glutamine, but is not limited thereto.

[0027] As another embodiment of the present invention, the composition may not include polyethylene glycol or ammonium ions, but is not limited thereto.

[0028] As another embodiment of the present invention, the composition may further include polyethylene glycol at a concentration of 0.1% to 0.5%, but is not limited thereto.

[0029] As another embodiment of the present invention, step (b) may not perform a run-off reaction or may additionally perform a run-off reaction of 30 minutes or less, but is not limited thereto.

[0030] As another embodiment of the present invention, step (c) may involve shaking and culturing at a speed of 100 rpm to 300 rpm, but is not limited thereto.

[0031] In addition, the present invention provides a cell-free protein synthesis kit used in the method of the present invention, comprising a bacterial cell extract, plasmid DNA, an amino acid mixture, a protein synthesis energy source comprising maltodextrin at a concentration of 20 mM to 50 mM, sodium hexametaphosphate (SHMP), and a salt. Effects of the invention

[0033] It has been confirmed that the cell-free protein synthesis reaction composition and the cell-free protein synthesis method according to the present invention increase the protein expression rate and reduce manufacturing costs compared to conventional methods. Accordingly, the present invention has the effect of obtaining protein at a high yield relative to cost in cell-free protein synthesis. Brief explanation of the drawing

[0035] FIG. 1 is a diagram showing the protein synthesis rate according to the presence or absence of shaking in a cell-free protein synthesis method according to one embodiment of the present invention. FIG. 2 is a diagram showing the protein synthesis rate depending on whether a run-off reaction is performed in a cell-free protein synthesis method according to one embodiment of the present invention. FIG. 3 is a diagram showing the protein synthesis rate according to culture temperature and time in a cell-free protein synthesis method according to one embodiment of the present invention. Figure 4 is a diagram showing the protein synthesis rate according to the concentration of ammonium ions in a cell-free protein synthesis method according to one embodiment of the present invention. FIG. 5 is a diagram showing the protein synthesis rate according to the concentration of potassium ions in a cell-free protein synthesis method according to one embodiment of the present invention. FIG. 6 is a diagram showing the protein synthesis rate according to the concentration of magnesium ions in a cell-free protein synthesis method according to one embodiment of the present invention. FIG. 7 is a diagram showing the protein synthesis rate depending on whether PEG-8000 is added in a cell-free protein synthesis method according to one embodiment of the present invention. FIG. 8 is a diagram showing the protein synthesis rate according to the concentration of HMP in a cell-free protein synthesis method according to one embodiment of the present invention. FIG. 9 is a diagram showing the protein synthesis rate according to the concentration of DNA in a cell-free protein synthesis method according to one embodiment of the present invention. Specific details for implementing the invention

[0036] In one experimental example of the present invention, the protein synthesis rate according to the cell-free protein synthesis method of the present invention was confirmed, and it was confirmed that the method of culturing while shaking at 29 ℃ at a speed of 300 rpm for 18 hours resulted in the highest protein synthesis rate (see Experimental Example 1).

[0037] In another experimental example of the present invention, the protein synthesis rate according to the cell-free protein synthesis reaction composition of the present invention was confirmed, and the concentration of the composition exhibiting the optimal protein synthesis rate was confirmed (see Experimental Example 2).

[0039] The present invention will be described in detail below.

[0041] The present invention provides a cell-free protein synthesis method comprising the following steps.

[0042] (a) A step of culturing and crushing bacteria in a medium;

[0043] (b) centrifuging the ruptured bacterial cell solution at 15,000xg to 35,000xg to obtain the supernatant and produce a bacterial cell extract; and

[0044] (c) A step of adding the cell extract to a cell-free protein synthesis reaction composition containing a protein synthesis energy source containing maltodextrin at a concentration of 20 mM to 50 mM, and shaking and culturing.

[0045] In the present invention, "cell-free protein synthesis" refers to performing protein synthesis that was previously carried out within a cell in vitro, such as in a test tube. It means producing a target protein in a short period of time by extracting only the components necessary for protein production—namely, the intracellular protein synthesis machinery and its factors—from the cell and artificially repeating only the protein synthesis process outside the cell while excluding the cell's physiological regulatory mechanisms. At this time, the protein biosynthetic machinery required for cell-free protein synthesis, namely ribosomes, initiation factors, elongation factors, termination factors, aminoacyl tRNA synthetase, RNA polymerase, etc., may be used from those contained in the cell extract, added separately, or produced separately using genetic recombination technology, but is not limited thereto.

