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    New England Biolabs purexpress
    Production and purification of USCTX. ( a ) Commercially available cell-free synthesis systems differ in their ability to produce USCTX (anticipated band size = 4.3 kDa). No such band was produced by the S30 Extract System (left) or the TnT T7 Insect Cell Extract Protein Expression System (right), but a band of the expected size was produced by the NEB <t>PURExpress</t> In Vitro Protein Synthesis System (middle, in duplicate to highlight reproducibility). ( b ) Purification of USCTX, showing the elution fractions E1–E3 from the His-Spin column. The red box indicates the area in which USCTX bands should appear.
    Purexpress, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A Spider Toxin Exemplifies the Promises and Pitfalls of Cell-Free Protein Production for Venom Biodiscovery"

    Article Title: A Spider Toxin Exemplifies the Promises and Pitfalls of Cell-Free Protein Production for Venom Biodiscovery

    Journal: Toxins

    doi: 10.3390/toxins13080575

    Production and purification of USCTX. ( a ) Commercially available cell-free synthesis systems differ in their ability to produce USCTX (anticipated band size = 4.3 kDa). No such band was produced by the S30 Extract System (left) or the TnT T7 Insect Cell Extract Protein Expression System (right), but a band of the expected size was produced by the NEB PURExpress In Vitro Protein Synthesis System (middle, in duplicate to highlight reproducibility). ( b ) Purification of USCTX, showing the elution fractions E1–E3 from the His-Spin column. The red box indicates the area in which USCTX bands should appear.
    Figure Legend Snippet: Production and purification of USCTX. ( a ) Commercially available cell-free synthesis systems differ in their ability to produce USCTX (anticipated band size = 4.3 kDa). No such band was produced by the S30 Extract System (left) or the TnT T7 Insect Cell Extract Protein Expression System (right), but a band of the expected size was produced by the NEB PURExpress In Vitro Protein Synthesis System (middle, in duplicate to highlight reproducibility). ( b ) Purification of USCTX, showing the elution fractions E1–E3 from the His-Spin column. The red box indicates the area in which USCTX bands should appear.

    Techniques Used: Purification, Produced, Expressing, In Vitro

    2) Product Images from "Cell-free synthesis of natural compounds from genomic DNA of biosynthetic gene clusters"

    Article Title: Cell-free synthesis of natural compounds from genomic DNA of biosynthetic gene clusters

    Journal: bioRxiv

    doi: 10.1101/2020.04.04.025353

    Synthesis of BpsA with the PURE cell-free system. (A) Expression control by Western blotting with anti-Strep antibodies performed in three independent reaction solutions (#1-3). BpsA was applied as holo -protein, produced by IVPS with simultaneous phosphopantetheinylation. Self-cast 9 % Tris-Tricine gel. Strep-tagged BpsA has a molecular weight of 142.7 kDa. For the uncropped blot, see Figure S2A. (B) SEC profiles and Western Blot detection of elution fractions. (top) Recombinantly produced BpsA and (bottom) IVPS reaction solution including phosphopantetheinylation. (C) Quantification of protein production yields and phosphopantetheinylation efficiency. BpsA was first produced by IVPS and then phosphopantetheinylated with Sfp and CoA-647 (purchased from NEB). Samples from three independent reactions (#1-3) were applied in repetition (a b). For calibration, recombinantly produced BpsA, diluted in the PURExpress reaction solution, was loaded in amounts of 1.25, 0.63, 0.31 and 0.16 pmol. 9 % Tris-Tricine gel as in panel A. For the uncropped gels, see Figure S2B. Overall, three times three reactions, each applied in duplicate (18 bands), were used for quantification of BpsA production and phosphopantetheinylation for the parallel and the sequential protocol, respectively (Figure S3 A-C).
    Figure Legend Snippet: Synthesis of BpsA with the PURE cell-free system. (A) Expression control by Western blotting with anti-Strep antibodies performed in three independent reaction solutions (#1-3). BpsA was applied as holo -protein, produced by IVPS with simultaneous phosphopantetheinylation. Self-cast 9 % Tris-Tricine gel. Strep-tagged BpsA has a molecular weight of 142.7 kDa. For the uncropped blot, see Figure S2A. (B) SEC profiles and Western Blot detection of elution fractions. (top) Recombinantly produced BpsA and (bottom) IVPS reaction solution including phosphopantetheinylation. (C) Quantification of protein production yields and phosphopantetheinylation efficiency. BpsA was first produced by IVPS and then phosphopantetheinylated with Sfp and CoA-647 (purchased from NEB). Samples from three independent reactions (#1-3) were applied in repetition (a b). For calibration, recombinantly produced BpsA, diluted in the PURExpress reaction solution, was loaded in amounts of 1.25, 0.63, 0.31 and 0.16 pmol. 9 % Tris-Tricine gel as in panel A. For the uncropped gels, see Figure S2B. Overall, three times three reactions, each applied in duplicate (18 bands), were used for quantification of BpsA production and phosphopantetheinylation for the parallel and the sequential protocol, respectively (Figure S3 A-C).

    Techniques Used: Expressing, Western Blot, Produced, Molecular Weight

    3) Product Images from "A simple, robust, and low-cost method to produce the PURE cell - free system"

    Article Title: A simple, robust, and low-cost method to produce the PURE cell - free system

    Journal: bioRxiv

    doi: 10.1101/420570

    Comparison of eGFP expression levels in PURExpress (Solution B) and OnePot PURE (EF-Tu 47%, replicate A) supplied with commercial energy solution (Solution A, PURExpress) and the OnePot energy solution used in this study. Each data point represents at least five technical replicates (mean ± s.d.)
    Figure Legend Snippet: Comparison of eGFP expression levels in PURExpress (Solution B) and OnePot PURE (EF-Tu 47%, replicate A) supplied with commercial energy solution (Solution A, PURExpress) and the OnePot energy solution used in this study. Each data point represents at least five technical replicates (mean ± s.d.)

    Techniques Used: Expressing

    Comparison of commercial ribosomes (ribosomes from PURExpress ? ribosome kit, NEB) and different batches of ribosomes purified in our laboratory. Batch 3 was used throughout this study. (a) Coomassie blue stained SDS-PAGE gels of different ribosomes. The amounts loaded onto the gel were 6.24 µ g for NEB ribosomes and 6.25 µ g in the case of purified ribosomes. (b) Comparison of expression levels in PURExpress ? ribosome kit and OnePot PURE (EF-Tu 47%, replicate A) supplied with PURExpress control ribosomes (2.4 µ M) and purified ribosomes (1.8 µ M). Each data point represents two technical replicates (mean ± s.d.)
    Figure Legend Snippet: Comparison of commercial ribosomes (ribosomes from PURExpress ? ribosome kit, NEB) and different batches of ribosomes purified in our laboratory. Batch 3 was used throughout this study. (a) Coomassie blue stained SDS-PAGE gels of different ribosomes. The amounts loaded onto the gel were 6.24 µ g for NEB ribosomes and 6.25 µ g in the case of purified ribosomes. (b) Comparison of expression levels in PURExpress ? ribosome kit and OnePot PURE (EF-Tu 47%, replicate A) supplied with PURExpress control ribosomes (2.4 µ M) and purified ribosomes (1.8 µ M). Each data point represents two technical replicates (mean ± s.d.)

    Techniques Used: Purification, Staining, SDS Page, Expressing

    OnePot PURE comparison to existing PURE systems. (a) SDS-PAGE gel of PURExpress, HomeMadePURE, OnePot PURE (EF-Tu 47%, replicate A). In the right panel, intensities of different replicates are plotted with molecular weight standards (kDa). (b) Comparison of eGFP expression activity (after 3 h) of different PURE systems. The different systems were tested in the same conditions except for TraMOS where the reported value was used ( 17 ). (c) Price comparison of the different PURE systems. Calculations are detailed in Supplementary Tables S1 , S2 , S3 , Supplementary Table S6 , S7 . (d) Yield of the different PURE systems plotted against their price per µ L. Mean values of the eGFP expression yield were plotted. (e) Cost-normalized yield of the different PURE systems. The mean value of the eGFP expression yield was used for the calculations.
    Figure Legend Snippet: OnePot PURE comparison to existing PURE systems. (a) SDS-PAGE gel of PURExpress, HomeMadePURE, OnePot PURE (EF-Tu 47%, replicate A). In the right panel, intensities of different replicates are plotted with molecular weight standards (kDa). (b) Comparison of eGFP expression activity (after 3 h) of different PURE systems. The different systems were tested in the same conditions except for TraMOS where the reported value was used ( 17 ). (c) Price comparison of the different PURE systems. Calculations are detailed in Supplementary Tables S1 , S2 , S3 , Supplementary Table S6 , S7 . (d) Yield of the different PURE systems plotted against their price per µ L. Mean values of the eGFP expression yield were plotted. (e) Cost-normalized yield of the different PURE systems. The mean value of the eGFP expression yield was used for the calculations.

    Techniques Used: SDS Page, Molecular Weight, Expressing, Activity Assay

    SDS-PAGE gel of proteins synthesized in PURExpress or OnePot (EF-Tu 47%, replicate A) (a) labeled with FluoroTect GreenLys, (b) Coomassie blue stained. Black arrows indicate the expected bands of synthesized proteins, GFP (26.9 kDa) T3 RNAP (98.8 kDa), β -galactosidase (116.5 kDa) and trehalase (63.7 kDa), DHFR (18 kDa)
    Figure Legend Snippet: SDS-PAGE gel of proteins synthesized in PURExpress or OnePot (EF-Tu 47%, replicate A) (a) labeled with FluoroTect GreenLys, (b) Coomassie blue stained. Black arrows indicate the expected bands of synthesized proteins, GFP (26.9 kDa) T3 RNAP (98.8 kDa), β -galactosidase (116.5 kDa) and trehalase (63.7 kDa), DHFR (18 kDa)

    Techniques Used: SDS Page, Synthesized, Labeling, Staining

    Activities of different proteins, expressed in PURExpress and OnePot (EF-Tu 47%, replicate A). Trehalase assay: (a) Absorbance change at 540 nm and (b) image of resulting color change due to the presence of trehalase in the reaction. Three reactions were measured for positive samples. Error bars represent standard deviation. β -galactosidase assay: (c) absorbance (580 nm) increase over time due to substrate cleavage, (d) slope of absorbance. Three and one OnePot PURE reactions were measured for positive and negative samples, respectively. Each reaction was measured in triplicate. Error bars represent standard deviation. Zinc-finger (ZF) repression: (e) Down-regulation of deGFP expression, due to binding of ZF to the target promoter. deGFP containing lambda PR promoter containing double ADD ZF binding sites was used as a reporter. The ADD ZF was co-expressed with deGFP (repressed state), and the CBD ZF was co-expressed as a negative control (unrepressed state). (f) Fold-repression, the ratio of unrepressed to repressed expression levels. Each data point represents three technical replicates (mean ± s.d.)
    Figure Legend Snippet: Activities of different proteins, expressed in PURExpress and OnePot (EF-Tu 47%, replicate A). Trehalase assay: (a) Absorbance change at 540 nm and (b) image of resulting color change due to the presence of trehalase in the reaction. Three reactions were measured for positive samples. Error bars represent standard deviation. β -galactosidase assay: (c) absorbance (580 nm) increase over time due to substrate cleavage, (d) slope of absorbance. Three and one OnePot PURE reactions were measured for positive and negative samples, respectively. Each reaction was measured in triplicate. Error bars represent standard deviation. Zinc-finger (ZF) repression: (e) Down-regulation of deGFP expression, due to binding of ZF to the target promoter. deGFP containing lambda PR promoter containing double ADD ZF binding sites was used as a reporter. The ADD ZF was co-expressed with deGFP (repressed state), and the CBD ZF was co-expressed as a negative control (unrepressed state). (f) Fold-repression, the ratio of unrepressed to repressed expression levels. Each data point represents three technical replicates (mean ± s.d.)

