purexpress system  (New England Biolabs)


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    New England Biolabs purexpress system
    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 <t>PURExpress</t> ® 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
    Purexpress System, 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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    Price from $9.99 to $1999.99
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    Images

    1) Product Images from "A minimal biochemical route towards de novo formation of synthetic phospholipid membranes"

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

    Journal: Nature Communications

    doi: 10.1038/s41467-018-08174-x

    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
    Figure Legend 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

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

    2) Product Images from "NusG-Dependent RNA Polymerase Pausing and Tylosin-Dependent Ribosome Stalling Are Required for Tylosin Resistance by Inducing 23S rRNA Methylation in Bacillus subtilis"

    Article Title: NusG-Dependent RNA Polymerase Pausing and Tylosin-Dependent Ribosome Stalling Are Required for Tylosin Resistance by Inducing 23S rRNA Methylation in Bacillus subtilis

    Journal: mBio

    doi: 10.1128/mBio.02665-19

    Tylosin-dependent ribosome stalling in the leader peptide. (A) Toeprint analysis of tylosin-induced ribosome stalling during translation of the leader peptide using WT and AYA mutant templates. The toeprint (TP) identified with the WT template in the presence of tylosin (+) is marked. Sequencing lanes (A, C, G, and U) are shown. The PURExpress kit containing T7 RNAP and E. coli ribosomes was used for this analysis. (B) yxjB leader region covered by the ribosome when tylosin induces stalling. The positions of the toeprint and the ribosome peptidyl (P) and aminoacyl (A) sites are shown. Additional details are as described in the Fig. 2A legend. Experiments were performed at least twice with comparable results.
    Figure Legend Snippet: Tylosin-dependent ribosome stalling in the leader peptide. (A) Toeprint analysis of tylosin-induced ribosome stalling during translation of the leader peptide using WT and AYA mutant templates. The toeprint (TP) identified with the WT template in the presence of tylosin (+) is marked. Sequencing lanes (A, C, G, and U) are shown. The PURExpress kit containing T7 RNAP and E. coli ribosomes was used for this analysis. (B) yxjB leader region covered by the ribosome when tylosin induces stalling. The positions of the toeprint and the ribosome peptidyl (P) and aminoacyl (A) sites are shown. Additional details are as described in the Fig. 2A legend. Experiments were performed at least twice with comparable results.

    Techniques Used: Mutagenesis, Sequencing

    3) Product Images from "(p)ppGpp directly regulates translation initiation during entry into quiescence"

    Article Title: (p)ppGpp directly regulates translation initiation during entry into quiescence

    Journal: bioRxiv

    doi: 10.1101/807917

    ppGpp directly inhibits translation in vitro Protein synthesis in the presence of increasing concentrations of ppGpp was measured using the PURExpress in vitro reconstituted, coupled transcription-translation system (NEB). Production of CotE-FLAG was measured via Western blot with α-FLAG (means ± SDs). n.s. p > 0.05, *p
    Figure Legend Snippet: ppGpp directly inhibits translation in vitro Protein synthesis in the presence of increasing concentrations of ppGpp was measured using the PURExpress in vitro reconstituted, coupled transcription-translation system (NEB). Production of CotE-FLAG was measured via Western blot with α-FLAG (means ± SDs). n.s. p > 0.05, *p

