Crude cell extract-based high-yield cell-free protein synthesis system and application thereof

Abstract

The invention discloses a high-yield cell-free protein synthesis system based on a cell crude extract and application of the high-yield cell-free protein synthesis system. The system comprises an escherichia coli extract, a DNA template, a buffer solution, an amino acid mixture, a nucleoside triphosphate mixture, an ATP regeneration system and antibiotics, the antibiotics are selected from compounds having inhibitory activity on metabolic processes except ribosome functions in prokaryotic microorganisms, including but not limited to one or more combinations of interfering DNA replication, DNA transcription, cell wall synthesis, energy metabolism, lipid synthesis or other non-translational related metabolic pathways. According to the method disclosed by the invention, the escherichia coli crude extract is used as a bacterial chassis, and non-essential metabolic pathways in a CFPS system are selectively inhibited by introducing different types of antibiotics, so that the yield of protein synthesis is increased.

Description

Technical Field

[0001] This invention specifically relates to a high-yield cell-free protein synthesis system based on crude cell extracts and its application, belonging to the field of protein synthesis technology. Background Technology

[0002] Cell-free protein synthesis (CFPS) is a groundbreaking technology that enables the efficient in vitro synthesis of recombinant proteins without the involvement of living cells, utilizing transcription and translation mechanisms preserved in cell extracts. Compared to traditional cell culture methods, CFPS offers advantages such as speed, efficiency, programmability, and high flexibility. It avoids the contamination risks associated with cell culture and lysis processes and allows for protein expression regulation in non-natural environments. Therefore, this technology shows broad application prospects in vaccine development, genetic engineering, and the synthesis of complex proteins.

[0003] However, the expression level of recombinant proteins is a core bottleneck restricting the practical application of CFPS technology. By optimizing the preparation of cell extracts and introducing specific cofactors, the synthesis efficiency of target proteins can be improved to some extent. For example, the Streptomyces-based CFPS system achieved a high protein yield after the addition of protein translation-related factors (Xu H, Liu W, Li J. Translation Related Factors Improve the Productivity of a Streptomyces-Based Cell-Free Protein Synthesis System[J]. ACS syntheticbiology, 2020, 9(5): 1221-1224.). In addition, using rare tRNA overexpressing strains as the source of extracts helps to optimize codon usage preferences, thereby further enhancing the expression level of target proteins (Kim J, Copeland CE, Seki K, et al. Tuning the Cell-Free Protein Synthesis System for Biomanufacturing of Monomeric Human Filaggrin[J]. Frontiers in bioengineering and biotechnology, 2020, 8: 590341.). Precise optimization of the reaction system has also been proven to be an important strategy for increasing CFPS yield. Optimizing the composition (e.g., magnesium ions, potassium ions, NAD, folic acid, etc.) and ratio of the reaction buffer can not only significantly improve the efficiency of protein synthesis, but also enhance the robustness of the reaction (Banks AM, Whitfield CJ, Brown SR, et al. Key reaction components affect the kinetics and performance robustness of cell-free protein synthesis reactions[J]. Computational and structural biotechnology journal, 2022, 20: 218-229.).

[0004] While these optimization strategies have improved CFPS expression yield to some extent, unresolved challenges remain regarding system scalability and energy efficiency. Currently, most mainstream cell-free protein synthesis systems use crude E. coli extracts as the reaction substrate. However, these extracts retain a significant amount of energy-consuming endogenous metabolic enzyme activity. These metabolic activities can not only cause non-specific consumption of substrates (such as ATP and amino acids) during the synthesis reaction but also compete for cofactors (such as NAD⁺ and FAD) and energy, thus interfering with the smooth progress of protein synthesis. These undesirable metabolic reactions are a major factor contributing to the poor stability and low energy efficiency of CFPS systems. To avoid energy competition, researchers have developed CFPS systems from other microorganisms, such as Vibrio vegetativeis, or engineered strains with minimized metabolic pathways, aiming to reduce interference from non-specific metabolism in the reaction system (Shrestha P, Zhang Y, Chen W, et al. Triclosan: antimicrobial mechanisms, antibiotics interactions, clinical applications, and human health[J]. J Environ Sci Health C Toxicol Carcinog,2020, 38(3): 245-268.). However, these strategies still face some challenges, such as the lack of standardization of cell lysis conditions for exogenous strains, the complexity of extract preparation processes, and significant differences in system stability and protein expression efficiency, which limit their promotion and large-scale development in practical applications. Therefore, to address the shortcomings of existing systems, we continue to construct CFPS systems based on Escherichia coli, and by reducing irrelevant metabolic activities and optimizing the competition for key resources, we hope to improve ATP utilization efficiency while ensuring stability and operability, thereby significantly enhancing protein expression levels. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a cell-free protein synthesis system and its application, aiming to improve the synthesis yield of recombinant proteins by effectively inhibiting non-essential metabolic pathways in the CFPS system.

