Cell-free protein synthesis method using photothermal effect

Abstract

The present invention relates to a cell-free protein synthesis method using a photothermal effect, wherein gold nanorods (GNR) having photothermal properties are introduced into a CFPS reaction mixture to induce a rapid and intensive local increase in temperature required for cell-free protein synthesis. The method of the present invention can improve protein yield within a shorter reaction time than a conventional incubation method. By using a PDMS well plat, the method allows for portable and efficient protein synthesis in various environments.

Description

Cell-free protein synthesis method using photothermal effect

[0001] The present invention relates to a cell-free protein synthesis method utilizing the photothermal effect. This invention was developed with support from the Defense Acquisition Program Administration's "Future Challenge Defense Technology Research and Development" project (Project No. UI220005TD), which focuses on the development of a multi-analysis technology for biological agents through the convergence of cell-free synthetic biology and artificial intelligence.

[0002] Amid growing global concerns about climate change and environmental pollution, the shift toward more sustainable solutions is accelerating. Biomanufacturing is emerging as an environmentally friendly and energy-efficient alternative to traditional chemical processes. Driven by advances in synthetic biology, which introduces new pathways, it is expected that in the near future, most of the chemical and biochemical products essential to modern civilization will be produced through biological means.

[0003] A key trend in modern biomanufacturing, particularly within the medical industry, is the shift from centralized mass production to decentralized, customized production of diverse products in smaller batches. In this context, cell-free protein synthesis (CFPS) technology is emerging as a valuable tool for the portable, on-demand production of recombinant proteins and other compounds. Using preassembled biosynthetic machinery and template DNA, it can bypass the complex steps required for traditional biomanufacturing processes, such as cloning and culturing. The inherent characteristics of CFPS allow for easy scalability of production, depending on the target product, without the need for large-scale facilities. Potential applications for CFPS technology include remote missions to battlefields, disaster zones, space, or deep-sea environments, all of which are ineffective for traditional supply chains. In these environments, preassembled reaction mixtures for cell-free protein synthesis, along with the DNA encoding the desired protein, can be readily transported. Simply mixing the reaction mixture with the target DNA allows for the synthesis of the desired protein within hours. However, several challenges remain to be addressed to fully utilize this technology, with the development of an effective heating method being paramount. Like many biochemical reactions, CFPS synthesis requires a specific temperature, typically ranging from 30 to 37°C. Laboratory-based CFPS reactions typically use water baths to maintain this temperature range, but this approach is impractical for decentralized, local production.

[0004] Meanwhile, as a technology related to cell-free protein synthesis, Korean Patent No. 0733712 discloses a method for preparing a cell extract for cell-free protein synthesis and a method for synthesizing proteins using the same. However, no technology has been disclosed yet regarding a cell-free protein synthesis method utilizing the photothermal effect of the present invention.

[0005] The present invention was derived from the above-mentioned needs, and provides a cell-free protein synthesis method utilizing a photothermal effect, and can increase the temperature of a reaction solution to a temperature of 30 to 37°C required for cell-free protein synthesis by using a PEG-GNRs (polyethylene glycol-conjugated gold nanorods) complex capable of exhibiting a photothermal effect, and has completed the present invention by confirming that not only is cell-free protein synthesis possible with the method of the present invention, but also proteins can be synthesized at a faster rate than with existing methods.

[0006] In order to achieve the above purpose, the present invention comprises the steps of (1) adding a cell-free protein synthesis reaction mixture containing a PEG-GNRs (polyethylene glycol-conjugated gold nanorods) solution, a cell extract, and a template DNA of a target protein to a PDMS (polydimethylsiloxane) well plate;

[0007] (2) After the above step (1), a method for synthesizing cell-free proteins using a photothermal effect is provided, including a step of irradiating a laser onto a well of a PDMS well plate to raise the temperature of the PDMS well plate to 30 to 37°C and synthesizing cell-free proteins for 2 to 4 hours.

