Cell-free protein synthesis system and application thereof

Abstract

The invention belongs to the technical field of bioengineering and enzyme engineering, and particularly relates to a cell-free protein synthesis system and application thereof.The cell-free protein synthesis system comprises a micro-fluidic chip reaction module used for adopting a micro-fluidic chip made of a polydimethylsiloxane material, the micro-fluidic chip integrates 1000-2000 independent micro-channels, and the micro-fluidic chip reaction module is connected with the micro-fluidic chip reaction module; each micro-channel serves as a protein synthesis chamber and an enzyme activity detection chamber at the same time, the inner wall of each micro-channel is modified with a hydrophilic coating, and an inlet of the micro-fluidic chip is of a double-channel liquid injection structure. By arranging the micro-fluidic chip reaction module integrated with 1000-2000 independent micro-channels and a double-channel liquid injection structure capable of synchronously injecting a cell-free reaction liquid and a substrate-hydrotropy system, the integrated connection of protein synthesis and enzyme activity detection is realized, the operation steps of centrifugation, enzyme liquid transfer and the like in a traditional scheme are omitted, and the detection efficiency is improved. And the screening process is simplified.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and enzyme engineering technology, specifically to a cell-free protein synthesis system and its applications. Background Technology

[0002] Cell-free protein synthesis systems (CFPS) are technologies that achieve efficient expression of exogenous genes by directly utilizing transcription-translation machinery (such as ribosomes, enzymes, and coenzymes) in cell extracts without relying on living cells. Compared to traditional live-cell expression systems, CFPS has advantages such as a short reaction cycle (usually 2-8 hours), the ability to synthesize toxic / hydrophobic proteins, and easily controllable reaction conditions, and has become an important tool for in vitro protein synthesis.

[0003] In vitro enzyme catalysis screening is a core component of enzyme engineering, widely used in industrial enzyme modification (e.g., improving the thermostability of lipases) and drug-metabolizing enzyme screening (e.g., screening for cytochrome P450 enzyme subtypes). Currently, the combination of CFPS and in vitro enzyme catalysis screening has become a research hotspot (e.g., as seen in patent literature).

[0004] CN114019169A aims to establish a rapid and simple method for quantitative detection of target protein yield in a CFPS system and for rapid screening of highly catalytically active enzyme proteins. However, existing technologies still have significant limitations and cannot meet the requirements for efficient and accurate screening, as detailed below:

[0005] 1. Low efficiency in reaction and screening integration, and cumbersome operation: Traditional methods require two steps: first, the target enzyme is synthesized in CFPS, then the enzyme solution is separated through centrifugation, ultrafiltration, and other steps, and finally transferred to a new system for enzyme activity detection. This process involves many steps, is time-consuming, and is prone to enzyme loss.

[0006] 2. Endogenous interfering substances in cell-free systems affect detection accuracy: Cell extracts of CFPS (such as E. coli extract and rabbit reticulocyte extract) contain a large number of endogenous proteins and metabolites, which will compete with the target enzyme for substrates or coenzymes, leading to distortion of enzyme activity detection results.

[0007] 3. Poor compatibility with hydrophobic / poorly soluble substrates, resulting in incomplete reactions: The natural substrates of most industrial enzymes (such as lipases and cholesterol esterases) are hydrophobic compounds, while the CFPS system is an aqueous environment. Hydrophobic substrates are prone to forming precipitates or emulsions, which cannot fully contact the target enzyme, leading to unstable enzyme activity detection results and poor repeatability. Summary of the Invention

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0009] A cell-free protein synthesis system comprising:

[0010] The microfluidic chip reaction module is used to employ a polydimethylsiloxane microfluidic chip, which integrates 1000-2000 independent microchannels. Each microchannel serves as both a protein synthesis chamber and an enzyme activity detection chamber. The inner wall of the microchannel is modified with a hydrophilic coating, and the inlet of the microfluidic chip is designed as a dual-channel liquid injection structure.

[0011] An optimized cell-free reaction solution is used to prepare E. coli extract as a base, with added interference-inhibiting components, transcription-translation-enhancing components, buffering and stabilizing components, and magnetic affinity trapping components.

[0012] The substrate-co-solubilizing system is used to employ a cyclodextrin-ethanol complex co-solubilizing strategy, in which the hydrophobic substrate is first dissolved in 5%-10% ethanol, and then mixed with 10-20mM hydroxypropyl-β-cyclodextrin to form a water-soluble cyclodextrin-substrate inclusion complex.

