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    Structured Review

    New England Biolabs purexpress in vitro
    IF2 is a target of ppGpp. IF2 was validated in vitro as a direct target of ppGpp using IF2 mutations that reduce ppGpp binding. ( A ) Affinity of B. subtilis ). (means ± SDs). ( B ) Alignment of G1 domains of B. subtilis ). Residues in red are those that differ in EF-G and IF2 and were used to engineer a mutant IF2 with reduced affinity for ppGpp (G226A H230A). ( C ) DRaCALA-based comparison of (p)ppGpp affinity for WT and mutant IF2 (means ± SDs). ( D ) In vitro sensitivity of WT and mutant IF2 was assessed using the <t>PURExpress</t> system (NEB). WT and mutant IF2 were added at equimolar amounts to separate PURExpress reactions in the presence of 1 mM ppGpp, and protein synthesis was monitored by Western blot (means ± SDs). ** P
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    1) Product Images from "The alarmones (p)ppGpp directly regulate translation initiation during entry into quiescence"

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

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

    doi: 10.1073/pnas.1920013117

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

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

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

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

    Journal: bioRxiv

    doi: 10.1101/807917

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

    Techniques Used: In Vitro, Western Blot

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

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

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

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

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

    doi: 10.1073/pnas.1920013117

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

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

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    New England Biolabs purexpress in vitro transcription translation system
    Determination of k R for each construct. (a) Scaled overlay of the individual plots in panels b-h. (b)-(h) Plot of the decrease in fraction arrested protein ( f A ) over time after chasing a 5 min <t>PURExpress</t> translation of the indicated construct. The data were fit to the first order equation in the main text to determine the rate of release ( k R ) of arrested protein from the ribosome. (i) Summary of the fitness of the equation for each construct: degrees of freedom ( d.f. ), R 2 , and goodness-of-fit calculation ( Sy.x ). (j) The goodness-of-fit calculation provided by Prism 8 (Graphpad software) where n is the number of data points and K is the degrees of freedom.
    Purexpress In Vitro Transcription Translation System, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs purexpress in vitro
    IF2 is a target of ppGpp. IF2 was validated in vitro as a direct target of ppGpp using IF2 mutations that reduce ppGpp binding. ( A ) Affinity of B. subtilis ). (means ± SDs). ( B ) Alignment of G1 domains of B. subtilis ). Residues in red are those that differ in EF-G and IF2 and were used to engineer a mutant IF2 with reduced affinity for ppGpp (G226A H230A). ( C ) DRaCALA-based comparison of (p)ppGpp affinity for WT and mutant IF2 (means ± SDs). ( D ) In vitro sensitivity of WT and mutant IF2 was assessed using the <t>PURExpress</t> system (NEB). WT and mutant IF2 were added at equimolar amounts to separate PURExpress reactions in the presence of 1 mM ppGpp, and protein synthesis was monitored by Western blot (means ± SDs). ** P
    Purexpress In Vitro, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    New England Biolabs purexpress in vitro translation system
    Quantification of the efficiency of ribosome sliding on mCherry reporters expressed in the <t>PURExpress</t> system. mCherry reporters ( Figure 1A : no insert, and various A stretches) were expressed in the PURExpress cell-free translation system ( Figure 5 ). The plot reports the percent of truncated peptide product expressed relative to total peptide product for each reporter (100% × (radioactivity in truncated band)/(radioactivity in truncated + full-length bands)). DOI: http://dx.doi.org/10.7554/eLife.05534.016
    Purexpress In Vitro Translation System, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    New England Biolabs vitro protein synthesis ivps kit
    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 <t>IVPS</t> 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 <t>PURExpress</t> 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).
    Vitro Protein Synthesis Ivps Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Determination of k R for each construct. (a) Scaled overlay of the individual plots in panels b-h. (b)-(h) Plot of the decrease in fraction arrested protein ( f A ) over time after chasing a 5 min PURExpress translation of the indicated construct. The data were fit to the first order equation in the main text to determine the rate of release ( k R ) of arrested protein from the ribosome. (i) Summary of the fitness of the equation for each construct: degrees of freedom ( d.f. ), R 2 , and goodness-of-fit calculation ( Sy.x ). (j) The goodness-of-fit calculation provided by Prism 8 (Graphpad software) where n is the number of data points and K is the degrees of freedom.

    Journal: bioRxiv

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

    doi: 10.1101/653360

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

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

    Techniques: Construct, Software

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

    Journal: bioRxiv

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

    doi: 10.1101/653360

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

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

    Techniques: Pulse Chase, Incubation, Construct

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

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

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

    doi: 10.1073/pnas.1920013117

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

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

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

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

    Journal: eLife

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

    doi: 10.7554/eLife.05534

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

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

    Techniques: Radioactivity

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

    Journal: eLife

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

    doi: 10.7554/eLife.05534

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

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

    Techniques: Radioactivity

    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