untagged shb17  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    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
    Buy from Supplier


    Structured Review

    New England Biolabs untagged shb17
    PURExpress In Vitro Protein Synthesis Kit
    PURExpress In Vitro Protein Synthesis Kit 100 rxns
    https://www.bioz.com/result/untagged shb17/product/New England Biolabs
    Average 99 stars, based on 474 article reviews
    Price from $9.99 to $1999.99
    untagged shb17 - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Riboneogenesis in yeast"

    Article Title: Riboneogenesis in yeast

    Journal: Cell

    doi: 10.1016/j.cell.2011.05.022

    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 .
    Figure Legend 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 .

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Quantitation Assay, Mass Spectrometry

    2) 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

    3) 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

    4) Product Images from "Campylobacter concisus pathotypes induce distinct global responses in intestinal epithelial cells"

    Article Title: Campylobacter concisus pathotypes induce distinct global responses in intestinal epithelial cells

    Journal: Scientific Reports

    doi: 10.1038/srep34288

    Transcriptional regulation in intestinal epithelial Caco-2 cells exposed to zonula occludens toxin. ( A ) In vitro expression of ZOT and confirmation of protein identity using mass spectrometry. ( B ) Number of significantly regulated transcripts following exposure of Caco-2 cells to ZOT. ( C ) Numbers of upregulated (red) and downregulated (green) transcripts visualised in the outer circle, and types of regulated transcripts visualised independently in the inner circle. ( D ) Comparison of the responses of Caco-2 cells to C. concisus BAA-1457 (outer circle) and ZOT (inner circle). Transcripts were mapped according to location on human chromosomes. Responses were visualised using circular Circos plot, with transcripts increased in expression in red and those decreased in blue.
    Figure Legend Snippet: Transcriptional regulation in intestinal epithelial Caco-2 cells exposed to zonula occludens toxin. ( A ) In vitro expression of ZOT and confirmation of protein identity using mass spectrometry. ( B ) Number of significantly regulated transcripts following exposure of Caco-2 cells to ZOT. ( C ) Numbers of upregulated (red) and downregulated (green) transcripts visualised in the outer circle, and types of regulated transcripts visualised independently in the inner circle. ( D ) Comparison of the responses of Caco-2 cells to C. concisus BAA-1457 (outer circle) and ZOT (inner circle). Transcripts were mapped according to location on human chromosomes. Responses were visualised using circular Circos plot, with transcripts increased in expression in red and those decreased in blue.

    Techniques Used: In Vitro, Expressing, Mass Spectrometry

    Pathways and processes significantly upregulated in intestinal epithelial Caco-2 cells exposed to zonula occludens toxin. Top 30 upregulated pathways ( A ) and processes ( B ) were selected. Pathways and processes were identified using MetaCore and inverse of log(FDR corrected P-value) was plotted. Full list of top 50 upregulated and downregulated pathways and processes can be found in Supplementary Data 2 .
    Figure Legend Snippet: Pathways and processes significantly upregulated in intestinal epithelial Caco-2 cells exposed to zonula occludens toxin. Top 30 upregulated pathways ( A ) and processes ( B ) were selected. Pathways and processes were identified using MetaCore and inverse of log(FDR corrected P-value) was plotted. Full list of top 50 upregulated and downregulated pathways and processes can be found in Supplementary Data 2 .

    Techniques Used:

    5) 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

    6) 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

    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 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

    9) 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

    10) Product Images from "Campylobacter concisus pathotypes induce distinct global responses in intestinal epithelial cells"

    Article Title: Campylobacter concisus pathotypes induce distinct global responses in intestinal epithelial cells

    Journal: Scientific Reports

    doi: 10.1038/srep34288

    Pathways significantly regulated in intestinal epithelial Caco-2 cells infected with Campylobacter concisus BAA-1457. Top 30 upregulated ( A ) and downregulated ( B ) pathways were selected. Pathways were identified using MetaCore and inverse of log(FDR corrected P-value) was plotted. Full list of top 50 upregulated and downregulated pathways can be found in Supplementary Data 2 .
    Figure Legend Snippet: Pathways significantly regulated in intestinal epithelial Caco-2 cells infected with Campylobacter concisus BAA-1457. Top 30 upregulated ( A ) and downregulated ( B ) pathways were selected. Pathways were identified using MetaCore and inverse of log(FDR corrected P-value) was plotted. Full list of top 50 upregulated and downregulated pathways can be found in Supplementary Data 2 .

    Techniques Used: Infection

    Transcriptional regulation in intestinal epithelial Caco-2 cells infected with Campylobacter concisus . ( A ) Number of significantly differentially expressed transcripts following infection with C. concisus BAA-1457 or UNSWCD. ( B ) Comparison of the responses of Caco-2 cells to C. concisus BAA-1457 (outer circle) and UNSWCD (inner circle). Transcripts were mapped according to location on human chromosomes. Responses were visualised using circular Circos plot, with transcripts increased in expression in red and those decreased in blue. ( C ) Numbers of upregulated (red) and downregulated (green) transcripts visualised in the outer circles, and types of regulated transcripts visualised independently in the inner circles.
    Figure Legend Snippet: Transcriptional regulation in intestinal epithelial Caco-2 cells infected with Campylobacter concisus . ( A ) Number of significantly differentially expressed transcripts following infection with C. concisus BAA-1457 or UNSWCD. ( B ) Comparison of the responses of Caco-2 cells to C. concisus BAA-1457 (outer circle) and UNSWCD (inner circle). Transcripts were mapped according to location on human chromosomes. Responses were visualised using circular Circos plot, with transcripts increased in expression in red and those decreased in blue. ( C ) Numbers of upregulated (red) and downregulated (green) transcripts visualised in the outer circles, and types of regulated transcripts visualised independently in the inner circles.

    Techniques Used: Infection, Expressing

    Processes significantly regulated in intestinal epithelial Caco-2 cells infected with Campylobacter concisus BAA-1457. Top 30 upregulated ( A ) and downregulated ( B ) processes were selected. Processes were identified using MetaCore and inverse of log(FDR corrected P-value) was plotted. Full list of top 50 upregulated and downregulated processes can be found in Supplementary Data 2 .
    Figure Legend Snippet: Processes significantly regulated in intestinal epithelial Caco-2 cells infected with Campylobacter concisus BAA-1457. Top 30 upregulated ( A ) and downregulated ( B ) processes were selected. Processes were identified using MetaCore and inverse of log(FDR corrected P-value) was plotted. Full list of top 50 upregulated and downregulated processes can be found in Supplementary Data 2 .