[0013] The real-time detection module integrates a fluorescence detector and an absorbance detector at the outlet of the microfluidic chip to monitor the characteristic signals of the enzyme catalytic reaction in real time. The detection frequency is 1 time / 5min, and the enzyme activity change curve is dynamically recorded.

[0014] As a preferred embodiment of the cell-free protein synthesis system described in this invention, each of the microchannels is further provided with an intelligent feedback regulation submodule;

[0015] The intelligent feedback regulation submodule includes a miniature pH sensor and an ion strength sensor integrated into the sidewall of the microchannel, a regulating liquid reservoir located at the edge of the chip, and a microvalve connecting the reservoir to the microchannel; the miniature pH sensor and the ion strength sensor are electrically connected to the control unit.

[0016] As a preferred embodiment of the cell-free protein synthesis system described in this invention, the interference inhibition component includes a 0.1-0.5 mM protease inhibitor to inhibit the degradation of the target enzyme by endogenous proteases and a 1-2 U / mL specific coenzyme protective enzyme.

[0017] As a preferred embodiment of the cell-free protein synthesis system described in this invention, the transcription-translation enhancement component comprises 0.5-1 mM cyclic adenosine monophosphate and 2-5 mM betaine.

[0018] As a preferred embodiment of the cell-free protein synthesis system described in this invention, the buffer stabilizing component is a mixed buffer solution of tris(hydroxymethyl)aminomethane hydrochloride and 4-hydroxyethylpiperazine ethanesulfonic acid, and the volume ratio of tris(hydroxymethyl)aminomethane hydrochloride to 4-hydroxyethylpiperazine ethanesulfonic acid is set to 1:1.

[0019] As a preferred embodiment of the cell-free protein synthesis system described in this invention, the magnetic affinity trapping component comprises 0.5-1 mg / mL of His-tagged specific magnetic microspheres, the magnetic microspheres having a diameter of 100-200 nm, a surface modified with Ni2+-NTA groups and coated with polyethylene glycol-2000; the magnetic microspheres specifically bind to the target enzyme with a 6×His tag through His-Ni2+ interaction.

[0020] As a preferred embodiment of the cell-free protein synthesis system of the present invention, the dual-channel injection structure includes an injection port, and the microfluidic chip inlet is provided with two sets of injection ports, and an injection channel is connected between the injection ports and the microfluidic chip inlet.

[0021] An application of a cell-free protein synthesis system in in vitro enzyme catalytic screening: When the optimized cell-free reaction solution, the substrate-solubilizing system, and the target enzyme are injected into the microchannel of the microfluidic chip, an intelligent feedback regulation submodule sets the target pH and ionic strength range, controls the temperature for incubation, and simultaneously performs protein synthesis and enzyme catalytic reaction. At the same time, enzyme catalytic characteristic signals are detected in real time, positive microchannels corresponding to highly active enzymes are screened, and the reaction solution of the positive microchannels is collected. Magnetic microspheres are adsorbed by a magnetic field and eluted to recover and purify the target enzyme.

[0022] As a preferred embodiment of the application of the cell-free protein synthesis system described in this invention in in vitro enzyme catalytic screening, wherein the target enzyme carries a 6×His tag.

[0023] Compared with existing technologies:

[0024] 1. By setting up a microfluidic chip reaction module integrating 1000-2000 independent microchannels (which simultaneously serve as protein synthesis chambers and enzyme activity detection chambers), and a dual-channel injection structure that can simultaneously inject cell-free reaction solution and substrate-solubilizing system, it has the advantage of realizing the integrated connection of protein synthesis and enzyme activity detection, eliminating the operation steps such as centrifugation and enzyme solution transfer in traditional solutions, and simplifying the screening process.

[0025] 2. By setting up an optimized cell-free reaction solution containing interference-inhibiting components (protease inhibitors and specific coenzyme protective enzymes), where the protease inhibitors can inhibit the degradation of the target enzyme by endogenous proteases and the specific coenzyme protective enzymes can maintain a stable coenzyme concentration, it has the advantage of eliminating the interference of endogenous proteins and metabolites on enzyme-catalyzed reactions in cell-free systems and ensuring the authenticity and reliability of enzyme activity detection results.

[0026] 3. By setting up a substrate-solubilizing system using a cyclodextrin-ethanol composite solubilizing strategy, hydrophobic substrates are first dissolved in ethanol and then mixed with cyclodextrin to form water-soluble inclusion complexes. This has the advantages of improving the compatibility of hydrophobic / poorly soluble substrates with cell-free aqueous solutions, reducing substrate precipitation or emulsification, improving the adequacy of substrate-target enzyme contact, and reducing detection errors between different batches. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below.