    Techniques Used: Standard Deviation, Expressing, Binding Assay, Negative Control

    4) Product Images from "Self-assembled nanoparticle-enzyme aggregates enhance functional protein production in pure transcription-translation systems"

    Article Title: Self-assembled nanoparticle-enzyme aggregates enhance functional protein production in pure transcription-translation systems

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0265274

    Characterization of PURExpress®–QD conjugates. (A) Chemical structure of the CL4 ligand used to make the QDs colloidally stable in aqueous shown in the open dithiol configuration. (B) Agarose gel electrophoretic mobility shift assay of 523 nm emitting CdSe/CdS/ZnS core/shell/shell QDs incubated without and with a series of decreasing concentrations of the PURExpress® protein solution. Less mobility is correlated with binding to enzyme and the magnitude of this is decreased as the protein solution is serially diluted. The dashed white line indicates the location of sample wells in the gel. (C) Left—High-resolution TEM micrograph of the 523 nm emitting CdSe/CdS/ZnS core/shell/shell QDs with an average diameter of 4.1 ± 0.5 nm. A single QD is circled in red for visualization. Right—High-resolution TEM micrograph of the 625 nm emitting CdSe/ZnS core/shell QDs utilized for nanoaggregation studies due to their larger size and higher electron density which makes for easier imaging. (D) TEM micrographs of the PURExpress® protein solution (i), 625 QDs in buffer (ii), and 625 QD mixed with 0.5× PURExpress® solution at two different magnifications (iii, iv). Only when the QDs are mixed with the PURExpress® solution is clustering seen. The grey shading around the QD clusters in (iii, iv) are believed to be the PURExpress® enzymes.
    Figure Legend Snippet: Characterization of PURExpress®–QD conjugates. (A) Chemical structure of the CL4 ligand used to make the QDs colloidally stable in aqueous shown in the open dithiol configuration. (B) Agarose gel electrophoretic mobility shift assay of 523 nm emitting CdSe/CdS/ZnS core/shell/shell QDs incubated without and with a series of decreasing concentrations of the PURExpress® protein solution. Less mobility is correlated with binding to enzyme and the magnitude of this is decreased as the protein solution is serially diluted. The dashed white line indicates the location of sample wells in the gel. (C) Left—High-resolution TEM micrograph of the 523 nm emitting CdSe/CdS/ZnS core/shell/shell QDs with an average diameter of 4.1 ± 0.5 nm. A single QD is circled in red for visualization. Right—High-resolution TEM micrograph of the 625 nm emitting CdSe/ZnS core/shell QDs utilized for nanoaggregation studies due to their larger size and higher electron density which makes for easier imaging. (D) TEM micrographs of the PURExpress® protein solution (i), 625 QDs in buffer (ii), and 625 QD mixed with 0.5× PURExpress® solution at two different magnifications (iii, iv). Only when the QDs are mixed with the PURExpress® solution is clustering seen. The grey shading around the QD clusters in (iii, iv) are believed to be the PURExpress® enzymes.

    Techniques Used: Agarose Gel Electrophoresis, Electrophoretic Mobility Shift Assay, Incubation, Binding Assay, Transmission Electron Microscopy, Imaging

    Enhancement of functional PTE production by QDs. (A) Reaction setup highlighting stopping of the CFPS reactions with kanamycin at different time points. Paraoxon hydrolysis tracked by measurement of the p -nitrophenol absorbance product. Schematic not to scale. (B) PURExpress® reaction with QDs produced functional PTE, the activity of which was monitored by absorbance. Kanamycin was added at various time points to quench translation. (C) Identical PURExpress® reaction without QDs treated in the same manner as panel (B) produced less functional PTE, resulting in less activity and p -nitrophenol product absorbance. PTE PDB ID: IPTA [ 76 ]. Other protein structures are the same as shown in Fig 1 .
    Figure Legend Snippet: Enhancement of functional PTE production by QDs. (A) Reaction setup highlighting stopping of the CFPS reactions with kanamycin at different time points. Paraoxon hydrolysis tracked by measurement of the p -nitrophenol absorbance product. Schematic not to scale. (B) PURExpress® reaction with QDs produced functional PTE, the activity of which was monitored by absorbance. Kanamycin was added at various time points to quench translation. (C) Identical PURExpress® reaction without QDs treated in the same manner as panel (B) produced less functional PTE, resulting in less activity and p -nitrophenol product absorbance. PTE PDB ID: IPTA [ 76 ]. Other protein structures are the same as shown in Fig 1 .

    Techniques Used: Functional Assay, Produced, Activity Assay

    sfGFP production is enhanced with QDs in diluted PURExpress® reaction conditions. (A) Production of sfGFP over time with a range of QD concentrations present versus a negative control as monitored by fluorescence. Samples were excited at 485 nm and fluorescence monitored at 510 nm [ 69 ]. Plot for all the QD concentrations can be found in S6 Fig . (B) Yield of functional sfGFP, as estimated by average fluorescence from the end-range of the reactions, over the range of QD concentrations tested (red) as compared to the QD-free reaction (grey). When tested, all samples were statistically different from the free reaction, see S7 Fig and S1 Appendix.
    Figure Legend Snippet: sfGFP production is enhanced with QDs in diluted PURExpress® reaction conditions. (A) Production of sfGFP over time with a range of QD concentrations present versus a negative control as monitored by fluorescence. Samples were excited at 485 nm and fluorescence monitored at 510 nm [ 69 ]. Plot for all the QD concentrations can be found in S6 Fig . (B) Yield of functional sfGFP, as estimated by average fluorescence from the end-range of the reactions, over the range of QD concentrations tested (red) as compared to the QD-free reaction (grey). When tested, all samples were statistically different from the free reaction, see S7 Fig and S1 Appendix.

    Techniques Used: Negative Control, Fluorescence, Functional Assay

    5) Product Images from "Plasmid replication-associated single-strand-specific methyltransferases"

    Article Title: Plasmid replication-associated single-strand-specific methyltransferases

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa1163

    Polymerase and MTase activities copurify when domains are fused. Panel ( A ): Size and purity of fusion proteins. For each MTase, both of the immunoreactive components of the MTase-PolI fusion proteins run at the same position, and comigrate with the Coomassie-stained purified proteins. Western blot (lanes 1, 5, 9 and 10) detected 1 μg of MTase-PolI fusion proteins; Coomassie (lanes 2, 3, 6, 7) visualized 1 μg or 20 μg of the same fractions. Western blots were probed separately with anti-Pol1 rabbit polyclonal or anti-6xHis (detecting the MTase) monoclonal antibodies and developed with horseradish peroxidase-labeled antirabbit or antimouse following kit instructions as detailed in Material and Methods. Dots on lane 1 correspond to the position of protein markers after Western blotting. The bands at the side of lane 10 are spillover from the adjacent lane, which were control 6xHis tagged proteins from a PurExpress extract. Panel ( B ): Activity copurification through two columns. Pooled HiTrapHepHP (#22–26) and HiTrapQHP (#15–19) protein fractions were tested for MTase activity on single-stranded M13mp18 DNA in the presence of [H 3 ]SAM and for DNA-polymerase activity on sonicated sperm-whale DNA in the presence of [H 3 ]TTP.
    Figure Legend Snippet: Polymerase and MTase activities copurify when domains are fused. Panel ( A ): Size and purity of fusion proteins. For each MTase, both of the immunoreactive components of the MTase-PolI fusion proteins run at the same position, and comigrate with the Coomassie-stained purified proteins. Western blot (lanes 1, 5, 9 and 10) detected 1 μg of MTase-PolI fusion proteins; Coomassie (lanes 2, 3, 6, 7) visualized 1 μg or 20 μg of the same fractions. Western blots were probed separately with anti-Pol1 rabbit polyclonal or anti-6xHis (detecting the MTase) monoclonal antibodies and developed with horseradish peroxidase-labeled antirabbit or antimouse following kit instructions as detailed in Material and Methods. Dots on lane 1 correspond to the position of protein markers after Western blotting. The bands at the side of lane 10 are spillover from the adjacent lane, which were control 6xHis tagged proteins from a PurExpress extract. Panel ( B ): Activity copurification through two columns. Pooled HiTrapHepHP (#22–26) and HiTrapQHP (#15–19) protein fractions were tested for MTase activity on single-stranded M13mp18 DNA in the presence of [H 3 ]SAM and for DNA-polymerase activity on sonicated sperm-whale DNA in the presence of [H 3 ]TTP.

    Techniques Used: Staining, Purification, Western Blot, Labeling, Activity Assay, Copurification, Sonication

    MTase activity requires single strands. Panels ( A ) and ( C ): M13 substrates stained with ethidium bromide. Panels ( B ) and ( D ): fluorograms of modification reactions using [H 3 ]SAM. M13 SS: virion DNA substrate. M13 RF cut: DS replication intermediate RFI was digested following the labelling reaction for visual simplification; NdeI (Panels A and B) or NdeI+BamHI (Panels C and D). The substrates were treated with MTase proteins obtained with PURExpress in vitro transcription-translation (Panels A and B) or were partially-purified (Ni-NTA purification) proteins synthesized in vivo (Panels C and D). Lanes 1) empty pSAPv6 vector, 2) M.BceJIII WT (pAF9), 3) M.EcoGIX WT (pAF10) and 4) M.EcoGIX APPA variant (pAF11). H 3 radiolabeled markers (M) are HindIII digested lambda DNA modified at A by M.EcoGII.
    Figure Legend Snippet: MTase activity requires single strands. Panels ( A ) and ( C ): M13 substrates stained with ethidium bromide. Panels ( B ) and ( D ): fluorograms of modification reactions using [H 3 ]SAM. M13 SS: virion DNA substrate. M13 RF cut: DS replication intermediate RFI was digested following the labelling reaction for visual simplification; NdeI (Panels A and B) or NdeI+BamHI (Panels C and D). The substrates were treated with MTase proteins obtained with PURExpress in vitro transcription-translation (Panels A and B) or were partially-purified (Ni-NTA purification) proteins synthesized in vivo (Panels C and D). Lanes 1) empty pSAPv6 vector, 2) M.BceJIII WT (pAF9), 3) M.EcoGIX WT (pAF10) and 4) M.EcoGIX APPA variant (pAF11). H 3 radiolabeled markers (M) are HindIII digested lambda DNA modified at A by M.EcoGII.

    Techniques Used: Activity Assay, Staining, Modification, In Vitro, Purification, Synthesized, In Vivo, Plasmid Preparation, Variant Assay, Lambda DNA Preparation

    6) Product Images from "Ribosomal protein S1 plays a critical role in horizontal gene transfer by mediating the expression of foreign mRNAs"

    Article Title: Ribosomal protein S1 plays a critical role in horizontal gene transfer by mediating the expression of foreign mRNAs

    Journal: bioRxiv

    doi: 10.1101/2021.10.13.464283

    PSIV IGR IRES translation efficiency is consistent over multiple growth phases. (A) Relative fluorescence in vivo time course of E. coli containing PSIV IGR IRES constructs as measured by flow cytometry. (B) The linear portion of sfGFP expression in panel A (C) sfGFP production in vitro time course using the PSIV IGR IRES constructs and the PURExpress® system (D) Relative fluorescence of PSIV IGR IRES constructs and the PURExpress® Δ ribosome system and ribosomes with and without ribosomal protein S1. Mean values of three biological replicates are plotted; error bars indicate one standard deviation.
    Figure Legend Snippet: PSIV IGR IRES translation efficiency is consistent over multiple growth phases. (A) Relative fluorescence in vivo time course of E. coli containing PSIV IGR IRES constructs as measured by flow cytometry. (B) The linear portion of sfGFP expression in panel A (C) sfGFP production in vitro time course using the PSIV IGR IRES constructs and the PURExpress® system (D) Relative fluorescence of PSIV IGR IRES constructs and the PURExpress® Δ ribosome system and ribosomes with and without ribosomal protein S1. Mean values of three biological replicates are plotted; error bars indicate one standard deviation.

    Techniques Used: Fluorescence, In Vivo, Construct, Flow Cytometry, Expressing, In Vitro, Standard Deviation

    7) Product Images from "PERSIA for Direct Fluorescence Measurements of Transcription, Translation, and Enzyme Activity in Cell-Free Systems"

    Article Title: PERSIA for Direct Fluorescence Measurements of Transcription, Translation, and Enzyme Activity in Cell-Free Systems

    Journal: ACS synthetic biology

    doi: 10.1021/acssynbio.8b00450

    Optimizing fluorophore concentrations for PERSIA. (A) Increasing amounts of DFHBI were added to the PURExpress reaction to determine an effective concentration for measuring mRNA present through DFHBI binding to the Spinach RNA tag. 50 µM was chosen as the standard amount of DFHBI to be used in future reactions due to a combination of low background and high signal. (B) Increasing amounts of ReAsH-EDT 2 were added to the PURExpress reaction to find an effective concentration to quantitate the amount of protein present through ReAsH-EDT 2 binding to the tetracysteine (TC) tag. 5 µM was chosen as the standard amount of ReAsH to be used in future reactions.
    Figure Legend Snippet: Optimizing fluorophore concentrations for PERSIA. (A) Increasing amounts of DFHBI were added to the PURExpress reaction to determine an effective concentration for measuring mRNA present through DFHBI binding to the Spinach RNA tag. 50 µM was chosen as the standard amount of DFHBI to be used in future reactions due to a combination of low background and high signal. (B) Increasing amounts of ReAsH-EDT 2 were added to the PURExpress reaction to find an effective concentration to quantitate the amount of protein present through ReAsH-EDT 2 binding to the tetracysteine (TC) tag. 5 µM was chosen as the standard amount of ReAsH to be used in future reactions.