    Techniques Used: Infection

    Transcriptional regulation in intestinal epithelial Caco-2 cells exposed to zonula occludens toxin. ( A ) In vitro expression of ZOT and confirmation of protein identity using mass spectrometry. ( B ) Number of significantly regulated transcripts following exposure of Caco-2 cells to ZOT. ( C ) Numbers of upregulated (red) and downregulated (green) transcripts visualised in the outer circle, and types of regulated transcripts visualised independently in the inner circle. ( D ) Comparison of the responses of Caco-2 cells to C. concisus BAA-1457 (outer circle) and ZOT (inner circle). Transcripts were mapped according to location on human chromosomes. Responses were visualised using circular Circos plot, with transcripts increased in expression in red and those decreased in blue.
    Figure Legend Snippet: Transcriptional regulation in intestinal epithelial Caco-2 cells exposed to zonula occludens toxin. ( A ) In vitro expression of ZOT and confirmation of protein identity using mass spectrometry. ( B ) Number of significantly regulated transcripts following exposure of Caco-2 cells to ZOT. ( C ) Numbers of upregulated (red) and downregulated (green) transcripts visualised in the outer circle, and types of regulated transcripts visualised independently in the inner circle. ( D ) Comparison of the responses of Caco-2 cells to C. concisus BAA-1457 (outer circle) and ZOT (inner circle). Transcripts were mapped according to location on human chromosomes. Responses were visualised using circular Circos plot, with transcripts increased in expression in red and those decreased in blue.

    Techniques Used: In Vitro, Expressing, Mass Spectrometry

    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 "Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)"

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003302

    Identification of the sialidase-encoding ORF on fosmid G7 and its in vitro expression. A , a map of fosmid G7 transposon insertion sites ( red lines ) in mutants with abolished sialidase activity. B , SDS–PAGE of ORF9 and ORF12 proteins expressed in vitro using the PURExpress system. C , sialidase activity produced in PURExpress reaction mixtures was assessed using the substrate 4MU-α-Neu5Ac as described under “Experimental procedures.” D , the deduced amino acid sequence of ORF12p. The nucleotide sequence and the deduced protein sequence for ORF12 are annotated in the fosmid G7 sequence record (GenBank TM accession number MH016668 ).
    Figure Legend Snippet: Identification of the sialidase-encoding ORF on fosmid G7 and its in vitro expression. A , a map of fosmid G7 transposon insertion sites ( red lines ) in mutants with abolished sialidase activity. B , SDS–PAGE of ORF9 and ORF12 proteins expressed in vitro using the PURExpress system. C , sialidase activity produced in PURExpress reaction mixtures was assessed using the substrate 4MU-α-Neu5Ac as described under “Experimental procedures.” D , the deduced amino acid sequence of ORF12p. The nucleotide sequence and the deduced protein sequence for ORF12 are annotated in the fosmid G7 sequence record (GenBank TM accession number MH016668 ).

    Techniques Used: In Vitro, Expressing, Activity Assay, SDS Page, Produced, Sequencing

    13) 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

    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

    14) Product Images from "Doc Toxin Is a Kinase That Inactivates Elongation Factor Tu *"

    Article Title: Doc Toxin Is a Kinase That Inactivates Elongation Factor Tu *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.544429

    Doc is a protein kinase that targets a single protein in E. coli , it is not an adenylyltransferase. A and B , PURExpress-coupled transcription/translation reactions (containing ∼90 factors required for transcription and translation) with added protein components as shown above each lane were performed with [α- 32 P]ATP to test for adenylylation activity (5-day exposure) ( A ) or [γ- 32 P]ATP to test for kinase activity (5-h exposure) ( B ). Molecular mass markers (in kDa) are on the left. C , reactions containing [γ- 32 P]ATP were incubated with E. coli cell lysate ( Lysate only ) or with lysate supplemented with purified proteins as indicated (18-h exposure). Molecular mass markers (in kDa) are on the left .
    Figure Legend Snippet: Doc is a protein kinase that targets a single protein in E. coli , it is not an adenylyltransferase. A and B , PURExpress-coupled transcription/translation reactions (containing ∼90 factors required for transcription and translation) with added protein components as shown above each lane were performed with [α- 32 P]ATP to test for adenylylation activity (5-day exposure) ( A ) or [γ- 32 P]ATP to test for kinase activity (5-h exposure) ( B ). Molecular mass markers (in kDa) are on the left. C , reactions containing [γ- 32 P]ATP were incubated with E. coli cell lysate ( Lysate only ) or with lysate supplemented with purified proteins as indicated (18-h exposure). Molecular mass markers (in kDa) are on the left .

    Techniques Used: Activity Assay, Incubation, Purification

    15) 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

    16) Product Images from "Structures of proline-rich peptides bound to the ribosome reveal a common mechanism of protein synthesis inhibition"

    Article Title: Structures of proline-rich peptides bound to the ribosome reveal a common mechanism of protein synthesis inhibition

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw018

    Toe-printing ( A ) and foot-printing ( B ) experiments reveal the mode of action and verify PrAMP binding mode in solution. (A) The 20-codon synthetic RST2 ORF containing codons for all 20 amino acids ( 49 ) was translated in the PURExpress cell-free transcription-translation system by Escherichia coli ribosomes in the presence of 100 μM of PrAMPs or 50 μM of the control antibiotic thiostrepton (Ths), and the position of the stalled ribosome was determined by primer extension. U- and A- specific reactions were used as a sequencing ladder. The toeprint band (marked by an arrow in the gel and in the sequence of the gene), which occurs at position +16 counting from the first nucleotide of the codon in the ribosomal P site, places the arrested ribosome at the initiator codon (boxed on the RST2 sequence on the left from the gel). (B) Foot-printing analysis of interaction of Bac7 1 –35 and Onc112 with the E. coli ribosome in solution. Ribosomes were pre-incubated with no PrAMP (’none’) or 50 μM of Bac7 1 –35 or Onc112 and subjected to modification with CMCT or DMS. Control sample remained unmodified. Some of the lanes in gels shown in A and B, which contained samples irrelevant to the current study, have been computationally removed.
    Figure Legend Snippet: Toe-printing ( A ) and foot-printing ( B ) experiments reveal the mode of action and verify PrAMP binding mode in solution. (A) The 20-codon synthetic RST2 ORF containing codons for all 20 amino acids ( 49 ) was translated in the PURExpress cell-free transcription-translation system by Escherichia coli ribosomes in the presence of 100 μM of PrAMPs or 50 μM of the control antibiotic thiostrepton (Ths), and the position of the stalled ribosome was determined by primer extension. U- and A- specific reactions were used as a sequencing ladder. The toeprint band (marked by an arrow in the gel and in the sequence of the gene), which occurs at position +16 counting from the first nucleotide of the codon in the ribosomal P site, places the arrested ribosome at the initiator codon (boxed on the RST2 sequence on the left from the gel). (B) Foot-printing analysis of interaction of Bac7 1 –35 and Onc112 with the E. coli ribosome in solution. Ribosomes were pre-incubated with no PrAMP (’none’) or 50 μM of Bac7 1 –35 or Onc112 and subjected to modification with CMCT or DMS. Control sample remained unmodified. Some of the lanes in gels shown in A and B, which contained samples irrelevant to the current study, have been computationally removed.