[0028] This invention provides a cell-free protein synthesis system, comprising:

[0029] The microfluidic chip reaction module uses a polydimethylsiloxane microfluidic chip, which integrates 1000-2000 independent microchannels. Each microchannel serves as both a protein synthesis chamber and an enzyme activity detection chamber, and the inner wall of the microchannel is modified with a hydrophilic coating (polyethylene glycol-4000). The chip inlet is designed with a dual-channel liquid injection structure. The dual-channel liquid injection structure includes two sets of injection ports at the microfluidic chip inlet, and a liquid injection channel connects the injection ports and the microfluidic chip inlet.

[0030] Each of the microchannels is also equipped with an intelligent feedback adjustment submodule;

[0031] The intelligent feedback regulation submodule includes a miniature pH sensor (accuracy ±0.05pH) and an ion strength sensor (accuracy ±1mM) integrated into the sidewall of the microchannel, a regulating solution reservoir (containing 0.1MHCl, 0.1MNaOH, and 1MNaCl regulating solutions) located at the edge of the chip, and a microvalve connecting the reservoir to the microchannel. The miniature pH sensor and the ion strength sensor are electrically connected to the control unit so that when the pH deviates from the target range (e.g., 7.0-7.5) or the ion strength is abnormal, the control unit drives the microvalve to inject the corresponding regulating solution (single injection volume 0.5-2nL) to dynamically correct the reaction conditions.

[0032] An optimized cell-free reaction solution is used to prepare E. coli extract as a base, with added interference-inhibiting components, transcription-translation-enhancing components, buffering and stabilizing components, and magnetic affinity trapping components.

[0033] The interference-inhibiting component includes a 0.1-0.5 mM protease inhibitor (such as benzyl sulfonyl fluoride) to inhibit the degradation of the target enzyme by endogenous proteases and a 1-2 U / mL specific coenzyme protective enzyme (such as glucose dehydrogenase against NADH).

[0034] The transcription-translation enhancing component includes 0.5-1 mM cyclic adenosine monophosphate and 2-5 mM betaine;

[0035] The buffer stabilizing component is a mixed buffer solution using tris(hydroxymethyl)aminomethane hydrochloride (pH 7.5) and 4-hydroxyethylpiperazine ethanesulfonic acid (pH 7.2), and the volume ratio of tris(hydroxymethyl)aminomethane hydrochloride to 4-hydroxyethylpiperazine ethanesulfonic acid is set to 1:1.

[0036] The magnetic affinity trap component comprises 0.5-1 mg / mL of His-tagged specific magnetic microspheres, the magnetic microspheres having a diameter of 100-200 nm, a surface modified with Ni2+-NTA groups and coated with polyethylene glycol-2000; the magnetic microspheres specifically bind to the target enzyme with a 6×His tag through His-Ni2+ interaction.

[0037] The substrate-co-solubilizing system is used to employ a cyclodextrin-ethanol complex co-solubilizing strategy, in which hydrophobic substrates (such as glyceryl tribanilate and p-nitrophenyl octanoate) are first dissolved in 5%-10% ethanol, and then mixed with 10-20mM hydroxypropyl-β-cyclodextrin to form a water-soluble cyclodextrin-substrate inclusion complex.

[0038] The real-time detection module integrates a fluorescence detector and an absorbance detector at the microfluidic chip outlet to monitor characteristic signals of enzyme-catalyzed reactions in real time (such as the 450nm fluorescence generated by esterase catalyzing the fluorescent substrate 4-methylumbelliferyl octanoate, and the 405nm absorbance generated by lipase catalyzing p-nitrophenylbutyrate). The detection frequency is once every 5 minutes, and the enzyme activity change curve is dynamically recorded.

[0039] An application of a cell-free protein synthesis system in in vitro enzyme catalytic screening: When the optimized cell-free reaction solution, the substrate-solubilizing system, and the target enzyme are injected into the microchannels of the microfluidic chip, an intelligent feedback regulation submodule sets the target pH and ionic strength range, controls the temperature for incubation, and simultaneously performs protein synthesis and enzyme catalytic reaction. Enzyme catalytic characteristic signals are detected in real time, and positive microchannels corresponding to highly active enzymes are screened. The reaction solution of the positive microchannels is collected, and magnetic microspheres are adsorbed and eluted using a magnetic field to recover and purify the target enzyme; wherein the target enzyme carries a 6×His tag.

