protein degradation machineries in nebexpress  (New England Biolabs)


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    Structured Review

    New England Biolabs protein degradation machineries in nebexpress
    DARPin molecules protect ribosomes from intoxication by Stx2a-A1 in vitro. Purified Stx2a-A1 (2.5 μM) was incubated with DARPin molecules (final 10 µM) in PBS supplemented with BSA (40 mg/mL) for 30 min at 37 °C before being added to the 1x <t>NEBExpress</t> <t>Cell-free</t> E. <t>coli</t> <t>protein</t> <t>synthesis</t> <t>system,</t> which contains both the translation and transcription machinery. The mixture was incubated at 37 °C for 30 min before the addition of a reporter plasmid encoding GFP, and the protein synthesis was continued at 37 °C for 6 h, followed by overnight incubation at 4 °C to ensure complete folding of GFP. The amount of GFP produced was quantified using a fluorescent plate reader and compared to that obtained from reactions lacking any toxin (positive control, PC) and reactions with toxin, but without any DARPin (negative control, NC).
    Protein Degradation Machineries In Nebexpress, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protein degradation machineries in nebexpress/product/New England Biolabs
    Average 94 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    protein degradation machineries in nebexpress - by Bioz Stars, 2022-12
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    Images

    1) Product Images from "A Multi-Specific DARPin Potently Neutralizes Shiga Toxin 2 via Simultaneous Modulation of Both Toxin Subunits"

    Article Title: A Multi-Specific DARPin Potently Neutralizes Shiga Toxin 2 via Simultaneous Modulation of Both Toxin Subunits

    Journal: Bioengineering

    doi: 10.3390/bioengineering9100511

    DARPin molecules protect ribosomes from intoxication by Stx2a-A1 in vitro. Purified Stx2a-A1 (2.5 μM) was incubated with DARPin molecules (final 10 µM) in PBS supplemented with BSA (40 mg/mL) for 30 min at 37 °C before being added to the 1x NEBExpress Cell-free E. coli protein synthesis system, which contains both the translation and transcription machinery. The mixture was incubated at 37 °C for 30 min before the addition of a reporter plasmid encoding GFP, and the protein synthesis was continued at 37 °C for 6 h, followed by overnight incubation at 4 °C to ensure complete folding of GFP. The amount of GFP produced was quantified using a fluorescent plate reader and compared to that obtained from reactions lacking any toxin (positive control, PC) and reactions with toxin, but without any DARPin (negative control, NC).
    Figure Legend Snippet: DARPin molecules protect ribosomes from intoxication by Stx2a-A1 in vitro. Purified Stx2a-A1 (2.5 μM) was incubated with DARPin molecules (final 10 µM) in PBS supplemented with BSA (40 mg/mL) for 30 min at 37 °C before being added to the 1x NEBExpress Cell-free E. coli protein synthesis system, which contains both the translation and transcription machinery. The mixture was incubated at 37 °C for 30 min before the addition of a reporter plasmid encoding GFP, and the protein synthesis was continued at 37 °C for 6 h, followed by overnight incubation at 4 °C to ensure complete folding of GFP. The amount of GFP produced was quantified using a fluorescent plate reader and compared to that obtained from reactions lacking any toxin (positive control, PC) and reactions with toxin, but without any DARPin (negative control, NC).

    Techniques Used: In Vitro, Purification, Incubation, Plasmid Preparation, Produced, Positive Control, Negative Control

    2) Product Images from "Characterizing and Improving pET Vectors for Cell-free Expression"

    Article Title: Characterizing and Improving pET Vectors for Cell-free Expression

    Journal: Frontiers in Bioengineering and Biotechnology

    doi: 10.3389/fbioe.2022.895069

    sfGFP expression from the CFAI-based, NEB Express, and NEB PURExpress CFPS systems. The sfGFP yields from (A) CFAI-based, (B) NEBExpress, and (C) NEB PURExpress CFPS systems determined by fluorescence. Data are presented as mean ± s.d. ( n = 3).
    Figure Legend Snippet: sfGFP expression from the CFAI-based, NEB Express, and NEB PURExpress CFPS systems. The sfGFP yields from (A) CFAI-based, (B) NEBExpress, and (C) NEB PURExpress CFPS systems determined by fluorescence. Data are presented as mean ± s.d. ( n = 3).

