Figures
Figure 1 with 1 supplement
Optimization of eCFPS components.
Protein expression levels from the eCFPS system were measured using a Nanoluciferase (NLuc) reporter DNA. Green area in the graphs indicate the common concentration range used in published protocols for eCFPS. Error bars represent the standard error (SE) of at least three independent reactions. (A–E) Protein expression levels of the eCFPS system supplemented with different concentrations of DTT (A), cAMP (B), PEG8000 (C), NH4+ (D), and folinic acid (E). Data present mean ± SEM, n=3 independent biological replicates. (F–I) Protein expression levels of eCFPS with various concentrations of tRNA (F), amino acids (G), CTP (H), and UTP (I). Data present mean ± SEM, n=3 independent biological replicates. (J) A summary of the supplement components before and after optimization. The quantitative data underlying the plots in this figure are provided in Figure 1—source data 1.
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Figure 1—source data 1
Plotted values in panels A—I.
- https://cdn.elifesciences.org/articles/109495/elife-109495-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
The roles of NTPs in the reaction efficiency of eCFPS.
(A) Reaction efficiency of eCFPS supplemented with and without NTPs. (B) Reaction efficiency of eCFPS supplemented with a complete mix of NTPs or individual NTPs (ATP, GTP, CTP or UTP). (C) Reaction efficiency of eCFPS supplemented with a mix of NTPs or a combination of ATP and GTP. Data from all panels present mean ± SEM, n = 3 independent biological replicates.The quantitative data underlying the plots in this figure are provided in Figure 1—figure supplement 1—source data 1.
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Figure 1—figure supplement 1—source data 1
Plotted values in panels A—C.
- https://cdn.elifesciences.org/articles/109495/elife-109495-fig1-figsupp1-data1-v1.xlsx
Figure 2 with 1 supplement
Optimization of essential components for eCFPS system.
(A) Protein expression levels of the eCFPS system measured at varying concentrations of KGlu and MgGlu2. (B) Protein expression levels of the eCFPS system measured at varying concentrations of MgGlu2 and PEG8000. (C) Protein expression levels of the eCFPS system measured at varying concentrations of ATP and GTP. (D) Protein expression levels of the eCFPS system measured at varying concentrations of CrK and CrP. (E) Protein expression levels of the eCFPS system measured at varying pH and buffer concentrations. Data from all panels present mean ± SEM, n=3 independent biological replicates. The quantitative data underlying the plots in this figure are provided in Figure 2—source data 1.
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Figure 2—source data 1
Plotted values in panels A—E.
- https://cdn.elifesciences.org/articles/109495/elife-109495-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Effects of CrK and CrP on the reaction efficiency of eCFPS.
(A) The reaction yield of optimized eCFPS at different concentrations of CrP. (B) Reaction efficiency of eCFPS at varying CrK concentrations and different reaction times. Data from all panels present mean ± SEM, n=3 independent biological replicates.The quantitative data underlying the plots in this figure are provided in Figure 2—figure supplement 1—source data 1.
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Figure 2—figure supplement 1—source data 1
Plotted values in panels A and B.
- https://cdn.elifesciences.org/articles/109495/elife-109495-fig2-figsupp1-data1-v1.xlsx
Figure 3 with 1 supplement
Characterization of the optimized eCFPS system.
(A) Kinetics of protein synthesis at 25°C, 30°C, and 37°C over a 60-min period. Data presented as mean ± SEM; n=3 independent biological replicates. (B) Protein expression levels of the eCFPS system measured at varying DNA concentrations for a reporter encoding a FLAG-tagged NLuc. The protein product was quantified via a luminescence assay and confirmed by western blotting (upper panel). Data presented as mean ± SEM; n=3 independent biological replicates. (C) Comparison of protein yield. The 'initial' system denotes the traditional 35-component reaction mixture prior to optimization, serving as a baseline control for benchmarking the streamlined system. Data presented as mean ± SEM; n=4 independent biological replicates. Statistical analysis was performed using unpaired t‑test, , with each optimized group compared against the initial control. All optimized lysate groups yielded significantly higher values than the initial group (P < 0.0001). Original blots and annotated blot images corresponding to (B) are provided in Figure 3—source data 1 and Figure 3—source data 2, respectively. The quantitative data underlying the plots in this figure are provided in Figure 3—source data 3.
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Figure 3—source data 1
Original files for western blot analysis displayed in Figure 3B.
- https://cdn.elifesciences.org/articles/109495/elife-109495-fig3-data1-v1.zip
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Figure 3—source data 2
PDF files containing original western blots for Figure 3B, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/109495/elife-109495-fig3-data2-v1.zip
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Figure 3—source data 3
Plotted values in panels A—C.
- https://cdn.elifesciences.org/articles/109495/elife-109495-fig3-data3-v1.xlsx
Figure 3—figure supplement 1
Performance evaluation and mechanistic analysis of transcriptional and translational efficiency.