[0040] Based on the above, the present invention includes, but is not limited to, the following embodiments (taking "lipase mutant library screening" as an example):

[0041] 1. Experimental Preparation

[0042] Construct a DNA library of lipase mutants (containing 1000 mutants, each mutant DNA being a 6×His tag fusion sequence of the C-terminus of the lipase gene, at a concentration of 10 ng / μL);

[0043] Prepare an optimized cell-free reaction solution: 30% v / v E. coli extract, 2 mM ATP, 1 mM GTP, 20 amino acids (0.5 mM each), 0.5 mM cyclic adenosine monophosphate, 3 mM betaine, 0.3 mM benzyl sulfonyl fluoride, 1.5 U / mL glucose dehydrogenase, 5 mM glucose, and Tris-HCl-HEPES mixed buffer (pH 7.3). Add 0.8 mg / mL Ni2+-NTA magnetic microspheres to the reaction solution and sonicate for 10 min (100 W) to ensure uniform suspension of the microspheres (no aggregation).

[0044] Preparation of substrate-solubilizing system: Dissolve glyceryl tribanilate in 8% ethanol (10mM concentration), then mix with 15mM hydroxypropyl-β-cyclodextrin and stir for 30min to form a transparent inclusion complex solution;

[0045] Set intelligent feedback adjustment parameters: Set the target pH range to 7.2-7.4 and the target NaCl concentration to 70-80mM through the control unit; inject 0.1M NaOH when pH < 7.2, inject 0.1M HCl when pH > 7.4; inject 1M NaCl when NaCl concentration < 70mM, inject 1nL of adjustment solution at a time, and collect sensor data once every 2 minutes.

[0046] 2. Integrated reaction and detection

[0047] Using a dual-channel injection structure of a microfluidic chip, 50 nL of cell-free reaction solution (containing magnetic microspheres) and 50 nL of substrate-co-solubilizing system are simultaneously injected into each microchannel, followed by the addition of 10 nL of lipase mutant DNA (one mutant corresponds to one microchannel).

[0048] The chip was placed in a 37℃ temperature control module and incubated for 3 hours (simultaneously completing lipase synthesis and catalytic reaction). During incubation, the intelligent feedback regulation submodule monitored the pH and NaCl concentration in real time. When the pH dropped to 7.15 at 120 minutes, 1 nL of 0.1 M NaOH was automatically injected to raise the pH back to 7.25. At 180 minutes, the NaCl concentration dropped to 68 mM, and 1 nL of 1 M NaCl was automatically injected to raise the concentration back to 75 mM.

[0049] The real-time detection module was activated, and the absorbance at 405 nm (characteristic absorbance of glyceryl tartrate hydrolysis product) of each microchannel was detected every 5 minutes. The absorbance change curve within 3 hours was recorded.

[0050] Using the rate of increase in absorbance (ΔA405 / min) as an indicator of enzyme activity, the top 10% of microchannels with ΔA405 / min were screened (a total of 100 positive microchannels).

[0051] 3. Validation of positive mutants

[0052] Positive enzyme recovery: The reaction solution (total 10 μL) from the 100 selected positive microchannels was collected into a centrifuge tube and placed next to a permanent magnet (magnetic field strength 1.2T) for 5 min. The magnetic microspheres (binding the target lipase) were adsorbed to the bottom of the tube. The supernatant was discarded, and 50 μL of elution buffer (PBS containing 250 mM imidazole, pH 7.4) was added. After vortexing for 1 min, the tube was placed next to the permanent magnet again for 5 min. The supernatant was the purified target lipase.

[0053] DNA was extracted from highly active microchannels, amplified by PCR, and then sequenced to determine the amino acid sequence of the highly active lipase mutant.