[0046] In the present invention, the bacteria E. coli It may be, but is not limited to.

[0047] In the present invention, the centrifugation speed of step (b) may be 15,000xg to 20,000xg, 15,000xg to 25,000xg, 15,000xg to 30,000xg, 15,000xg to 35,000xg, 20,000xg to 25,000xg, 20,000xg to 30,000xg, 20,000xg to 35,000xg, 25,000xg to 30,000xg, 25,000xg to 35,000xg, or 35,000xg to 35,000xg.

[0048] In the present invention, the concentration of the maltodextrin may be 20 mM to 30 mM, 20 mM to 35 mM, 20 mM to 40 mM, 20 mM to 45 mM, 20 mM to 50 mM, 25 mM to 30 mM, 25 mM to 35 mM, 25 mM to 40 mM, 25 mM to 45 mM, 25 mM to 50 mM, 30 mM to 35 mM, 30 mM to 40 mM, 30 mM to 45 mM, 30 mM to 50 mM, 35 mM to 40 mM, 35 mM to 45 mM, 35 mM to 50 mM, or 35 mM.

[0049] In the present invention, the protein synthesis energy source may additionally include one or more selected from the group consisting of HEPES, NTP, tRNA, coenzyme A, NAD, cAMP, polynic acid, and spermidine, but is not limited thereto.

[0050] In the present invention, the composition may additionally include one or more selected from the group consisting of plasmid DNA, amino acid mixture, sodium hexametaphosphate (SHMP), potassium glutamate, and magnesium glutamate, but is not limited thereto.

[0051] In the present invention, the amino acid mixture may be one or more mixtures selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, cysteine, methionine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, and glutamine, but is not limited thereto.

[0052] In the present invention, the concentration of the HMP may be 0.1 mM to 0.3 mM, 0.1 mM to 0.5 mM, 0.1 mM to 0.7 mM, 0.1 mM to 1 mM, 0.3 mM to 0.5 mM, 0.3 mM to 0.7 mM, 0.3 mM to 1 mM, 0.5 mM to 0.7 mM, 0.5 mM to 1 mM, or 0.7 mM to 1 mM, but is not limited thereto.

[0053] In the present invention, the concentration of the plasmid DNA may be 1 nM to 3 nM, 1 nM to 5 nM, 1 nM to 7 nM, 1 nM to 10 nM, 3 nM to 5 nM, 3 nM to 7 nM, 3 nM to 10 nM, 5 nM to 7 nM, 5 nM to 10 nM, or 7 nM to 10 nM, but is not limited thereto.

[0054] In the present invention, the composition may not include polyethylene glycol or ammonium ions, but is not limited thereto.

[0055] In the present invention, the composition may further include polyethylene glycol at a concentration of 0.5% or less relative to the total composition volume, but is not limited thereto. For example, the concentration of polyethylene glycol may be 0.1% to 0.2%, 0.1% to 0.3%, 0.1% to 0.4%, 0.1% to 0.5%, 0.2% to 0.3%, 0.2% to 0.4%, 0.2% to 0.5%, 0.3% to 0.4%, 0.3% to 0.5%, or 0.4% to 0.5%, but is not limited thereto.

[0056] In the present invention, the polyethylene glycol may be PEG-8000, but is not limited thereto.

[0057] In the present invention, the % concentration may be w / v % or v / v %, but is not limited thereto.

[0058] In the present invention, step (b) may additionally perform a run-off reaction of 30 minutes or less, but is not limited thereto. For example, the run-off reaction may be performed for 0.1 seconds to 30 minutes, 1 second to 30 minutes, 1 minute to 5 minutes, 1 minute to 10 minutes, 1 minute to 15 minutes, 1 minute to 20 minutes, 1 minute to 25 minutes, 1 minute to 30 minutes, 5 minutes to 10 minutes, 5 minutes to 15 minutes, 5 minutes to 20 minutes, 5 minutes to 25 minutes, 5 minutes to 30 minutes, 10 minutes to 15 minutes, 10 minutes to 20 minutes, 10 minutes to 25 minutes, 10 minutes to 30 minutes, 15 minutes to 20 minutes, 15 minutes to 25 minutes, 15 minutes to 30 minutes, 20 minutes to 25 minutes, 20 minutes to 30 minutes, or 25 minutes to 30 minutes.

[0059] In the present invention, step (b) may not perform a run-off reaction, but is not limited thereto.