(A) Comparison between the initial and optimized systems at three different concentrations of DNA and mRNA templates to assess transcription and translation efficiency. Data are presented as mean ± SEM, n = 3 independent biological replicates. Statistical analysis was performed using unpaired t‑test. At all tested DNA template concentrations, the initial system exhibited significantly higher relative luciferase units than the optimized system (P < 0.0001). At all tested mRNA template concentrations, the optimized system exhibited significantly higher relative luciferase units than the initial system (P < 0.0001). (B) Quantitative RT-qPCR analysis of reporter transcript levels in the initial and optimized systems. Numbers above bars denote fold‑change relative to the 100 ng/μL T7 condition. Data present mean ± SEM, n = 3 independent biological replicates. Statistical significance is indicated: **p < 0.01, ***p < 0.001. (C) Comparison between the initial system supplemented with varying concentrations of T7 RNA polymerase (0–1600 ng/μL) and the optimized system. Data present mean ± SEM, n = 3 independent biological replicates. Statistical analysis was performed using unpaired t‑test. Both 50% and 70% fast‑lysate groups of the optimized system were significantly higher to all T7‑polymerase concentrations tested in the initial system (P < 0.0001). The quantitative data underlying the plots in this figure are provided in Figure 3—figure supplement 1—source data 1.
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Figure 3—figure supplement 1—source data 1
Plotted values in panels A—C.
- https://cdn.elifesciences.org/articles/109495/elife-109495-fig3-figsupp1-data1-v1.xlsx
Figure 4 with 1 supplement
Benchmarking and expression of challenging proteins.
(A) NLuc protein expression kinetics over time, comparing the PEP-based, initial CrP/CrK-based, and optimized CrP/CrK-based energy regeneration systems. Data present mean ± SEM, n=4 independent biological replicates. Statistical analysis was performed using unpaired t‑tests. The optimized system was significantly higher than both initial and PEP‑based groups across 10 min, 30 min and 60 min time points (P < 0.0001). (B) sfGFP protein expression kinetics over time from the three energy regeneration systems. Data present mean ± SEM, n=3 independent biological replicates. Statistical analysis was performed using unpaired t‑tests. The optimized system was significantly higher than both initial and PEP‑based groups across 10 min, 30 min and 60 min time points (P < 0.0001). (C, D) Western blot validation of protein expression for NLuc (C) and sfGFP (D) from the different eCFPS system shown in (A, B). Protein products were detected using an anti-FLAG antibody. The asterisk (*) indicates a non-specific band. (E) Western blot detection of His-FLAG-BsaI expressed by the optimized eCFPS system using an anti-FLAG antibody. (F) Agarose gel electrophoresis confirming the functional activity of eCFPS-synthesized BsaI via cleavage of a substrate plasmid. A 10-fold serial dilution of BsaI was with 1x representing 0.05 mg/mL. NC (negative control) indicates no plasmid in the eCFPS reaction. S, L, and O indicate the respective position of the supercoiled, linear, and open circular forms of the plasmid. (G) Western blot analysis of vimentin expressed by the optimized eCFPS system using an anti-vimentin antibody. (H) Negative-stain electron microscopy image showing that vimentin expressed via eCFPS can successfully self-assemble into filaments in vitro. Scale bars: 500 nm (main panel), 200 nm (inset panels). Original blots and annotated blot images corresponding to Figure 4C/D/E/F/G are provided in Figure 4—source data 1 and Figure 4—source data 2, respectively. The quantitative data underlying the plots are provided in Figure 4—source data 3.
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Figure 4—source data 1
Original files for western blot analysis displayed in Figure 4C, D, E, G.
- https://cdn.elifesciences.org/articles/109495/elife-109495-fig4-data1-v1.zip
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Figure 4—source data 2
PDF files containing original western blots for Figure 4C, D, E, G, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/109495/elife-109495-fig4-data2-v1.zip
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Figure 4—source data 3
Plotted values in panels A and B.
- https://cdn.elifesciences.org/articles/109495/elife-109495-fig4-data3-v1.xlsx
Figure 4—figure supplement 1
Applications of eCFPS.
