purexpress in vitro protein synthesis kit  (New England Biolabs)


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    Structured Review

    New England Biolabs purexpress in vitro protein synthesis kit
    <t>In</t> vitro translation assays. (A) Schematic of in vitro transcription/translation and translation assays using the <t>PurExpress</t> <t>kit,</t> a NanoLuc reporter, and varying levels of 2′,3′-cNMPs. (B) Addition of 2′,3′-cNMPs inhibits production of NanoLuc in a coupled transcription/translation assay, but high levels of 2′,3′-cAMP result in greater luminescence. (C) 2′,3′-cNMPs inhibit NanoLuc production in an in vitro translation assay (mRNA transcribed separately). Error bars represent standard deviation. Levels of inhibition are significant, relative to control (0 mM), for all concentrations except 1 mM 2′,3′-cAMP and 100 μM 2′,3′-cCMP. 2′,3′-cAMP: 10–100 mM, P
    Purexpress In Vitro Protein Synthesis Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 40 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 97 stars, based on 40 article reviews
    Price from $9.99 to $1999.99
    purexpress in vitro protein synthesis kit - by Bioz Stars, 2022-12
    97/100 stars

    Images

    1) Product Images from "Binding of 2′,3′-Cyclic Nucleotide Monophosphates to Bacterial Ribosomes Inhibits Translation"

    Article Title: Binding of 2′,3′-Cyclic Nucleotide Monophosphates to Bacterial Ribosomes Inhibits Translation

    Journal: ACS Central Science

    doi: 10.1021/acscentsci.2c00681

    In vitro translation assays. (A) Schematic of in vitro transcription/translation and translation assays using the PurExpress kit, a NanoLuc reporter, and varying levels of 2′,3′-cNMPs. (B) Addition of 2′,3′-cNMPs inhibits production of NanoLuc in a coupled transcription/translation assay, but high levels of 2′,3′-cAMP result in greater luminescence. (C) 2′,3′-cNMPs inhibit NanoLuc production in an in vitro translation assay (mRNA transcribed separately). Error bars represent standard deviation. Levels of inhibition are significant, relative to control (0 mM), for all concentrations except 1 mM 2′,3′-cAMP and 100 μM 2′,3′-cCMP. 2′,3′-cAMP: 10–100 mM, P
    Figure Legend Snippet: In vitro translation assays. (A) Schematic of in vitro transcription/translation and translation assays using the PurExpress kit, a NanoLuc reporter, and varying levels of 2′,3′-cNMPs. (B) Addition of 2′,3′-cNMPs inhibits production of NanoLuc in a coupled transcription/translation assay, but high levels of 2′,3′-cAMP result in greater luminescence. (C) 2′,3′-cNMPs inhibit NanoLuc production in an in vitro translation assay (mRNA transcribed separately). Error bars represent standard deviation. Levels of inhibition are significant, relative to control (0 mM), for all concentrations except 1 mM 2′,3′-cAMP and 100 μM 2′,3′-cCMP. 2′,3′-cAMP: 10–100 mM, P

    Techniques Used: In Vitro, Standard Deviation, Inhibition

    2) Product Images from "ADP-ribosylation of RNA in mammalian cells is mediated by TRPT1 and multiple PARPs"

    Article Title: ADP-ribosylation of RNA in mammalian cells is mediated by TRPT1 and multiple PARPs