    Techniques Used: Binding Assay, Sequencing, Incubation, Modification

    17) Product Images from "Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle"

    Article Title: Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky560

    ItaT inhibits translation by acetylation of tRNA. ( A ) Activity of firefly luciferase synthesized in in vitro coupled transcription-translation PURExpress system in the absence or presence of ItaT, acetyl-CoA (AcCoA) or both. The graph shows the relative luminescence of samples obtained from three independent experiments expressed in arbitrary units, AU. The error bars represent standard deviation. ( B ) SDS-PAGE analysis of DHFR produced in the transcription-translation reaction described in (A). Reactions were supplemented with [ 14 C] acetyl-CoA and carried out in the absence (left) or presence of ItaT (right). Reaction products were visualized with Coomassie blue staining. The position of the DHFR protein band is indicated by the red arrowhead on the left. Lane M shows the molecular weight markers. ( C ) Analysis of tRNAs extracted from the samples shown in (B) by acid-urea PAGE followed by methylene blue staining (top panel) and autoradiography (lower panel). The position of tRNAs on the gel is indicated by the black arrowhead on the right.
    Figure Legend Snippet: ItaT inhibits translation by acetylation of tRNA. ( A ) Activity of firefly luciferase synthesized in in vitro coupled transcription-translation PURExpress system in the absence or presence of ItaT, acetyl-CoA (AcCoA) or both. The graph shows the relative luminescence of samples obtained from three independent experiments expressed in arbitrary units, AU. The error bars represent standard deviation. ( B ) SDS-PAGE analysis of DHFR produced in the transcription-translation reaction described in (A). Reactions were supplemented with [ 14 C] acetyl-CoA and carried out in the absence (left) or presence of ItaT (right). Reaction products were visualized with Coomassie blue staining. The position of the DHFR protein band is indicated by the red arrowhead on the left. Lane M shows the molecular weight markers. ( C ) Analysis of tRNAs extracted from the samples shown in (B) by acid-urea PAGE followed by methylene blue staining (top panel) and autoradiography (lower panel). The position of tRNAs on the gel is indicated by the black arrowhead on the right.

    Techniques Used: Activity Assay, Luciferase, Synthesized, In Vitro, Standard Deviation, SDS Page, Produced, Staining, Molecular Weight, Polyacrylamide Gel Electrophoresis, Autoradiography

    18) Product Images from "Doc Toxin Is a Kinase That Inactivates Elongation Factor Tu *"

    Article Title: Doc Toxin Is a Kinase That Inactivates Elongation Factor Tu *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.544429

    Doc is a protein kinase that targets a single protein in E. coli , it is not an adenylyltransferase. A and B , PURExpress-coupled transcription/translation reactions (containing ∼90 factors required for transcription and translation) with added protein components as shown above each lane were performed with [α- 32 P]ATP to test for adenylylation activity (5-day exposure) ( A ) or [γ- 32 P]ATP to test for kinase activity (5-h exposure) ( B ). Molecular mass markers (in kDa) are on the left. C , reactions containing [γ- 32 P]ATP were incubated with E. coli cell lysate ( Lysate only ) or with lysate supplemented with purified proteins as indicated (18-h exposure). Molecular mass markers (in kDa) are on the left .
    Figure Legend Snippet: Doc is a protein kinase that targets a single protein in E. coli , it is not an adenylyltransferase. A and B , PURExpress-coupled transcription/translation reactions (containing ∼90 factors required for transcription and translation) with added protein components as shown above each lane were performed with [α- 32 P]ATP to test for adenylylation activity (5-day exposure) ( A ) or [γ- 32 P]ATP to test for kinase activity (5-h exposure) ( B ). Molecular mass markers (in kDa) are on the left. C , reactions containing [γ- 32 P]ATP were incubated with E. coli cell lysate ( Lysate only ) or with lysate supplemented with purified proteins as indicated (18-h exposure). Molecular mass markers (in kDa) are on the left .

    Techniques Used: Activity Assay, Incubation, Purification

    19) Product Images from "Clostridium difficile Cell Wall Protein CwpV Undergoes Enzyme-independent Intramolecular Autoproteolysis *"

    Article Title: Clostridium difficile Cell Wall Protein CwpV Undergoes Enzyme-independent Intramolecular Autoproteolysis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.302463

    CwpV undergoes enzyme-independent intramolecular autoprocessing. Plasmids encoding a Strep -tagged fragment of CwpV (pCwpVfr (WT), pCwpVfr(T413A), and pCwpVfr(GGG)) were used as templates in PURExpress in vitro protein synthesis reactions. Proteins of interest were purified on Strep Tactin resin separated on 12% SDS-polyacrylamide gels and analyzed via Coomassie Blue staining and Western blotting. In the pCwpVfr (WT) sample, two products corresponding to the N-terminal domain and a fragment of the repeat domain could be seen aside from the full-length CwpV fragment. In both pCwpVfr(T413A) and pCwpVfr(GGG)samples, no cleavage was observed as only the full-length CwpV fragment could be seen. ◂, repeat domain; ◁, anchoring domain.
    Figure Legend Snippet: CwpV undergoes enzyme-independent intramolecular autoprocessing. Plasmids encoding a Strep -tagged fragment of CwpV (pCwpVfr (WT), pCwpVfr(T413A), and pCwpVfr(GGG)) were used as templates in PURExpress in vitro protein synthesis reactions. Proteins of interest were purified on Strep Tactin resin separated on 12% SDS-polyacrylamide gels and analyzed via Coomassie Blue staining and Western blotting. In the pCwpVfr (WT) sample, two products corresponding to the N-terminal domain and a fragment of the repeat domain could be seen aside from the full-length CwpV fragment. In both pCwpVfr(T413A) and pCwpVfr(GGG)samples, no cleavage was observed as only the full-length CwpV fragment could be seen. ◂, repeat domain; ◁, anchoring domain.

    Techniques Used: In Vitro, Purification, Staining, Western Blot

    20) Product Images from "Posttranscriptional Regulation of tnaA by Protein-RNA Interaction Mediated by Ribosomal Protein L4 in Escherichia coli"

    Article Title: Posttranscriptional Regulation of tnaA by Protein-RNA Interaction Mediated by Ribosomal Protein L4 in Escherichia coli