[0006] The technical solution of the present invention is as follows: A cell-free protein synthesis system, comprising: Prokaryotic microbial extract, wherein the prokaryotic microbial extract is derived from Escherichia coli; The DNA template contains promoter regions and ribosome binding sites (RBS) and other elements required for transcription and translation that can be recognized by RNA polymerases such as T7 RNA polymerase and SP6 RNA polymerase. Buffer solution, wherein the buffer solution contains compounds such as HEPES or Tris necessary for maintaining the pH environment; An amino acid mixture comprising 20 known natural amino acids and artificially synthesized amino acids; A mixture of nucleoside triphosphates or a mixture of nucleoside monophosphates, wherein the nucleoside triphosphates contain UTP, ATP, CTP and GTP, and the nucleoside monophosphates contain UMP, AMP, CMP and GMP; An ATP regeneration system, comprising enzymes, coenzymes and compounds necessary for the ATP regeneration reaction, such as creatine kinase, pyruvate kinase, phosphoenolpyruvate (PEP), or rNMP. Antibiotics, wherein the antibiotics are selected from compounds that have inhibitory activity against metabolic processes in prokaryotic microorganisms other than ribosome function, including but not limited to one or more combinations of interfering with microbial DNA replication, DNA transcription, cell wall synthesis, energy metabolism, lipid synthesis or other non-translation-related metabolic pathways, for the purpose of improving the expression efficiency of target proteins.

[0007] Furthermore, the concentration of the antibiotic added ranges from 0.05 μg / mL to 20 mg / mL.

[0008] Furthermore, the antibiotic, in a cell-free protein synthesis reaction with added recombinant RNA polymerase, is used to inhibit endogenous metabolic activity without inhibiting recombinant RNA polymerase (such as T7 RNA polymerase, SP6 RNA polymerase, etc.) and ribosome function.

[0009] Furthermore, the types of antibiotics include, but are not limited to, the following: Novobiocin, Daunorubicin hydrochloride, Orbifloxacin, Nalidixic Acid, Rifamycin, Trimethoprim, Isoniazid, Ciprofloxacin hydrochloride, 5-Fluorouracil, Fosfomycin Sodium, Azacitidine, Mizoribine, Bacitracin zinc, Triptolide, and Triclosan.

[0010] Furthermore, the antibiotic is preferably one or a combination of Isoniazid, 5-Fluorouracil, Fosfomycin Sodium, and Mizoribine.

[0011] Furthermore, the DNA template may encode recombinant proteins of animal, plant, or microbial origin, wherein the recombinant proteins are selected from structural proteins, membrane proteins, enzymes, receptor proteins, antigen proteins, or polypeptides with specific functions.

[0012] Furthermore, the prokaryotic microbial extract is derived from an engineered strain of *Escherichia coli* possessing endogenous recombinant RNA polymerase, wherein the strain is selected from BL21, BL21(DE3), BL21 Star™(DE3), Rosetta(DE3), C41(DE3), C43(DE3) or their functionally equivalent derivatives.

[0013] Optionally, the prokaryotic microbial extract may also be derived from an engineered strain of Escherichia coli that does not possess endogenous recombinant RNA polymerase. The RNA polymerase in this system is added later, such as T7 RNA polymerase. Alternatively, Escherichia coli's own RNA polymerase and sigma factor may be used, along with antibiotics other than transcription inhibitors such as Ansamycin, to increase the yield of recombinant protein expression.

[0014] The cell-free protein synthesis system can be used in a non-dialysis mode or a dialysis mode. In the dialysis mode, a dialysis membrane with a molecular cutoff of 2000-25000 Da is used, and the reaction is carried out at 20℃-40℃ for 5-100 hours. The reaction is carried out in a static or agitated manner.