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkac711

    ADP-ribosylated RNA is protected from degradation but cannot be translated. ( A ) Schematic representation of Gaussia Luciferase reporter ( G Luc reporter). G Luc reporter RNA was produced by in vitro transcription, followed by poly-A tailing to prevent 3′-degradation. The T7-promotor and Kozak-sequence were introduced to enable transcription and translation, respectively. ( B ) In vitro transcribed and poly-A tailed G Luc reporter RNA was monophosphorylated with RppH, purified and ADP-ribosylated with TRPT1 (left panel). In parallel, a synthetic 5′-phosphorylated and 3′-Cy3 labelled ssRNA oligo was modified with TRPT1 and treated with MACROD1 (right panel). Monophosphorylated or ADPr-capped G Luc reporter RNA (left panel), or synthetic RNA (right panel), were incubated with the 5′-monophosphate dependent 5′→3′ exoribonuclease XRN1 at 37°C for 1 h. After proteinase K treatment, reactions were resolved via urea-PAGE. RNA was visualised with SYBR gold nucleic acid gel stain (left panel) or by the detection of in-gel fluorescence of the Cy3-label (right panel). ( C ) ADPr-capped G Luc reporter RNA was produced as described in (B) followed by removal of non-modified species by XRN1 mediated degradation and purification. m 7 G-capped RNA was generated using the Vaccinia Capping System after in vitro transcription and poly-A tailing of the G Luc reporter RNA. 0.5 μg differently capped or triphosphorylated G Luc reporter RNAs were transfected into HeLa cells (96-well plates) using MessengerMax. Cells were incubated for different periods to allow translation of the reporter mRNA and secretion of luciferase. The medium was harvested and analysed using luminescence-assays. ( D ) 2 μg of each G Luc reporter RNA that was generated in (C) were utilised in an in vitro translation assay using the PURExpress ® in vitro protein synthesis kit. Measured luminescence reflects the rate of translation of G Luc constructs after 2 h. In (C) and (D), mean and standard deviation of three independent experiments are shown.
    Figure Legend Snippet: ADP-ribosylated RNA is protected from degradation but cannot be translated. ( A ) Schematic representation of Gaussia Luciferase reporter ( G Luc reporter). G Luc reporter RNA was produced by in vitro transcription, followed by poly-A tailing to prevent 3′-degradation. The T7-promotor and Kozak-sequence were introduced to enable transcription and translation, respectively. ( B ) In vitro transcribed and poly-A tailed G Luc reporter RNA was monophosphorylated with RppH, purified and ADP-ribosylated with TRPT1 (left panel). In parallel, a synthetic 5′-phosphorylated and 3′-Cy3 labelled ssRNA oligo was modified with TRPT1 and treated with MACROD1 (right panel). Monophosphorylated or ADPr-capped G Luc reporter RNA (left panel), or synthetic RNA (right panel), were incubated with the 5′-monophosphate dependent 5′→3′ exoribonuclease XRN1 at 37°C for 1 h. After proteinase K treatment, reactions were resolved via urea-PAGE. RNA was visualised with SYBR gold nucleic acid gel stain (left panel) or by the detection of in-gel fluorescence of the Cy3-label (right panel). ( C ) ADPr-capped G Luc reporter RNA was produced as described in (B) followed by removal of non-modified species by XRN1 mediated degradation and purification. m 7 G-capped RNA was generated using the Vaccinia Capping System after in vitro transcription and poly-A tailing of the G Luc reporter RNA. 0.5 μg differently capped or triphosphorylated G Luc reporter RNAs were transfected into HeLa cells (96-well plates) using MessengerMax. Cells were incubated for different periods to allow translation of the reporter mRNA and secretion of luciferase. The medium was harvested and analysed using luminescence-assays. ( D ) 2 μg of each G Luc reporter RNA that was generated in (C) were utilised in an in vitro translation assay using the PURExpress ® in vitro protein synthesis kit. Measured luminescence reflects the rate of translation of G Luc constructs after 2 h. In (C) and (D), mean and standard deviation of three independent experiments are shown.

    Techniques Used: Luciferase, Produced, In Vitro, Sequencing, Purification, Modification, Incubation, Polyacrylamide Gel Electrophoresis, Staining, Fluorescence, Generated, Transfection, Construct, Standard Deviation

    3) Product Images from "Hydrophobic mismatch drives self-organization of designer proteins into synthetic membranes"

    Article Title: Hydrophobic mismatch drives self-organization of designer proteins into synthetic membranes

    Journal: bioRxiv

    doi: 10.1101/2022.06.01.494374

    The expression and insertion of transmembrane pore proteins enables the release of calcein dye. 50 mM calcein, a self-quenching dye, was encapsulated into DOPC vesicles. Vesicles were then added to a cell free reaction without DNA, or DNA encoding the 40 Å hairpin or pore protein. Upon expression and insertion of the 40 Å pore protein, an increase in fluorescence because of calcein leakage and subsequent dequenching was observed. This demonstrates that calcein leakage is specific to pore protein expression and integration into vesicle membranes, and not due to interactions with PURExpress or insertion of non-pore proteins.
    Figure Legend Snippet: The expression and insertion of transmembrane pore proteins enables the release of calcein dye. 50 mM calcein, a self-quenching dye, was encapsulated into DOPC vesicles. Vesicles were then added to a cell free reaction without DNA, or DNA encoding the 40 Å hairpin or pore protein. Upon expression and insertion of the 40 Å pore protein, an increase in fluorescence because of calcein leakage and subsequent dequenching was observed. This demonstrates that calcein leakage is specific to pore protein expression and integration into vesicle membranes, and not due to interactions with PURExpress or insertion of non-pore proteins.