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00799-19

    L4 downregulates TnaA but not TnaC protein synthesis in an in vitro transcription/translation assay. (A) Schematic representation of a synthetic tna operon for in vitro transcription and translation with the PURExpress in vitro protein synthesis kit (New England BioLabs, UK). The upstream T7 promoter (P), ribosome binding site (RBS), NdeI and BamHI cloning sites, and the T7 terminator (TERM) downstream of the stop codon are indicated, respectively. Internal deletion variants (indicated by Δspacer, Δ5′-end, and Δ3′-end) of the tnaC-tnaA spacer in the synthetic tna operon were generated as described in the Materials and Methods and further used for in vitro transcription/translation in the absence and presence of FLAG-L4. (B) L4 downregulates TnaA protein synthesis. Shown are Western blot analysis (a) and quantitative data (b) for TnaA protein produced from the wild-type (wt) synthetic tna operon and FLAG-L4 upon detection by TnaA polyclonal antibody (α-TnaA) or FLAG peptide monoclonal antibody (α-FLAG) on 10% SDS-PAGE. (a) Increasing concentrations of FLAG-L4 (0.75, 1.5, 2.25, 3, and 3.75 μM, lanes 2 to 6) inhibited TnaA translation. Lane 1 shows the reaction control (without FLAG-L4). (C) Comparison of RNA sequences of tnaC-tnaA spacer (220 nt) and S10 upstream regions (172 nt) by means of a CLUSTAL O (1.2.4) multiple sequence alignment ( 27 ). Solid and dotted lines box nucleotides involved in RNA binding of L4 with S10 UTR to regulate transcription and both transcription and translation, respectively ( 11 , 53 , 57 ). Asterisks indicate identical nucleotides. The Shine-Dalgarno (SD) sequence of TnaA is shown in red. (D to F) The inhibitory effect of L4 on the synthesis of TnaA but not TnaC by the synthetic tna operon. Shown are Western blots of TnaA (Da) and a 15% Bis-Tris polyacrylamide gel (Ea) of TnaC produced from the wt and various deletion constructs (see panel A) of the synthetic tna operon in the absence of L4 (lanes 1, 3, 5, and 7) compared with a positive-control plasmid in the presence of L4 (lanes 2, 4, 6, and 8). The reaction was stopped using 2× SDS loading dye 60 min after adding template DNA, and we then conducted 10% SDS-PAGE and detection with antibodies against FLAG peptide and TnaA or a 15% Bis-Tris polyacrylamide gel to resolve the very small molecular weight TnaC protein (∼3 kDa). The concentration of FLAG-L4 was 1.5 μM. The TnaC protein was further confirmed by peptide sequencing. (Db and Eb) Quantification of the data obtained in panels Da and Ea is presented, and TnaA and TnaC protein levels were compared from different spacer deletion constructs as indicated. n = 3; mean ± SEM; ****, P
    Figure Legend Snippet: L4 downregulates TnaA but not TnaC protein synthesis in an in vitro transcription/translation assay. (A) Schematic representation of a synthetic tna operon for in vitro transcription and translation with the PURExpress in vitro protein synthesis kit (New England BioLabs, UK). The upstream T7 promoter (P), ribosome binding site (RBS), NdeI and BamHI cloning sites, and the T7 terminator (TERM) downstream of the stop codon are indicated, respectively. Internal deletion variants (indicated by Δspacer, Δ5′-end, and Δ3′-end) of the tnaC-tnaA spacer in the synthetic tna operon were generated as described in the Materials and Methods and further used for in vitro transcription/translation in the absence and presence of FLAG-L4. (B) L4 downregulates TnaA protein synthesis. Shown are Western blot analysis (a) and quantitative data (b) for TnaA protein produced from the wild-type (wt) synthetic tna operon and FLAG-L4 upon detection by TnaA polyclonal antibody (α-TnaA) or FLAG peptide monoclonal antibody (α-FLAG) on 10% SDS-PAGE. (a) Increasing concentrations of FLAG-L4 (0.75, 1.5, 2.25, 3, and 3.75 μM, lanes 2 to 6) inhibited TnaA translation. Lane 1 shows the reaction control (without FLAG-L4). (C) Comparison of RNA sequences of tnaC-tnaA spacer (220 nt) and S10 upstream regions (172 nt) by means of a CLUSTAL O (1.2.4) multiple sequence alignment ( 27 ). Solid and dotted lines box nucleotides involved in RNA binding of L4 with S10 UTR to regulate transcription and both transcription and translation, respectively ( 11 , 53 , 57 ). Asterisks indicate identical nucleotides. The Shine-Dalgarno (SD) sequence of TnaA is shown in red. (D to F) The inhibitory effect of L4 on the synthesis of TnaA but not TnaC by the synthetic tna operon. Shown are Western blots of TnaA (Da) and a 15% Bis-Tris polyacrylamide gel (Ea) of TnaC produced from the wt and various deletion constructs (see panel A) of the synthetic tna operon in the absence of L4 (lanes 1, 3, 5, and 7) compared with a positive-control plasmid in the presence of L4 (lanes 2, 4, 6, and 8). The reaction was stopped using 2× SDS loading dye 60 min after adding template DNA, and we then conducted 10% SDS-PAGE and detection with antibodies against FLAG peptide and TnaA or a 15% Bis-Tris polyacrylamide gel to resolve the very small molecular weight TnaC protein (∼3 kDa). The concentration of FLAG-L4 was 1.5 μM. The TnaC protein was further confirmed by peptide sequencing. (Db and Eb) Quantification of the data obtained in panels Da and Ea is presented, and TnaA and TnaC protein levels were compared from different spacer deletion constructs as indicated. n = 3; mean ± SEM; ****, P

    Techniques Used: In Vitro, Binding Assay, Clone Assay, Generated, Western Blot, Produced, SDS Page, Sequencing, RNA Binding Assay, Construct, Positive Control, Plasmid Preparation, Molecular Weight, Concentration Assay

    21) Product Images from "Posttranscriptional Regulation of tnaA by Protein-RNA Interaction Mediated by Ribosomal Protein L4 in Escherichia coli"

    Article Title: Posttranscriptional Regulation of tnaA by Protein-RNA Interaction Mediated by Ribosomal Protein L4 in Escherichia coli

