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    New England Biolabs purexpress in vitro protein synthesis kit
    Chromozyme cleavage assay (Roche) for a truncated version of tissue plasminogen activator protein (vtPA) synthesized in the <t>PURExpress</t> reactions with and without PURExpress Disulfide Bond Enhancer (PDBE). This tissue plasminogen activator contains 9 disulfide
    Purexpress In Vitro Protein Synthesis Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 540 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Chromozyme cleavage assay (Roche) for a truncated version of tissue plasminogen activator protein (vtPA) synthesized in the <t>PURExpress</t> reactions with and without PURExpress Disulfide Bond Enhancer (PDBE). This tissue plasminogen activator contains 9 disulfide
    Amylose Resin, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 14717 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Chromozyme cleavage assay (Roche) for a truncated version of tissue plasminogen activator protein (vtPA) synthesized in the <t>PURExpress</t> reactions with and without PURExpress Disulfide Bond Enhancer (PDBE). This tissue plasminogen activator contains 9 disulfide
    Gfp Emerald Gfpem, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Chromozyme cleavage assay (Roche) for a truncated version of tissue plasminogen activator protein (vtPA) synthesized in the PURExpress reactions with and without PURExpress Disulfide Bond Enhancer (PDBE). This tissue plasminogen activator contains 9 disulfide

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

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

    doi: 10.1002/0471142727.mb1631s108

    Figure Lengend Snippet: Chromozyme cleavage assay (Roche) for a truncated version of tissue plasminogen activator protein (vtPA) synthesized in the PURExpress reactions with and without PURExpress Disulfide Bond Enhancer (PDBE). This tissue plasminogen activator contains 9 disulfide

    Article Snippet: A PURExpress In vitro Protein Synthesis kit (New England Biolabs, ) containing: Solution A (yellow tube) Solution B (red tube) DHFR control template PURExpress Disulfide Bond Enhancer (New England Biolabs, ) containing: Disulfide Bond Enhancer Solution 1 Disulfide Bond Enhancer Solution 2 microcentrifuge tubes or microtiter plate Nuclease-free H2 O (Integrated DNA technologies) Template DNA (See Support Protocol 1 and 2) or mRNA (See Support Protocol 3) Murine RNase inhibitor (40 U/µl, New England Biolabs) or RNasin Ribonuclease inhibitor (20–40 U/µl, Promega) Microcentrifuge Air incubator set at 37°C 3x SDS-PAGE loading buffer (New England Biolabs, CPMB Chapter X) SDS-PAGE gel (4–20% Tris-glycine, Life Technologies, CPMB Chapter X)

    Techniques: Cleavage Assay, Synthesized

    SDS-PAGE analysis of the reverse purification of the DHFR control protein synthesized in the PURExpress reaction. M: molecular weight standards (kDa); Lane 1: Control PURExpress reaction with no input template, Lane 2: PURExpress reaction with the DHFR

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

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

    doi: 10.1002/0471142727.mb1631s108

    Figure Lengend Snippet: SDS-PAGE analysis of the reverse purification of the DHFR control protein synthesized in the PURExpress reaction. M: molecular weight standards (kDa); Lane 1: Control PURExpress reaction with no input template, Lane 2: PURExpress reaction with the DHFR

    Article Snippet: A PURExpress In vitro Protein Synthesis kit (New England Biolabs, ) containing: Solution A (yellow tube) Solution B (red tube) DHFR control template PURExpress Disulfide Bond Enhancer (New England Biolabs, ) containing: Disulfide Bond Enhancer Solution 1 Disulfide Bond Enhancer Solution 2 microcentrifuge tubes or microtiter plate Nuclease-free H2 O (Integrated DNA technologies) Template DNA (See Support Protocol 1 and 2) or mRNA (See Support Protocol 3) Murine RNase inhibitor (40 U/µl, New England Biolabs) or RNasin Ribonuclease inhibitor (20–40 U/µl, Promega) Microcentrifuge Air incubator set at 37°C 3x SDS-PAGE loading buffer (New England Biolabs, CPMB Chapter X) SDS-PAGE gel (4–20% Tris-glycine, Life Technologies, CPMB Chapter X)

