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    Name:
    PURExpress In Vitro Protein Synthesis Kit
    Description:
    PURExpress In Vitro Protein Synthesis Kit 100 rxns
    Catalog Number:
    e6800l
    Price:
    2292
    Size:
    100 rxns
    Category:
    Transcription Translation Systems
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    Structured Review

    New England Biolabs purexpress
    PURExpress In Vitro Protein Synthesis Kit
    PURExpress In Vitro Protein Synthesis Kit 100 rxns
    https://www.bioz.com/result/purexpress/product/New England Biolabs
    Average 97 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    purexpress - by Bioz Stars, 2021-01
    97/100 stars

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    Images

    1) 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

    2) 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

    3) 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

    4) 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

    5) 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

    6) 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

    7) 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

    8) 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

    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 "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

    11) 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

    12) 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

    13) 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

    14) 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

    15) 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

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    Mutagenesis:

    Article Title: Identification of functional cis-acting RNA elements in the hepatitis E virus genome required for viral replication
    Article Snippet: .. In vitro transcription assay and viral RNA transfection HEV Kernow-C1/p6, HEV Kernow-C1/p6-Gluc, and the truncated mutant plasmids were linearized by MluI. pSAR55-Gluc was linearized by BglII, pGEM-9Zf-pSHEV3-Gluc was linearized by XbaI, and pGEM-7Zf(-)-TW6196E and pGEM-7Zf(-)-TW6196E/Gluc were linearized by SpeI. .. Viral RNAs were transcribed in vitro from linearized plasmid using HiScribe T7 antireverse cap analog (ARCA) mRNA kit (New England Biolabs, Ipswich, MA) according to the manufacturer’s instructions.

    Activity Assay:

    Article Title: Riboneogenesis in yeast
    Article Snippet: .. Initial enzymatic screens for enzymatic activity were performed using in vitro synthesized, untagged Shb17 (New England Biolabs Inc. PURExpress® In Vitro Protein Synthesis Kit). .. Subsequent studies were performed using N-terminal His-tagged recombinant protein purified from E. coli .

    SDS Page:

    Article Title: Protein Synthesis Using A Reconstituted Cell-Free System
    Article Snippet: .. A PURExpress In vitro Protein Synthesis kit (New England Biolabs, ) containing: Solution A (yellow tube) Solution B (red tube) DHFR control template PURExpress Disulfide Bond Enhancer (New England Biolabs, ) containing: Disulfide Bond Enhancer Solution 1 Disulfide Bond Enhancer Solution 2 microcentrifuge tubes or microtiter plate Nuclease-free H2 O (Integrated DNA technologies) Template DNA (See Support Protocol 1 and 2) or mRNA (See Support Protocol 3) Murine RNase inhibitor (40 U/µl, New England Biolabs) or RNasin Ribonuclease inhibitor (20–40 U/µl, Promega) Microcentrifuge Air incubator set at 37°C 3x SDS-PAGE loading buffer (New England Biolabs, CPMB Chapter X) SDS-PAGE gel (4–20% Tris-glycine, Life Technologies, CPMB Chapter X) ..

    Article Title: Protein Synthesis Using A Reconstituted Cell-Free System
    Article Snippet: .. PURExpress In vitro Protein Synthesis kit (New England Biolabs) containing: Solution A (yellow tube) Solution B (red tube) DHFR control template Nuclease free microcentrifuge tubes or microtiter plates Nuclease free H2 O Template DNA (See Support Protocol 1 and 2) or mRNA (See Support Protocol 3) Murine RNase inhibitor (40 U/µl, New England Biolabs) [35 S]-L-Methionine (15 mCi/ml, 1000 Ci/mmol) (PerkinElmer) Microcentrifuge Air incubator set at 37°C 3x SDS-PAGE loading buffer (New England Biolabs) SDS-PAGE gel (4–20 % Tris-glycine, Life Technologies) Filter paper (Whatman) Vacuum gel dryer X-ray film or phosphorimager ..