[0054] The activity of the recovered purified lipases was verified using the traditional CFPS-centrifugation-detection protocol (i.e., the standard two-step method of "CFPS synthesizing enzyme → centrifugation → enzyme activity detection"). These positive enzymes were retested, and the activity value of each mutant was measured and ranked. The results showed that the activity ranking obtained by the traditional method was 98% consistent with the activity ranking obtained by this system, proving that the screening results of high-activity mutants by this system are accurate and reliable. Simultaneously, the lipase recovery rate in this system was 88%, significantly higher than the 35% of traditional nickel column chromatography. The recovery step took only 5 minutes and cost approximately 8 yuan, far lower than the 200 yuan of the traditional method. The comparison is shown in the table below:

[0055]

[0056]

[0057] Although the present invention has been described above with reference to embodiments, various modifications can be made and components can be replaced with equivalents without departing from the scope of the invention. In particular, as long as there is no structural conflict, the features in the disclosed embodiments can be combined with each other in any manner. The lack of an exhaustive description of these combinations in this specification is merely for the sake of brevity and resource conservation. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A cell-free protein synthesis system, characterized by, The application relates to a microfluidic chip reaction module, an optimized cell-free reaction liquid, a substrate-solubilizing system and a real-time detection module. The microfluidic chip reaction module is made of polydimethylsiloxane and integrates 1000-2000 independent microchannels; each microchannel simultaneously serves as a protein synthesis chamber and an enzyme activity detection chamber; the inner wall of the microchannel is coated with a hydrophilic coating; and the microfluidic chip inlet is provided with a double-channel liquid injection structure. The optimized cell-free reaction liquid is based on an Escherichia coli extract and is added with interference inhibition components, transcription-translation enhancement components, buffer stabilization components and a magnetic affinity capture subassembly. The substrate-solubilizing system adopts a cyclodextrin-ethanol complex solubilizing strategy, in which a hydrophobic substrate is first dissolved in 5%-10% ethanol, then mixed with 10-20 mM hydroxypropyl-beta-cyclodextrin to form a cyclodextrin-substrate inclusion compound which can be dissolved in water. The real-time detection module is provided with a fluorescence detector and an absorbance detector at the outlet end of the microfluidic chip to monitor the characteristic signals of enzyme catalytic reactions in real time, dynamically record enzyme activity change curves, and screen positive microchannels corresponding to high-activity enzymes.

2. The cell-free protein synthesis system of claim 1, wherein, An intelligent feedback adjustment sub-module is further arranged in each microchannel. The intelligent feedback adjustment sub-module comprises a micro pH sensor and an ion strength sensor integrated in the side wall of the microchannel, an adjustment liquid storage pool arranged at the edge of the chip, and a micro valve connecting the storage pool and the microchannel; the micro pH sensor and the ion strength sensor are electrically connected with a control unit.

3. The cell-free protein synthesis system of claim 1, wherein, The interference inhibition components comprise 0.1-0.5 mM protease inhibitors and 1-2 U / mL specific coenzyme protection enzymes.

4. The cell-free protein synthesis system of claim 1, wherein, The transcription-translation enhancement components comprise 0.5-1 mM cyclic adenosine monophosphate and 2-5 mM betaine.

5. The cell-free protein synthesis system of claim 1, wherein, The buffer stabilization components are a mixed buffer of tris-hydroxymethyl aminomethane hydrochloride and 4-hydroxyethyl piperazine ethanesulfonic acid, and the volume ratio of tris-hydroxymethyl aminomethane hydrochloride to 4-hydroxyethyl piperazine ethanesulfonic acid is 1:

1.

6. The cell-free protein synthesis system of claim 1, wherein, The magnetic affinity capture subassembly comprises 0.5-1 mg / mL His-tag specific magnetic microspheres, the magnetic microspheres have a diameter of 100-200 nm, are modified with Ni2+-NTA groups on the surface and are coated with polyethylene glycol-2000, and the magnetic microspheres are specifically combined with the target enzyme with a 6xHis tag through His-Ni2+ interaction.

7. The cell-free protein synthesis system of claim 1, wherein, The double-channel liquid injection structure comprises a liquid injection port, two groups of liquid injection ports are arranged at the inlet of the microfluidic chip, and a liquid injection channel is connected between the liquid injection ports and the microfluidic chip inlet.

8. Use of the cell-free protein synthesis system of claim 1 in an in vitro enzymatic screening, characterized in that, When the optimized cell-free reaction liquid, the substrate-solubilizing system and the target enzyme are injected into the microchannels of the microfluidic chip, the intelligent feedback adjustment sub-module sets a target pH and ion strength range, controls temperature incubation, synchronously performs protein synthesis and enzyme catalytic reaction, simultaneously detects enzyme catalytic characteristic signals in real time, screens positive microchannels corresponding to high-activity enzymes, collects the reaction liquid of the positive microchannels, and recovers and purifies the target enzyme through magnetic field adsorption and elution.

9. Use of a cell-free protein synthesis system according to claim 8 in an in vitro enzymatic screening, characterized in that, The target enzyme has a 6xHis tag.

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