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00799-19

    L4 downregulates TnaA but not TnaC protein synthesis in an in vitro transcription/translation assay. (A) Schematic representation of a synthetic tna operon for in vitro transcription and translation with the PURExpress in vitro protein synthesis kit (New England BioLabs, UK). The upstream T7 promoter (P), ribosome binding site (RBS), NdeI and BamHI cloning sites, and the T7 terminator (TERM) downstream of the stop codon are indicated, respectively. Internal deletion variants (indicated by Δspacer, Δ5′-end, and Δ3′-end) of the tnaC-tnaA spacer in the synthetic tna operon were generated as described in the Materials and Methods and further used for in vitro transcription/translation in the absence and presence of FLAG-L4. (B) L4 downregulates TnaA protein synthesis. Shown are Western blot analysis (a) and quantitative data (b) for TnaA protein produced from the wild-type (wt) synthetic tna operon and FLAG-L4 upon detection by TnaA polyclonal antibody (α-TnaA) or FLAG peptide monoclonal antibody (α-FLAG) on 10% SDS-PAGE. (a) Increasing concentrations of FLAG-L4 (0.75, 1.5, 2.25, 3, and 3.75 μM, lanes 2 to 6) inhibited TnaA translation. Lane 1 shows the reaction control (without FLAG-L4). (C) Comparison of RNA sequences of tnaC-tnaA spacer (220 nt) and S10 upstream regions (172 nt) by means of a CLUSTAL O (1.2.4) multiple sequence alignment ( 27 ). Solid and dotted lines box nucleotides involved in RNA binding of L4 with S10 UTR to regulate transcription and both transcription and translation, respectively ( 11 , 53 , 57 ). Asterisks indicate identical nucleotides. The Shine-Dalgarno (SD) sequence of TnaA is shown in red. (D to F) The inhibitory effect of L4 on the synthesis of TnaA but not TnaC by the synthetic tna operon. Shown are Western blots of TnaA (Da) and a 15% Bis-Tris polyacrylamide gel (Ea) of TnaC produced from the wt and various deletion constructs (see panel A) of the synthetic tna operon in the absence of L4 (lanes 1, 3, 5, and 7) compared with a positive-control plasmid in the presence of L4 (lanes 2, 4, 6, and 8). The reaction was stopped using 2× SDS loading dye 60 min after adding template DNA, and we then conducted 10% SDS-PAGE and detection with antibodies against FLAG peptide and TnaA or a 15% Bis-Tris polyacrylamide gel to resolve the very small molecular weight TnaC protein (∼3 kDa). The concentration of FLAG-L4 was 1.5 μM. The TnaC protein was further confirmed by peptide sequencing. (Db and Eb) Quantification of the data obtained in panels Da and Ea is presented, and TnaA and TnaC protein levels were compared from different spacer deletion constructs as indicated. n = 3; mean ± SEM; ****, P
    Figure Legend Snippet: L4 downregulates TnaA but not TnaC protein synthesis in an in vitro transcription/translation assay. (A) Schematic representation of a synthetic tna operon for in vitro transcription and translation with the PURExpress in vitro protein synthesis kit (New England BioLabs, UK). The upstream T7 promoter (P), ribosome binding site (RBS), NdeI and BamHI cloning sites, and the T7 terminator (TERM) downstream of the stop codon are indicated, respectively. Internal deletion variants (indicated by Δspacer, Δ5′-end, and Δ3′-end) of the tnaC-tnaA spacer in the synthetic tna operon were generated as described in the Materials and Methods and further used for in vitro transcription/translation in the absence and presence of FLAG-L4. (B) L4 downregulates TnaA protein synthesis. Shown are Western blot analysis (a) and quantitative data (b) for TnaA protein produced from the wild-type (wt) synthetic tna operon and FLAG-L4 upon detection by TnaA polyclonal antibody (α-TnaA) or FLAG peptide monoclonal antibody (α-FLAG) on 10% SDS-PAGE. (a) Increasing concentrations of FLAG-L4 (0.75, 1.5, 2.25, 3, and 3.75 μM, lanes 2 to 6) inhibited TnaA translation. Lane 1 shows the reaction control (without FLAG-L4). (C) Comparison of RNA sequences of tnaC-tnaA spacer (220 nt) and S10 upstream regions (172 nt) by means of a CLUSTAL O (1.2.4) multiple sequence alignment ( 27 ). Solid and dotted lines box nucleotides involved in RNA binding of L4 with S10 UTR to regulate transcription and both transcription and translation, respectively ( 11 , 53 , 57 ). Asterisks indicate identical nucleotides. The Shine-Dalgarno (SD) sequence of TnaA is shown in red. (D to F) The inhibitory effect of L4 on the synthesis of TnaA but not TnaC by the synthetic tna operon. Shown are Western blots of TnaA (Da) and a 15% Bis-Tris polyacrylamide gel (Ea) of TnaC produced from the wt and various deletion constructs (see panel A) of the synthetic tna operon in the absence of L4 (lanes 1, 3, 5, and 7) compared with a positive-control plasmid in the presence of L4 (lanes 2, 4, 6, and 8). The reaction was stopped using 2× SDS loading dye 60 min after adding template DNA, and we then conducted 10% SDS-PAGE and detection with antibodies against FLAG peptide and TnaA or a 15% Bis-Tris polyacrylamide gel to resolve the very small molecular weight TnaC protein (∼3 kDa). The concentration of FLAG-L4 was 1.5 μM. The TnaC protein was further confirmed by peptide sequencing. (Db and Eb) Quantification of the data obtained in panels Da and Ea is presented, and TnaA and TnaC protein levels were compared from different spacer deletion constructs as indicated. n = 3; mean ± SEM; ****, P

    Techniques Used: In Vitro, Binding Assay, Clone Assay, Generated, Western Blot, Produced, SDS Page, Sequencing, RNA Binding Assay, Construct, Positive Control, Plasmid Preparation, Molecular Weight, Concentration Assay

    22) 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

    23) Product Images from "OxyR senses sulfane sulfur and activates the genes for its removal in Escherichia coli"

    Article Title: OxyR senses sulfane sulfur and activates the genes for its removal in Escherichia coli

    Journal: Redox Biology

    doi: 10.1016/j.redox.2019.101293

    In vitro transcription-translation analysis of H 2 S n activation of OxyR and its mutants. Purified OxyR and its mutants were treated with DTT to ensure their thiols were in the reduce form; The proteins were then treated with H 2 S n to generate H 2 S n modified protein. The in vitro transcription-translation system contained P trxC -mKate DNA fragment (200 ng) and DTT-reduced or H 2 S n -treated OxyR (500 ng), and the expressed mKate was analyzed with the fluorescence photometer Synergy H1. (n ≥ 3 for each group) Data information: Data are presented as mean ± SEM.
    Figure Legend Snippet: In vitro transcription-translation analysis of H 2 S n activation of OxyR and its mutants. Purified OxyR and its mutants were treated with DTT to ensure their thiols were in the reduce form; The proteins were then treated with H 2 S n to generate H 2 S n modified protein. The in vitro transcription-translation system contained P trxC -mKate DNA fragment (200 ng) and DTT-reduced or H 2 S n -treated OxyR (500 ng), and the expressed mKate was analyzed with the fluorescence photometer Synergy H1. (n ≥ 3 for each group) Data information: Data are presented as mean ± SEM.

    Techniques Used: In Vitro, Activation Assay, Purification, Modification, Fluorescence

    Comparison of the activation effect of H 2 S n and H 2 O 2 . A-C H 2 S n or H 2 O 2 (100–600 μM) was used to treat E. coli wt strains containing reporter plasmids. (n ≥ 3 for each group) D Purified OxyR and its mutants were treated with DTT to ensure their thiols were in the reduce form; The proteins were then treated with H 2 S n or H 2 O 2 to generate H 2 S n - or H 2 O 2 -modified OxyR. The in vitro transcription-translation system contained P trxC -mKate DNA fragment (200 ng) and DTT-reduced, H 2 S n - or H 2 O 2 -treated OxyR (500 ng) and the expressed mKate was analyzed with the fluorescence photometer Synergy H1. (n ≥ 3 for each group) E H 2 S n or H 2 O 2 (400 μM) was used to treat E. coli wt. RT-qPCR was used to quantify the expression of trxC . (n ≥ 3 for each group).
    Figure Legend Snippet: Comparison of the activation effect of H 2 S n and H 2 O 2 . A-C H 2 S n or H 2 O 2 (100–600 μM) was used to treat E. coli wt strains containing reporter plasmids. (n ≥ 3 for each group) D Purified OxyR and its mutants were treated with DTT to ensure their thiols were in the reduce form; The proteins were then treated with H 2 S n or H 2 O 2 to generate H 2 S n - or H 2 O 2 -modified OxyR. The in vitro transcription-translation system contained P trxC -mKate DNA fragment (200 ng) and DTT-reduced, H 2 S n - or H 2 O 2 -treated OxyR (500 ng) and the expressed mKate was analyzed with the fluorescence photometer Synergy H1. (n ≥ 3 for each group) E H 2 S n or H 2 O 2 (400 μM) was used to treat E. coli wt. RT-qPCR was used to quantify the expression of trxC . (n ≥ 3 for each group).