[0060] In the present invention, the shaking speed of step (c) may be 100 rpm to 150 rpm, 100 rpm to 200 rpm, 100 rpm to 250 rpm, 100 rpm to 300 rpm, 150 rpm to 200 rpm, 150 rpm to 250 rpm, 150 rpm to 300 rpm, 200 rpm to 250 rpm, 200 rpm to 300 rpm, or 250 rpm to 300 rpm, but is not limited thereto.

[0061] In one experimental example of the present invention, the protein synthesis rate according to the shaking speed was confirmed in cell-free protein synthesis. As a result, it was confirmed that the protein synthesis rate increased when shaking was performed compared to when shaking was not performed, and that the protein synthesis rate increased significantly starting from 100 rpm (see Experimental Example 1-1 and Fig. 1).

[0062] In the present invention, the temperature of step (c) is 18°C ​​to 23°C, 18°C ​​to 25°C, 18°C ​​to 27°C, 18°C ​​to 29°C, 18°C ​​to 31°C, 18°C ​​to 33°C, 18°C ​​to 35°C, 23°C to 25°C, 23°C to 27°C, 23°C to 29°C, 23°C to 31°C, 23°C to 33°C, 23°C to 35°C, 25°C to 27°C, 25°C to 29°C, 25°C to 31°C, 25°C to 33°C, 25°C to 35°C, 27°C to 29°C, 27°C to 31°C, 27°C to 33°C, 27°C to 35°C, 29°C to 31°C, 29°C It may be up to 33°C, 29°C to 35°C, 31°C to 33°C, 31°C to 35°C, or 33°C to 35°C, but is not limited thereto.

[0063] In the present invention, the culture time of step (c) may be 15 to 17 hours, 15 to 18 hours, 15 to 19 hours, 15 to 20 hours, 15 to 20 hours, 17 to 18 hours, 17 to 19 hours, 17 to 20 hours, 17 to 21 hours, 17 to 22 hours, 18 to 19 hours, 18 to 20 hours, 18 to 21 hours, 18 to 22 hours, 19 to 20 hours, 19 to 21 hours, 19 to 22 hours, 20 to 21 hours, 20 to 22 hours, or 21 to 22 hours, but is not limited thereto.

[0064] In addition, the present invention provides a cell-free protein synthesis kit used in the method of the present invention, comprising a bacterial cell extract, plasmid DNA, an amino acid mixture, a protein synthesis energy source comprising maltodextrin at a concentration of 20 mM to 50 mM, sodium hexametaphosphate (SHMP), and a salt.

[0066] Hereinafter, preferred embodiments and experimental examples are presented to aid in understanding the present invention. However, the following embodiments and experimental examples are provided merely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following embodiments and experimental examples.

[0068] [Example]

[0069] Example 1. Preparation of bacterial cell extract

[0070] Example 1-1. Preparation of cultured cells

[0071] E. coli A single colony of BL21 (DE3) was inoculated into a 250 mL flask containing 50 mL of LB (Luria-Berani) medium (10 g / L Yeast extract, 5 g / L Tryptone, 10 g / L Sodium chloride) and cultured with shaking at 200 rpm for 16 hours at 37 ℃. The cultured E. coli 5 mL was transferred to a 2 L baffled flask containing 500 mL of TB (Terrific broth) medium (24 g / L Yeast extract, 12 g / L Tryptone, 0.4% Glycerol, 89 mM Potassium phosphate, pH 7.2), and shaken at 37 ℃ at a speed of 260 rpm until the OD600 reached 0.6. Subsequently, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a concentration of 0.84 mM, and induction culture was performed at 37 ℃ at a speed of 260 rpm until the OD600 reached 4.0. E. coli was centrifuged at 5000 xg for 15 minutes at a temperature of 4 ℃. The centrifuged E. coliThe pellet was thoroughly dissolved in 200 mL of Buffer S30A (14 mM Magnesium-acetate, 60 mM Potassium-acetate, 10 mM Tris-acetate, pH 8.2), 7 mM β-mercaptoethanol (βME), and 1 mM 1,4-dithiothreitol (DTT), and subsequently centrifuged at 5000 xg for 15 minutes at 4 ℃, and the above step was repeated once more. The centrifuged E. coli The pellet was thoroughly dissolved in 100 mL of Buffer S30B and 2 mM 1,4-dithiothreitol (DTT), and then centrifuged at 3500 xg for 15 minutes at 4 ℃. E. coli The pellet wet mass was weighed, rapidly cooled with liquid nitrogen, and stored at -80 ℃.