    Techniques Used: Expressing, Fluorescence

    3) Product Images from "Substrate recognition and cryo-EM structure of the ribosome-bound TAC toxin of Mycobacterium tuberculosis"

    Article Title: Substrate recognition and cryo-EM structure of the ribosome-bound TAC toxin of Mycobacterium tuberculosis

    Journal: Nature Communications

    doi: 10.1038/s41467-022-30373-w

    HigB TAC interactions with the ribosome and with cspA mRNA. Interaction between HigB TAC [K95A] and: a helix 18 of 16S rRNA; b the region delimited by helices 30 and 31 of 16S rRNA; c helix 34 of 16S rRNA; and d helix 44 of 16S rRNA and helix 69 of 23S mRNA. e Interactions between the HigB TAC [K95A] catalytic site and the CCA codon of the cspA mRNA. In all figures, HigB TAC [K95A] is red, the cspA mRNA is purple, the 16S rRNA is light yellow, and the 23S rRNA is light blue. Residues and nucleotides within 4 Å of each other are indicated. f M. smegmatis transformed with pGMC-vector (−), HigB TAC wild-type or its mutant derivatives were serial diluted, spotted on LB streptomycin agar plates with or without anhydrotetracycline (Atc ng/mL) inducer and incubated 3 days at 37 °C. Representative results of three independent experiments are shown.
    Figure Legend Snippet: HigB TAC interactions with the ribosome and with cspA mRNA. Interaction between HigB TAC [K95A] and: a helix 18 of 16S rRNA; b the region delimited by helices 30 and 31 of 16S rRNA; c helix 34 of 16S rRNA; and d helix 44 of 16S rRNA and helix 69 of 23S mRNA. e Interactions between the HigB TAC [K95A] catalytic site and the CCA codon of the cspA mRNA. In all figures, HigB TAC [K95A] is red, the cspA mRNA is purple, the 16S rRNA is light yellow, and the 23S rRNA is light blue. Residues and nucleotides within 4 Å of each other are indicated. f M. smegmatis transformed with pGMC-vector (−), HigB TAC wild-type or its mutant derivatives were serial diluted, spotted on LB streptomycin agar plates with or without anhydrotetracycline (Atc ng/mL) inducer and incubated 3 days at 37 °C. Representative results of three independent experiments are shown.

    Techniques Used: Transformation Assay, Plasmid Preparation, Mutagenesis, Incubation

    The C-terminal extension of the TAC toxin interacts with P-site fMet-tRNA fMet and is critical for its function. a Contrary to other ribosome-dependent toxins, HigB TAC strongly interacts with the P-site tRNA and ribosomal protein S13 (uS13). Left, Overview of the contacts between HigB TAC [K95A], the P-site fMet-tRNA fMet , uS13, and the cspA mRNA. Right, Close up of the interactions observed between the third α-helix of HigB TAC [K95A], the P-site fMet-tRNA fMet , and uS13. Residues and nucleotides within 4 Å of each other are indicated, and the cryo-electron density map is displayed as a gray mesh. b Known toxins observed in pre-cleavage state with their respective mRNA targets and the P-site tRNA are shown here with a canonical translation reference. The structures are aligned on the P-site tRNA and presented in the same orientation as in a . Shown are (left to right): E. coli RelE [R45A-R81A] (PDB 4V7J); Proteus vulgaris HigB (PDB 4ZSN); E. coli YoeB dimer (PDB 4V8X); and a cognate tRNA observed in the A-site during canonical translation (PDB 7K00). As above, the P-site tRNA is orange and the mRNA is purple. c Positively charged residues of HigB TAC helix α3 in contact with P-site tRNA are important for its function. M. smegmatis transformed with pGMC-vector (-), HigB TAC wild-type or its mutant derivatives (Alanine substitution of residue R61, E106, D110, K113, or R117) were serial diluted, spotted on LB streptomycin agar plates with or without anhydrotetracycline (Atc ng/mL) inducer and incubated 3 days at 37 °C. Alanine substitution in R61 and K113 affects HigB TAC inhibition of CspA synthesis ( d ) and cleavage ( e ) in vitro. CspA was expressed in a cell-free translation system with or without HigB TAC wild-type, R61A, or K113A substitution, and analyzed as described in Fig. 3a for CspA protein synthesis and Fig. 3b for cspA cleavage. HigB TAC [K95A] is shown as inactive control. Arrows show the uncleaved (A, 126 nt) and cleaved (A*, 95 nt) cspA . Representative results of three independent experiments are shown.
    Figure Legend Snippet: The C-terminal extension of the TAC toxin interacts with P-site fMet-tRNA fMet and is critical for its function. a Contrary to other ribosome-dependent toxins, HigB TAC strongly interacts with the P-site tRNA and ribosomal protein S13 (uS13). Left, Overview of the contacts between HigB TAC [K95A], the P-site fMet-tRNA fMet , uS13, and the cspA mRNA. Right, Close up of the interactions observed between the third α-helix of HigB TAC [K95A], the P-site fMet-tRNA fMet , and uS13. Residues and nucleotides within 4 Å of each other are indicated, and the cryo-electron density map is displayed as a gray mesh. b Known toxins observed in pre-cleavage state with their respective mRNA targets and the P-site tRNA are shown here with a canonical translation reference. The structures are aligned on the P-site tRNA and presented in the same orientation as in a . Shown are (left to right): E. coli RelE [R45A-R81A] (PDB 4V7J); Proteus vulgaris HigB (PDB 4ZSN); E. coli YoeB dimer (PDB 4V8X); and a cognate tRNA observed in the A-site during canonical translation (PDB 7K00). As above, the P-site tRNA is orange and the mRNA is purple. c Positively charged residues of HigB TAC helix α3 in contact with P-site tRNA are important for its function. M. smegmatis transformed with pGMC-vector (-), HigB TAC wild-type or its mutant derivatives (Alanine substitution of residue R61, E106, D110, K113, or R117) were serial diluted, spotted on LB streptomycin agar plates with or without anhydrotetracycline (Atc ng/mL) inducer and incubated 3 days at 37 °C. Alanine substitution in R61 and K113 affects HigB TAC inhibition of CspA synthesis ( d ) and cleavage ( e ) in vitro. CspA was expressed in a cell-free translation system with or without HigB TAC wild-type, R61A, or K113A substitution, and analyzed as described in Fig. 3a for CspA protein synthesis and Fig. 3b for cspA cleavage. HigB TAC [K95A] is shown as inactive control. Arrows show the uncleaved (A, 126 nt) and cleaved (A*, 95 nt) cspA . Representative results of three independent experiments are shown.