[0008] In addition, the present invention provides a cell-free protein synthesis kit utilizing the photothermal effect, which includes a cell-free protein synthesis reaction mixture containing a PEG-GNRs (polyethylene glycol-conjugated gold nanorods) solution, a PDMS (polydimethylsiloxane) well plate loaded with a cell extract and a template DNA of a target protein, and a battery-powered portable laser module.

[0009] The present invention relates to a cell-free protein synthesis method utilizing the photothermal effect. Direct heat generation through photothermal energy conversion within a cell-free protein synthesis solution induces a rapid and concentrated local temperature increase. Therefore, a large amount of protein can be produced within a shorter reaction time than conventional incubation methods, and since it can be utilized as a portable cell-free synthesis system using a PDMS well plate, the photothermal effect-mediated cell-free protein synthesis (CFPS) system has the effect of expanding the scope of existing CFPS applications.

[0010] Figure 1 shows the results confirming the temperature dependence of cell-free protein synthesis.

[0011] Figure 2 is a schematic diagram of a portable photothermal cell-free protein synthesis system.

[0012] Figure 3 illustrates a PEG-GNR and portable laser module-based investigation system. (A) UV / Vis absorption spectrum of PEG-GNR, (B) transmission electron microscopy image of PEG-GNRs, and (C) battery-powered portable laser module and PDMS well plate used for GNR-mediated photothermal heating.

[0013] Figure 4 shows the photothermal characteristics of a PEG-GNR solution in a PDMS well plate. (A) This is the result of confirming the degree of temperature increase according to the PEG-GNR concentration and reaction time upon laser irradiation, and (B) this shows the plateau temperature of the plate in the PEG-GNR solution according to the concentration.

[0014] Figure 5 shows the results of confirming the temperature increase effect of the PEG-GNR solution in the PDMS well plate according to the heating method. (A) A photograph of a PDMS well plate, (B) A thermal photograph of a PDMS well plate when photothermal heating is performed at 30°C, where the dash line indicates the area irradiated using a portable laser module, (C) A thermal photograph of a PDMS well plate in an incubator at 30°C, and (D) A comparison of the temperature increase for photothermal heating and incubator heating.

[0015] Figure 6 shows the photothermal effect of PEG-GNR on cell-free protein synthesis, with an OD of 0.33 810 Comparison of cell-free protein synthesis reactions of sfGFP with GNRs, heated to 30°C, or performed in an incubator at 30°C without laser irradiation. Error bars represent the standard deviation of three independent experiments.

[0016] Figure 7 compares the efficiency of cell-free protein synthesis using photothermal heating and the conventional incubator heating method. (A) shows the result of comparing the fluorescence signal of sfGFP synthesized cell-free protein using photothermal heating by room temperature, 30℃ incubator, and PEG-GNR, and (B) shows the result of quantifying the fluorescence signal of sfGFP synthesized cell-free protein using photothermal heating by PEG-GNR in (A). The results are expressed as the mean ± SD, and statistical analysis was performed using two-way ANOVA, Tukey (A) or Sidak (B) multiple comparison test using Prism 8.4 (GraphPad Software) (*p<0.05; **p<0.01; ***p<0.001, ****p<0.0001).

[0017] In order to achieve the object of the present invention, the present invention comprises the steps of (1) adding a cell-free protein synthesis reaction mixture containing a PEG-GNRs (polyethylene glycol-conjugated gold nanorods) solution, a cell extract, and a template DNA of a target protein to a PDMS (polydimethylsiloxane) well plate;

[0018] (2) A method for synthesizing cell-free proteins using a photothermal effect, comprising the step of, after the above step (1), irradiating a laser to a well of a PDMS well plate to raise the temperature of the PDMS well plate to 30 to 37°C and synthesizing cell-free proteins for 30 to 90 minutes.