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    • purexpress δribosome kit  (New England Biolabs)
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    New England Biolabs purexpress δribosome kit
    Ribosome analysis of extracts from NM522 Escherichia coli cells. a Sedimentation profiles of NM522 E. coli ribosomes. Wild-type NM522 E. coli cells (black curve) and cells expressing viral bS21 (red), bL12 (green) or HPF (blue) were lysed and their ribosomes were purified using a 10–50% sucrose gradient (see experimental section). The dotted lines indicate the fractions that were pooled and further analyzed by mass spectrometry. b Quantification of in vitro translation of GFP by E. coli 70S ribosomes carrying either E. coli wt bS21 (control), E. coli streptavidin-tagged bS21 (70S-eS21) or viral streptavidin-tagged bS21 (70S-pS21). Translation assay was performed using <t>PURExpress®</t> <t>ΔRibosome</t> Kit, complemented with 10 pmol of purified ribosomes and 250 ng of a PCR product encoding for GFP under control of T7 promoter. Fluorescence signal was detected by spectrofluorimetry at 510 nm with an excitation at 485 nm. The percentage of fluorescence was measured with respect to the translation control. The error bar represents the standard deviation measured over three independent experiments. The variance was analyzed using Kruskal–Wallis test followed by a Dunn’s multiple comparison test ( p -value for 70S-pS21 = 0.0219)
    Purexpress δribosome Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
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    purexpress in vitro translation system  (New England Biolabs)
    99
    New England Biolabs purexpress in vitro translation system
    Quantification of the efficiency of ribosome sliding on mCherry reporters expressed in the <t>PURExpress</t> system. mCherry reporters ( Figure 1A : no insert, and various A stretches) were expressed in the PURExpress cell-free translation system ( Figure 5 ). The plot reports the percent of truncated peptide product expressed relative to total peptide product for each reporter (100% × (radioactivity in truncated band)/(radioactivity in truncated + full-length bands)). DOI: http://dx.doi.org/10.7554/eLife.05534.016
    Purexpress In Vitro Translation System, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 33 article reviews
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    Ribosome analysis of extracts from NM522 Escherichia coli cells. a Sedimentation profiles of NM522 E. coli ribosomes. Wild-type NM522 E. coli cells (black curve) and cells expressing viral bS21 (red), bL12 (green) or HPF (blue) were lysed and their ribosomes were purified using a 10–50% sucrose gradient (see experimental section). The dotted lines indicate the fractions that were pooled and further analyzed by mass spectrometry. b Quantification of in vitro translation of GFP by E. coli 70S ribosomes carrying either E. coli wt bS21 (control), E. coli streptavidin-tagged bS21 (70S-eS21) or viral streptavidin-tagged bS21 (70S-pS21). Translation assay was performed using PURExpress® ΔRibosome Kit, complemented with 10 pmol of purified ribosomes and 250 ng of a PCR product encoding for GFP under control of T7 promoter. Fluorescence signal was detected by spectrofluorimetry at 510 nm with an excitation at 485 nm. The percentage of fluorescence was measured with respect to the translation control. The error bar represents the standard deviation measured over three independent experiments. The variance was analyzed using Kruskal–Wallis test followed by a Dunn’s multiple comparison test ( p -value for 70S-pS21 = 0.0219)

    Journal: Nature Communications

    Article Title: Numerous cultivated and uncultivated viruses encode ribosomal proteins

    doi: 10.1038/s41467-019-08672-6

    Figure Lengend Snippet: Ribosome analysis of extracts from NM522 Escherichia coli cells. a Sedimentation profiles of NM522 E. coli ribosomes. Wild-type NM522 E. coli cells (black curve) and cells expressing viral bS21 (red), bL12 (green) or HPF (blue) were lysed and their ribosomes were purified using a 10–50% sucrose gradient (see experimental section). The dotted lines indicate the fractions that were pooled and further analyzed by mass spectrometry. b Quantification of in vitro translation of GFP by E. coli 70S ribosomes carrying either E. coli wt bS21 (control), E. coli streptavidin-tagged bS21 (70S-eS21) or viral streptavidin-tagged bS21 (70S-pS21). Translation assay was performed using PURExpress® ΔRibosome Kit, complemented with 10 pmol of purified ribosomes and 250 ng of a PCR product encoding for GFP under control of T7 promoter. Fluorescence signal was detected by spectrofluorimetry at 510 nm with an excitation at 485 nm. The percentage of fluorescence was measured with respect to the translation control. The error bar represents the standard deviation measured over three independent experiments. The variance was analyzed using Kruskal–Wallis test followed by a Dunn’s multiple comparison test ( p -value for 70S-pS21 = 0.0219)

    Article Snippet: In vitro translation assays In vitro translation was performed using PURExpress® ΔRibosome Kit (New England Biolabs).