    Techniques Used: Activation Assay, Purification, Modification, In Vitro, Fluorescence, Quantitative RT-PCR, Expressing

    24) 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

    25) Product Images from "Fragments of the Nonlytic Proline-Rich Antimicrobial Peptide Bac5 Kill Escherichia coli Cells by Inhibiting Protein Synthesis"

    Article Title: Fragments of the Nonlytic Proline-Rich Antimicrobial Peptide Bac5 Kill Escherichia coli Cells by Inhibiting Protein Synthesis

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00534-18

    Effect of Bac5 fragments on in vitro transcription and translation assays. (A) Effect of Bac5 fragments in in vitro prokaryotic coupled transcription/translation assays ( E. coli lysate). The presence of luciferase was checked and quantified using luminescence. As a negative control, a reaction was performed in the absence of DNA template (No DNA). (B) Effect of Bac5(1-25) homologues on E. coli in vitro transcription/translation reactions. The cow [cBac5(1-25)], sheep [sBac5(1-25)], and goat [gBac5(1-25)] orthologues were used. As a negative control, a reaction was performed in the absence of DNA template (No DNA). (C) In vitro mRNA synthesis in the presence of Bac5 fragments. The presence of two different RNA products (arrows) was checked and quantified by agarose gel electrophoresis (top) and spectrophotometric quantification ( A 260 ) (bottom). As a negative control, a reaction was performed in the absence of T7 RNA polymerase (No T7 pol.). (D) Effect of Bac5 fragments on in vitro prokaryotic translation assay ( T. thermophilus lysate). The luciferase was quantified by luminescence. As a negative control, a reaction was performed in the absence of RNA template (No RNA). Bac7(1-35) was used for comparison. Error bars represent the standard deviation from the average from three independent experiments. Results are expressed as a percentage of the value for the positive controls, namely, reactions performed in the presence of water instead of peptides, for which the results were defined as 100%.
    Figure Legend Snippet: Effect of Bac5 fragments on in vitro transcription and translation assays. (A) Effect of Bac5 fragments in in vitro prokaryotic coupled transcription/translation assays ( E. coli lysate). The presence of luciferase was checked and quantified using luminescence. As a negative control, a reaction was performed in the absence of DNA template (No DNA). (B) Effect of Bac5(1-25) homologues on E. coli in vitro transcription/translation reactions. The cow [cBac5(1-25)], sheep [sBac5(1-25)], and goat [gBac5(1-25)] orthologues were used. As a negative control, a reaction was performed in the absence of DNA template (No DNA). (C) In vitro mRNA synthesis in the presence of Bac5 fragments. The presence of two different RNA products (arrows) was checked and quantified by agarose gel electrophoresis (top) and spectrophotometric quantification ( A 260 ) (bottom). As a negative control, a reaction was performed in the absence of T7 RNA polymerase (No T7 pol.). (D) Effect of Bac5 fragments on in vitro prokaryotic translation assay ( T. thermophilus lysate). The luciferase was quantified by luminescence. As a negative control, a reaction was performed in the absence of RNA template (No RNA). Bac7(1-35) was used for comparison. Error bars represent the standard deviation from the average from three independent experiments. Results are expressed as a percentage of the value for the positive controls, namely, reactions performed in the presence of water instead of peptides, for which the results were defined as 100%.

    Techniques Used: In Vitro, Luciferase, Negative Control, Agarose Gel Electrophoresis, Standard Deviation

    26) 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

    27) Product Images from "Accurate target identification for Mycobacterium tuberculosis endoribonuclease toxins requires expression in their native host"

    Article Title: Accurate target identification for Mycobacterium tuberculosis endoribonuclease toxins requires expression in their native host

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-41548-9

    VapC-mt11 inhibits translation in vitro and in M. smegmatis . ( A ) The PURExpress translation reaction was incubated with (+) or without (−) VapC-mt11. Production of the control DHFR template was assayed (yellow arrow). Uncropped image shown in Supplementary Information Fig. 1 ( B ) [ 35 S]-Methionine incorporation in M. smegmatis mc 2 155 cells grown to exponential phase and split into uninduced (−VapC-mt11) and induced (+VapC-mt11) cultures. Cell aliquots were collected for up to 6 h post induction. Equivalent amounts of cell lysate (resuspended in appropriate Laemmli buffer volumes to normalize for differences in OD 600 ) were subjected to SDS-PAGE and visualized on a phosphorimager. Uncropped image shown in Supplementary Information Fig. 1 .
    Figure Legend Snippet: VapC-mt11 inhibits translation in vitro and in M. smegmatis . ( A ) The PURExpress translation reaction was incubated with (+) or without (−) VapC-mt11. Production of the control DHFR template was assayed (yellow arrow). Uncropped image shown in Supplementary Information Fig. 1 ( B ) [ 35 S]-Methionine incorporation in M. smegmatis mc 2 155 cells grown to exponential phase and split into uninduced (−VapC-mt11) and induced (+VapC-mt11) cultures. Cell aliquots were collected for up to 6 h post induction. Equivalent amounts of cell lysate (resuspended in appropriate Laemmli buffer volumes to normalize for differences in OD 600 ) were subjected to SDS-PAGE and visualized on a phosphorimager. Uncropped image shown in Supplementary Information Fig. 1 .