[0073] Example 1-2. Production of cell extract

[0074] stored E. coli The pellet was removed and thawed on ice for about 10 minutes. E. coli Dissolve 1 g of wet pellet in 0.9 mL of Buffer S30B, and the dissolved E. coli The material was crushed four times at 25,000 psi using an EmulsFlex-C3 homogenizer. The crushed material above E. coli The cell suspension was centrifuged at 30,000 xg for 30 minutes at 4°C, and the supernatant was centrifuged once more under the same conditions. After centrifugation, a run-off reaction was performed at 250 rpm at 37°C for 0, 30, 60, 90, and 120 minutes. When a run-off reaction was performed, the sample was centrifuged at 15,000 xg for 15 minutes at 4°C; when no run-off reaction was performed, centrifugation was omitted. Subsequently, the supernatant was dispensed into microcentrifuge tubes to the desired volume, rapidly cooled with liquid nitrogen, and stored at -80°C.

[0076] Example 2. Cell-free protein synthesis

[0077] Stored at -80 ℃ E. coli The lysis solution, energy solution, HMP, and amino acid solution were removed, thawed at room temperature, and immediately placed on ice. The DNA (pRSET-SG-sfGFP) and essential inorganic cations (ammonium, potassium, magnesium) stored at -20 ℃ were removed, thawed at room temperature, and immediately placed on ice. Then, E. coli The lysate, energy solution, HMP, amino acid solution, and essential inorganic cations were mixed, and finally, DNA was added. After that, the CFPS reaction was started in a shaking incubator under the conditions of Table 1 below.

[0078] condition Shaking ammonium potassium magnesium Run-off reaction time PEG-8000 CFPS response time temperature HMP DNA rpm mM mM mM minute % hour o C mM nM 1 0 ~ 300 6 70 10.7 0 2 18 29 0.565 6 2 300 0 ~ 10 70 10.7 0 2 18 29 0.565 6 3 300 6 20 ~ 150 10.7 0 2 18 29 0.565 6 4 300 6 70 6.7 ~ 16.7 0 2 18 29 0.565 6 5 300 0 70 10.7 0 ~ 120 2 18 29 0.565 6 6 300 0 70 10.7 0 0 ~ 2 18 29 0.565 6 7 300 0 70 10.7 0 0 0 ~22 18 ~ 29 0.565 6 8 300 0 70 10.7 0 0 18 18 ~ 35 0.565 6 9 300 0 70 10.7 0 0 18 29 0 ~ 1.553 6 10 300 0 70 10.7 0 0 18 29 0.565 2 ~ 26

[0080] The volume of the reaction solution is 12 μL, and the specific composition for cell-free protein synthesis is shown in Table 2 below.

[0081] composition Final concentration volume Stock concentration BL21 T7 cell extract 33.3333% 4μL 100% A mixture of 20 amino acids 70mM 3.135μL 268mM Plasmid DNA 6nM 2μL 72nM HMP 0.56mM 0.2μL 96mM K-glutamate 50mM 0.323μL 1860mM Mg-glutamate 6mM 0.129μL 186mM Energy Solution HEPES-KOH 50mM 2μL 2,000mM ATP 1.5mM 100mM GTP 0.9mM 100mM CTP 0.9mM 100mM UTP 0.9mM 100mM tRNA 0.2 mg / ml 20mg / ml CoA 0.26mM 20mM NAD 0.33mM 30mM cAMP 0.75mM 91.12724mM folinic acid 0.068mM 20mM spermidine 1mM 1000mM maltodextrin 35mM 660mM DIW - 0.213μL - Total - 12μL -

[0083] Example 3. Evaluation of protein synthesis ability

[0084] To evaluate the protein synthesis ability of the cell-free protein synthesis method of the present invention, the sfGFP expression rate was measured.