    Techniques: Sedimentation, Expressing, Purification, Mass Spectrometry, In Vitro, Polymerase Chain Reaction, Fluorescence, Standard Deviation

    Quantification of the efficiency of ribosome sliding on mCherry reporters expressed in the PURExpress system. mCherry reporters ( Figure 1A : no insert, and various A stretches) were expressed in the PURExpress cell-free translation system ( Figure 5 ). The plot reports the percent of truncated peptide product expressed relative to total peptide product for each reporter (100% × (radioactivity in truncated band)/(radioactivity in truncated + full-length bands)). DOI: http://dx.doi.org/10.7554/eLife.05534.016

    Journal: eLife

    Article Title: Ribosomes slide on lysine-encoding homopolymeric A stretches

    doi: 10.7554/eLife.05534

    Figure Lengend Snippet: Quantification of the efficiency of ribosome sliding on mCherry reporters expressed in the PURExpress system. mCherry reporters ( Figure 1A : no insert, and various A stretches) were expressed in the PURExpress cell-free translation system ( Figure 5 ). The plot reports the percent of truncated peptide product expressed relative to total peptide product for each reporter (100% × (radioactivity in truncated band)/(radioactivity in truncated + full-length bands)). DOI: http://dx.doi.org/10.7554/eLife.05534.016

    Article Snippet: Expression of reporters in the PURExpress in vitro translation system The Thrdx-HA-mCherry and Thrdx-HA-insert-mCherry reporters were expressed in the PURExpress in vitro translation system (NEB, Ipswitch, MA) from PCR products.

    Techniques: Radioactivity

    Truncated product release is independent of RF3 in the PURExpress cell-free translation system. mCherry reporters ( Figure 1A : no insert, AAG 12 , AAA 12 ) were expressed in the PURExpress cell-free translation system lacking release factors (RFs) (light gray). RFs were added back to the reactions individually (RF1 in green, RF2 in purple), and in combination (RF1/3 in red and Rf2/3 dark gray). The plot displays the fraction of protein in the truncated band (100% × (radioactivity in truncated band)/(radioactivity in truncated + full-length bands)). DOI: http://dx.doi.org/10.7554/eLife.05534.013

    Journal: eLife

    Article Title: Ribosomes slide on lysine-encoding homopolymeric A stretches

    doi: 10.7554/eLife.05534

    Figure Lengend Snippet: Truncated product release is independent of RF3 in the PURExpress cell-free translation system. mCherry reporters ( Figure 1A : no insert, AAG 12 , AAA 12 ) were expressed in the PURExpress cell-free translation system lacking release factors (RFs) (light gray). RFs were added back to the reactions individually (RF1 in green, RF2 in purple), and in combination (RF1/3 in red and Rf2/3 dark gray). The plot displays the fraction of protein in the truncated band (100% × (radioactivity in truncated band)/(radioactivity in truncated + full-length bands)). DOI: http://dx.doi.org/10.7554/eLife.05534.013

    Article Snippet: Expression of reporters in the PURExpress in vitro translation system The Thrdx-HA-mCherry and Thrdx-HA-insert-mCherry reporters were expressed in the PURExpress in vitro translation system (NEB, Ipswitch, MA) from PCR products.

    Techniques: Radioactivity

    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.

    Journal: Biochemistry

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

    doi: 10.1021/acs.biochem.7b01170

    Figure Lengend 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.

    Article Snippet: The PURExpress probing experiments without ribosomes were carried out in a similar fashion using the PURExpress ΔRibosome Kit (New England Biolabs), with each 10 μL reaction containing 4 μL kit Solution A, 1.2 μL Factor Mix, and 3.24 picomoles of mRNA.

    Techniques: Purification, Fluorescence, Construct, Standard Deviation

    Shb17 feeds carbon into the non-oxidative pentose phosphate pathway. (A) Flux through Shb17 into S7P can be measured using [6- 13 C 1 ]-glucose. [6- 13 C 1 ]-glucose leads to [7- 13 C 1 ]-S7P when S7P is made via the oxidative PPP or the non-oxidative PPP. However, when S7P is produced from SBP via Shb17, a fraction of the S7P pool is doubly labeled: [1,7- 13 C 2 ]-S7P. Flux is calculated based on the measured isotopic distribution of SBP and S7P. (B) Flux through Shb17 is increased by supplementation with nutrients whose endogenous production requires NADPH, and thus drives oxidative PPP flux. All measurements are performed in wild type yeast. YNB is yeast nitrogen base without amino acids plus 2% glucose. Supplementation with amino acids includes 17 amino acids. Data shown is the arithmetic mean ± SE of N=3 technical replicates. (C) Effects of PPP gene deletions on Shb17 flux. Deletions are: glucose 6-phosphate dehydrogenase zwf1 Δ; transketolase tkl1 Δ/ tkl2 Δ; transaldolase is tal1 Δ/ nqm1 Δ. Less than 1% doubly labeled S7P was observed in any shb17 Δ strain in all measured conditions. All strains were grown in YNB + 2% glucose and supplements as required: methionine for zwf1 Δ; synthetic complete media including aromatic amino acids for tkl1 Δ/ tkl2 Δ. (C) Triple deletion of the sedoheptulose bisphosphatase SHB17 , the glucose-6-phosphate dehydrogenase ZWF1 , and the transaldolase TAL1 .