    Techniques Used: In Vitro, Incubation, SDS Page

    28) Product Images from "Clostridium difficile Cell Wall Protein CwpV Undergoes Enzyme-independent Intramolecular Autoproteolysis *"

    Article Title: Clostridium difficile Cell Wall Protein CwpV Undergoes Enzyme-independent Intramolecular Autoproteolysis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.302463

    CwpV undergoes enzyme-independent intramolecular autoprocessing. Plasmids encoding a Strep -tagged fragment of CwpV (pCwpVfr (WT), pCwpVfr(T413A), and pCwpVfr(GGG)) were used as templates in PURExpress in vitro protein synthesis reactions. Proteins of interest were purified on Strep Tactin resin separated on 12% SDS-polyacrylamide gels and analyzed via Coomassie Blue staining and Western blotting. In the pCwpVfr (WT) sample, two products corresponding to the N-terminal domain and a fragment of the repeat domain could be seen aside from the full-length CwpV fragment. In both pCwpVfr(T413A) and pCwpVfr(GGG)samples, no cleavage was observed as only the full-length CwpV fragment could be seen. ◂, repeat domain; ◁, anchoring domain.
    Figure Legend Snippet: CwpV undergoes enzyme-independent intramolecular autoprocessing. Plasmids encoding a Strep -tagged fragment of CwpV (pCwpVfr (WT), pCwpVfr(T413A), and pCwpVfr(GGG)) were used as templates in PURExpress in vitro protein synthesis reactions. Proteins of interest were purified on Strep Tactin resin separated on 12% SDS-polyacrylamide gels and analyzed via Coomassie Blue staining and Western blotting. In the pCwpVfr (WT) sample, two products corresponding to the N-terminal domain and a fragment of the repeat domain could be seen aside from the full-length CwpV fragment. In both pCwpVfr(T413A) and pCwpVfr(GGG)samples, no cleavage was observed as only the full-length CwpV fragment could be seen. ◂, repeat domain; ◁, anchoring domain.

    Techniques Used: In Vitro, Purification, Staining, Western Blot

    29) Product Images from "Translation Enhancing ACA Motifs and Their Silencing by a Bacterial Small Regulatory RNA"

    Article Title: Translation Enhancing ACA Motifs and Their Silencing by a Bacterial Small Regulatory RNA

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004026

    Effect of yifK 5′ UTR's changes on mRNA translation. The CA 49,50 to UC 49,50 change in yifK mRNA affects expression levels in vivo (A) and mRNA translation in vitro (B). In vitro translation was performed using the coupled transcription/translation PURExpress kit (see Materials and Methods ). DNA templates were from plasmids carrying the entire yifK 5′ UTR from wild-type, or from the UC 49,50 mutant, fused to the coding sequence of a 3×FLAG epitope-tagged version of the cat gene. Fusions were initially obtained as chromosomal constructs using DNA fragments amplified from strain MA7224 with primer pairs ppL50/ppL52 (wt) and ppL51/ppL52 (UC 49,50 ). Subsequently, the fusions were cloned into plasmid DHFR following amplification (ppM29/ppM30) and double Xba I/Pst I digestion. Transcription/translation reactions were carried out at a template DNA concentration of 0.5 pM for 90 min or 5 pM for 30 min and products analyzed by Western blotting using anti-FLAG monoclonal antibodies [53] . Under both conditions, higher amounts of cat -3×FLAG protein were synthesized from the construct with the wild-type yifK sequence than from the construct harboring the CA 49,50 to UC 49,50 change.
    Figure Legend Snippet: Effect of yifK 5′ UTR's changes on mRNA translation. The CA 49,50 to UC 49,50 change in yifK mRNA affects expression levels in vivo (A) and mRNA translation in vitro (B). In vitro translation was performed using the coupled transcription/translation PURExpress kit (see Materials and Methods ). DNA templates were from plasmids carrying the entire yifK 5′ UTR from wild-type, or from the UC 49,50 mutant, fused to the coding sequence of a 3×FLAG epitope-tagged version of the cat gene. Fusions were initially obtained as chromosomal constructs using DNA fragments amplified from strain MA7224 with primer pairs ppL50/ppL52 (wt) and ppL51/ppL52 (UC 49,50 ). Subsequently, the fusions were cloned into plasmid DHFR following amplification (ppM29/ppM30) and double Xba I/Pst I digestion. Transcription/translation reactions were carried out at a template DNA concentration of 0.5 pM for 90 min or 5 pM for 30 min and products analyzed by Western blotting using anti-FLAG monoclonal antibodies [53] . Under both conditions, higher amounts of cat -3×FLAG protein were synthesized from the construct with the wild-type yifK sequence than from the construct harboring the CA 49,50 to UC 49,50 change.

    Techniques Used: Expressing, In Vivo, In Vitro, Mutagenesis, Sequencing, Construct, Amplification, Clone Assay, Plasmid Preparation, Concentration Assay, Western Blot, Synthesized

    30) Product Images from "CsrA Participates in a PNPase Autoregulatory Mechanism by Selectively Repressing Translation of pnp Transcripts That Have Been Previously Processed by RNase III and PNPase"

    Article Title: CsrA Participates in a PNPase Autoregulatory Mechanism by Selectively Repressing Translation of pnp Transcripts That Have Been Previously Processed by RNase III and PNPase

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00721-15

    Effect of CsrA on pnp translation. Coupled transcription-translation reactions were performed with a PURExpress kit using WT pnp Fl , WT pnp Pr , and BS1 mutant pnp Pr DNA templates containing pnp ′-′ lacZ translational fusions. Purified CsrA protein was added prior to starting the reaction. β-Galactosidase activity normalized to 0 μM CsrA for each template is shown. Each experiment was performed at least twice, with representative results shown.
    Figure Legend Snippet: Effect of CsrA on pnp translation. Coupled transcription-translation reactions were performed with a PURExpress kit using WT pnp Fl , WT pnp Pr , and BS1 mutant pnp Pr DNA templates containing pnp ′-′ lacZ translational fusions. Purified CsrA protein was added prior to starting the reaction. β-Galactosidase activity normalized to 0 μM CsrA for each template is shown. Each experiment was performed at least twice, with representative results shown.

    Techniques Used: Mutagenesis, Purification, Activity Assay

    31) Product Images from "The IntXO-PSL Recombination System Is a Key Component of the Second Maintenance System for Bacillus anthracis Plasmid pXO1"

    Article Title: The IntXO-PSL Recombination System Is a Key Component of the Second Maintenance System for Bacillus anthracis Plasmid pXO1

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01004-15

    Recombinant IntXO protein activity in vitro . (A) Profiles of proteins (5 μl in each lane) synthesized in vitro by use of a PURExpress kit (New England BioLabs). Lane 1, no template DNA; lane 2, DHFR control plasmid used as template DNA; lane 3,
    Figure Legend Snippet: Recombinant IntXO protein activity in vitro . (A) Profiles of proteins (5 μl in each lane) synthesized in vitro by use of a PURExpress kit (New England BioLabs). Lane 1, no template DNA; lane 2, DHFR control plasmid used as template DNA; lane 3,

    Techniques Used: Recombinant, Activity Assay, In Vitro, Synthesized, Plasmid Preparation

    32) Product Images from "Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle"