    Techniques Used: Concentration Assay, Binding Assay

    8) Product Images from "Circuitry Linking the Global Csr- and σE-Dependent Cell Envelope Stress Response Systems"

    Article Title: Circuitry Linking the Global Csr- and σE-Dependent Cell Envelope Stress Response Systems

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00484-17

    CsrA represses translation of rpoE . (A and B) Schematic representations of the fusions used in this analysis are shown at the top. T7 RNAP drives transcription from the P2 (A) or P1 (B) transcription start sites. The start codon (ATG) driving the translation of each fusion is shown. The rpoE promoter and leader regions are depicted with a thin black line, while the rpoE and lacZ coding sequences are depicted with thick black and red lines, respectively. GGA motif mutations in BS1 and/or BS3, as well as an ORF51 stop codon mutant, are indicated with a red X. Relative β-galactosidase activity ± standard deviation as a function of CsrA concentration from at least three experiments was determined in vitro with PURExpress. A phoB ′-′ lacZ translational fusion was used as a negative control. (A) Expression of a WT T7(P2)- rpoE ′-′ lacZ translational fusion, as well as mutant fusions containing GGA-to-CCA mutations in BS1, or both BS1 and BS3. (B) Expression of a WT T7(P1)- rpoE ′-′ lacZ translational fusion, as well as mutant fusions containing a GGA-to-GCA mutation in BS1, or a stop codon mutation in codon 12 of ORF51.
    Figure Legend Snippet: CsrA represses translation of rpoE . (A and B) Schematic representations of the fusions used in this analysis are shown at the top. T7 RNAP drives transcription from the P2 (A) or P1 (B) transcription start sites. The start codon (ATG) driving the translation of each fusion is shown. The rpoE promoter and leader regions are depicted with a thin black line, while the rpoE and lacZ coding sequences are depicted with thick black and red lines, respectively. GGA motif mutations in BS1 and/or BS3, as well as an ORF51 stop codon mutant, are indicated with a red X. Relative β-galactosidase activity ± standard deviation as a function of CsrA concentration from at least three experiments was determined in vitro with PURExpress. A phoB ′-′ lacZ translational fusion was used as a negative control. (A) Expression of a WT T7(P2)- rpoE ′-′ lacZ translational fusion, as well as mutant fusions containing GGA-to-CCA mutations in BS1, or both BS1 and BS3. (B) Expression of a WT T7(P1)- rpoE ′-′ lacZ translational fusion, as well as mutant fusions containing a GGA-to-GCA mutation in BS1, or a stop codon mutation in codon 12 of ORF51.

    Techniques Used: Mutagenesis, Activity Assay, Standard Deviation, Concentration Assay, In Vitro, Negative Control, Expressing

    9) Product Images from "Diblock copolymers enhance folding of a mechanosensitive membrane protein during cell-free expression"

    Article Title: Diblock copolymers enhance folding of a mechanosensitive membrane protein during cell-free expression

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1814775116

    Lipid vesicles improve the production of MscL during cell-free protein synthesis. ( A ) Schematic of a cell-free reaction in which DNA and vesicles were mixed with PURExpress kit components. ( B ) Schematic of the plasmid used to generate an MscLGFP fusion protein. MscL is tagged C-terminally with mEGFP: the proper folding of MscL allows GFP folding and fluorescence ( Right ) while the misfolding or aggregation of MscL does not permit GFP folding ( Left ). ( C ) Fluorescence of MscLGFP and soluble GFP 3.5 h after cell-free reactions with varying concentrations of DOPC vesicles, normalized to the maximum GFP fluorescence value observed for each protein. ( D ) Quantitative Western blot of MscLGFP from cell-free reactions shown in C . Densitometry values were normalized to reactions performed in water. **** P ≤ 0.0001 ( P values were generated by ANOVA using the Dunnett test for multiple comparisons to the sample performed in water). n = 3; error bars represent standard error of the mean (SEM); ns, nonsignificant, P > 0.05.
    Figure Legend Snippet: Lipid vesicles improve the production of MscL during cell-free protein synthesis. ( A ) Schematic of a cell-free reaction in which DNA and vesicles were mixed with PURExpress kit components. ( B ) Schematic of the plasmid used to generate an MscLGFP fusion protein. MscL is tagged C-terminally with mEGFP: the proper folding of MscL allows GFP folding and fluorescence ( Right ) while the misfolding or aggregation of MscL does not permit GFP folding ( Left ). ( C ) Fluorescence of MscLGFP and soluble GFP 3.5 h after cell-free reactions with varying concentrations of DOPC vesicles, normalized to the maximum GFP fluorescence value observed for each protein. ( D ) Quantitative Western blot of MscLGFP from cell-free reactions shown in C . Densitometry values were normalized to reactions performed in water. **** P ≤ 0.0001 ( P values were generated by ANOVA using the Dunnett test for multiple comparisons to the sample performed in water). n = 3; error bars represent standard error of the mean (SEM); ns, nonsignificant, P > 0.05.

    Techniques Used: Plasmid Preparation, Fluorescence, Western Blot, Generated

    10) Product Images from "Cell-free synthesis of natural compounds from genomic DNA of biosynthetic gene clusters"

    Article Title: Cell-free synthesis of natural compounds from genomic DNA of biosynthetic gene clusters

    Journal: bioRxiv

    doi: 10.1101/2020.04.04.025353

    Synthesis of BpsA with the PURE cell-free system. (A) Expression control by Western blotting with anti-Strep antibodies performed in three independent reaction solutions (#1-3). BpsA was applied as holo -protein, produced by IVPS with simultaneous phosphopantetheinylation. Self-cast 9 % Tris-Tricine gel. Strep-tagged BpsA has a molecular weight of 142.7 kDa. For the uncropped blot, see Figure S2A. (B) SEC profiles and Western Blot detection of elution fractions. (top) Recombinantly produced BpsA and (bottom) IVPS reaction solution including phosphopantetheinylation. (C) Quantification of protein production yields and phosphopantetheinylation efficiency. BpsA was first produced by IVPS and then phosphopantetheinylated with Sfp and CoA-647 (purchased from NEB). Samples from three independent reactions (#1-3) were applied in repetition (a b). For calibration, recombinantly produced BpsA, diluted in the PURExpress reaction solution, was loaded in amounts of 1.25, 0.63, 0.31 and 0.16 pmol. 9 % Tris-Tricine gel as in panel A. For the uncropped gels, see Figure S2B. Overall, three times three reactions, each applied in duplicate (18 bands), were used for quantification of BpsA production and phosphopantetheinylation for the parallel and the sequential protocol, respectively (Figure S3 A-C).
    Figure Legend Snippet: Synthesis of BpsA with the PURE cell-free system. (A) Expression control by Western blotting with anti-Strep antibodies performed in three independent reaction solutions (#1-3). BpsA was applied as holo -protein, produced by IVPS with simultaneous phosphopantetheinylation. Self-cast 9 % Tris-Tricine gel. Strep-tagged BpsA has a molecular weight of 142.7 kDa. For the uncropped blot, see Figure S2A. (B) SEC profiles and Western Blot detection of elution fractions. (top) Recombinantly produced BpsA and (bottom) IVPS reaction solution including phosphopantetheinylation. (C) Quantification of protein production yields and phosphopantetheinylation efficiency. BpsA was first produced by IVPS and then phosphopantetheinylated with Sfp and CoA-647 (purchased from NEB). Samples from three independent reactions (#1-3) were applied in repetition (a b). For calibration, recombinantly produced BpsA, diluted in the PURExpress reaction solution, was loaded in amounts of 1.25, 0.63, 0.31 and 0.16 pmol. 9 % Tris-Tricine gel as in panel A. For the uncropped gels, see Figure S2B. Overall, three times three reactions, each applied in duplicate (18 bands), were used for quantification of BpsA production and phosphopantetheinylation for the parallel and the sequential protocol, respectively (Figure S3 A-C).

    Techniques Used: Expressing, Western Blot, Produced, Molecular Weight

    11) Product Images from "CsrA maximizes expression of the AcrAB multidrug resistance transporter"

    Article Title: CsrA maximizes expression of the AcrAB multidrug resistance transporter

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx929

    Effect of CsrA on acrA -GFP translation. Coupled transcription-translation reactions were performed with a PURExpress kit using pT7- acrA -GFP and pT7- acrA -GFP Mut CsrA BS translational fusions in the presence and absence of purified CsrA-His protein (320 nM). Fluorescence was measured at excitation and emission wavelengths of 492 and 520 nm, respectively using a FLUOstar Optima. Each experiment was performed three times.
    Figure Legend Snippet: Effect of CsrA on acrA -GFP translation. Coupled transcription-translation reactions were performed with a PURExpress kit using pT7- acrA -GFP and pT7- acrA -GFP Mut CsrA BS translational fusions in the presence and absence of purified CsrA-His protein (320 nM). Fluorescence was measured at excitation and emission wavelengths of 492 and 520 nm, respectively using a FLUOstar Optima. Each experiment was performed three times.

    Techniques Used: Purification, Fluorescence

    12) Product Images from "Cell-free expression tools to study co-translational folding of alpha helical membrane transporters"

    Article Title: Cell-free expression tools to study co-translational folding of alpha helical membrane transporters

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-66097-4

    Cell-free expression of individual MFS domains as two separate polypeptides. ( a ) The DNA for each domain of LacY and XylE were cloned into two separate plasmids to be expressed as independent domains. LacY was split at residue L212, XylE at V275. The N and C domain of each MFS transporter could then be expressed separately as two separate polypeptides in PURExpress. ( b ) The N and C domains of LacY and XylE were expressed individually using PURExpress in the presence of 25:50:25 DOPC:DOPE:DOPG liposomes, and the inserted protein was separated from aggregates by sucrose flotation. When the inserted protein was analysed by SDS-PAGE and visualised by [ 35 S] Met phosphorimaging, a band was observed for the N and C domains of each transporter, indicating that each can insert spontaneously into liposomes when produced separately. A small amount of oligomer was also observed for the LacY C domain and the N and C domains of XylE. The N and C domains of each transporter were also expressed as two separate polypeptides in the same reaction and floated on a sucrose gradient (lanes 3 and 6). The original, uncropped, image is in the Supplementary Information, and is otherwise unadjusted. ( c ) As in (b), but the amount of spontaneous insertion was quantified via LSC of incorporated [ 35 S] Met. Around 40% of the protein expressed inserts spontaneously into liposomes under these conditions (LacY N 37.9 ± 3.0%, LacY C 41.2 ± 2.8%, XylE N 39.0 ± 3.2%, XylE C 43.4 ± 3.0%. Error is SEM, n = 3).
    Figure Legend Snippet: Cell-free expression of individual MFS domains as two separate polypeptides. ( a ) The DNA for each domain of LacY and XylE were cloned into two separate plasmids to be expressed as independent domains. LacY was split at residue L212, XylE at V275. The N and C domain of each MFS transporter could then be expressed separately as two separate polypeptides in PURExpress. ( b ) The N and C domains of LacY and XylE were expressed individually using PURExpress in the presence of 25:50:25 DOPC:DOPE:DOPG liposomes, and the inserted protein was separated from aggregates by sucrose flotation. When the inserted protein was analysed by SDS-PAGE and visualised by [ 35 S] Met phosphorimaging, a band was observed for the N and C domains of each transporter, indicating that each can insert spontaneously into liposomes when produced separately. A small amount of oligomer was also observed for the LacY C domain and the N and C domains of XylE. The N and C domains of each transporter were also expressed as two separate polypeptides in the same reaction and floated on a sucrose gradient (lanes 3 and 6). The original, uncropped, image is in the Supplementary Information, and is otherwise unadjusted. ( c ) As in (b), but the amount of spontaneous insertion was quantified via LSC of incorporated [ 35 S] Met. Around 40% of the protein expressed inserts spontaneously into liposomes under these conditions (LacY N 37.9 ± 3.0%, LacY C 41.2 ± 2.8%, XylE N 39.0 ± 3.2%, XylE C 43.4 ± 3.0%. Error is SEM, n = 3).