[0086] Example 3-1. sfGFP standard curve experiment for measuring super folder GFP (sfGFP) expression rate

[0087] pET28a-sfGFP plasmid E. coli The BL21 (DE3) strain was transformed and plated on LB agar. The transformed E. coli A single colony was inoculated into 50 mL of LB medium in a 250 mL flask at 37°C at a speed of 200 rpm for 16 hours. The cultured E. coli5 mL was transferred to 500 mL of LB medium in a 2 L flask, and after incubating at 37°C at a speed of 260 rpm until the OD600 reached 0.6, IPTG was added to a concentration of 0.84 mM. Then, expression was induced at 37°C at a speed of 260 rpm until the OD600 reached 4.0. The induced E. coli was centrifuged at 5,000 xg for 15 minutes at a temperature of 4 ℃. The centrifuged E. coli The pellet was completely dissolved in 30 mL of buffer solution S (150 mM NaCl, 50 mM Tris-HCl, pH 7.0), and the dissolved E. coli was fragmented using sonication (Branson, USA). The fragmented E. coli It was centrifuged at 30,000 xg for 30 minutes at a temperature of 4 ℃. 700 μL Ni 2+ -NTA (Qiagen, USA) resin was added, and a gentle mixing reaction was carried out at 4°C for 2 hours. Next, the mixture was poured into a Gravity empty column (Bio-Rad, USA), packed, and washed twice with 5 mL of buffer solution S containing 25 mM imidazole. Then, 5 mL of buffer solution S containing 250 mM imidazole was added for elution. The eluted protein (sfGFP) was dialyzed three times with 3 mL of buffer solution S using an amicon ultra centrifugal filter unit (10 MWCO). Finally, the absorbance of the purified sfGFP was measured at 485 nm using a UV-VIS spectrophotometer, and the extinction coefficient of sfGFP (833,300 M) -1 cm -1The concentration was calculated using the protein size (29 kDa). 20 μL aliquots of sfGFP at various concentrations were transferred to a black 384-well plate, and the fluorescence intensity (FI) of sfGFP was measured using a 2300 EnSpire Multimode Plate Reader (PerkinElmer, USA) (Excitation: 485 nm, Emission: 540 nm). A standard curve of fluorescence intensity against sfGFP concentration was calculated.

[0089] Example 3-2. Quantification of sfGFP concentration expressed after cell-free protein synthesis

[0090] The expression rate of cell-free protein synthesis was quantified according to the sfGFP concentration. After the cell-free protein synthesis reaction, 400 μL of buffer solution S was added and mixed, and 20 μL of the diluted solution was transferred to a black 384-well plate. Fluorescence intensity was measured using a 2300 EnSpire Multimode Plate Reader, and the sfGFP concentration was quantified using a standard curve function. Independent experiments (n = 3–6) were conducted, and the mean and standard deviation were calculated.

[0092] [Experimental Example]

[0093] Experimental Example 1. Confirmation of protein synthesis rate according to cell-free protein synthesis method

[0094] Experimental Example 1-1. Comparison of protein synthesis rates with and without shaking

[0095] In a cell-free protein synthesis method, E. coli Since the protein expression rate varies depending on the oxygen concentration when culturing, it was determined that the oxygen concentration is important. Therefore, when culturing a mixture of cell extract and a cell-free protein synthesis reaction composition, the protein synthesis rate was confirmed with or without shaking.

[0096] As a result, as shown in Figure 1, it was confirmed that the protein synthesis rate of the group cultured with shaking was higher than that of the group cultured without shaking, and that the protein synthesis rate increased when shaking was performed compared to when shaking was not performed, and that the protein synthesis rate increased significantly starting from 100 rpm. In particular, it was confirmed that at a speed of 300 rpm, the synthesis rate increased by approximately 2.3 times compared to 0 rpm.

[0098] Experimental Example 1-2. Comparison of protein synthesis rates according to run-off reaction time

[0099] In general, in cell-free protein synthesis, the run-off reaction is essential as a process for removing ribosome mRNA contained in E. coli lysates.

[0100] The present invention is characterized by not performing a run-off reaction in a cell-free protein synthesis method, and compared the protein synthesis rate according to the run-off reaction time.

[0101] As a result, as shown in Figure 2, the protein synthesis rate decreased as the reaction time of the run-off reaction increased, and it was confirmed that the protein synthesis rate was highest in the method that does not perform the run-off reaction.

[0103] Experimental Example 1-3. Confirmation of protein synthesis rate according to reaction temperature and time

[0104] In the cell-free protein synthesis method, the protein synthesis rate according to temperature and time was verified to find the optimal temperature and reaction time when the cell extract was mixed with the cell-free protein synthesis reaction composition and cultured.

[0105] As a result, as shown in Figure 3, it was confirmed that culturing at 29°C for 18 hours is the optimal condition for protein expression.

[0107] Experimental Example 2. Confirmation of protein synthesis rate according to cell-free protein synthesis reaction composition

[0108] Experimental Example 2-1. Comparison of Protein Synthesis Rates According to Inorganic Cation Concentration

[0109] The efficiency of cell-free protein synthesis can vary depending on the concentration of inorganic cations, and it is known that ammonium ions, potassium ions, and magnesium ions are particularly required.