    Article Title: Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky560

    ItaT inhibits translation by acetylation of tRNA. ( A ) Activity of firefly luciferase synthesized in in vitro coupled transcription-translation PURExpress system in the absence or presence of ItaT, acetyl-CoA (AcCoA) or both. The graph shows the relative luminescence of samples obtained from three independent experiments expressed in arbitrary units, AU. The error bars represent standard deviation. ( B ) SDS-PAGE analysis of DHFR produced in the transcription-translation reaction described in (A). Reactions were supplemented with [ 14 C] acetyl-CoA and carried out in the absence (left) or presence of ItaT (right). Reaction products were visualized with Coomassie blue staining. The position of the DHFR protein band is indicated by the red arrowhead on the left. Lane M shows the molecular weight markers. ( C ) Analysis of tRNAs extracted from the samples shown in (B) by acid-urea PAGE followed by methylene blue staining (top panel) and autoradiography (lower panel). The position of tRNAs on the gel is indicated by the black arrowhead on the right.
    Figure Legend Snippet: ItaT inhibits translation by acetylation of tRNA. ( A ) Activity of firefly luciferase synthesized in in vitro coupled transcription-translation PURExpress system in the absence or presence of ItaT, acetyl-CoA (AcCoA) or both. The graph shows the relative luminescence of samples obtained from three independent experiments expressed in arbitrary units, AU. The error bars represent standard deviation. ( B ) SDS-PAGE analysis of DHFR produced in the transcription-translation reaction described in (A). Reactions were supplemented with [ 14 C] acetyl-CoA and carried out in the absence (left) or presence of ItaT (right). Reaction products were visualized with Coomassie blue staining. The position of the DHFR protein band is indicated by the red arrowhead on the left. Lane M shows the molecular weight markers. ( C ) Analysis of tRNAs extracted from the samples shown in (B) by acid-urea PAGE followed by methylene blue staining (top panel) and autoradiography (lower panel). The position of tRNAs on the gel is indicated by the black arrowhead on the right.

    Techniques Used: Activity Assay, Luciferase, Synthesized, In Vitro, Standard Deviation, SDS Page, Produced, Staining, Molecular Weight, Polyacrylamide Gel Electrophoresis, Autoradiography

    33) 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

    Related Articles

    In Vitro:

    Article Title: Using Group II Introns for Attenuating the In Vitro and In Vivo Expression of a Homing Endonuclease
    Article Snippet: .. The PURExpress In Vitro Protein Synthesis Kit (New England Biolab, MA, USA) which is a cell-free transcription/translation system was utilized to assess if the proteins can be expressed in an “E . coli ” environment. .. Although the PURExpress kit is designed for coupled transcription and translation from an expression construct, direct translation from an mRNA template is also possible provided purified RNA (1–5 μg) is added to the reaction mixture, albeit a proper ribosome binding site must be present for efficient translation.

    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 ..

    Article Title: Intramolecular chaperone-mediated secretion of an Rhs effector toxin by a type VI secretion system
    Article Snippet: .. In vitro protein expression was performed with a PURExpress® In Vitro Protein Synthesis Kit (NEB) following the instruction of the manufacturer. .. For TseI purification under denaturing conditions, His-tagged proteins were first purified with Ni-NTA resin, eluted with elution buffer A (50 mM NaH2 PO4 pH 8.0, 300 mM NaCl, 250 mM imidazole), and dialyzed with dialysis buffer (50 mM NaH2 PO4 pH 8.0, 300 mM NaCl) three times to remove imidazole.

    Article Title: Cell-free synthesis of natural compounds from genomic DNA of biosynthetic gene clusters
    Article Snippet: .. In evaluating the PURE system for the cell-free synthesis of natural compounds from genomic DNA, we worked with the commercially available E. coli -based PURExpress In Vitro Protein Synthesis (IVPS) Kit as a “reaction solution” for gene expression and product formation (New England Biolabs, USA) . .. We posited that the E. coli -based system provides a suited setting, because a multitude of proteins from biosynthetic gene clusters were successfully produced in E. coli - .

    Article Title: Cell-free synthesis of natural compounds from genomic DNA of biosynthetic gene clusters
    Article Snippet: .. Results & Discussion To establish an in vitro platform for megasynthase production and biosynthesis, the monomodular NRPS protein BpsA was produced in vitro using the commercially available PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, USA) . ..

    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 .

    Article Title: Protein Synthesis Using A Reconstituted Cell-Free System
    Article Snippet: .. A PURExpress In vitro Protein Synthesis kit (New England Biolabs, ) 10 mM magnesium acetate Amicon Ultracel −0.5 ml-100 K MW cut off spin concentrator (Millipore) Microcentrifuge at 4°C Ni-NTA agarose (Qiagen) Microcentrifuge tubes Bio-Rad micro-spin column (Bio-Rad) ..

    Synthesized:

    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 .

    Produced:

    Article Title: Cell-free synthesis of natural compounds from genomic DNA of biosynthetic gene clusters
    Article Snippet: .. Results & Discussion To establish an in vitro platform for megasynthase production and biosynthesis, the monomodular NRPS protein BpsA was produced in vitro using the commercially available PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, USA) . ..

    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 .

    Expressing:

    Article Title: Intramolecular chaperone-mediated secretion of an Rhs effector toxin by a type VI secretion system
    Article Snippet: .. In vitro protein expression was performed with a PURExpress® In Vitro Protein Synthesis Kit (NEB) following the instruction of the manufacturer. .. For TseI purification under denaturing conditions, His-tagged proteins were first purified with Ni-NTA resin, eluted with elution buffer A (50 mM NaH2 PO4 pH 8.0, 300 mM NaCl, 250 mM imidazole), and dialyzed with dialysis buffer (50 mM NaH2 PO4 pH 8.0, 300 mM NaCl) three times to remove imidazole.

    Article Title: Cell-free synthesis of natural compounds from genomic DNA of biosynthetic gene clusters
    Article Snippet: .. In evaluating the PURE system for the cell-free synthesis of natural compounds from genomic DNA, we worked with the commercially available E. coli -based PURExpress In Vitro Protein Synthesis (IVPS) Kit as a “reaction solution” for gene expression and product formation (New England Biolabs, USA) . .. We posited that the E. coli -based system provides a suited setting, because a multitude of proteins from biosynthetic gene clusters were successfully produced in 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 ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs purexpress in vitro protein synthesis kit
    Chromozyme cleavage assay (Roche) for a truncated version of tissue plasminogen activator protein (vtPA) synthesized in the <t>PURExpress</t> reactions with and without PURExpress Disulfide Bond Enhancer (PDBE). This tissue plasminogen activator contains 9 disulfide
    Purexpress In Vitro Protein Synthesis Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/purexpress in vitro protein synthesis kit/product/New England Biolabs
    Average 99 stars, based on 86 article reviews
    Price from $9.99 to $1999.99
    purexpress in vitro protein synthesis kit - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    Image Search Results


    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

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

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

    doi: 10.1002/0471142727.mb1631s108

    Figure Lengend 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

    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)

    Techniques: 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

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

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

    doi: 10.1002/0471142727.mb1631s108

    Figure Lengend 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

    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)

    Techniques: 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.

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

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

    doi: 10.1002/0471142727.mb1631s108

    Figure Lengend 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.

    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)

    Techniques: SDS Page, Synthesized, Labeling, Luciferase

    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