    Techniques Used: Transformation Assay, Plasmid Preparation, Mutagenesis, Incubation, Inhibition, In Vitro

    Impact of HigB toxins on growth and protein synthesis. a Schematic representation of the three HigBA-like systems of M. tuberculosis . Toxins are shown in red, antitoxins in blue and the chaperone of the TAC system (SecB TA ) in green. The amino acid length of each protein is given as a subscript and the locus tag for each gene within color arrows. b Phylogenetic tree of M. tuberculosis HigB toxins (red) and E. coli RelE (orange). The percentage of sequence identity between pairs of toxins is shown on the right (colored brackets). c Expression of HigB toxins in vivo. M. smegmatis transformed with pGMC-vector (-), HigB TAC , HigB TAC [K95A], HigB2 or HigB3 or TAC (HigB/HigA/SecB) TAC were serial diluted and spotted on LB agar plates supplemented with or without Atc inducer at the indicated concentration. Plates were incubated for 3 days at 37 °C. d In vitro transcription/translation reactions assessing the synthesis of GFP protein in the absence (-) or presence of increasing concentrations of HigB toxins, (0.3, 3, and 6 µM). Samples were separated by SDS-PAGE and revealed by western blot using an anti-GFP antibody. Representative results of three independent experiments are shown.
    Figure Legend Snippet: Impact of HigB toxins on growth and protein synthesis. a Schematic representation of the three HigBA-like systems of M. tuberculosis . Toxins are shown in red, antitoxins in blue and the chaperone of the TAC system (SecB TA ) in green. The amino acid length of each protein is given as a subscript and the locus tag for each gene within color arrows. b Phylogenetic tree of M. tuberculosis HigB toxins (red) and E. coli RelE (orange). The percentage of sequence identity between pairs of toxins is shown on the right (colored brackets). c Expression of HigB toxins in vivo. M. smegmatis transformed with pGMC-vector (-), HigB TAC , HigB TAC [K95A], HigB2 or HigB3 or TAC (HigB/HigA/SecB) TAC were serial diluted and spotted on LB agar plates supplemented with or without Atc inducer at the indicated concentration. Plates were incubated for 3 days at 37 °C. d In vitro transcription/translation reactions assessing the synthesis of GFP protein in the absence (-) or presence of increasing concentrations of HigB toxins, (0.3, 3, and 6 µM). Samples were separated by SDS-PAGE and revealed by western blot using an anti-GFP antibody. Representative results of three independent experiments are shown.