    Techniques: SDS Page, Purification, Synthesized, Molecular Weight

    Scanned image of a SDS-PAGE gel of proteins synthesized in the PURExpress reactions and labeled with FluoroTect™ Green Lys . Lane 1: DHFR; lane 2: GFP; lane 3: Renilla luciferase; lane 4: Firefly luciferase; lane 5: E. coli β-galactosidase.

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

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

    doi: 10.1002/0471142727.mb1631s108

    Figure Lengend Snippet: Scanned image of a SDS-PAGE gel of proteins synthesized in the PURExpress reactions and labeled with FluoroTect™ Green Lys . Lane 1: DHFR; lane 2: GFP; lane 3: Renilla luciferase; lane 4: Firefly luciferase; lane 5: E. coli β-galactosidase.

    Article Snippet: A PURExpress In vitro Protein Synthesis kit (New England Biolabs, ) containing: Solution A (yellow tube) Solution B (red tube) DHFR control template PURExpress Disulfide Bond Enhancer (New England Biolabs, ) containing: Disulfide Bond Enhancer Solution 1 Disulfide Bond Enhancer Solution 2 microcentrifuge tubes or microtiter plate Nuclease-free H2 O (Integrated DNA technologies) Template DNA (See Support Protocol 1 and 2) or mRNA (See Support Protocol 3) Murine RNase inhibitor (40 U/µl, New England Biolabs) or RNasin Ribonuclease inhibitor (20–40 U/µl, Promega) Microcentrifuge Air incubator set at 37°C 3x SDS-PAGE loading buffer (New England Biolabs, CPMB Chapter X) SDS-PAGE gel (4–20% Tris-glycine, Life Technologies, CPMB Chapter X)

    Techniques: SDS Page, Synthesized, Labeling, Luciferase

    Characterization of TseI cleavage and key residues. a Cleavage sites determined by N-terminal sequencing. Each band was excised for N-terminal Edman sequencing as well as LC-MS/MS identification (see also Supplementary Fig. 3 A). b Weblogo depicting conserved residues of Rhs N-/C-terminal sequences deriving from sequence alignment of 48 representative Rhs homologs. Sequences are provided in Supplementary Data 1 . Black arrows indicate the predicted key activity residues that are mutated in this study while gray arrows indicate the first residue of Rhs and VIRC post cleavage, respectively. c Western blotting analysis of TseI and its cleavage-defective mutants. All constructs were cloned to pETDUET1 vectors with an N-terminal FLAG tag and a C-terminal 3V5 tag. Proteins were induced in E. coli with 0.01 mM IPTG. The nontoxic HFH-AAA TseI mutant is used as the parental construct. The same pETDUET1 constructs were also used for in vitro expression shown in d . In vitro expression was performed with a PURExpress ® In Vitro Protein Synthesis Kit following the manufacturer's instruction. Synthesized proteins were subject to SDS-PAGE analysis, followed by western blot analysis with anti-FLAG and anti-V5 antisera. Source data are provided as a Source Data file. Data in a , c , d are representative of at least two replications.

    Journal: Nature Communications

    Article Title: Intramolecular chaperone-mediated secretion of an Rhs effector toxin by a type VI secretion system