    Journal: Cell

    Article Title: Riboneogenesis in yeast

    doi: 10.1016/j.cell.2011.05.022

    Figure Lengend Snippet: Shb17 feeds carbon into the non-oxidative pentose phosphate pathway. (A) Flux through Shb17 into S7P can be measured using [6- 13 C 1 ]-glucose. [6- 13 C 1 ]-glucose leads to [7- 13 C 1 ]-S7P when S7P is made via the oxidative PPP or the non-oxidative PPP. However, when S7P is produced from SBP via Shb17, a fraction of the S7P pool is doubly labeled: [1,7- 13 C 2 ]-S7P. Flux is calculated based on the measured isotopic distribution of SBP and S7P. (B) Flux through Shb17 is increased by supplementation with nutrients whose endogenous production requires NADPH, and thus drives oxidative PPP flux. All measurements are performed in wild type yeast. YNB is yeast nitrogen base without amino acids plus 2% glucose. Supplementation with amino acids includes 17 amino acids. Data shown is the arithmetic mean ± SE of N=3 technical replicates. (C) Effects of PPP gene deletions on Shb17 flux. Deletions are: glucose 6-phosphate dehydrogenase zwf1 Δ; transketolase tkl1 Δ/ tkl2 Δ; transaldolase is tal1 Δ/ nqm1 Δ. Less than 1% doubly labeled S7P was observed in any shb17 Δ strain in all measured conditions. All strains were grown in YNB + 2% glucose and supplements as required: methionine for zwf1 Δ; synthetic complete media including aromatic amino acids for tkl1 Δ/ tkl2 Δ. (C) Triple deletion of the sedoheptulose bisphosphatase SHB17 , the glucose-6-phosphate dehydrogenase ZWF1 , and the transaldolase TAL1 .

    Article Snippet: Initial enzymatic screens for enzymatic activity were performed using in vitro synthesized, untagged Shb17 (New England Biolabs Inc. PURExpress® In Vitro Protein Synthesis Kit).

    Techniques: Produced, Labeling

    Structure of the Shb17/SBP complex. (A) Overall fold of the Shb17 (H13A) in complex with SBP (PDB 3OI7, grey ribbon) shown in two orientations with secondary structural elements being labeled. The SBP molecule (magenta carbon atoms) is shown in a stick representation. (B) Close-up view of the active site of Shb17 in complex with SBP. The side chains of residues in contact with SBP are displayed in a stick representation (green carbon atoms) and labeled. SBP is shown in a stick representation (magenta carbon atoms) and labeled, whereas the Mg 2+ ion is shown as a purple sphere and labeled. (C) Active site of Shb17 in complex with FBP, a similar view as (B). The red sphere denotes a water molecule. Y102 makes two hydrogen bonds with SBP, whereas only one hydrogen bond can be formed between this residue and FBP. These hydrogen bonds are shown by dashed lines in parts B and C.

    Journal: Cell

    Article Title: Riboneogenesis in yeast

    doi: 10.1016/j.cell.2011.05.022

    Figure Lengend Snippet: Structure of the Shb17/SBP complex. (A) Overall fold of the Shb17 (H13A) in complex with SBP (PDB 3OI7, grey ribbon) shown in two orientations with secondary structural elements being labeled. The SBP molecule (magenta carbon atoms) is shown in a stick representation. (B) Close-up view of the active site of Shb17 in complex with SBP. The side chains of residues in contact with SBP are displayed in a stick representation (green carbon atoms) and labeled. SBP is shown in a stick representation (magenta carbon atoms) and labeled, whereas the Mg 2+ ion is shown as a purple sphere and labeled. (C) Active site of Shb17 in complex with FBP, a similar view as (B). The red sphere denotes a water molecule. Y102 makes two hydrogen bonds with SBP, whereas only one hydrogen bond can be formed between this residue and FBP. These hydrogen bonds are shown by dashed lines in parts B and C.

    Article Snippet: Initial enzymatic screens for enzymatic activity were performed using in vitro synthesized, untagged Shb17 (New England Biolabs Inc. PURExpress® In Vitro Protein Synthesis Kit).