    Techniques Used: Sequencing, Expressing, In Vivo, Transformation Assay, Plasmid Preparation, Concentration Assay, Incubation, In Vitro, SDS Page, Western Blot

    Structure of the ribosome-associated TAC toxin with its native cspA substrate. a Electron density map of the complex (transparent gray), showing HigB TAC [K95A] (red), its target cspA mRNA (purple), fMet-tRNA fMet (orange), and the atomic models for the 50S (blue) and 30S (yellow) ribosomal subunits. b Crystal structure of HigB TAC [K95A]. The N-terminus, C-terminus, and the secondary structure elements are indicated. The sequence of the toxin is shown underneath with the secondary structure element highlighted (α-helix in pink, β-strand in yellow). The first six residues (light gray) are not visible in the crystal structure. c Close-up of the interactions between HigB TAC [K95A], the cspA mRNA, and the translating ribosome, rotated by 90° with respect to the structure in a . The HigB TAC [K95A] is red; the P-site fMet-tRNA fMet is orange; the cspA mRNA is purple; the uS13 ribosomal protein is green; the 16S rRNA is light yellow; the 23S rRNA is light blue; and the cryo-electron density map is a gray mesh. For clarity, 16S and 23S α-helices are labeled.
    Figure Legend Snippet: Structure of the ribosome-associated TAC toxin with its native cspA substrate. a Electron density map of the complex (transparent gray), showing HigB TAC [K95A] (red), its target cspA mRNA (purple), fMet-tRNA fMet (orange), and the atomic models for the 50S (blue) and 30S (yellow) ribosomal subunits. b Crystal structure of HigB TAC [K95A]. The N-terminus, C-terminus, and the secondary structure elements are indicated. The sequence of the toxin is shown underneath with the secondary structure element highlighted (α-helix in pink, β-strand in yellow). The first six residues (light gray) are not visible in the crystal structure. c Close-up of the interactions between HigB TAC [K95A], the cspA mRNA, and the translating ribosome, rotated by 90° with respect to the structure in a . The HigB TAC [K95A] is red; the P-site fMet-tRNA fMet is orange; the cspA mRNA is purple; the uS13 ribosomal protein is green; the 16S rRNA is light yellow; the 23S rRNA is light blue; and the cryo-electron density map is a gray mesh. For clarity, 16S and 23S α-helices are labeled.

    Techniques Used: Sequencing, Labeling

    HigB TAC cleaves cspA mRNA at CCA codon during translation in vitro. a HigB TAC inhibits synthesis of CspA wild-type (WT with codon CCA at Pro2) but not of CspA with +1 (OOF1) and +2 (OOF2) out of frame CCA motifs. CspA WT, OOF1, and OOF2 were independently expressed in a cell-free translation system with or without HigB TAC (1.5 µM) as described in Fig. 1d . CspA translation products were labeled with [ 35 S]methionine and reactions were performed for 1 h 30 min at 37 °C. After translation, samples were separated on SDS–PAGE and visualized by phosphorimager. b Cleavage of cspA wild-type is ribosome-dependent. cspA wild-type (CCA), OOF1, and OOF2 were independently expressed in a cell-free translation system for 2 h with or without HigB TAC (1.5 µM). RNA was extracted and subjected to a primer extension with [ 32 P]-labeled cspA primer. In parallel, cspA mRNA was incubated for the same time with or without HigB TAC (1.5 µM) in the absence of ribosomes (CCA-∆ ribo ), and also used for primer extension. The obtained labeled cDNAs were separated on denaturing urea-polyacrylamide gel and revealed by autoradiography. Arrows show the uncleaved (A, 126 nt) and cleaved (A*, 95 nt) cspA , and (m) stands for molecular ladder. Mutations in the CCA codon prevent both inhibition of CspA synthesis ( c ) and cleavage ( d ) of cspA by HigB TAC in vitro. cspA wild type (CCA) and its mutant derivatives (mutations depicted in red) were independently expressed in a cell-free translation system with or without HigB TAC (1.5 µM) and analyzed as described in panel ( a ) for CspA protein synthesis and in panel ( b ) for cspA cleavage. Representative results of triplicate experiments are shown.
    Figure Legend Snippet: HigB TAC cleaves cspA mRNA at CCA codon during translation in vitro. a HigB TAC inhibits synthesis of CspA wild-type (WT with codon CCA at Pro2) but not of CspA with +1 (OOF1) and +2 (OOF2) out of frame CCA motifs. CspA WT, OOF1, and OOF2 were independently expressed in a cell-free translation system with or without HigB TAC (1.5 µM) as described in Fig. 1d . CspA translation products were labeled with [ 35 S]methionine and reactions were performed for 1 h 30 min at 37 °C. After translation, samples were separated on SDS–PAGE and visualized by phosphorimager. b Cleavage of cspA wild-type is ribosome-dependent. cspA wild-type (CCA), OOF1, and OOF2 were independently expressed in a cell-free translation system for 2 h with or without HigB TAC (1.5 µM). RNA was extracted and subjected to a primer extension with [ 32 P]-labeled cspA primer. In parallel, cspA mRNA was incubated for the same time with or without HigB TAC (1.5 µM) in the absence of ribosomes (CCA-∆ ribo ), and also used for primer extension. The obtained labeled cDNAs were separated on denaturing urea-polyacrylamide gel and revealed by autoradiography. Arrows show the uncleaved (A, 126 nt) and cleaved (A*, 95 nt) cspA , and (m) stands for molecular ladder. Mutations in the CCA codon prevent both inhibition of CspA synthesis ( c ) and cleavage ( d ) of cspA by HigB TAC in vitro. cspA wild type (CCA) and its mutant derivatives (mutations depicted in red) were independently expressed in a cell-free translation system with or without HigB TAC (1.5 µM) and analyzed as described in panel ( a ) for CspA protein synthesis and in panel ( b ) for cspA cleavage. Representative results of triplicate experiments are shown.

