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    (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 <t>PURExpress</t> 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).
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    (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).

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0150097

    Figure Lengend 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).

    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.

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

    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

    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 .

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M113.544429

    Figure Lengend 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 .

    Article Snippet: To test whether addition of EF-Tu could rescue translation activity after inhibition by Doc ( ) we used the PURExpress kit following the manufacturer's instructions.

    Techniques: Activity Assay, Incubation, Purification

    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

    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).

    Journal: bioRxiv

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

    doi: 10.1101/2020.04.04.025353

    Figure Lengend 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).

    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) .

    Techniques: Expressing, Western Blot, Produced, Molecular Weight

    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw018

    Figure Lengend 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.

    Article Snippet: Cell free translation reactions, carried out in the PURExpress in vitro protein synthesis system (New England Biolabs, Ipswich, MA, USA) were supplemented with 50 μM thiostrepton or 100 μM of peptides which were added in water and samples (5 μl volume) were incubated for 15 min at 37°C prior to the primer extension phase of the procedure.

    Techniques: Binding Assay, Sequencing, Incubation, Modification