purexpress in vitro protein synthesis kit  (New England Biolabs)


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    Structured Review

    New England Biolabs purexpress in vitro protein synthesis kit
    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 <t>PURExpress</t> 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.
    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
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    Images

    1) 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

    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 "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

    4) 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

    5) 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

    6) 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

    7) 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

    8) 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

    9) 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

    10) 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

    11) 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

    12) 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

    13) 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

    14) 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

    15) 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

    16) 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

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    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).
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    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

    In vitro translation (IVT) of peptides initiated with monomers 7 (Z = L-NH 2 ), 13 (Z = -SH); 14 (Z = -COOH); and 15 (Z = L-NHCHO) via codon skipping. A. Workflow for in vitro translation. B. Extracted ion chromatograms (EICs) and mass spectra of peptide products obtained using Ma -tRNA Pyl -ACC charged with monomers 7 , 13 - 15 by Ma FRS1 ( 7 and 15 ) or Ma FRS2 ( 13 and 14 ). Insets show mass spectra for major ions used to generate the EIC of the translated peptide initiated with the indicated monomer. Expected (exp) and observed (obs) m/z peaks in mass spectra are as follows: L-Phe 7 (M+3H), exp: 420.51906, obs: 420.52249; α-SH 13 (M+2H), exp: 638.75554, obs: 638.75614; N -lPhe 15 (M+2H), exp: 644.27242, obs: 644.27167; and 2-BMA 14 (M+2H), exp: 644.76442, obs: 644.76498.

    Journal: bioRxiv

    Article Title: Orthogonal synthetases for polyketide precursors

    doi: 10.1101/2022.02.28.482149

    Figure Lengend Snippet: In vitro translation (IVT) of peptides initiated with monomers 7 (Z = L-NH 2 ), 13 (Z = -SH); 14 (Z = -COOH); and 15 (Z = L-NHCHO) via codon skipping. A. Workflow for in vitro translation. B. Extracted ion chromatograms (EICs) and mass spectra of peptide products obtained using Ma -tRNA Pyl -ACC charged with monomers 7 , 13 - 15 by Ma FRS1 ( 7 and 15 ) or Ma FRS2 ( 13 and 14 ). Insets show mass spectra for major ions used to generate the EIC of the translated peptide initiated with the indicated monomer. Expected (exp) and observed (obs) m/z peaks in mass spectra are as follows: L-Phe 7 (M+3H), exp: 420.51906, obs: 420.52249; α-SH 13 (M+2H), exp: 638.75554, obs: 638.75614; N -lPhe 15 (M+2H), exp: 644.27242, obs: 644.27167; and 2-BMA 14 (M+2H), exp: 644.76442, obs: 644.76498.

    Article Snippet: The acylated Ma -tRNAPyl -ACC was added with a DNA template encoding a short MGV-FLAG peptide (MGVDYKDDDDK) ( ) to a commercial in vitro translation kit (PURExpress®Δ (aa, tRNA), NEB).

    Techniques: In Vitro

    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.

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M111.302463

    Figure Lengend 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.

    Article Snippet: In an attempt to reduce the effect of endogenous proteases, the PURExpress in vitro protein synthesis kit (NEB) was used.

    Techniques: In Vitro, Purification, Staining, Western Blot

    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, ) 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)

    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, ) 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)

    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, ) 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)

    Techniques: SDS Page, Synthesized, Labeling, Luciferase