[0015] The cell-free protein synthesis system can be used in conjunction with equipment such as pipetting robots and high-throughput metal shaking baths to achieve high-throughput protein expression.

[0016] Beneficial effects: This invention uses crude Escherichia coli extract as a bacterial chassis and selectively inhibits non-essential metabolic pathways in the CFPS system by introducing different types of antibiotics, thereby increasing the yield of protein synthesis. Attached Figure Description

[0017] Figure 1 The effect of adding different concentrations and types of antibiotics to the system on protein expression: specifically, the expression of eGFP under antibiotic treatment. The eGFP expression level is expressed as a relative percentage (%) of the untreated control group (without antibiotics). The control group is set as 100%. All values ​​are expressed as the mean ± standard error (SEM) of at least three independent replicate experiments (n ≥ 3). Detailed Implementation

[0018] To better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below.

[0019] A cell-free protein synthesis method includes the following steps.

[0020] I. Preparation of cell extracts (for cell-free protein synthesis) Cell extracts of Escherichia coli BL21 Star™ (DE3) (ThermoFisher, catalog number: C601003) were prepared using a modified known method.

[0021] The specific steps are as follows: 500 mL of BL21 Star™ (DE3) cells were seeded into 2 L Erlenmeyer flasks containing LB medium and cultured overnight at 37°C with circular shaking at 160 rpm. The overnight culture was then transferred to a 10 L fermenter and mixed with 9 L of Z medium, which consisted of 40 mM KH₂PO₄, 165 mM K₂HPO₄, 100 g yeast extract, 6 mM glucose, and 100 mg vitamin B1. The fermenter was then incubated at 37°C with stirring at 250 rpm and continuous aeration to maintain a dissolved oxygen concentration above 15%.

[0022] When cell density reaches the logarithmic mid-term (OD) 600 = 1), 2 g IPTG was added to the culture system to induce high-level expression of T7 RNA polymerase. After the induction reaction lasted for 3 hours, cells were collected by centrifugation at 4000×g (4°C) and washed three times with S30 buffer, which consisted of 10 mM Tris-acetic acid buffer (pH 8.2) containing 14 mM Mg(OAc)2, 60 mM KOAc, 1 mM DTT and 0.5 mL / L 2-mercaptoethanol.

[0023] Resuspend the cell paste in S30 buffer B (same formulation as S30 buffer) at a ratio of 1 g cells to 1.3 mL buffer, and perform a single-pass homogenization using a Thermo Spectronic French high-pressure homogenizer (model FA-078) at 300 bar. Centrifuge the homogenate at 30,000 × g for 30 minutes at 4°C, repeat twice, and collect the supernatant.

[0024] After incubating the supernatant at 37°C for 80 minutes, it was dialyzed three times at 4°C using S30 buffer B, each dialyzing for 1 hour, with the buffer volume being 50 times the sample volume. After dialyzing, the sample was centrifuged at 4000×g for 10 minutes, and the supernatant was collected as the cell extract, which was used for subsequent cell-free protein expression reactions.

[0025] II. Cell-Free Protein Synthesis (CFPS) Reaction System The total volume of the non-dialysis mode CFPS reaction system was 200 μL, composed of the following components: 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 1.2 mM ATP, 0.5 mM CTP, GTP and UTP, 0.03 mg / mL leucovorin, 0.17 mg / mL E. coli tRNA, 0.5 mg / mL creatine kinase, 57 mM HEPES buffer (pH 7.5), 1 mM of a mixture of 20 amino acids, 20% (v / v) E. coli extract, and an appropriate amount of template (10-20 ng / μL plasmid or PCR product of the same concentration; mRNA of the same concentration can also be added). The template has the nucleotide sequence shown in SEQ ID No. 1 (encoding the eGFP recombinant protein). The system also contained four different concentrations of antibiotics (10 μg / mL, 100 μg / mL, 1...). Comparative experiments were conducted using antibiotics at concentrations of 1 mg / mL and 10 mg / mL, including Novobiocin, Daunorubicin hydrochloride, Orbifloxacin, Nalidixic Acid, Rifamycin, Trimethoprim, Isoniazid, Ciprofloxacin hydrochloride, 5-Fluorouracil, Fosfomycin Sodium, Azacitidine, Mizoribine, Bacitracin zinc, Triptolide, and Triclosan. A system without added antibiotics served as the control.