    Techniques Used: Expressing, Clone Assay, SDS Page, Produced

    13) Product Images from "Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology"

    Article Title: Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology

    Journal: Frontiers in Bioengineering and Biotechnology

    doi: 10.3389/fbioe.2020.00213

    Compartmentalized cell-free reactions. Schematic representation of the different strategies used to compartmentalize cell-free transcription translation reactions. (A) Emulsion-based compartments: polydisperse water-in-oil droplets obtained by mechanical agitation, and microfluidic production of monodisperse droplets. (B) Liquid-liquid phase separation: aqueous multiphase systems containing cell-free transcription translation machinery (Torre et al., 2014 ), and representation of a complex coacervate. (C) Hydrogels: X-DNA linking template DNA and forming a DNA hydrogel (Park et al., 2009a , b ), a DNA-clay hydrogel (Yang et al., 2013 ), hyaluronic acid (Thiele et al., 2014 ), or agarose (Aufinger and Simmel, 2018 ) functionalized with DNA template, polyacrylamide hydrogel functionalized with Ni 2+ -NTA binding PURExpress His-tagged proteins (Zhou et al., 2018 ). (D) Liposomes: rehydration of lipid films with an aqueous solution containing TX-TL, droplet transfer method where a lipid-stabilized W/O emulsion is layered on top of a feeding buffer and liposomes transferred to the bottom by centrifugation (Noireaux and Libchaber, 2004 ), double-emulsions with ultrathin shells containing lipids in organic solvent (Ho et al., 2015 , 2017 ), and octanol-assisted assembly (Deshpande et al., 2016 ; Deshpande and Dekker, 2018 ). (E) Other compartments: polymersomes with membrane formed by amphiphilic polymers, proteinosomes with amphiphilic peptides (Vogele et al., 2018 ), alginate hydrogel coated with various polymers, artificial cells with polymeric shell and liquid core containing a DNA-clay “nucleus” (Niederholtmeyer et al., 2018 ).
    Figure Legend Snippet: Compartmentalized cell-free reactions. Schematic representation of the different strategies used to compartmentalize cell-free transcription translation reactions. (A) Emulsion-based compartments: polydisperse water-in-oil droplets obtained by mechanical agitation, and microfluidic production of monodisperse droplets. (B) Liquid-liquid phase separation: aqueous multiphase systems containing cell-free transcription translation machinery (Torre et al., 2014 ), and representation of a complex coacervate. (C) Hydrogels: X-DNA linking template DNA and forming a DNA hydrogel (Park et al., 2009a , b ), a DNA-clay hydrogel (Yang et al., 2013 ), hyaluronic acid (Thiele et al., 2014 ), or agarose (Aufinger and Simmel, 2018 ) functionalized with DNA template, polyacrylamide hydrogel functionalized with Ni 2+ -NTA binding PURExpress His-tagged proteins (Zhou et al., 2018 ). (D) Liposomes: rehydration of lipid films with an aqueous solution containing TX-TL, droplet transfer method where a lipid-stabilized W/O emulsion is layered on top of a feeding buffer and liposomes transferred to the bottom by centrifugation (Noireaux and Libchaber, 2004 ), double-emulsions with ultrathin shells containing lipids in organic solvent (Ho et al., 2015 , 2017 ), and octanol-assisted assembly (Deshpande et al., 2016 ; Deshpande and Dekker, 2018 ). (E) Other compartments: polymersomes with membrane formed by amphiphilic polymers, proteinosomes with amphiphilic peptides (Vogele et al., 2018 ), alginate hydrogel coated with various polymers, artificial cells with polymeric shell and liquid core containing a DNA-clay “nucleus” (Niederholtmeyer et al., 2018 ).

    Techniques Used: Binding Assay, Centrifugation

    14) Product Images from "Intramolecular chaperone-mediated secretion of an Rhs effector toxin by a type VI secretion system"

    Article Title: Intramolecular chaperone-mediated secretion of an Rhs effector toxin by a type VI secretion system

    Journal: Nature Communications

    doi: 10.1038/s41467-020-15774-z

    Characterization of TseI cleavage and key residues. a Cleavage sites determined by N-terminal sequencing. Each band was excised for N-terminal Edman sequencing as well as LC-MS/MS identification (see also Supplementary Fig. 3 A). b Weblogo depicting conserved residues of Rhs N-/C-terminal sequences deriving from sequence alignment of 48 representative Rhs homologs. Sequences are provided in Supplementary Data 1 . Black arrows indicate the predicted key activity residues that are mutated in this study while gray arrows indicate the first residue of Rhs and VIRC post cleavage, respectively. c Western blotting analysis of TseI and its cleavage-defective mutants. All constructs were cloned to pETDUET1 vectors with an N-terminal FLAG tag and a C-terminal 3V5 tag. Proteins were induced in E. coli with 0.01 mM IPTG. The nontoxic HFH-AAA TseI mutant is used as the parental construct. The same pETDUET1 constructs were also used for in vitro expression shown in d . In vitro expression was performed with a PURExpress ® In Vitro Protein Synthesis Kit following the manufacturer's instruction. Synthesized proteins were subject to SDS-PAGE analysis, followed by western blot analysis with anti-FLAG and anti-V5 antisera. Source data are provided as a Source Data file. Data in a , c , d are representative of at least two replications.
    Figure Legend Snippet: Characterization of TseI cleavage and key residues. a Cleavage sites determined by N-terminal sequencing. Each band was excised for N-terminal Edman sequencing as well as LC-MS/MS identification (see also Supplementary Fig. 3 A). b Weblogo depicting conserved residues of Rhs N-/C-terminal sequences deriving from sequence alignment of 48 representative Rhs homologs. Sequences are provided in Supplementary Data 1 . Black arrows indicate the predicted key activity residues that are mutated in this study while gray arrows indicate the first residue of Rhs and VIRC post cleavage, respectively. c Western blotting analysis of TseI and its cleavage-defective mutants. All constructs were cloned to pETDUET1 vectors with an N-terminal FLAG tag and a C-terminal 3V5 tag. Proteins were induced in E. coli with 0.01 mM IPTG. The nontoxic HFH-AAA TseI mutant is used as the parental construct. The same pETDUET1 constructs were also used for in vitro expression shown in d . In vitro expression was performed with a PURExpress ® In Vitro Protein Synthesis Kit following the manufacturer's instruction. Synthesized proteins were subject to SDS-PAGE analysis, followed by western blot analysis with anti-FLAG and anti-V5 antisera. Source data are provided as a Source Data file. Data in a , c , d are representative of at least two replications.

    Techniques Used: Sequencing, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Western Blot, Construct, Clone Assay, FLAG-tag, Mutagenesis, In Vitro, Expressing, Synthesized, SDS Page

    15) Product Images from "Cell-free synthesis of natural compounds from genomic DNA of biosynthetic gene clusters"

    Article Title: Cell-free synthesis of natural compounds from genomic DNA of biosynthetic gene clusters

    Journal: bioRxiv

    doi: 10.1101/2020.04.04.025353

    Synthesis of BpsA with the PURE cell-free system. (A) Expression control by Western blotting with anti-Strep antibodies performed in three independent reaction solutions (#1-3). BpsA was applied as holo -protein, produced by IVPS with simultaneous phosphopantetheinylation. Self-cast 9 % Tris-Tricine gel. Strep-tagged BpsA has a molecular weight of 142.7 kDa. For the uncropped blot, see Figure S2A. (B) SEC profiles and Western Blot detection of elution fractions. (top) Recombinantly produced BpsA and (bottom) IVPS reaction solution including phosphopantetheinylation. (C) Quantification of protein production yields and phosphopantetheinylation efficiency. BpsA was first produced by IVPS and then phosphopantetheinylated with Sfp and CoA-647 (purchased from NEB). Samples from three independent reactions (#1-3) were applied in repetition (a b). For calibration, recombinantly produced BpsA, diluted in the PURExpress reaction solution, was loaded in amounts of 1.25, 0.63, 0.31 and 0.16 pmol. 9 % Tris-Tricine gel as in panel A. For the uncropped gels, see Figure S2B. Overall, three times three reactions, each applied in duplicate (18 bands), were used for quantification of BpsA production and phosphopantetheinylation for the parallel and the sequential protocol, respectively (Figure S3 A-C).
    Figure Legend Snippet: Synthesis of BpsA with the PURE cell-free system. (A) Expression control by Western blotting with anti-Strep antibodies performed in three independent reaction solutions (#1-3). BpsA was applied as holo -protein, produced by IVPS with simultaneous phosphopantetheinylation. Self-cast 9 % Tris-Tricine gel. Strep-tagged BpsA has a molecular weight of 142.7 kDa. For the uncropped blot, see Figure S2A. (B) SEC profiles and Western Blot detection of elution fractions. (top) Recombinantly produced BpsA and (bottom) IVPS reaction solution including phosphopantetheinylation. (C) Quantification of protein production yields and phosphopantetheinylation efficiency. BpsA was first produced by IVPS and then phosphopantetheinylated with Sfp and CoA-647 (purchased from NEB). Samples from three independent reactions (#1-3) were applied in repetition (a b). For calibration, recombinantly produced BpsA, diluted in the PURExpress reaction solution, was loaded in amounts of 1.25, 0.63, 0.31 and 0.16 pmol. 9 % Tris-Tricine gel as in panel A. For the uncropped gels, see Figure S2B. Overall, three times three reactions, each applied in duplicate (18 bands), were used for quantification of BpsA production and phosphopantetheinylation for the parallel and the sequential protocol, respectively (Figure S3 A-C).

    Techniques Used: Expressing, Western Blot, Produced, Molecular Weight

    16) Product Images from "Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame"

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame

    Journal: mBio

    doi: 10.1128/mBio.01355-17

    Effects of csrA :: kan and BS2 mutations on iraD expression. (A) β-Galactosidase activities (Miller units) ± standard deviations of P1-P2- iraD ' - ' lacZ translational fusions were determined throughout growth. Experiments were performed at least three times. A representative growth curve is shown with a dashed line (Klett). Symbols: black, WT fusion WT csrA strain; orange, WT fusion csrA :: kan strain; blue, BS2 mutant fusion WT csrA strain; gray, BS2 mutant fusion csrA :: kan strain. (B) β-Galactosidase activities ± standard deviations of P1-P2- iraD ' - ' lacZ and P2- iraD ' - ' lacZ translational fusions in the presence of the indicated CsrA concentration were determined usinjg a PURExpress system. Experiments were performed at least three times. Values for samples without CsrA were set to 100. Symbols: black, WT P1-P2- iraD ' - ' lacZ fusion; orange, BS2 mutant P1-P2- iraD ' - ' lacZ fusion; blue, WT P2- iraD ' - ' lacZ fusion; gray, BS2 mutant P2- iraD ' - ' lacZ fusion; green, control pnp ' - ' lacZ translational fusion that is not repressed by CsrA ( 19 ). (C) Cultures were grown to mid-exponential phase prior to the addition of rifampin. Samples were harvested at the indicated times and then analyzed by primer extension for iraD ' - ' lacZ mRNA levels. mRNA half-lives (T1/2) are shown at the bottom of the gel. This experiment was performed twice.
    Figure Legend Snippet: Effects of csrA :: kan and BS2 mutations on iraD expression. (A) β-Galactosidase activities (Miller units) ± standard deviations of P1-P2- iraD ' - ' lacZ translational fusions were determined throughout growth. Experiments were performed at least three times. A representative growth curve is shown with a dashed line (Klett). Symbols: black, WT fusion WT csrA strain; orange, WT fusion csrA :: kan strain; blue, BS2 mutant fusion WT csrA strain; gray, BS2 mutant fusion csrA :: kan strain. (B) β-Galactosidase activities ± standard deviations of P1-P2- iraD ' - ' lacZ and P2- iraD ' - ' lacZ translational fusions in the presence of the indicated CsrA concentration were determined usinjg a PURExpress system. Experiments were performed at least three times. Values for samples without CsrA were set to 100. Symbols: black, WT P1-P2- iraD ' - ' lacZ fusion; orange, BS2 mutant P1-P2- iraD ' - ' lacZ fusion; blue, WT P2- iraD ' - ' lacZ fusion; gray, BS2 mutant P2- iraD ' - ' lacZ fusion; green, control pnp ' - ' lacZ translational fusion that is not repressed by CsrA ( 19 ). (C) Cultures were grown to mid-exponential phase prior to the addition of rifampin. Samples were harvested at the indicated times and then analyzed by primer extension for iraD ' - ' lacZ mRNA levels. mRNA half-lives (T1/2) are shown at the bottom of the gel. This experiment was performed twice.