[0019] The above cell-free protein synthesis reaction mixture preferably includes, but is not limited to, HEPES-KOH; tRNA mixture; cAMP; magnesium acetate; potassium glutamate; ammonium acetate; DTT; ATP; CTP, GTP, and UTP; folinic acid; 20 amino acids; creatine phosphate; creatine kinase; and PEG (polyethylene glycol).

[0020] The above cell extract is preferably an S12 cell extract extracted from E. coli BL21Star (DE3) (see Korean Patent No. 10-1841081), and may be an extract of one or more cells selected from among Bacillus subtilis, wheat germ, rice germ, barley germ, CHO cells, hybridoma cells, and reticulocytes, but is not limited thereto.

[0021] In the above step (2), the laser irradiation is preferably, but not limited to, laser irradiation by a 300 mW dot beam diode laser module of 808 nm near infrared (NIR) or a battery-powered portable laser module.

[0022] The zeta potential of the above PEG-GNRs is characterized by being -3.5±0.4 mV.

[0023] The present invention relates to a cell-free protein synthesis kit utilizing a photothermal effect, which comprises a cell-free protein synthesis reaction mixture containing a PEG-GNRs (polyethylene glycol-conjugated gold nanorods) solution, a PDMS (polydimethylsiloxane) well plate loaded with a cell extract and a template DNA of a target protein, and a battery-powered portable laser module.

[0024]

[0025] Hereinafter, the present invention will be described in more detail using examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0026]

[0027] 1. Preparation of materials

[0028] ATP, GTP, UTP, CTP, creatine phosphate, creatine kinase, and tRNA mixture of Escherichia coli (E. coli) strain MRE600 were supplied by Roche Applied Sciences (Indianapolis, IN, USA). PCR reagents were obtained from Violin (London, UK), and restriction enzymes and T4 DNA ligase were obtained from Engenomics (Daejeon, Korea). L-[U- 14C] Leucine was purchased from PerkinElmer (Waltham, MA, USA). Poly(ethylene glycol)methyl ether thiol (MPEG, average Mn 2,000) was purchased from Raysan Bio (Alab, AL, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). S12 extract was prepared from Escherichia coli BL21Star (DE3) strain according to a previously reported procedure (see Korean Patent No. 10-1841081).

[0029]

[0030] 2. Preparation and characterization of polyethylene glycol-conjugated gold nanorods (PEG-GNR)

[0031] PEG-GNRs were prepared from cetyl trimethyl ammonium bromide-stabilized GNRs (CTAB-GNRs) by exchanging CTAB with methoxy-PEG-thiol (mPEG-thiol). CTAB-GNRs were synthesized using a previously known 'seed-mediated growth method.'

[0032] The seed solution was prepared by mixing 5 ml of 0.5 mM HAuCl4 and 5 ml of 0.2 M CTAB solution and sonicating. During sonication, 600 μl of ice-cold 0.01 M NaBH4 was added to the mixture and sonicated for an additional 4 minutes at 28°C. The seed solution was then incubated at 28°C for 2 hours. The growth solution was prepared by sequentially adding 2.6 ml of 4 mM AgNO3, 50 ml of 1 mM HAuCl4, and 700 μl of 78.8 mM ascorbic acid to 50 ml of 0.2 M CTAB solution. After gentle mixing, 120 μl of seed solution was added to the mixture to initiate growth and incubated at 28°C for at least 1 hour.

[0033] CTAB-GNRs were centrifuged at 10,000×g for 25 minutes. The supernatant was discarded, and CTAB-GNRs were resuspended in distilled water. 25 mg of mPEG-thiol was added to 1 mL of the CTAB-GNRs solution, and the solution was stirred vigorously overnight. PEG-GNRs were then prepared by centrifuging three times at 10,000×g for 25 minutes each to remove unincorporated mPEG-thiol.

[0034] The optical spectra of GNRs were recorded using a UV / Vis spectrophotometer (SpectraMax Plus 384, Molecular Devices, Sunnyvale, USA), and the zeta potential of GNRs was measured using dynamic light scattering (DLS; Zetasizer Nano ZS90, Malvern Instruments, Malvern, UK). The morphology and size distribution of GNRs were investigated using transmission electron microscopy (TEM; 200 kV, JEOL Ltd., Tokyo, Japan).