    Techniques Used: In Vitro, Labeling, SDS Page, Incubation, Autoradiography, Inhibition, Mutagenesis

    4) Product Images from "The ribosome modulates folding inside the ribosomal exit tunnel"

    Article Title: The ribosome modulates folding inside the ribosomal exit tunnel

    Journal: Communications Biology

    doi: 10.1038/s42003-021-02055-8

    Experimental setup. a Structure of zinc-finger protein domain ADR1a (PDB: 2ADR). The Zn 2+ ion is depicted as a blue sphere. b The construct used in this study. ADR1a was synthesized in a cell-free system in two reaction steps. Transcription/translation reaction 1 (R1) incorporated biotin at the N-terminal amber stop codon with protein synthesis stalled at the His-tag. After addition of histidine, protein synthesis continued and transcription/translation reaction 2 (R2) incorporated the donor dye TAMRA and the acceptor dye Atto633 at the two termini of ADR1a. The SecMstr arrest peptide ensured that the fully synthesized protein remained bound following translation. c Cartoon of experimental setup at the optical tweezers, where a depict the DNA handles, b the mRNA of the stalled construct, and c the SecMstr-stalled nascent chain.
    Figure Legend Snippet: Experimental setup. a Structure of zinc-finger protein domain ADR1a (PDB: 2ADR). The Zn 2+ ion is depicted as a blue sphere. b The construct used in this study. ADR1a was synthesized in a cell-free system in two reaction steps. Transcription/translation reaction 1 (R1) incorporated biotin at the N-terminal amber stop codon with protein synthesis stalled at the His-tag. After addition of histidine, protein synthesis continued and transcription/translation reaction 2 (R2) incorporated the donor dye TAMRA and the acceptor dye Atto633 at the two termini of ADR1a. The SecMstr arrest peptide ensured that the fully synthesized protein remained bound following translation. c Cartoon of experimental setup at the optical tweezers, where a depict the DNA handles, b the mRNA of the stalled construct, and c the SecMstr-stalled nascent chain.

    Techniques Used: Construct, Synthesized

    5) Product Images from "The ribosome modulates folding inside the ribosomal exit tunnel"