[0110] In this invention, the protein synthesis rate was checked at different concentrations to determine the optimal concentration of essential inorganic cations.

[0111] As a result, as shown in Figure 4, it was confirmed that a sufficient protein synthesis rate was achieved even without the addition of ammonium ions.

[0112] As shown in Figure 5, it was confirmed that there is no significant difference in the synthesis rate depending on the concentration of potassium ions.

[0113] In addition, as shown in Figure 6, the highest protein synthesis rate was observed at magnesium ions of 8.7 to 14.7 mM, and the optimal concentration of magnesium ions as a coenzyme was determined to be 10.7 mM.

[0115] Experimental Example 2-2. Comparison of protein expression rates according to PEG-8000 concentration

[0116] It is known that in cell-free protein synthesis, an appropriate crowding effect is required, and thus, adding PEG-8000 stabilizes cell-free protein synthesis.

[0117] Accordingly, the protein synthesis rate was checked according to the concentration of PEG-8000.

[0118] As a result, as shown in Figure 7, it was confirmed that the protein expression rate was highest under conditions not containing PEG-8000.

[0120] Experimental Example 2-3. Comparison of Protein Synthesis Rates According to HMP Concentration

[0121] In cell-free protein synthesis, HMP is an energy source that supplies orthophosphate (iP), which is essential for energy metabolism. To determine the optimal concentration of the cell-free protein synthesis reaction composition of the present invention, the protein synthesis rate according to concentration was confirmed.

[0122] As a result, as shown in Figure 8, HMP showed a maximum synthesis rate at 0.42 to 0.84 mM, and the optimal concentration was determined to be 0.56 mM.

[0124] Experimental Example 2-4. Comparison of protein synthesis rates according to DNA concentration

[0125] In cell-free protein synthesis, since the protein synthesis rate varies depending on the DNA concentration, the protein synthesis rate according to concentration was verified to determine the optimal concentration of the cell-free protein synthesis reaction composition of the present invention.

[0126] As a result, as shown in Figure 9, it was confirmed that the optimal concentration of DNA is 6 nM.

[0128] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments and experimental examples described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 A method for synthesizing a cell-free protein, comprising: (a) a step of culturing and lysing bacteria in a medium; (b) a step of centrifuging the lysed bacterial cell solution at 15,000xg to 35,000xg and obtaining a supernatant to produce a bacterial cell extract; and (c) a step of adding the cell extract to a cell-free protein synthesis reaction composition comprising a protein synthesis energy source comprising maltodextrin at a concentration of 20mM to 50mM and culturing while shaking, wherein step (b) is characterized by not performing a run-off reaction or additionally performing a run-off reaction of 30 minutes or less. Claim 2 In paragraph 1, the bacteria E. coli A method characterized by being. Claim 3 A method according to claim 1, characterized in that the maltodextrin has a concentration of 35 mM. Claim 4 A method according to claim 1, characterized in that the protein synthesis energy source further comprises one or more selected from the group consisting of HEPES, NTP, tRNA, coenzyme A, NAD, cAMP, polynic acid, and spermidine. Claim 5 The method according to claim 1, characterized in that the composition further comprises one or more selected from the group consisting of plasmid DNA, an amino acid mixture, sodium hexametaphosphate (SHMP), potassium glutamate, and magnesium glutamate. Claim 6 A method according to claim 5, wherein the amino acid mixture is one or more mixtures selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, cysteine, methionine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, and glutamine. Claim 7 A method according to claim 1, characterized in that the composition does not contain polyethylene glycol or ammonium ions. Claim 8 A method according to claim 1, characterized in that the composition does not contain polyethylene glycol or additionally contains polyethylene glycol at a concentration of 0.5% or less. Claim 9 delete Claim 10 A method according to claim 1, wherein step (c) is characterized by culturing while shaking at a speed of 100 rpm to 300 rpm. Claim 11 The method according to claim 1, wherein step (c) is characterized by incubating at a temperature of 18°C ​​to 35°C for 15 to 22 hours. Claim 12 A cell-free protein synthesis kit used in the method described in any one of claims 1 to 8, 10 and 11, comprising a bacterial cell extract, plasmid DNA, an amino acid mixture, a protein synthesis energy source comprising maltodextrin at a concentration of 20 mM to 50 mM, sodium hexametaphosphate (SHMP), and a salt.

Images