linear dna templates  (TaKaRa)

Journal: bioRxiv

Article Title: PURE makes PURE: reconstitution of the PURE cell-free system from self-synthesized non-ribosomal proteins

doi: 10.64898/2025.12.17.694911

Figure Lengend Snippet: a , Workflow for the expression and purification of PURE proteins by the PURE system and their functional evaluation. Each his-tagged protein in a given subset was expressed using PUREfrex by adding the corresponding DNA template to the reaction and incubating at 37°C. Protein expression was confirmed by BODIPY-lysine fluorescence on SDS–PAGE. Reactions corresponding to each subset were pooled and purified using Ni-charged magnetic beads, followed by buffer exchange using mini dialysis devices and concentrated by ultra-filtration. For the functional assay, the purified subset was added to the corresponding ΔPURE reaction (a PURE reaction lacking the corresponding proteins) containing an eGFP template as a reporter for protein synthesis. Full PURE is included as the positive control, ΔPURE + purification control as the negative control, ΔPURE + EE subset as the concentration adjusted control (containing E. coli synthesized proteins at the same concentration as the PUREfrex-expressed subsets as determined by Bradford assay), and the ΔPURE + PE subset (PUREfrex expressed proteins). The fluorescence signal was measured using a plate reader. b , The table summarizes the PUREfrex reaction volumes used for each protein and the final volume and concentration obtained after purification of each pooled subset. c , SDS–PAGE analysis of individual PUREfrex reactions expressing PURE proteins. The gel shows the BODIPY-lysine fluorescence of each synthesized PURE protein. d , Functional assay result of each subset. The plot shows eGFP fluorescence in each reaction (n=2 for all conditions). e , Yield and rate of eGFP synthesis calculated from panel d.

Article Snippet: Linear DNA templates with identical non-coding regions for all 36 PURE proteins as well as the eGFP linear template, were synthesized by Twist Bioscience, amplified by PCR using Q5 High-Fidelity 2X Master Mix (NEB, catalog no. M0492), and purified using a commercial kit (Zymo Research, catalog no. D4014).

Techniques: Expressing, Purification, Functional Assay, Fluorescence, SDS Page, Magnetic Beads, Buffer Exchange, Filtration, Positive Control, Control, Negative Control, Concentration Assay, Synthesized, Bradford Assay

Journal: bioRxiv

Article Title: PURE makes PURE: reconstitution of the PURE cell-free system from self-synthesized non-ribosomal proteins

doi: 10.64898/2025.12.17.694911

Figure Lengend Snippet: a , Titration of the different protein subsets in PURE. Each subset was titrated in its corresponding 10-fold diluted ΔPURE reaction (containing a 10-fold lower concentration of non-ribosomal proteins), and the optimal mixing ratio was determined based on the effect of each subset’s volume on the reaction rate. Orange dots show synthesis rate data points, and blue crosses represent their average. b , Reconstitution of PURE by combining functional subsets. After producing the five PURE synthesized protein subsets, a new PURE reaction was reconstituted by combining all subsets. The mixing ratios are indicated on the arrows. Subsequently, 2.6 µL of the reconstituted PURE protein solution was added to a ΔPURE reaction (lacking all non-ribosomal proteins) containing an eGFP template for a functional test. c , Functional assay result of reconstituted PURE assembled from the five subsets. The plot shows eGFP fluorescence generated in each reaction ( n = 2 for all conditions). Full PURE is included as the positive control, ΔPURE supplemented with a purification control as the negative control, and the ad-justed control was prepared from E. coli expressed proteins at equivalent concentrations, combined at the same ratios, and added to the ΔPURE reaction. d , Yield and rate of eGFP synthesis calculated from panel c. e , Single-reaction regeneration of PURE proteins in PURE. All 36 non-ribosomal proteins were expressed together in a single PURE reaction by including all DNA templates encoding the respective proteins. For EF-Tu, a 35-fold excess of DNA template was used, and for EF-Ts, EF-G, and T7 RNAP, a 5-fold excess was used. After incubation at 37°C, magnetic beads were used for purification, followed by buffer exchange and concentration. Subsequently, 2.6 µL of the single-reaction regenerated PURE was added to a ΔPURE reaction (lacking all non-ribosomal proteins) containing an eGFP template for a functional test. f , SDS–PAGE of single-reaction regenerated PURE proteins visualized by BODIPY-lysine fluorescence. g , Coomassie-stained SDS-PAGE of final obtained single-reaction regenerated PURE proteins next to concentration adjusted E. coli expressed full PURE. h , Functional assay result of single-reaction regenerated PURE. The plot shows eGFP fluorescence generated in each reaction ( n = 2 for all conditions). Full PURE is included as the positive control, ΔPURE supplemented with a purification control as the negative control, and the adjusted control was prepared from homemade full PURE diluted to the same final total concentration and added to the ΔPURE reaction. i , Yield and rate of eGFP synthesis calculated from panel h.

Article Snippet: Linear DNA templates with identical non-coding regions for all 36 PURE proteins as well as the eGFP linear template, were synthesized by Twist Bioscience, amplified by PCR using Q5 High-Fidelity 2X Master Mix (NEB, catalog no. M0492), and purified using a commercial kit (Zymo Research, catalog no. D4014).

Techniques: Titration, Concentration Assay, Functional Assay, Synthesized, Fluorescence, Generated, Positive Control, Purification, Control, Negative Control, Incubation, Magnetic Beads, Buffer Exchange, SDS Page, Staining