    Article Title: The ribosome modulates folding inside the ribosomal exit tunnel

    Journal: bioRxiv

    doi: 10.1101/2020.06.30.180224

    Experimental setup. (A) Structure of zinc-finger protein domain ADR1a (PDB: 2ADR). The Zn 2+ ion is depicted as a blue sphere. (B) The construct used in this study. ADR1a was synthesized in a cell-free system in two reaction steps. Transcription/translation reaction 1 (R1) incorporated biotin at the N-terminal amber stop codon with protein synthesis stalled at the His tag. After addition of histidine, protein synthesis continued and transcription/translation reaction 2 (R2) incorporated the donor dye TAMRA and the acceptor dye Atto633 at the two termini of ADR1a. The SecMstr arrest peptide ensured that the fully synthesized protein remained bound following translation. (C) Cartoon of experimental setup at the optical tweezers, where a depicts the DNA handles, b the mRNA of the stalled construct and c the SecMstr-stalled nascent chain.
    Figure Legend Snippet: Experimental setup. (A) Structure of zinc-finger protein domain ADR1a (PDB: 2ADR). The Zn 2+ ion is depicted as a blue sphere. (B) The construct used in this study. ADR1a was synthesized in a cell-free system in two reaction steps. Transcription/translation reaction 1 (R1) incorporated biotin at the N-terminal amber stop codon with protein synthesis stalled at the His tag. After addition of histidine, protein synthesis continued and transcription/translation reaction 2 (R2) incorporated the donor dye TAMRA and the acceptor dye Atto633 at the two termini of ADR1a. The SecMstr arrest peptide ensured that the fully synthesized protein remained bound following translation. (C) Cartoon of experimental setup at the optical tweezers, where a depicts the DNA handles, b the mRNA of the stalled construct and c the SecMstr-stalled nascent chain.

    Techniques Used: Construct, Synthesized

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    New England Biolabs protein degradation machineries in nebexpress
    DARPin molecules protect ribosomes from intoxication by Stx2a-A1 in vitro. Purified Stx2a-A1 (2.5 μM) was incubated with DARPin molecules (final 10 µM) in PBS supplemented with BSA (40 mg/mL) for 30 min at 37 °C before being added to the 1x <t>NEBExpress</t> <t>Cell-free</t> E. <t>coli</t> <t>protein</t> <t>synthesis</t> <t>system,</t> which contains both the translation and transcription machinery. The mixture was incubated at 37 °C for 30 min before the addition of a reporter plasmid encoding GFP, and the protein synthesis was continued at 37 °C for 6 h, followed by overnight incubation at 4 °C to ensure complete folding of GFP. The amount of GFP produced was quantified using a fluorescent plate reader and compared to that obtained from reactions lacking any toxin (positive control, PC) and reactions with toxin, but without any DARPin (negative control, NC).
    Protein Degradation Machineries In Nebexpress, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protein degradation machineries in nebexpress/product/New England Biolabs
    Average 94 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    protein degradation machineries in nebexpress - by Bioz Stars, 2022-12
    94/100 stars
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    DARPin molecules protect ribosomes from intoxication by Stx2a-A1 in vitro. Purified Stx2a-A1 (2.5 μM) was incubated with DARPin molecules (final 10 µM) in PBS supplemented with BSA (40 mg/mL) for 30 min at 37 °C before being added to the 1x NEBExpress Cell-free E. coli protein synthesis system, which contains both the translation and transcription machinery. The mixture was incubated at 37 °C for 30 min before the addition of a reporter plasmid encoding GFP, and the protein synthesis was continued at 37 °C for 6 h, followed by overnight incubation at 4 °C to ensure complete folding of GFP. The amount of GFP produced was quantified using a fluorescent plate reader and compared to that obtained from reactions lacking any toxin (positive control, PC) and reactions with toxin, but without any DARPin (negative control, NC).

    Journal: Bioengineering

    Article Title: A Multi-Specific DARPin Potently Neutralizes Shiga Toxin 2 via Simultaneous Modulation of Both Toxin Subunits

    doi: 10.3390/bioengineering9100511

    Figure Lengend Snippet: DARPin molecules protect ribosomes from intoxication by Stx2a-A1 in vitro. Purified Stx2a-A1 (2.5 μM) was incubated with DARPin molecules (final 10 µM) in PBS supplemented with BSA (40 mg/mL) for 30 min at 37 °C before being added to the 1x NEBExpress Cell-free E. coli protein synthesis system, which contains both the translation and transcription machinery. The mixture was incubated at 37 °C for 30 min before the addition of a reporter plasmid encoding GFP, and the protein synthesis was continued at 37 °C for 6 h, followed by overnight incubation at 4 °C to ensure complete folding of GFP. The amount of GFP produced was quantified using a fluorescent plate reader and compared to that obtained from reactions lacking any toxin (positive control, PC) and reactions with toxin, but without any DARPin (negative control, NC).

    Article Snippet: Next, 1.6 μL of this mixture was added to 1× NEBExpress Cell-free E. coli Protein Synthesis System (NEB, Cat# E5360S) containing 3 μL of S30 extract, 6 μL of Protein Synthesis Buffer, 0.2 μL T7 RNA polymerase, and 0.2 μL RNase Inhibitor, Murine.

    Techniques: In Vitro, Purification, Incubation, Plasmid Preparation, Produced, Positive Control, Negative Control