(A) Schematic overview of the vectors or expression cassettes used in this study, as described in the Methods section. (B-C) Total protein analysis by SDS-PAGE. SDS-PAGE gels for eCFPS reactions using NLuc (B) and sfGFP (C) as DNA templates. The SDS-PAGE results are shown as loading controls for western blot analysis of Figure 4C, D. (D) Validation of antibiotic-mediated reaction inhibition using the eCFPS system. Data present mean ± SEM, n = 3 independent biological replicates. (E) Standard curve correlating sfGFP fluorescence intensity with absolute protein yield. (F) Quantitative comparison of sfGFP protein yields between the initial and optimized systems following short (0.5 h) and long (4 h) incubation. Data present mean ± SEM, n = 3 independent biological replicates. Statistical analysis was performed using unpaired t‑tests. At both the 0.5 h and 4 h time points, the 50 % and 70 % lysate conditions of the optimized system produced significantly higher sfGFP yields than the initial system (P < 0.0001). (G) Benchmarking of absolute protein productivity for the optimized system against a high-end commercial cell-free system at different time points using sfGFP as a reporter. Data present mean ± SEM, n = 3 independent biological replicates. Statistical analysis was performed using unpaired t‑tests. The optimized system exhibited significantly higher sfGFP productivity relative to the commercial system at 2 h (***, P < 0.001), 4 h (***, P < 0.001), 6 h (****, P < 0.0001), and 8 h (*, P < 0.05).Original uncropped gels and annotated gel images corresponding to Figure 4—figure supplement 1B, C are provided in Figure 4—figure supplement 1—source data 1 and 2, respectively. The quantitative data underlying the plots in this figure are provided in Figure 4—figure supplement 1—source data 3.
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Figure 4—figure supplement 1—source data 1
Original files for SDS‑PAGE gel displayed in Figure 4—figure supplement 1B, C.
- https://cdn.elifesciences.org/articles/109495/elife-109495-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
PDF files containing original SDS‑PAGE gel for Figure 4—figure supplement 1B, C, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/109495/elife-109495-fig4-figsupp1-data2-v1.zip
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Figure 4—figure supplement 1—source data 3
Plotted values in panels D—G.
- https://cdn.elifesciences.org/articles/109495/elife-109495-fig4-figsupp1-data3-v1.xlsx
Figure 5 with 1 supplement
Preparation of eCFPS from cultured E. coli cells.
(A) Flowchart of the eCFPS preparation procedures. (B) Comparison of reaction efficiency in eCFPS using lysate with bacteria cells harvested at different optical density. Data present mean ± SEM, n=3 independent biological replicates. (C) Sucrose gradient sedimentation analysis of different lysates used for eCFPS, revealing the presence of ribosome monomers. (D) Comparison of protein expression levels in eCFPS system using lysates prepared by runoff, dialysis, and rapid endogenous T7 RNA polymerase induction. Data present mean ± SEM, n=4 independent biological replicates. Statistical analysis was performed using unpaired t‑tests. The fast lysate (rapid endogenous T7 induction) group exhibited significantly higher units than both the runoff and dialysis groups (P < 0.0001). (E) Comparison of reaction efficiency in eCFPS using lysates after different numbers of freeze–thaw cycles. Data present mean ± SEM, n=3 independent biological replicates. The quantitative data underlying the plots are provided in Figure 5—source data 1.
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Figure 5—source data 1
Plotted values in panels B—E.
- https://cdn.elifesciences.org/articles/109495/elife-109495-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Comparison of different cell lysate preparation methods.
(A) Sucrose gradient sedimentation analysis of cell lysates harvested at different cell densities (Optical density, was measured by absorbance at 600 nm). Peaks correspond to small ribosomal subunit (SSU), large ribosomal subunit (LSU), ribosome monomer, and disome. (B) Negative-staining TEM images of ribosomes isolated from different lysates. Scale bar, 100 nm. (C) Comparison of reaction efficiency in different eCFPS systems. Data present mean ± SEM, n=4 independent biological replicates. Statistical analysis was performed using unpaired t‑tests. The optimized system produced significantly higher units than the initial system across all tested time points from 10 min to 120 min (P < 0.0001). The quantitative data underlying the plots in this figure are provided in Figure 5—figure supplement 1—source data 1.
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Figure 5—figure supplement 1—source data 1
Plotted values in panels A and C.
- https://cdn.elifesciences.org/articles/109495/elife-109495-fig5-figsupp1-data1-v1.xlsx
Additional files
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Supplementary file 1
Chemical composition of eCFPS buffers from previous studies (xlsx).
- https://cdn.elifesciences.org/articles/109495/elife-109495-supp1-v1.xlsx
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Supplementary file 2
Plasmid or expression cassette construct and primers (xlsx).
- https://cdn.elifesciences.org/articles/109495/elife-109495-supp2-v1.xlsx
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Supplementary file 3
Chemical composition of eCFPS buffers used in this study (xlsx).
- https://cdn.elifesciences.org/articles/109495/elife-109495-supp3-v1.xlsx
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Supplementary file 4
Key reagents and resources (xlsx).
- https://cdn.elifesciences.org/articles/109495/elife-109495-supp4-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/109495/elife-109495-mdarchecklist1-v1.docx
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A simplified and highly efficient cell-free protein synthesis system for prokaryotes
eLife 14:RP109495.
https://doi.org/10.7554/eLife.109495.5