    Techniques Used: In Vitro, Western Blot

    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 EF-G and IF2 for (p)ppGpp was compared using the differential radial capillary action of a ligand assay (DRaCALA) ( Roelofs et al., 2011 ). (means ± SDs). (B) Alignment of G1 domains of B. subtilis IF2 and EF-G. Residues in blue denote those whose chemical shifts were previously identified to be most shifted upon binding of ppGpp versus GDP. Residues in red are those that were different in EF-G versus IF2 and that were used to engineer a mutant IF2 with reduced affinity for ppGpp (G226A H230A). (C) DRaCALA-based comparison of ppGpp affinity for WT and mutant IF2 (means ± SDs). (D) in vitro sensitivity of WT and mutant IF2 was assessed using the PURExpress in vitro reconstituted, coupled transcription-translation system (NEB). WT and mutant IF2 were added at equimolar amounts to separate PURExpress reactions in the presence of 1mM ppGpp and protein synthesis was monitored by Western blot (means ± SDs). n.s. p > 0.05, *p
    Figure Legend 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 EF-G and IF2 for (p)ppGpp was compared using the differential radial capillary action of a ligand assay (DRaCALA) ( Roelofs et al., 2011 ). (means ± SDs). (B) Alignment of G1 domains of B. subtilis IF2 and EF-G. Residues in blue denote those whose chemical shifts were previously identified to be most shifted upon binding of ppGpp versus GDP. Residues in red are those that were different in EF-G versus IF2 and that were used to engineer a mutant IF2 with reduced affinity for ppGpp (G226A H230A). (C) DRaCALA-based comparison of ppGpp affinity for WT and mutant IF2 (means ± SDs). (D) in vitro sensitivity of WT and mutant IF2 was assessed using the PURExpress in vitro reconstituted, coupled transcription-translation system (NEB). WT and mutant IF2 were added at equimolar amounts to separate PURExpress reactions in the presence of 1mM ppGpp and protein synthesis was monitored by Western blot (means ± SDs). n.s. p > 0.05, *p

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

    4) Product Images from "Protein Synthesis Using A Reconstituted Cell-Free System"

    Article Title: Protein Synthesis Using A Reconstituted Cell-Free System

    Journal: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]

    doi: 10.1002/0471142727.mb1631s108

    Chromozyme cleavage assay (Roche) for a truncated version of tissue plasminogen activator protein (vtPA) synthesized in the PURExpress reactions with and without PURExpress Disulfide Bond Enhancer (PDBE). This tissue plasminogen activator contains 9 disulfide
    Figure Legend Snippet: Chromozyme cleavage assay (Roche) for a truncated version of tissue plasminogen activator protein (vtPA) synthesized in the PURExpress reactions with and without PURExpress Disulfide Bond Enhancer (PDBE). This tissue plasminogen activator contains 9 disulfide

    Techniques Used: Cleavage Assay, Synthesized

    SDS-PAGE analysis of the reverse purification of the DHFR control protein synthesized in the PURExpress reaction. M: molecular weight standards (kDa); Lane 1: Control PURExpress reaction with no input template, Lane 2: PURExpress reaction with the DHFR
    Figure Legend Snippet: SDS-PAGE analysis of the reverse purification of the DHFR control protein synthesized in the PURExpress reaction. M: molecular weight standards (kDa); Lane 1: Control PURExpress reaction with no input template, Lane 2: PURExpress reaction with the DHFR

    Techniques Used: SDS Page, Purification, Synthesized, Molecular Weight

    Scanned image of a SDS-PAGE gel of proteins synthesized in the PURExpress reactions and labeled with FluoroTect™ Green Lys . Lane 1: DHFR; lane 2: GFP; lane 3: Renilla luciferase; lane 4: Firefly luciferase; lane 5: E. coli β-galactosidase.
    Figure Legend Snippet: Scanned image of a SDS-PAGE gel of proteins synthesized in the PURExpress reactions and labeled with FluoroTect™ Green Lys . Lane 1: DHFR; lane 2: GFP; lane 3: Renilla luciferase; lane 4: Firefly luciferase; lane 5: E. coli β-galactosidase.

    Techniques Used: SDS Page, Synthesized, Labeling, Luciferase

    5) 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

    6) Product Images from "The alarmones (p)ppGpp directly regulate translation initiation during entry into quiescence"

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

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

    doi: 10.1073/pnas.1920013117

    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
    Figure Legend 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

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

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    New England Biolabs purexpress system
    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 <t>PURExpress</t> ® 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
    Purexpress System, 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 system/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    New England Biolabs purexpress in vitro transcription translation system
    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.
    Purexpress In Vitro Transcription Translation System, 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 in vitro transcription translation system/product/New England Biolabs
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    New England Biolabs purexpress in vitro
    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
    Purexpress In Vitro, 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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    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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    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