[0035]

[0036] 3. Cell-free protein synthesis and analysis

[0037] For cell-free protein synthesis (CFPS), the standard reaction mixture contained 57 mM HEPES-KOH (pH 8.2); 1.2 mM ATP; 0.85 mM each of CTP, GTP, and UTP; 2 mM DTT; 0.17 mg / mL E. coli total tRNA mixture; 0.64 mM cAMP; 90 mM potassium glutamate; 80 mM ammonium acetate; 12 mM magnesium acetate; 34 μg / mL L-5-formyl-5,6,7,8-tetrahydrofolic acid (folinic acid); 1.0 mM each of 20 amino acids; 2% (w / v) polyethylene glycol 8000; 67 mM creatine phosphate; 3.2 μg / mL creatine kinase; 10 μM L-[U- 14C]leucine (11.9 GBq / mmol); 6.7 μg / ml pK7SFGFP (Superfolder Green fluorescent protein, sfGFP); 4 μl of S12 cell extract from Escherichia coli BL21Star (DE3).

[0038] The standard CFPS reaction was performed in a water bath at 30°C, and CFPS was performed at λ excitation = 472 nm, λ emission The fluorescence of the reaction solution was monitored using a Gemini XPS fluorescence spectrophotometer (Molecular Devices, Sunnyvale, USA) set to 507 nm. For quantitative analysis of the synthetic protein, a liquid scintillation counter (Wallac 1410, Perkinelmer, Waltham, MA, USA) was used to precipitate the protein with trichloroacetic acid. 14 The radioactivity of C-leucine was measured.

[0039] For the photothermal cell-free synthesis reaction, 10 μl of the reaction mixture containing PEG-GNRs was loaded into each well of a PDMS (polydimethylsiloxane) well plate, and the cell-free protein synthesis reaction was performed under the corresponding conditions.

[0040] To induce photothermal heating, a near-infrared (NIR) 808 nm 300 mW dot beam diode laser module (Laser Lab, Yongin, Korea) was irradiated to one well within a PDMS well plate. The surface temperature of the laser-irradiated PDMS was monitored using a thermal conductivity infrared camera (FLIR, Wilsonville, USA) to confirm the photothermal effect.

[0041]

[0042] Example 1. Temperature dependence of cell-free protein synthesis

[0043] The efficiency of CFPS is significantly affected by reaction temperature. As shown in Figure 1, the rate and duration of CFPS varied depending on the temperature of the bath for the standard reaction mixture. Compared to 30°C, the temperature commonly used for cell-free protein synthesis, low reaction temperatures significantly reduced the initial rate of sfGFP synthesis, resulting in very low fluorescence in the reaction mixture.

[0044] When considering systems for on-site protein production, the temperature dependence of CFPS efficiency is a noteworthy factor. To enable protein synthesis at ambient room temperature (approximately 25°C) without supplemental heating, we sought to perform protein synthesis by internally heating the reaction mixture using light in a compact device.

[0045] Gold nanorods (GNRs) with longitudinal plasmonic absorption in the near-infrared (NIR) window are promising photoactivatable nanomaterials for biological applications. They efficiently convert absorbed light, primarily in the NIR region, into heat through nonradiative decay, providing localized temperature control. The size and aspect ratio of these GNRs can be tuned to enable light absorption at specific wavelengths, making them suitable for a variety of applications.

[0046] Furthermore, when coated with a stabilizer such as polyethylene glycol (PEG), GNRs exhibit excellent colloidal stability and minimal protein adsorption in biological solutions. Taking advantage of these properties, the present invention employed photoactivatable PEG-GNRs as an internal heating source during the CFPS reaction (Fig. 2).