[0026] The CFPS reaction system in dialysis mode was operated using a commercial dialysis device (Hangzhou Jin'ao Biotechnology Co., Ltd., catalog number: G-DIA-001, molecular weight cutoff ≥6000Da). The internal fluid volume was 200 μL, and the composition was the same as that in the non-dialysis mode described above; the external fluid (2 mL) consisted of: 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 1.2 mM ATP, 0.5 mM CTP, GTP and UTP, 0.03 mg / mL leucovorin, 57 mM HEPES (pH 7.5), and 1 mM amino acid mixture. One or more of the following four concentrations (10 μg / mL, 100 μg / mL, 1 mg / mL, 10 mg / mL) of antibiotics were added to the external solution: Novobiocin, Daunorubicin hydrochloride, Orbifloxacin, Nalidixic Acid, Rifamycin, Trimethoprim, Isoniazid, Ciprofloxacin hydrochloride, 5-Fluorouracil, Fosfomycin Sodium, Azacitidine, Mizoribine, Bacitracin zinc, Triptolide, and Triclosan. The antibiotic types and concentrations were consistent between the internal and external solutions. A system without added antibiotics served as a control.

[0027] III. Reaction Conditions The CFPS reaction was carried out at 30°C. The reaction time was 5 hours in non-dialysis mode and 12 hours in dialysis mode, with slow shaking during the reaction. In this example, the recombinant fluorescent protein generated in the reaction system can be quantified by fluorescence intensity. If other non-fluorescent proteins are generated, SDS-PAGE can be used to separate the proteins and then observe the expression yield of the recombinant protein. The non-dialysis mode is simple to operate and has high throughput; the dialysis mode is more complex to operate, but its protein yield is more than 5 times higher than that of the non-dialysis mode.

[0028] IV. Results and Discussion To minimize the interference of E. coli's own energy consumption on the system, we selected eGFP (enhanced green fluorescent protein) as the target recombinant protein and quantified its expression level by fluorescence intensity. We selected 15 antibiotics acting on different stages of protein translation and investigated their effects on protein expression levels in the CFPS system. We used a CFPS system based on T7 RNA polymerase and crude E. coli S30 lysate to ensure the system contained the essential components required for protein expression, thus enabling the analysis of the impact of host energy consumption on cell-free protein synthesis efficiency. Experimental results are as follows: Figure 1 As shown in the figure, all the antibiotics tested promoted protein expression, with the most significant promotion being Isoniazid, 5-Fluorouracil, Fosfomycin Sodium, and Mizoribine. Specifically, 5-Fluorouracil at a concentration of 100 μg / mL increased protein expression levels by 2.7 times compared to the untreated condition.

[0029] The possible mechanisms of action of various antibiotics are shown in the table below: antibiotic Mechanism of action Novobiocin Inhibit GyrB ATPase activity to block DNA replication; activate LptB ATPase to interfere with lipopolysaccharide transport; inhibit Polθ ATPase and its binding to nucleic acids. Daunorubicin hydrochloride (Doxorubicin) It disrupts cell membranes, inhibits the transfer of resistance genes, and suppresses topoisomerase II activity. Orbifloxacin Inhibition of DNA gyrase and topoisomerase IV in Gram-negative bacteria Nalidixic Acid Inhibition of DNA gyrase and topoisomerase IV Rifamycin It specifically binds to the β subunit of bacterial DNA-dependent RNA polymerase, blocking strand elongation after DNA binding. Ciprofloxacin hydrochloride Inhibition of DNA gyrase and topoisomerase IV Bacitracin zinc Inhibiting the recycling of bacterial cell wall synthesis precursors Triclosan Antibiotic efficacy is enhanced by inhibiting enoyl-apolipoprotein reductase (FASII pathway) to block fatty acid synthesis; disrupting cell wall synthesis; and increasing membrane permeability and altering lipid composition. Trimethoprim Inhibits nucleic acid synthesis by binding to dihydrofolate reductase. Isoniazid By targeting enoyl-apolipoprotein reductase to inhibit branched acid synthesis, defects in lipid layer formation lead to impaired cell wall integrity. 5-Fluorouracil Uracil substitution inhibits RNA and DNA synthesis; it interferes with the expression of genes related to metabolism, DNA repair, and cell wall synthesis. Fosfomycin Sodium By covalently binding to MurA, it inhibits peptidoglycan precursor synthesis and disrupts cell wall construction and barrier function. Azacitidine Inhibiting DNA methylation by incorporating into DNA and capturing DNA methyltransferases; interfering with gene regulation and epigenetic control; inducing DNA damage and disrupting repair mechanisms; and affecting bacterial adaptability. Mizoribine Inhibiting inosine nucleotide dehydrogenase interferes with purine nucleotide synthesis and reduces the supply of DNA and RNA precursors. Triptolide (Tripterygium wilfordii lactone alcohol) Inhibits ribosome function and protein synthesis; interferes with oxidative stress response and DNA repair mechanisms. .