    doi: 10.1038/s41467-020-15774-z

    Figure Lengend Snippet: Characterization of TseI cleavage and key residues. a Cleavage sites determined by N-terminal sequencing. Each band was excised for N-terminal Edman sequencing as well as LC-MS/MS identification (see also Supplementary Fig. 3 A). b Weblogo depicting conserved residues of Rhs N-/C-terminal sequences deriving from sequence alignment of 48 representative Rhs homologs. Sequences are provided in Supplementary Data 1 . Black arrows indicate the predicted key activity residues that are mutated in this study while gray arrows indicate the first residue of Rhs and VIRC post cleavage, respectively. c Western blotting analysis of TseI and its cleavage-defective mutants. All constructs were cloned to pETDUET1 vectors with an N-terminal FLAG tag and a C-terminal 3V5 tag. Proteins were induced in E. coli with 0.01 mM IPTG. The nontoxic HFH-AAA TseI mutant is used as the parental construct. The same pETDUET1 constructs were also used for in vitro expression shown in d . In vitro expression was performed with a PURExpress ® In Vitro Protein Synthesis Kit following the manufacturer's instruction. Synthesized proteins were subject to SDS-PAGE analysis, followed by western blot analysis with anti-FLAG and anti-V5 antisera. Source data are provided as a Source Data file. Data in a , c , d are representative of at least two replications.

    Article Snippet: In vitro protein expression was performed with a PURExpress® In Vitro Protein Synthesis Kit (NEB) following the instruction of the manufacturer.

    Techniques: Sequencing, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Western Blot, Construct, Clone Assay, FLAG-tag, Mutagenesis, In Vitro, Expressing, Synthesized, SDS Page

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M113.544429

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

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

    Techniques: Activity Assay, Incubation, Purification

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw018

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

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

    Techniques: Binding Assay, Sequencing, Incubation, Modification

    Full-length HEV genome with synonymous mutations in the cis -acting RNA elements is significantly impaired in its ability to produce infectious virus. (A) Schematic diagrams of the protocol to determine the production of infectious virus. For the cis -acting element mutants, synonymous mutations were introduced in the ORF1 coding region (G113C or G113T) or ORF2 coding region (G7335A). (B-C) Transfection of in vitro -transcribed WT, synonymous mutants or GAD (Pol-) RNA of full-length Kernow C1/p6 (GT 3) into S10-3 cells. Cell lysate supernatant was collected 7 days after transfection, and virus was titrated by infecting HepG2C3A cells. Cells were stained with anti-HEV ORF2 mAbs at 5 days post of infection. (B) to quantify infectious viral particles by foci-forming assay (C). Values are means plus SD (n = 3). IF, immunofluorescence; FFU, foci-forming units; LOD, limit of detection.

    Journal: PLoS Pathogens

    Article Title: Identification of functional cis-acting RNA elements in the hepatitis E virus genome required for viral replication

    doi: 10.1371/journal.ppat.1008488

    Figure Lengend Snippet: Full-length HEV genome with synonymous mutations in the cis -acting RNA elements is significantly impaired in its ability to produce infectious virus. (A) Schematic diagrams of the protocol to determine the production of infectious virus. For the cis -acting element mutants, synonymous mutations were introduced in the ORF1 coding region (G113C or G113T) or ORF2 coding region (G7335A). (B-C) Transfection of in vitro -transcribed WT, synonymous mutants or GAD (Pol-) RNA of full-length Kernow C1/p6 (GT 3) into S10-3 cells. Cell lysate supernatant was collected 7 days after transfection, and virus was titrated by infecting HepG2C3A cells. Cells were stained with anti-HEV ORF2 mAbs at 5 days post of infection. (B) to quantify infectious viral particles by foci-forming assay (C). Values are means plus SD (n = 3). IF, immunofluorescence; FFU, foci-forming units; LOD, limit of detection.

    Article Snippet: In vitro transcription assay and viral RNA transfection HEV Kernow-C1/p6, HEV Kernow-C1/p6-Gluc, and the truncated mutant plasmids were linearized by MluI. pSAR55-Gluc was linearized by BglII, pGEM-9Zf-pSHEV3-Gluc was linearized by XbaI, and pGEM-7Zf(-)-TW6196E and pGEM-7Zf(-)-TW6196E/Gluc were linearized by SpeI.

    Techniques: Transfection, In Vitro, Staining, Infection, Immunofluorescence