[0047]

[0048] Example 2. Characterization of PEG-GNR

[0049] CTAB-stabilized GNRs (CTAB-GNRs) with a longitudinal absorption peak at 810 nm were synthesized using a seed-mediated growth method as described in Materials and Methods.

[0050] Colloidal stability was improved by exchanging the surface stabilizer CTAB with thiolated PEG, minimizing interactions with components within the cell-free synthesis reaction mixture. Replacing CTAB with PEG stabilized GNRs, as evidenced by a decrease in surface charge from +30.1±8.0 mV (CTAB-GNRs) to -3.5±0.4 mV (PEG-GNRs) (Table 1).

[0051] S1 zeta potential of CTAB-GNRs and PEG-GNRs. Sample zeta potential (mV) CTAB-GNR + 30.1 ± 8.0 PEG-GNRs - 3.5 ± 0.4

[0052] PEG-GNRs exhibited a longitudinal absorption peak at approximately 810 nm and a high longitudinal-to-transverse surface plasmon resonance peak ratio suitable for photothermal applications (Fig. 3A). Transmission electron microscopy (TEM) of PEG-GNRs revealed a rod-shaped morphology with an aspect ratio of 4.3 (Fig. 3B).

[0053] Example 3. Portable laser module-based investigation system

[0054] To commercialize CFPS, a simple laser module-based irradiation system was established as a light source. A battery-powered portable 808 nm laser module with a diameter of 22 mm and a length of 65 mm was used to induce the photothermal effect of PEG-GNR, which has a longitudinal absorption peak wavelength of 810 nm (Fig. 3C).

[0055] Additionally, photothermal CFPS reactions were performed using polydimethylsiloxane (PDMS) well plates with a well diameter of 3 mm (Figs. 3C and 5A). PDMS well plates, widely used in microfluidic point-of-care devices, can be easily combined with portable laser modules to implement CFPS in resource-constrained environments.

[0056]

[0057] Example 4. Photothermal effect of PEG-GNR on PDMS well plate

[0058] In this Example 4, the photothermal capabilities of PEG-GNRs were investigated on PDMS well plates. To determine the degree of temperature increase according to PEG-GNR concentration, each well containing different concentrations of PEG-GNRs was illuminated with a portable laser module while monitoring the solution surface temperature of the well using a thermal imaging infrared camera.

[0059] As a result, as disclosed in Fig. 4, the wells containing PEG-GNRs showed a temperature increase upon laser irradiation. The maximum temperature achieved through photothermal heating was 3.3 OD. 810 It was shown in PEG-GNR, and the degree of temperature increase varied depending on the GNR concentration.

[0060] Considering that the preferred temperature for CFPS is in the range of 30 to 37°C, in the present invention, PEG-GNRs 0.33 OD is used as an internal heating source to maintain the reaction temperature for CFPS at 30°C. 810 was used.

[0061] Photothermal heating offers several advantages over conventional heating methods, such as incubators, water baths, and heating blocks. These conventional heating methods all employ indirect heating, transferring heat energy through a container. In contrast, photothermal heating rapidly increases solution temperature by generating heat energy within the solution. To determine whether photothermal heating increases solution temperature more rapidly than conventional heating methods, the entire well plate was heated in an incubator at 30°C while irradiating with a laser module or monitoring the temperature using a thermal imaging infrared camera (Figure 5). The thermal images showed that while the entire PDMS well plate was heated in the incubator, a temperature increase was observed only in the laser-irradiated wells (Figures 5B and 5C). Furthermore, as expected, the temperature profile confirmed that photothermal heating increased the solution temperature within the well to 30°C more rapidly than incubator heating (Figure 5D).

[0062]

[0063] Example 5. Photothermal cell-free protein synthesis

[0064] Finally, we investigated the efficacy of CFPS via photothermal heating. Superfolder green fluorescent protein (sfGFP) was used as a reporter protein because protein synthesis can be easily monitored by fluorescence. We first tested whether the presence of PEG-GNR interfered with the translational activity of the reaction mixture.