    Techniques Used: Expressing, Fluorescence

    4) Product Images from "Cell-Free Gene Expression Dynamics in Synthetic Cell Populations"

    Article Title: Cell-Free Gene Expression Dynamics in Synthetic Cell Populations

    Journal: ACS Synthetic Biology

    doi: 10.1021/acssynbio.1c00376

    Monitoring transcription and translation dynamics in cell-free expression. (A) Construct of the pEXP5-NT/6xHis mCherry F30-2xdBroccoli plasmid containing a constitutive T7 RNAP-mediated promoter expressing 6xHis mCherry with an F30-2xdBroccoli RNA aptamer tag. The small-molecule dye DFHBI becomes fluorescent upon binding with the Broccoli RNA aptamer. (B) mRNA and mCherry protein expression levels over time from bulk PURExpress CFES titrated with varying concentrations of pEXP5-NT/6xHis mCherry F30-2xdBroccoli DNA plasmid. (C) mRNA and mCherry protein expression levels over time from bulk PURExpress CFESs titrated with varying concentrations of purified 6xHis mCherry F30-2xdBroccoli RNA transcripts. Solid lines and shaded areas correspond to mean and standard deviation values from triplicate experiments, respectively. Dashed lines are resource-limited CFES model fits. (D) Illustration of the resource-limited gene expression model for CFESs. Parameters are k r : RNA transcription rate, K r : dissociation constant between RNAP and DNA, δ r : RNA degradation rate, k p : protein translation rate, K p : dissociation constant between ribosome and RNA, k mat : mCherry maturation rate, δ TsR : TsR degradation rate, δ TlR : TlR degradation rate, K l : Michaelis–Menten constant for TlR degradation, a : scaling factor for consumption of TsR with transcription, b : scaling factor for consumption of TlR with translation, and τ d : time delay for protein translation.
    Figure Legend Snippet: Monitoring transcription and translation dynamics in cell-free expression. (A) Construct of the pEXP5-NT/6xHis mCherry F30-2xdBroccoli plasmid containing a constitutive T7 RNAP-mediated promoter expressing 6xHis mCherry with an F30-2xdBroccoli RNA aptamer tag. The small-molecule dye DFHBI becomes fluorescent upon binding with the Broccoli RNA aptamer. (B) mRNA and mCherry protein expression levels over time from bulk PURExpress CFES titrated with varying concentrations of pEXP5-NT/6xHis mCherry F30-2xdBroccoli DNA plasmid. (C) mRNA and mCherry protein expression levels over time from bulk PURExpress CFESs titrated with varying concentrations of purified 6xHis mCherry F30-2xdBroccoli RNA transcripts. Solid lines and shaded areas correspond to mean and standard deviation values from triplicate experiments, respectively. Dashed lines are resource-limited CFES model fits. (D) Illustration of the resource-limited gene expression model for CFESs. Parameters are k r : RNA transcription rate, K r : dissociation constant between RNAP and DNA, δ r : RNA degradation rate, k p : protein translation rate, K p : dissociation constant between ribosome and RNA, k mat : mCherry maturation rate, δ TsR : TsR degradation rate, δ TlR : TlR degradation rate, K l : Michaelis–Menten constant for TlR degradation, a : scaling factor for consumption of TsR with transcription, b : scaling factor for consumption of TlR with translation, and τ d : time delay for protein translation.

    Techniques Used: Expressing, Construct, Plasmid Preparation, Binding Assay, Purification, Standard Deviation

    5) Product Images from "Combining functional metagenomics and glycoanalytics to identify enzymes that facilitate structural characterization of sulfated N-glycans"

    Article Title: Combining functional metagenomics and glycoanalytics to identify enzymes that facilitate structural characterization of sulfated N-glycans

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-021-01652-w

    In vitro expression of hexosaminidase clones F3-ORF26 and F10-ORF19. A SDS-PAGE of 2 hexosaminidases produced with the PURExpress® system. Expressed enzymes are shown with a red triangle. B Activity of in vitro expressed hexosaminidases. Hexosaminidases were assayed on 4MU-GlcNAc-6-SO 4 and its asulfated counterpart 4MU-GlcNAc
    Figure Legend Snippet: In vitro expression of hexosaminidase clones F3-ORF26 and F10-ORF19. A SDS-PAGE of 2 hexosaminidases produced with the PURExpress® system. Expressed enzymes are shown with a red triangle. B Activity of in vitro expressed hexosaminidases. Hexosaminidases were assayed on 4MU-GlcNAc-6-SO 4 and its asulfated counterpart 4MU-GlcNAc

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

    In vitro expression of sulfatase candidates. A SDS-PAGE of 8 sulfatases produced with the PURExpress® system. Expressed enzymes are shown with a red triangle. B Activity of in vitro expressed sulfatases. Sulfatases were assayed on 4MU-GlcNAc-6-SO 4 in the presence and absence of exogenous hexosaminidase
    Figure Legend Snippet: In vitro expression of sulfatase candidates. A SDS-PAGE of 8 sulfatases produced with the PURExpress® system. Expressed enzymes are shown with a red triangle. B Activity of in vitro expressed sulfatases. Sulfatases were assayed on 4MU-GlcNAc-6-SO 4 in the presence and absence of exogenous hexosaminidase

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

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    New England Biolabs purexpress in vitro protein synthesis kit
    Purexpress In Vitro Protein Synthesis Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 80 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 97 stars, based on 80 article reviews
    Price from $9.99 to $1999.99
    purexpress in vitro protein synthesis kit - by Bioz Stars, 2022-12
    97/100 stars
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