    Techniques Used: Expressing, Mutagenesis, Concentration Assay

    CsrA represses translation of ORF27 and iraD via translation coupling. (A) Translational coupling model. CsrA-mediated repression of ORF27 translation leads to repression of iraD translation via translational coupling. Critical GGA motifs of CsrA binding sites BS1 to BS4 (red) and the iraD start codon are also marked. The ORF27 start and stop codons are boxed in yellow. The ORF27 and iraD SD sequences are marked. (B) β-Galactosidase activities ± standard deviations of WT and stop codon mutant ORF27' - ' lacZ and iraD ' - ' lacZ translational fusions determined in vitro with PURExpress.
    Figure Legend Snippet: CsrA represses translation of ORF27 and iraD via translation coupling. (A) Translational coupling model. CsrA-mediated repression of ORF27 translation leads to repression of iraD translation via translational coupling. Critical GGA motifs of CsrA binding sites BS1 to BS4 (red) and the iraD start codon are also marked. The ORF27 start and stop codons are boxed in yellow. The ORF27 and iraD SD sequences are marked. (B) β-Galactosidase activities ± standard deviations of WT and stop codon mutant ORF27' - ' lacZ and iraD ' - ' lacZ translational fusions determined in vitro with PURExpress. "stop" indicates an ORF27 start-to-stop codon mutation. Experiments were performed at least three times. Values are reported as (1,000)(optical density at 420 nm [OD 420 ]) in a 12.5-min reaction. Symbols: solid blue, wild-type (WT) ORF27' - ' lacZ fusion; striped blue, stop codon mutant ORF27' - ' lacZ fusion; solid black, WT iraD ' - ' lacZ fusion; striped black, stop codon mutant iraD ' - ' lacZ fusion. (C) β-Galactosidase activities (Miller units) ± standard deviations of P1-P2-ORF27' - ' lacZ and P1-P2- iraD ' - ' lacZ translational fusions determined in WT (+) and csrA :: kan mutant (–) strains during exponential-phase and stationary-phase growth. Experiments were performed at least three times. Symbols: blue, WT ORF27' - ' lacZ fusion; black, WT iraD ' - ' lacZ fusion; red, iraD ' - ' lacZ fusion with an ORF27 stop codon mutation.

    Techniques Used: Binding Assay, Mutagenesis, In Vitro

    17) Product Images from "Using Group II Introns for Attenuating the In Vitro and In Vivo Expression of a Homing Endonuclease"

    Article Title: Using Group II Introns for Attenuating the In Vitro and In Vivo Expression of a Homing Endonuclease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0150097

    (A) The effect of MgCl 2 on in vitro protein expression. A 12.5% SDS-PAGE showing in vitro protein expression for constructs I-CthI-[IIA1]-pET28b (+) [left] and I-CthI-[IIB]-pET28b (+) [right] in the presence of various concentrations of external MgCl 2 in the culture media. Lane 1 represents the E . coli dihydrofolate reductase (marked with arrow) when 125 ng/μL was used as the template (positive control) for the PURExpress In Vitro Protein Synthesis kit. Lanes 2 and 10 show the in vitro protein expression profiles when empty pET28b (+) vectors (without the above constructs) were used as the negative control. Lanes 3 and 11 represent the in vitro protein expression profile when RNA (extracted from the culture in the absence of MgCl 2 ) was used as the template. Lanes 4 through 7 represent the protein expression profiles when RNA (extracted from the cultures in the presence of 1 mM, 5 mM, 10 mM and 20 mM respectively) was used as the template for the in vitro protein synthesis. The expression of the protein (I-CthI) has been marked with arrows. For in vitro expression from the I-CthI-[IIB]-pET28b (+) construct, lanes 12 through 15 follow the same order as depicted for the I-CthI-[IIA1]-pET28b (+) construct (i.e. lanes 4–7). Lanes 8 and 9 represent the Blueye prestained protein ladder (FroggaBio, North York, Ontario). (B) The effect of MgCl 2 on in vivo protein expression. A 12.5% SDS-PAGE showing in vivo protein expression for constructs I-CthI-[IIA1]-pET28b (+) [left] and I-CthI-[IIB]-pET28b (+) [right] in the presence of various concentrations of external MgCl 2 in the culture media. Lanes 1 and 9 represent the in vivo protein expression profiles from the empty pET28b (+) vector (without the constructs). Lanes 2 through 6 represent the protein expression profiles when I-CthI-[IIA1]-pET28b (+) [BL21] was grown under increasing concentrations of external MgCl 2 starting from 0 mM, 1 mM, 5 mM, 10 mM and 20 mM. Lane 10 through 14 represent the protein expression profiles when I-CthI-[IIB]-pET28b (+) (BL21) was grown under increasing concentrations of external MgCl 2 . Lanes 10 through 14 follow the same order as for the protein expression profiles when I-CthI-[IIA1]-pET28b (+) [BL21] was grown under increasing concentrations of external MgCl 2 (i.e. lanes 2–6). The overexpressed I-CthI (migrate at ~29 kDa) has been marked with arrows. Lanes 7 and 8 represent the Blueye prestained protein ladder (FroggaBio, North York, Ontario).
    Figure Legend Snippet: (A) The effect of MgCl 2 on in vitro protein expression. A 12.5% SDS-PAGE showing in vitro protein expression for constructs I-CthI-[IIA1]-pET28b (+) [left] and I-CthI-[IIB]-pET28b (+) [right] in the presence of various concentrations of external MgCl 2 in the culture media. Lane 1 represents the E . coli dihydrofolate reductase (marked with arrow) when 125 ng/μL was used as the template (positive control) for the PURExpress In Vitro Protein Synthesis kit. Lanes 2 and 10 show the in vitro protein expression profiles when empty pET28b (+) vectors (without the above constructs) were used as the negative control. Lanes 3 and 11 represent the in vitro protein expression profile when RNA (extracted from the culture in the absence of MgCl 2 ) was used as the template. Lanes 4 through 7 represent the protein expression profiles when RNA (extracted from the cultures in the presence of 1 mM, 5 mM, 10 mM and 20 mM respectively) was used as the template for the in vitro protein synthesis. The expression of the protein (I-CthI) has been marked with arrows. For in vitro expression from the I-CthI-[IIB]-pET28b (+) construct, lanes 12 through 15 follow the same order as depicted for the I-CthI-[IIA1]-pET28b (+) construct (i.e. lanes 4–7). Lanes 8 and 9 represent the Blueye prestained protein ladder (FroggaBio, North York, Ontario). (B) The effect of MgCl 2 on in vivo protein expression. A 12.5% SDS-PAGE showing in vivo protein expression for constructs I-CthI-[IIA1]-pET28b (+) [left] and I-CthI-[IIB]-pET28b (+) [right] in the presence of various concentrations of external MgCl 2 in the culture media. Lanes 1 and 9 represent the in vivo protein expression profiles from the empty pET28b (+) vector (without the constructs). Lanes 2 through 6 represent the protein expression profiles when I-CthI-[IIA1]-pET28b (+) [BL21] was grown under increasing concentrations of external MgCl 2 starting from 0 mM, 1 mM, 5 mM, 10 mM and 20 mM. Lane 10 through 14 represent the protein expression profiles when I-CthI-[IIB]-pET28b (+) (BL21) was grown under increasing concentrations of external MgCl 2 . Lanes 10 through 14 follow the same order as for the protein expression profiles when I-CthI-[IIA1]-pET28b (+) [BL21] was grown under increasing concentrations of external MgCl 2 (i.e. lanes 2–6). The overexpressed I-CthI (migrate at ~29 kDa) has been marked with arrows. Lanes 7 and 8 represent the Blueye prestained protein ladder (FroggaBio, North York, Ontario).

    Techniques Used: In Vitro, Expressing, SDS Page, Construct, Positive Control, Negative Control, In Vivo, Plasmid Preparation

    18) Product Images from "Characterizing the structure-function relationship of a naturally-occurring RNA thermometer"

    Article Title: Characterizing the structure-function relationship of a naturally-occurring RNA thermometer

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.7b01170

    Testing agsA thermometer function in a cell-free protein synthesis system using purified pre-transcribed mRNA. (a) Fluorescence trajectories over time for translation of SFGFP from agsA constructs and control mRNA in the PURExpress protein synthesis system at 30 °C and 42 °C. Shading represents standard deviation over three replicates. (b) SFGFP production rates, calculated from the trajectories in (a), during the linear synthesis regime for agsA constructs and control at 30 °C (45–50 minutes) and 42 °C (30–35 minutes), with error bars representing standard deviation.
    Figure Legend Snippet: Testing agsA thermometer function in a cell-free protein synthesis system using purified pre-transcribed mRNA. (a) Fluorescence trajectories over time for translation of SFGFP from agsA constructs and control mRNA in the PURExpress protein synthesis system at 30 °C and 42 °C. Shading represents standard deviation over three replicates. (b) SFGFP production rates, calculated from the trajectories in (a), during the linear synthesis regime for agsA constructs and control at 30 °C (45–50 minutes) and 42 °C (30–35 minutes), with error bars representing standard deviation.

    Techniques Used: Purification, Fluorescence, Construct, Standard Deviation

    19) Product Images from "In vitro characterisation of the MS2 RNA polymerase complex reveals host factors that modulate emesviral replicase activity"

    Article Title: In vitro characterisation of the MS2 RNA polymerase complex reveals host factors that modulate emesviral replicase activity

    Journal: Communications Biology

    doi: 10.1038/s42003-022-03178-2

    Effect of different PURE co-factors on [F30-Bro(+)] UTR(+) synthesis by MS2rep. a The broccoli aptamer (F30-Bro) for fluorescence readout is incorporated into a replication scaffold based on the MS2 UTRs. Using [F30-Bro(−)] UTR(−) as template, the MS2 replicase complex synthesises [F30-Bro(+)] UTR(+) and the broccoli aptamer therein binds fluorogenic DFHBI-1T. b Replicase activity in commercially available PURExpress (blue), in an in-house PURE 3.0 (purple), as well as in negative controls either lacking MS2rep (black dark grey) or the RNA template (light grey). Reactions were incubated at 37 °C for 30 min and fluorescence was measured every minute. c Endpoint fluorescence of depletion permutation assay for in-house PURE-based replication system, programmed with 50 nM [F30-Bro(−)] UTR(−) RNA. Reactions contained all components except MS2rep (NC, dark grey), all components (PC, blue), or all components except the indicated ones. Fluorescence was measured every minute over a 30-min time course at 37 °C. Error bars indicate standard deviation, based on three independent technical replicates.
    Figure Legend Snippet: Effect of different PURE co-factors on [F30-Bro(+)] UTR(+) synthesis by MS2rep. a The broccoli aptamer (F30-Bro) for fluorescence readout is incorporated into a replication scaffold based on the MS2 UTRs. Using [F30-Bro(−)] UTR(−) as template, the MS2 replicase complex synthesises [F30-Bro(+)] UTR(+) and the broccoli aptamer therein binds fluorogenic DFHBI-1T. b Replicase activity in commercially available PURExpress (blue), in an in-house PURE 3.0 (purple), as well as in negative controls either lacking MS2rep (black dark grey) or the RNA template (light grey). Reactions were incubated at 37 °C for 30 min and fluorescence was measured every minute. c Endpoint fluorescence of depletion permutation assay for in-house PURE-based replication system, programmed with 50 nM [F30-Bro(−)] UTR(−) RNA. Reactions contained all components except MS2rep (NC, dark grey), all components (PC, blue), or all components except the indicated ones. Fluorescence was measured every minute over a 30-min time course at 37 °C. Error bars indicate standard deviation, based on three independent technical replicates.

    Techniques Used: Fluorescence, Activity Assay, Incubation, Standard Deviation

    Full-length MS2 genome replication in PURExpress. a Replication scheme of [F30-Bro(+/−)] MS2(+/−) RNA by the MS2rep complex. b Fluorescence change over the 6-h incubation at 37 °C for replication analysis of 15 nM [F30-Bro(+)] MS2(+) in PURExpress with (blue) and without (red) 1 µM MS2rep. Grey lines indicate average fluorescence levels of reference concentrations of in vitro transcribed [F30-Bro(+)] MS2(+) in PURExpress. Standard deviations are derived from three independent replicates.
    Figure Legend Snippet: Full-length MS2 genome replication in PURExpress. a Replication scheme of [F30-Bro(+/−)] MS2(+/−) RNA by the MS2rep complex. b Fluorescence change over the 6-h incubation at 37 °C for replication analysis of 15 nM [F30-Bro(+)] MS2(+) in PURExpress with (blue) and without (red) 1 µM MS2rep. Grey lines indicate average fluorescence levels of reference concentrations of in vitro transcribed [F30-Bro(+)] MS2(+) in PURExpress. Standard deviations are derived from three independent replicates.