[0065] 0.33 OD 810CFPS reaction mixtures containing or without PEG-GNR were incubated at 30°C for 30 min without laser irradiation. Fluorescence measurements of the mixtures showed that although the fluorescence intensity was slightly reduced in the reaction mixture containing PEG-GNR, PEG-GNR did not significantly affect the efficiency of protein synthesis (Fig. 6). This decrease in the presence of PEG-GNR is probably due to the absorption of the excitation and emission light of SFGFP by PEG-GNR.

[0066] Subsequently, the CFPS efficacy through photothermal heating at 30°C was compared with that when heated in a temperature-controlled incubator at 30°C and when left unheated at room temperature (~24°C). Fluorescence measurements of the reaction mixtures revealed significantly higher fluorescence in the photothermally heated wells from 20 minutes after irradiation compared to the wells heated in the incubator and wells heated at room temperature (Fig. 7A). The fluorescence signal of the photothermally heated wells was 1.60-fold higher at 20 minutes and 1.63-fold higher at 30 minutes than that of the wells heated in the incubator. The CFPS efficacy at room temperature was less than half that of the reaction mixture heated at 30°C. Subsequently, we quantified the amount of sfGFP synthesized through both photothermal heating and incubator heating using a radioactive protein label. The photothermal heating of the reaction mixture was confirmed to be 334.11 μg / mL and 454.78 μg / mL of sfGFP at 20 and 30 minutes after irradiation, respectively, which were 5.6- and 3.3-fold higher, respectively, than when heated in an incubator. In summary, the above results suggest that GNR-mediated photothermal heating can accelerate CFPS by rapidly increasing the temperature of the mixture during the initial reaction.

Claims

1. (1) A step of adding a cell-free protein synthesis reaction mixture containing a PEG-GNRs (polyethylene glycol-conjugated gold nanorods) solution, a cell extract, and a template DNA of a target protein to a PDMS (polydimethylsiloxane) well plate; (2) A method for synthesizing cell-free proteins using a photothermal effect, comprising: after the above step (1), irradiating a laser onto a well of a PDMS well plate to increase the temperature of the PDMS well plate to 30 to 37°C, and synthesizing cell-free proteins for 30 to 90 minutes.

2. A cell-free protein synthesis method utilizing the photothermal effect in claim 1, characterized in that the cell-free protein synthesis reaction mixture is a mixture containing HEPES-KOH; tRNA mixture; cAMP; magnesium acetate; potassium glutamate; ammonium acetate; DTT; ATP; CTP, GTP and UTP; folinic acid; 20 kinds of amino acids; creatine phosphate; creatine kinase; and PEG (polyethylene glycol).

3. A cell-free protein synthesis method utilizing the photothermal effect, characterized in that in paragraph 1, the cell extract is an extract of one or more cells selected from Escherichia coli, Bacillus subtilis, wheat germ, rice germ, barley germ, CHO cells, hybridoma cells, and reticulocytes.

4. A cell-free protein synthesis method utilizing the photothermal effect, characterized in that in the third paragraph, the cell extract is an S12 cell extract extracted from E. coli BL21Star (DE3).

5. A cell-free protein synthesis method utilizing the photothermal effect, characterized in that in the first paragraph, the laser irradiation in the step (2) is laser irradiation by a near-infrared (NIR) 300 mW dot beam diode laser module or a battery-powered portable laser module.

6. A cell-free protein synthesis method utilizing the photothermal effect, characterized in that in the first paragraph, the zeta potential of the PEG-GNRs is -3.5±0.4 mV.

7. A cell-free protein synthesis kit utilizing the photothermal effect, comprising a cell-free protein synthesis reaction mixture containing a PEG-GNRs (polyethylene glycol-conjugated gold nanorods) solution, a PDMS (polydimethylsiloxane) well plate loaded with a cell extract and a template DNA of a target protein, and a battery-powered portable laser module.

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