    Techniques: Labeling

    SBP and OBP are synthesized in vivo by C 3 + C 4 and C 3 + C 5 subunits via fructose bisphosphate aldolase. (A) Cells were switched from unlabeled to 70:30 unlabeled glucose:[U- 13 C]-glucose. Labeling patterns of erythrose-4-phosphate (E4P), dihydroxyacetone-phosphate (DHAP), ribose-5-phosphate (R5P), SBP and OBP were measured in shb17 Δ, where SBP and OBP accumulate and hence are more readily quantitated. The reaction products sedoheptulose-7-phosphate (S7P) and octulose 8-phosphate (O8P) were measured in wild type (for data on S7P in shb17 ). Labeling is reported 20 minutes after nutrient switch for all compounds except OBP, where data is taken at 120 min due to its slower labeling. (B) Kinetics of labeling of SBP after switching shb17 Δ cells with wild type fructose bisphosphate aldolase ( FBA1-wt ), or the Decreased Abundance by mRNA Perturbation allele ( FBA1-DAmP ) into [U- 13 C 6 . (C) Kinetics of labeling of SBP and S1P after switching shb17 Δ cells into [U- 13 C 6 ]-glucose.

    Journal: Cell

    Article Title: Riboneogenesis in yeast

    doi: 10.1016/j.cell.2011.05.022

    Figure Lengend Snippet: SBP and OBP are synthesized in vivo by C 3 + C 4 and C 3 + C 5 subunits via fructose bisphosphate aldolase. (A) Cells were switched from unlabeled to 70:30 unlabeled glucose:[U- 13 C]-glucose. Labeling patterns of erythrose-4-phosphate (E4P), dihydroxyacetone-phosphate (DHAP), ribose-5-phosphate (R5P), SBP and OBP were measured in shb17 Δ, where SBP and OBP accumulate and hence are more readily quantitated. The reaction products sedoheptulose-7-phosphate (S7P) and octulose 8-phosphate (O8P) were measured in wild type (for data on S7P in shb17 ). Labeling is reported 20 minutes after nutrient switch for all compounds except OBP, where data is taken at 120 min due to its slower labeling. (B) Kinetics of labeling of SBP after switching shb17 Δ cells with wild type fructose bisphosphate aldolase ( FBA1-wt ), or the Decreased Abundance by mRNA Perturbation allele ( FBA1-DAmP ) into [U- 13 C 6 . (C) Kinetics of labeling of SBP and S1P after switching shb17 Δ cells into [U- 13 C 6 ]-glucose.

    Article Snippet: Initial enzymatic screens for enzymatic activity were performed using in vitro synthesized, untagged Shb17 (New England Biolabs Inc. PURExpress® In Vitro Protein Synthesis Kit).

    Techniques: Synthesized, In Vivo, Labeling

    Metabolomic phenotype of shb17 Δ. (A) Metabolite structures associated with metabolic phenotype of shb17Δ . (B) Relative quantitation of metabolites. Data shown is arithmetic mean ± SE of N=4 independent biological replicates. (C)The negative ionization mode extracted ion chromatogram for SBP in shb17 Δ and wild type S. cerevisiae . Inset: Mass spectrum displaying the accurate mass for the parent ion (M) and natural 13 C abundance ion (M+1) observed for SBP in negative ionization mode via LC/Exactive Orbitrap MS. (D) Table of [M-H] ions with altered abundance between shb17 Δ and wild type.

    Journal: Cell

    Article Title: Riboneogenesis in yeast

    doi: 10.1016/j.cell.2011.05.022

    Figure Lengend Snippet: Metabolomic phenotype of shb17 Δ. (A) Metabolite structures associated with metabolic phenotype of shb17Δ . (B) Relative quantitation of metabolites. Data shown is arithmetic mean ± SE of N=4 independent biological replicates. (C)The negative ionization mode extracted ion chromatogram for SBP in shb17 Δ and wild type S. cerevisiae . Inset: Mass spectrum displaying the accurate mass for the parent ion (M) and natural 13 C abundance ion (M+1) observed for SBP in negative ionization mode via LC/Exactive Orbitrap MS. (D) Table of [M-H] ions with altered abundance between shb17 Δ and wild type.

    Article Snippet: Initial enzymatic screens for enzymatic activity were performed using in vitro synthesized, untagged Shb17 (New England Biolabs Inc. PURExpress® In Vitro Protein Synthesis Kit).

    Techniques: Quantitation Assay, Mass Spectrometry