    Techniques Used: Fluorescence, Incubation, In Vitro, Derivative Assay

    20) Product Images from "Characterizing the structure-function relationship of a naturally-occurring RNA thermometer"

    Article Title: Characterizing the structure-function relationship of a naturally-occurring RNA thermometer

    Journal: bioRxiv

    doi: 10.1101/142141

    SHAPE-Seq characterization of the agsA thermometer in vitro in PURExpress. ( a ) SHAPE-Seq normalized reactivity plots for agsA WT mRNA from triplicate experiments in vitro performed on mRNAs at 30 °C and 42 °C, graphed alongside in vivo reactivities from Figure 2a for comparison. Error bars represent standard deviations over replicates. Stars denote statistically significant differences between in vivo and in vitro as determined by heteroscedastic Welch’s T-tests, while the difference plot shows the magnitude of reactivity change (in vitro reactitivies minus in vivo reactivities). ( b ) Experimentally-informed minimum free energy (MFE) RNA structures as predicted using the RNAStructure Fold program with the in vitro reactivity values from (a) taken as pseudoenergy folding restraints. Nucleotides are color-coded according to normalized SHAPE reactivity (ϱ), with red indicating high reactivity and black low reactivity.
    Figure Legend Snippet: SHAPE-Seq characterization of the agsA thermometer in vitro in PURExpress. ( a ) SHAPE-Seq normalized reactivity plots for agsA WT mRNA from triplicate experiments in vitro performed on mRNAs at 30 °C and 42 °C, graphed alongside in vivo reactivities from Figure 2a for comparison. Error bars represent standard deviations over replicates. Stars denote statistically significant differences between in vivo and in vitro as determined by heteroscedastic Welch’s T-tests, while the difference plot shows the magnitude of reactivity change (in vitro reactitivies minus in vivo reactivities). ( b ) Experimentally-informed minimum free energy (MFE) RNA structures as predicted using the RNAStructure Fold program with the in vitro reactivity values from (a) taken as pseudoenergy folding restraints. Nucleotides are color-coded according to normalized SHAPE reactivity (ϱ), with red indicating high reactivity and black low reactivity.

    Techniques Used: In Vitro, In Vivo

    Testing agsA thermometer function in a cell-free protein synthesis system using purified pre-transcribed mRNA. ( a ) Fluorescence trajectories over time for translation of SFGFP from agsA constructs and control mRNA in the PURExpress protein synthesis system at 30 °C and 42 °C. Shading represents standard deviation over three replicates. ( b ) SFGFP production rates, calculated from the trajectories in (a), during the linear synthesis regime for agsA constructs and control at 30 °C (45-50 minutes) and 42 °C (30-35 minutes), with error bars representing standard deviation.
    Figure Legend Snippet: Testing agsA thermometer function in a cell-free protein synthesis system using purified pre-transcribed mRNA. ( a ) Fluorescence trajectories over time for translation of SFGFP from agsA constructs and control mRNA in the PURExpress protein synthesis system at 30 °C and 42 °C. Shading represents standard deviation over three replicates. ( b ) SFGFP production rates, calculated from the trajectories in (a), during the linear synthesis regime for agsA constructs and control at 30 °C (45-50 minutes) and 42 °C (30-35 minutes), with error bars representing standard deviation.

    Techniques Used: Purification, Fluorescence, Construct, Standard Deviation

    Investigating the effect of translation and ribosome occupancy on agsA WT structure and reactivity. ( a ) agsA WT SHAPE-Seq reactivity plot for triplicate experiments carried out in vitro in PURExpress without ribosomes. ( b ) agsA WT SHAPE-Seq reactivity plot in PURExpress with active translation and difference plot of reactivities showing the change from the no ribosome case in part (a). ( c ) agsA WT SHAPE-Seq reactivity plot in PURExpress with stalled translation initiation complexes generated by supplying only methionine and difference plot of reactivities showing the change from the no ribosome case in part (a). Error bars represent standard deviations over replicates. Stars represent statistical significance as in Figure 5 .
    Figure Legend Snippet: Investigating the effect of translation and ribosome occupancy on agsA WT structure and reactivity. ( a ) agsA WT SHAPE-Seq reactivity plot for triplicate experiments carried out in vitro in PURExpress without ribosomes. ( b ) agsA WT SHAPE-Seq reactivity plot in PURExpress with active translation and difference plot of reactivities showing the change from the no ribosome case in part (a). ( c ) agsA WT SHAPE-Seq reactivity plot in PURExpress with stalled translation initiation complexes generated by supplying only methionine and difference plot of reactivities showing the change from the no ribosome case in part (a). Error bars represent standard deviations over replicates. Stars represent statistical significance as in Figure 5 .

    Techniques Used: In Vitro, Generated

    21) Product Images from "In vitro gene expression and detergent-free reconstitution of active proteorhodopsin in lipid vesicles"

    Article Title: In vitro gene expression and detergent-free reconstitution of active proteorhodopsin in lipid vesicles

    Journal: Experimental Biology and Medicine

    doi: 10.1177/1535370218820290

    Proteorhodopsin activity in POPC lipid vesicles was characterized by fluorescence spectroscopic measurements of pH-sensitive pyranine dye (5 mM) in the inner lumen (pH 6.2) of multilamellar vesicles with a pH of 8.5 on the outside of the lipid vesicles. Lipid vesicles containing pyranine were either excited with UV light for 15 min (+) or kept in the dark (–). Fluorescence emission was measured by excitation at λ exc = 404 nm (bandwidth 15 nm) and λ exc = 454 nm (bandwidth 15 nm) and the emission measured at λ emiss = 514 nm (bandwidth 20 nm). The ratio of λ emiss = 514 nm from λ exc (454)/λ exc (404) nm was obtained and then converted to pH units via a calibration curve (Supplementary Figure 4), and the difference in the pH with and without UV radiation was determined (a) (i) proteorhodopsin expressed using PURExpress at 37°C, incubated with POPC lipid vesicles and heat shocked, ΔpH = –0.32 ± 0.07 (ii) CALML3 expressed using PURExpress at 37°C, incubated with POPC lipid vesicles and heat shocked Δ pH = +0.13 ± 0.08 (iii) proteorhodopsin (PR) expressed using PURExpress at 37°C, incubated with POPC lipid vesicles with no heat shock, ΔpH = +0.53 ± 0.08 (iv) proteorhodopsin (PR) expressed using PURExpress at 37°C with 0.4 wt.% digitonin and incubated with POPC lipid vesicles, ΔpH = –0.06 ± 0.05. (v) proteorhodopsin (PR) expressed using PURExpress at 37°C, incubated with POPC lipid vesicles, heat shocked and then incubated with trypsin (0.017 v/v %, 25°C, 3 min), ΔpH = –0.51 ± 0.08 (b) control experiments showing the effect of 15 min of UV light illumination (+) on POPC lipid vesicles filled with pyranine (5 mM) compared to storage of vesicles in the dark for 15 min (–) at (i) equal pH in the inner lumen compared to the extravesicular space (pH 6.2) and (ii) with a pH gradient across the membrane, pH 6.2 in the interlumen space and pH 8.5 on the outside of the vesicles. All measurements were undertaken on lipid vesicles and were incubated with retinal (10 μM) prior to incubation with lipid vesicles with an optical density (OD) at 600 nm of 1. Error bars are obtained from three repeats of three different experiments. (A color version of this figure is available in the online journal.)
    Figure Legend Snippet: Proteorhodopsin activity in POPC lipid vesicles was characterized by fluorescence spectroscopic measurements of pH-sensitive pyranine dye (5 mM) in the inner lumen (pH 6.2) of multilamellar vesicles with a pH of 8.5 on the outside of the lipid vesicles. Lipid vesicles containing pyranine were either excited with UV light for 15 min (+) or kept in the dark (–). Fluorescence emission was measured by excitation at λ exc = 404 nm (bandwidth 15 nm) and λ exc = 454 nm (bandwidth 15 nm) and the emission measured at λ emiss = 514 nm (bandwidth 20 nm). The ratio of λ emiss = 514 nm from λ exc (454)/λ exc (404) nm was obtained and then converted to pH units via a calibration curve (Supplementary Figure 4), and the difference in the pH with and without UV radiation was determined (a) (i) proteorhodopsin expressed using PURExpress at 37°C, incubated with POPC lipid vesicles and heat shocked, ΔpH = –0.32 ± 0.07 (ii) CALML3 expressed using PURExpress at 37°C, incubated with POPC lipid vesicles and heat shocked Δ pH = +0.13 ± 0.08 (iii) proteorhodopsin (PR) expressed using PURExpress at 37°C, incubated with POPC lipid vesicles with no heat shock, ΔpH = +0.53 ± 0.08 (iv) proteorhodopsin (PR) expressed using PURExpress at 37°C with 0.4 wt.% digitonin and incubated with POPC lipid vesicles, ΔpH = –0.06 ± 0.05. (v) proteorhodopsin (PR) expressed using PURExpress at 37°C, incubated with POPC lipid vesicles, heat shocked and then incubated with trypsin (0.017 v/v %, 25°C, 3 min), ΔpH = –0.51 ± 0.08 (b) control experiments showing the effect of 15 min of UV light illumination (+) on POPC lipid vesicles filled with pyranine (5 mM) compared to storage of vesicles in the dark for 15 min (–) at (i) equal pH in the inner lumen compared to the extravesicular space (pH 6.2) and (ii) with a pH gradient across the membrane, pH 6.2 in the interlumen space and pH 8.5 on the outside of the vesicles. All measurements were undertaken on lipid vesicles and were incubated with retinal (10 μM) prior to incubation with lipid vesicles with an optical density (OD) at 600 nm of 1. Error bars are obtained from three repeats of three different experiments. (A color version of this figure is available in the online journal.)

    Techniques Used: Activity Assay, Fluorescence, Incubation

    In vitro transcription translation using PUREexpress at 37°C from pIVEX2.3d construct. (a) Time-resolved fluorescence spectroscopy of in vitro transcription and translation with PURExpress at 37°C for the expression of sfGFP (solid black line), proteorhodopsin (PR) (dotted black line), sfGFP-tagged proteorhodopsin (PR-sfGFP) (solid grey line) and a positive control calmodulin (CALML3) (dotted grey line) with λ exc = 485 nm (bandwidth=20 nm) and λ em = 535 nm (bandwidth = 20 nm) every 10 min. Plot shows that sfGFP reaches a steady state after 120 min with no GFP fluorescence observed from PR-sfGFP or the positive control CALML3 (b) End point fluorescence spectra of sfGFP (grey), PR-sfGFP (black) after 4 h of expression with PUREexpress without digitonin for sfGFP (solid grey line) and PR-sfGFP (solid black line) and in the presence of 0.4 wt.% digitonin for sfGFP (dotted grey line) and PR-sfGFP (dotted black line) shows characteristic spectra for sfGFP. Spectra were measured with λ exc = 480 nm (bandwidth = 15 nm), λ emiss = 505 nm--650 nm (bandwidth (5 nm) with 1 nm step size. (c) Western blot analysis of 10 µl of in vitro transcription translation of calmodulin (CALM3) (i) proteorhodopsin-His tag (PR) (ii) characterized using a primary antibody anti His-tag from mouse and secondary antibody anti-mouse shows bands at approximately 25 kDa for CALML3 (i) and 23 kDa for proteorhodopsin (d) Western blot analysis from 10 µl of in vitro transcription translation of proteorhodopsin-sfGFP (PR-sfGFP) (i) sfGFP) (ii) or calmodulin (CALM3) (iii), PR-sfGFP diluted 1:2 with milli Q water (iv) or sfGFP diluted 1:2 with milli Q water (v) or CALM3 diluted 1:2 in milli q water (vi), using primary antibody anti His-GFP from mouse and secondary antibody anti-mouse show bands at 50 kDa and 27 kDa for PR-sfGFP and sfGFP, respectively. (A color version of this figure is available in the online journal.)
    Figure Legend Snippet: In vitro transcription translation using PUREexpress at 37°C from pIVEX2.3d construct. (a) Time-resolved fluorescence spectroscopy of in vitro transcription and translation with PURExpress at 37°C for the expression of sfGFP (solid black line), proteorhodopsin (PR) (dotted black line), sfGFP-tagged proteorhodopsin (PR-sfGFP) (solid grey line) and a positive control calmodulin (CALML3) (dotted grey line) with λ exc = 485 nm (bandwidth=20 nm) and λ em = 535 nm (bandwidth = 20 nm) every 10 min. Plot shows that sfGFP reaches a steady state after 120 min with no GFP fluorescence observed from PR-sfGFP or the positive control CALML3 (b) End point fluorescence spectra of sfGFP (grey), PR-sfGFP (black) after 4 h of expression with PUREexpress without digitonin for sfGFP (solid grey line) and PR-sfGFP (solid black line) and in the presence of 0.4 wt.% digitonin for sfGFP (dotted grey line) and PR-sfGFP (dotted black line) shows characteristic spectra for sfGFP. Spectra were measured with λ exc = 480 nm (bandwidth = 15 nm), λ emiss = 505 nm--650 nm (bandwidth (5 nm) with 1 nm step size. (c) Western blot analysis of 10 µl of in vitro transcription translation of calmodulin (CALM3) (i) proteorhodopsin-His tag (PR) (ii) characterized using a primary antibody anti His-tag from mouse and secondary antibody anti-mouse shows bands at approximately 25 kDa for CALML3 (i) and 23 kDa for proteorhodopsin (d) Western blot analysis from 10 µl of in vitro transcription translation of proteorhodopsin-sfGFP (PR-sfGFP) (i) sfGFP) (ii) or calmodulin (CALM3) (iii), PR-sfGFP diluted 1:2 with milli Q water (iv) or sfGFP diluted 1:2 with milli Q water (v) or CALM3 diluted 1:2 in milli q water (vi), using primary antibody anti His-GFP from mouse and secondary antibody anti-mouse show bands at 50 kDa and 27 kDa for PR-sfGFP and sfGFP, respectively. (A color version of this figure is available in the online journal.)