[0030] In summary, this invention uses crude Escherichia coli extract as a bacterial chassis. By introducing different types and concentrations of antibiotics into the reaction system, it was found that these antibiotics can selectively inhibit non-essential metabolic pathways in the CFPS system, thereby increasing the yield of target recombinant protein synthesis. This opens up a new and effective way to improve the expression level and efficiency of recombinant proteins in the CFPS system.

[0031] The cell-free reaction system provided by this invention is a modular and scalable system. Its components are stable and controllable, and its volume is flexible, making it suitable for standard 96-well or 384-well plate formats. Therefore, it possesses high compatibility with automated pipetting platforms (such as pipetting robots) and high-throughput processing equipment (such as metal shaking baths). Without relying on manual sample loading, the system can achieve high-throughput parallel reactions, adapting to automated systems for rapid and efficient protein expression screening and optimization.

Claims

1. A cell-free protein synthesis system, wherein the system uses prokaryotic microbial extracts as the chassis material, characterized in that, The system also includes antibiotics.

2. The cell-free protein synthesis system according to claim 1, characterized in that, The antibiotics mentioned include, but are not limited to, the following: Novobiocin, Daunorubicin hydrochloride, Orbifloxacin, Nalidixic Acid, Rifamycin, Trimethoprim, Isoniazid, Ciprofloxacin hydrochloride, 5-Fluorouracil, Fosfomycin Sodium, Azacitidine, Mizoribine, Bacitracin zinc, Triptolide, and Triclosan.

3. The cell-free protein synthesis system according to claim 1, characterized in that, The concentration of the antibiotic added is from 0.05 μg / mL to 20 mg / mL.

4. The cell-free protein synthesis system according to claim 1, characterized in that, The prokaryotic microbial extract is derived from Escherichia coli possessing endogenous recombinant RNA polymerase. The Escherichia coli strain is selected from BL21, BL21(DE3), BL21Star™(DE3), Rosetta(DE3), C41(DE3), C43(DE3) or their functionally equivalent derivatives.

5. The cell-free protein synthesis system according to claim 1, characterized in that, The prokaryotic microbial extract can also be derived from engineered strains of Escherichia coli that do not possess endogenous recombinant RNA polymerase. In this system, Escherichia coli RNA polymerase and Sigma factor need to be added.

6. The cell-free protein synthesis system according to claim 1, characterized in that, The system also includes: DNA template, buffer solution, amino acid mixture, nucleoside triphosphate mixture, and ATP regeneration system.

7. The cell-free protein synthesis system according to claim 6, characterized in that, The DNA template may encode recombinant proteins of animal, plant, or microbial origin, etc., and the recombinant proteins are selected from structural proteins, membrane proteins, enzymes, receptor proteins, antigen proteins, or polypeptides with specific functions.

8. The cell-free protein synthesis system according to claim 6, characterized in that, The DNA template encodes the eGFP recombinant protein, and the nucleotide sequence of the DNA template is shown in SEQ ID No.

1.

9. The cell-free protein synthesis system according to any one of claims 1-8, characterized in that, The reaction system can be carried out in a non-dialysis mode or in a dialysis mode; in the dialysis mode, a dialysis membrane with a molecular cutoff of 2000-25000 Da is used, the reaction is carried out at 20℃-40℃, the reaction time is 5-100 hours, and the reaction is carried out in a static or oscillating manner.

10. The application of the cell-free protein synthesis system as described in claims 1-8, wherein the system, in conjunction with a pipetting robot and a high-throughput metal shaking bath device, enables high-throughput protein expression.

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