    Techniques Used: In Vitro, Construct, Fluorescence, Spectroscopy, Expressing, Positive Control, Western Blot

    Proteorhodopsin folding and insertion into POPC lipid vesicles. (a) End point fluorescence spectroscopy of PR-sfGFP expressed using PURExpress at 37°C with 0.4 wt.% digitonin (dotted black line) and without digitonin (solid black line) with lipid vesicles. The in vitro translation/transcription reaction mixture was incubated with POPC lipid vesicles (dotted black line) or subjected to heat shock to insert the protein into the lipid vesicle (solid black line). Spectra were measured at λ exc = 480 nm (bandwidth = 15 nm), λ emiss = 505 nm--650 nm (bandwidth = 5 nm) with 1 nm step size show characteristic emission profiles for sfGFP indicating that a hydrophobic environment is required for the folding of sfGFP tagged to proteorhodopsin. The fluoresence spectra of only lipid vesicles are shown with the solid grey line. (b) The absorbance spectroscopy of proteorhodopsin (PR) expressed using PURExpress at 37°C measured between 310 nm and 900 nm with 1 nm step size before (dotted grey line) and after heat shock into lipid vesicles (black). An increase in absorbance intensity after heat shock can be attributed to the inclusion of retinal into a folded proteorhodopsin (c) Western blot analysis of lipid vesicles subjected to heat shock (+) and without heat shock (−) of lipid vesicles incubated with in vitro translation transcription reaction mix. (i) Gel of lipid vesicles washed at least five times to remove any protein not associated to the membrane increased chemiluminesence for reaction mixtures which had been subjected to heat shock (+) compared to without heatshock (−) at 23 kDa for proteorhodopsin with 6× histidine tag (ii) Western blot of the supernatant removed after pelleting lipid vesicles shock shows weak chemiluminescence without heat shock (−) compared to with heat shock (+) at 23 kDa, indicating that there is an increased of PR associated to the lipid vesicle and a decrease in the supernatant after heat shock. (A color version of this figure is available in the online journal.)
    Figure Legend Snippet: Proteorhodopsin folding and insertion into POPC lipid vesicles. (a) End point fluorescence spectroscopy of PR-sfGFP expressed using PURExpress at 37°C with 0.4 wt.% digitonin (dotted black line) and without digitonin (solid black line) with lipid vesicles. The in vitro translation/transcription reaction mixture was incubated with POPC lipid vesicles (dotted black line) or subjected to heat shock to insert the protein into the lipid vesicle (solid black line). Spectra were measured at λ exc = 480 nm (bandwidth = 15 nm), λ emiss = 505 nm--650 nm (bandwidth = 5 nm) with 1 nm step size show characteristic emission profiles for sfGFP indicating that a hydrophobic environment is required for the folding of sfGFP tagged to proteorhodopsin. The fluoresence spectra of only lipid vesicles are shown with the solid grey line. (b) The absorbance spectroscopy of proteorhodopsin (PR) expressed using PURExpress at 37°C measured between 310 nm and 900 nm with 1 nm step size before (dotted grey line) and after heat shock into lipid vesicles (black). An increase in absorbance intensity after heat shock can be attributed to the inclusion of retinal into a folded proteorhodopsin (c) Western blot analysis of lipid vesicles subjected to heat shock (+) and without heat shock (−) of lipid vesicles incubated with in vitro translation transcription reaction mix. (i) Gel of lipid vesicles washed at least five times to remove any protein not associated to the membrane increased chemiluminesence for reaction mixtures which had been subjected to heat shock (+) compared to without heatshock (−) at 23 kDa for proteorhodopsin with 6× histidine tag (ii) Western blot of the supernatant removed after pelleting lipid vesicles shock shows weak chemiluminescence without heat shock (−) compared to with heat shock (+) at 23 kDa, indicating that there is an increased of PR associated to the lipid vesicle and a decrease in the supernatant after heat shock. (A color version of this figure is available in the online journal.)

    Techniques Used: Fluorescence, Spectroscopy, In Vitro, Incubation, Western Blot

    22) Product Images from "A simple, robust, and low-cost method to produce the PURE cell - free system"

    Article Title: A simple, robust, and low-cost method to produce the PURE cell - free system

    Journal: bioRxiv

    doi: 10.1101/420570

    Comparison of eGFP expression levels in PURExpress (Solution B) and OnePot PURE (EF-Tu 47%, replicate A) supplied with commercial energy solution (Solution A, PURExpress) and the OnePot energy solution used in this study. Each data point represents at least five technical replicates (mean ± s.d.)
    Figure Legend Snippet: Comparison of eGFP expression levels in PURExpress (Solution B) and OnePot PURE (EF-Tu 47%, replicate A) supplied with commercial energy solution (Solution A, PURExpress) and the OnePot energy solution used in this study. Each data point represents at least five technical replicates (mean ± s.d.)

    Techniques Used: Expressing

    Comparison of commercial ribosomes (ribosomes from PURExpress ? ribosome kit, NEB) and different batches of ribosomes purified in our laboratory. Batch 3 was used throughout this study. (a) Coomassie blue stained SDS-PAGE gels of different ribosomes. The amounts loaded onto the gel were 6.24 µ g for NEB ribosomes and 6.25 µ g in the case of purified ribosomes. (b) Comparison of expression levels in PURExpress ? ribosome kit and OnePot PURE (EF-Tu 47%, replicate A) supplied with PURExpress control ribosomes (2.4 µ M) and purified ribosomes (1.8 µ M). Each data point represents two technical replicates (mean ± s.d.)
    Figure Legend Snippet: Comparison of commercial ribosomes (ribosomes from PURExpress ? ribosome kit, NEB) and different batches of ribosomes purified in our laboratory. Batch 3 was used throughout this study. (a) Coomassie blue stained SDS-PAGE gels of different ribosomes. The amounts loaded onto the gel were 6.24 µ g for NEB ribosomes and 6.25 µ g in the case of purified ribosomes. (b) Comparison of expression levels in PURExpress ? ribosome kit and OnePot PURE (EF-Tu 47%, replicate A) supplied with PURExpress control ribosomes (2.4 µ M) and purified ribosomes (1.8 µ M). Each data point represents two technical replicates (mean ± s.d.)

    Techniques Used: Purification, Staining, SDS Page, Expressing

    OnePot PURE comparison to existing PURE systems. (a) SDS-PAGE gel of PURExpress, HomeMadePURE, OnePot PURE (EF-Tu 47%, replicate A). In the right panel, intensities of different replicates are plotted with molecular weight standards (kDa). (b) Comparison of eGFP expression activity (after 3 h) of different PURE systems. The different systems were tested in the same conditions except for TraMOS where the reported value was used ( 17 ). (c) Price comparison of the different PURE systems. Calculations are detailed in Supplementary Tables S1 , S2 , S3 , Supplementary Table S6 , S7 . (d) Yield of the different PURE systems plotted against their price per µ L. Mean values of the eGFP expression yield were plotted. (e) Cost-normalized yield of the different PURE systems. The mean value of the eGFP expression yield was used for the calculations.
    Figure Legend Snippet: OnePot PURE comparison to existing PURE systems. (a) SDS-PAGE gel of PURExpress, HomeMadePURE, OnePot PURE (EF-Tu 47%, replicate A). In the right panel, intensities of different replicates are plotted with molecular weight standards (kDa). (b) Comparison of eGFP expression activity (after 3 h) of different PURE systems. The different systems were tested in the same conditions except for TraMOS where the reported value was used ( 17 ). (c) Price comparison of the different PURE systems. Calculations are detailed in Supplementary Tables S1 , S2 , S3 , Supplementary Table S6 , S7 . (d) Yield of the different PURE systems plotted against their price per µ L. Mean values of the eGFP expression yield were plotted. (e) Cost-normalized yield of the different PURE systems. The mean value of the eGFP expression yield was used for the calculations.

    Techniques Used: SDS Page, Molecular Weight, Expressing, Activity Assay

    SDS-PAGE gel of proteins synthesized in PURExpress or OnePot (EF-Tu 47%, replicate A) (a) labeled with FluoroTect GreenLys, (b) Coomassie blue stained. Black arrows indicate the expected bands of synthesized proteins, GFP (26.9 kDa) T3 RNAP (98.8 kDa), β -galactosidase (116.5 kDa) and trehalase (63.7 kDa), DHFR (18 kDa)
    Figure Legend Snippet: SDS-PAGE gel of proteins synthesized in PURExpress or OnePot (EF-Tu 47%, replicate A) (a) labeled with FluoroTect GreenLys, (b) Coomassie blue stained. Black arrows indicate the expected bands of synthesized proteins, GFP (26.9 kDa) T3 RNAP (98.8 kDa), β -galactosidase (116.5 kDa) and trehalase (63.7 kDa), DHFR (18 kDa)

    Techniques Used: SDS Page, Synthesized, Labeling, Staining

    Activities of different proteins, expressed in PURExpress and OnePot (EF-Tu 47%, replicate A). Trehalase assay: (a) Absorbance change at 540 nm and (b) image of resulting color change due to the presence of trehalase in the reaction. Three reactions were measured for positive samples. Error bars represent standard deviation. β -galactosidase assay: (c) absorbance (580 nm) increase over time due to substrate cleavage, (d) slope of absorbance. Three and one OnePot PURE reactions were measured for positive and negative samples, respectively. Each reaction was measured in triplicate. Error bars represent standard deviation. Zinc-finger (ZF) repression: (e) Down-regulation of deGFP expression, due to binding of ZF to the target promoter. deGFP containing lambda PR promoter containing double ADD ZF binding sites was used as a reporter. The ADD ZF was co-expressed with deGFP (repressed state), and the CBD ZF was co-expressed as a negative control (unrepressed state). (f) Fold-repression, the ratio of unrepressed to repressed expression levels. Each data point represents three technical replicates (mean ± s.d.)
    Figure Legend Snippet: Activities of different proteins, expressed in PURExpress and OnePot (EF-Tu 47%, replicate A). Trehalase assay: (a) Absorbance change at 540 nm and (b) image of resulting color change due to the presence of trehalase in the reaction. Three reactions were measured for positive samples. Error bars represent standard deviation. β -galactosidase assay: (c) absorbance (580 nm) increase over time due to substrate cleavage, (d) slope of absorbance. Three and one OnePot PURE reactions were measured for positive and negative samples, respectively. Each reaction was measured in triplicate. Error bars represent standard deviation. Zinc-finger (ZF) repression: (e) Down-regulation of deGFP expression, due to binding of ZF to the target promoter. deGFP containing lambda PR promoter containing double ADD ZF binding sites was used as a reporter. The ADD ZF was co-expressed with deGFP (repressed state), and the CBD ZF was co-expressed as a negative control (unrepressed state). (f) Fold-repression, the ratio of unrepressed to repressed expression levels. Each data point represents three technical replicates (mean ± s.d.)

    Techniques Used: Standard Deviation, Expressing, Binding Assay, Negative Control

    23) Product Images from "Cell-Free Expression to Probe Co-Translational Insertion of an Alpha Helical Membrane Protein"

    Article Title: Cell-Free Expression to Probe Co-Translational Insertion of an Alpha Helical Membrane Protein

    Journal: Frontiers in Molecular Biosciences

    doi: 10.3389/fmolb.2022.795212

    LeuT expressed in PURExpress ® IVTT systems in the presence of liposomes comprising various lipid compositions as presented in Table 1 and Table 2 . Spontaneously inserted protein was quantified using LSC counting of Methionine, L-[ 35 S] incorporated into LeuT. Insertion efficiency is represented as a percentage of protein in the top fraction over the total yield of protein synthesized. Highlighted here are the results of lipid optimization on LeuT spontaneous insertion efficiencies, comparing various lipid species derived from DO, DPh and Cardiolipin liposome compositions. Errors presented are SEM calculated from ≥3 repeats.
    Figure Legend Snippet: LeuT expressed in PURExpress ® IVTT systems in the presence of liposomes comprising various lipid compositions as presented in Table 1 and Table 2 . Spontaneously inserted protein was quantified using LSC counting of Methionine, L-[ 35 S] incorporated into LeuT. Insertion efficiency is represented as a percentage of protein in the top fraction over the total yield of protein synthesized. Highlighted here are the results of lipid optimization on LeuT spontaneous insertion efficiencies, comparing various lipid species derived from DO, DPh and Cardiolipin liposome compositions. Errors presented are SEM calculated from ≥3 repeats.

    Techniques Used: Synthesized, Derivative Assay

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    Determination of k R for each construct. (a) Scaled overlay of the individual plots in panels b-h. (b)-(h) Plot of the decrease in fraction arrested protein ( f A ) over time after chasing a 5 min <t>PURExpress</t> translation of the indicated construct. The data were fit to the first order equation in the main text to determine the rate of release ( k R ) of arrested protein from the ribosome. (i) Summary of the fitness of the equation for each construct: degrees of freedom ( d.f. ), R 2 , and goodness-of-fit calculation ( Sy.x ). (j) The goodness-of-fit calculation provided by Prism 8 (Graphpad software) where n is the number of data points and K is the degrees of freedom.
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    IF2 is a target of ppGpp. IF2 was validated in vitro as a direct target of ppGpp using IF2 mutations that reduce ppGpp binding. ( A ) Affinity of B. subtilis ). (means ± SDs). ( B ) Alignment of G1 domains of B. subtilis ). Residues in red are those that differ in EF-G and IF2 and were used to engineer a mutant IF2 with reduced affinity for ppGpp (G226A H230A). ( C ) DRaCALA-based comparison of (p)ppGpp affinity for WT and mutant IF2 (means ± SDs). ( D ) In vitro sensitivity of WT and mutant IF2 was assessed using the <t>PURExpress</t> system (NEB). WT and mutant IF2 were added at equimolar amounts to separate PURExpress reactions in the presence of 1 mM ppGpp, and protein synthesis was monitored by Western blot (means ± SDs). ** P
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    Production and purification of USCTX. ( a ) Commercially available cell-free synthesis systems differ in their ability to produce USCTX (anticipated band size = 4.3 kDa). No such band was produced by the S30 Extract System (left) or the TnT T7 Insect Cell Extract Protein Expression System (right), but a band of the expected size was produced by the NEB <t>PURExpress</t> In Vitro Protein Synthesis System (middle, in duplicate to highlight reproducibility). ( b ) Purification of USCTX, showing the elution fractions E1–E3 from the His-Spin column. The red box indicates the area in which USCTX bands should appear.
    Purexpress, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/purexpress/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    purexpress - by Bioz Stars, 2022-05
    86/100 stars
    		  Buy from Supplier

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    Linking gene expression of FadD10 to phospholipid synthesis. a Schematic representation of the cell-free expression of FadD10 and subsequent assembly of the de novo synthesized phospholipid into vesicles in the presence of appropriate reactive precursors [TX-TL: transcription/translation]. b SDS–PAGE analysis of the expression of FadD10 in the PURExpress ® System. Lane L1: No DNA; Lane L2: DHFR DNA; Lane L3: FadD10 DNA. c HPLC/ELSD traces monitoring the formation of phospholipid 3 by incubation of PURExpress ® System with an aqueous solution of dodecanoic acid, lysolipid 2 , ATP and MgCl 2 at 37 °C in the absence (gray line) or presence (orange line) of plasmid DNA coding for FadD10. d Spinning disk confocal microscopy of the in situ formed phospholipid vesicles in the PURExpress ® System driven by FadD10 expression. Membranes were stained using 0.1 mol% Texas Red ® DHPE dye. Scale bar: 5 µm. e Localization of sfGFP-FadD10 to the membrane of the vesicles formed upon addition of the plasmid encoding the former into PURE system. External proteins were digested by Proteinase K. Scale bar: 5 µm

    Journal: Nature Communications

    Article Title: A minimal biochemical route towards de novo formation of synthetic phospholipid membranes

    doi: 10.1038/s41467-018-08174-x

    Figure Lengend Snippet: Linking gene expression of FadD10 to phospholipid synthesis. a Schematic representation of the cell-free expression of FadD10 and subsequent assembly of the de novo synthesized phospholipid into vesicles in the presence of appropriate reactive precursors [TX-TL: transcription/translation]. b SDS–PAGE analysis of the expression of FadD10 in the PURExpress ® System. Lane L1: No DNA; Lane L2: DHFR DNA; Lane L3: FadD10 DNA. c HPLC/ELSD traces monitoring the formation of phospholipid 3 by incubation of PURExpress ® System with an aqueous solution of dodecanoic acid, lysolipid 2 , ATP and MgCl 2 at 37 °C in the absence (gray line) or presence (orange line) of plasmid DNA coding for FadD10. d Spinning disk confocal microscopy of the in situ formed phospholipid vesicles in the PURExpress ® System driven by FadD10 expression. Membranes were stained using 0.1 mol% Texas Red ® DHPE dye. Scale bar: 5 µm. e Localization of sfGFP-FadD10 to the membrane of the vesicles formed upon addition of the plasmid encoding the former into PURE system. External proteins were digested by Proteinase K. Scale bar: 5 µm

    Article Snippet: De novo phospholipid formation in PURExpress® System In a typical 10 µL protein expression reaction, the following components were added in the given order: 4 µL of Solution A (containing amino acids, energy factors, etc.), 3 µL of Solution B (containing ribosomes, aminoacyl tRNA synthetases, etc), 0.2 µL (4 U) murine RNase inhibitor (New England Biolabs), x µL nuclease-free H2 O, y µL DNA.

    Techniques: Expressing, Synthesized, SDS Page, High Performance Liquid Chromatography, Incubation, Plasmid Preparation, Confocal Microscopy, In Situ, Staining

    Determination of k R for each construct. (a) Scaled overlay of the individual plots in panels b-h. (b)-(h) Plot of the decrease in fraction arrested protein ( f A ) over time after chasing a 5 min PURExpress translation of the indicated construct. The data were fit to the first order equation in the main text to determine the rate of release ( k R ) of arrested protein from the ribosome. (i) Summary of the fitness of the equation for each construct: degrees of freedom ( d.f. ), R 2 , and goodness-of-fit calculation ( Sy.x ). (j) The goodness-of-fit calculation provided by Prism 8 (Graphpad software) where n is the number of data points and K is the degrees of freedom.

    Journal: bioRxiv

    Article Title: Cotranslational folding cooperativity of contiguous domains of α-spectrin

    doi: 10.1101/653360

    Figure Lengend Snippet: Determination of k R for each construct. (a) Scaled overlay of the individual plots in panels b-h. (b)-(h) Plot of the decrease in fraction arrested protein ( f A ) over time after chasing a 5 min PURExpress translation of the indicated construct. The data were fit to the first order equation in the main text to determine the rate of release ( k R ) of arrested protein from the ribosome. (i) Summary of the fitness of the equation for each construct: degrees of freedom ( d.f. ), R 2 , and goodness-of-fit calculation ( Sy.x ). (j) The goodness-of-fit calculation provided by Prism 8 (Graphpad software) where n is the number of data points and K is the degrees of freedom.

    Article Snippet: The PURExpress in vitro transcription-translation system was purchased from NEB.

    Techniques: Construct, Software

    Release rates and estimation of pulling forces. (a) The rate of release ( k R ) obtained from pulse-chase experiments (see Supplementary Fig. S4 and Supplementary Table S2), the fraction full-length protein ( f FL ) measured under standard experimental conditions (20 min. incubation in PURExpress in the continuous presence of [ 35 S] Met), and the pulling force F calculated using Eq. [1] ( k 0 = 3.0 × 10 −4 s −1 , Δ x ‡ = 0.65 nm). The constructs are from Kudva et al. ( 12 ), and are colored to match those in panel b and Supplementary Figs. S4 and S5. (b) F values calculated from Eq. [1] plotted against the standard f FL values, with constructs colored as in panel a . The least-squares fit line is indicated by the blue line, and the analytic relation Eq. [3] between F and f FL , assuming an average delay time Δ t = 550 s (approximately equal to half the standard incubation time), is shown as a red curve.

    Journal: bioRxiv

    Article Title: Cotranslational folding cooperativity of contiguous domains of α-spectrin

    doi: 10.1101/653360

    Figure Lengend Snippet: Release rates and estimation of pulling forces. (a) The rate of release ( k R ) obtained from pulse-chase experiments (see Supplementary Fig. S4 and Supplementary Table S2), the fraction full-length protein ( f FL ) measured under standard experimental conditions (20 min. incubation in PURExpress in the continuous presence of [ 35 S] Met), and the pulling force F calculated using Eq. [1] ( k 0 = 3.0 × 10 −4 s −1 , Δ x ‡ = 0.65 nm). The constructs are from Kudva et al. ( 12 ), and are colored to match those in panel b and Supplementary Figs. S4 and S5. (b) F values calculated from Eq. [1] plotted against the standard f FL values, with constructs colored as in panel a . The least-squares fit line is indicated by the blue line, and the analytic relation Eq. [3] between F and f FL , assuming an average delay time Δ t = 550 s (approximately equal to half the standard incubation time), is shown as a red curve.

    Article Snippet: The PURExpress in vitro transcription-translation system was purchased from NEB.

    Techniques: Pulse Chase, Incubation, Construct

    IF2 is a target of ppGpp. IF2 was validated in vitro as a direct target of ppGpp using IF2 mutations that reduce ppGpp binding. ( A ) Affinity of B. subtilis ). (means ± SDs). ( B ) Alignment of G1 domains of B. subtilis ). Residues in red are those that differ in EF-G and IF2 and were used to engineer a mutant IF2 with reduced affinity for ppGpp (G226A H230A). ( C ) DRaCALA-based comparison of (p)ppGpp affinity for WT and mutant IF2 (means ± SDs). ( D ) In vitro sensitivity of WT and mutant IF2 was assessed using the PURExpress system (NEB). WT and mutant IF2 were added at equimolar amounts to separate PURExpress reactions in the presence of 1 mM ppGpp, and protein synthesis was monitored by Western blot (means ± SDs). ** P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The alarmones (p)ppGpp directly regulate translation initiation during entry into quiescence

    doi: 10.1073/pnas.1920013117

    Figure Lengend Snippet: IF2 is a target of ppGpp. IF2 was validated in vitro as a direct target of ppGpp using IF2 mutations that reduce ppGpp binding. ( A ) Affinity of B. subtilis ). (means ± SDs). ( B ) Alignment of G1 domains of B. subtilis ). Residues in red are those that differ in EF-G and IF2 and were used to engineer a mutant IF2 with reduced affinity for ppGpp (G226A H230A). ( C ) DRaCALA-based comparison of (p)ppGpp affinity for WT and mutant IF2 (means ± SDs). ( D ) In vitro sensitivity of WT and mutant IF2 was assessed using the PURExpress system (NEB). WT and mutant IF2 were added at equimolar amounts to separate PURExpress reactions in the presence of 1 mM ppGpp, and protein synthesis was monitored by Western blot (means ± SDs). ** P

    Article Snippet: We extended these in vivo observations by using the PURExpress in vitro reconstituted, coupled transcription-translation system (New England Biolabs, NEB) that utilizes a defined mix of purified transcription and E. coli translation components to transcribe and translate a specific mRNA ( ).

    Techniques: In Vitro, Binding Assay, Mutagenesis, Western Blot

    Production and purification of USCTX. ( a ) Commercially available cell-free synthesis systems differ in their ability to produce USCTX (anticipated band size = 4.3 kDa). No such band was produced by the S30 Extract System (left) or the TnT T7 Insect Cell Extract Protein Expression System (right), but a band of the expected size was produced by the NEB PURExpress In Vitro Protein Synthesis System (middle, in duplicate to highlight reproducibility). ( b ) Purification of USCTX, showing the elution fractions E1–E3 from the His-Spin column. The red box indicates the area in which USCTX bands should appear.

    Journal: Toxins

    Article Title: A Spider Toxin Exemplifies the Promises and Pitfalls of Cell-Free Protein Production for Venom Biodiscovery

    doi: 10.3390/toxins13080575

    Figure Lengend Snippet: Production and purification of USCTX. ( a ) Commercially available cell-free synthesis systems differ in their ability to produce USCTX (anticipated band size = 4.3 kDa). No such band was produced by the S30 Extract System (left) or the TnT T7 Insect Cell Extract Protein Expression System (right), but a band of the expected size was produced by the NEB PURExpress In Vitro Protein Synthesis System (middle, in duplicate to highlight reproducibility). ( b ) Purification of USCTX, showing the elution fractions E1–E3 from the His-Spin column. The red box indicates the area in which USCTX bands should appear.

    Article Snippet: We tested three different commercially available cell-free expression systems: the PURExpress In Vitro Protein Synthesis System (New England Biolabs, Ipswich, MA, USA), the S30 Extract System (Promega, Madison, WI, USA), and the TnT T7 Insect Cell Extract Protein Expression System (Promega) based on the S. frugiperda cell line Sf21.

    Techniques: Purification, Produced, Expressing, In Vitro