shuffle express competent escherichia coli  (New England Biolabs)


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    SHuffle Express Competent E coli
    Description:
    SHuffle Express Competent E coli 12x0 05 ml
    Catalog Number:
    c3028j
    Price:
    228
    Size:
    12x0 05 ml
    Category:
    Competent Bacteria
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    New England Biolabs shuffle express competent escherichia coli
    SHuffle Express Competent E coli
    SHuffle Express Competent E coli 12x0 05 ml
    https://www.bioz.com/result/shuffle express competent escherichia coli/product/New England Biolabs
    Average 95 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    shuffle express competent escherichia coli - by Bioz Stars, 2020-02
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    Clone Assay:

    Article Title: Structural basis of ribosomal peptide macrocyclization in plants
    Article Snippet: .. Protein expression and purification DNA sequence encoding residues 28–491 of HaAEP1 (accession code: KJ147147), Ricinus communis AEP (RcAEP1) residues 58–497 (accession code: D17401) and Arabidopsis thaliana AEP (AtAEP2) residues 47–486 (accession code: Q39044) were cloned into a pQE30 (QIAGEN, Hilden, Germany) expression vector with an N-terminal six-histidine tag and expressed in the SHuffle strain E. coli (New England Biolabs) transformed with pREP4 (QIAGEN). .. Briefly, cultures were grown at 30°C to an OD600 of 0.8–1.0 in Luria Broth medium containing 100 μg/mL ampicillin and 35 μg/mL kanamycin with expression induced at 16°C with 0.1 mM isopropyl β-D-1-thiogalactopyranoside then cells cultured overnight.

    Article Title: Structural basis of ribosomal peptide macrocyclization in plants
    Article Snippet: .. DNA sequence encoding residues 28–491 of HaAEP1 (accession code: ), Ricinus communis AEP (RcAEP1) residues 58–497 (accession code: ) and Arabidopsis thaliana AEP (AtAEP2) residues 47–486 (accession code: ) were cloned into a pQE30 (QIAGEN, Hilden, Germany) expression vector with an N-terminal six-histidine tag and expressed in the SHuffle strain E. coli (New England Biolabs) transformed with pREP4 (QIAGEN). .. Briefly, cultures were grown at 30°C to an OD600 of 0.8–1.0 in Luria Broth medium containing 100 μg/mL ampicillin and 35 μg/mL kanamycin with expression induced at 16°C with 0.1 mM isopropyl β-D-1-thiogalactopyranoside then cells cultured overnight.

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: PCR products were inserted in frame between the Factor Xa recognition site and the stop codon of pCold I vector (Takara Bio, Japan) by In-Fusion cloning (Clontech, USA). .. Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA).

    Article Title: Exploring Codon Optimization and Response Surface Methodology to Express Biologically Active Transmembrane RANKL in E. coli
    Article Snippet: For preliminary screening experiments, E. coli BL21 (DE3) (Stratagene, USA), E. coli BL21 (Stratagene, USA), TOP10F'E. coli (Invitrogen, USA) and SHuffle Express E.coli (New England Biolabs, UK) cells were tested for expression of proteins. .. While pGEM-T Easy vector (Promega, USA) was used for the cloning of PCR products, pMAL-c5X vector (New England Biolabs, UK), pET32a (+) vector (Novagen, USA), and pGEX-5X-1 (GE Healthcare, UK) were used as expression vectors.

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: PCR products were inserted in frame between the Factor Xa recognition site and the stop codon of pCold I vector (Takara Bio, Japan) by In-Fusion cloning (Clontech, USA). .. Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA).

    Article Title: Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae
    Article Snippet: .. The amplified DNA was purified, digested, and cloned into the BamHI site of pTrcHisA (Invitrogen Life Technologies), resulting in pTrcHisA-pPhlyP, which was transformed into Shuffle Express competent E. coli cells (New England BioLabs [NEB]). ..

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
    Article Snippet: With engineering of the SHuffle E. coli strain (New England Biolabs), with a more oxidizing cytoplasm, the four most abundant proteases were successfully solubly expressed in a bacterial system. .. Therefore, here we describe the cloning and successful recombinant soluble expression of wild type zymogen (inactive no leader sequence) forms of the most abundant Ae. aegypti mosquito midgut proteases using SHuffle E. coli cells.

    Article Title: Mining the biomass deconstructing capabilities of rice yellow stem borer symbionts
    Article Snippet: .. The encoding gene was cloned (without the signal peptide sequence) into the expression vector pET30a (Fig. b) and recombinant protein expressed in E. coli strain shuffle (NEB), purified by metal affinity chromatography (Fig. c). .. The purified protein was active towards beechwood xylan and we found that the recombinant xylanase showed maximum activity at 60 °C, a pH optimum of 7.0 (Fig. d, e) and V max and K M values were found to be 72.2 µmol/min/mg and 2.859 mg/mL, respectively.

    Centrifugation:

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: Similarly, nanobodies were expressed in E. coli SHuffle Express bacteria (New England Biolabs) at 25 °C overnight, 120 rpm. .. The next day, bacteria were harvested by centrifugation for 20 minutes at 3200 x g. The pellet was resuspended in lysis buffer containing 50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 500 µg/ml lysozyme and 100 µg/ml DNaseI (all from Sigma-Aldrich) at pH 8.0.

    Article Title: Structural basis of ribosomal peptide macrocyclization in plants
    Article Snippet: Protein expression and purification DNA sequence encoding residues 28–491 of HaAEP1 (accession code: KJ147147), Ricinus communis AEP (RcAEP1) residues 58–497 (accession code: D17401) and Arabidopsis thaliana AEP (AtAEP2) residues 47–486 (accession code: Q39044) were cloned into a pQE30 (QIAGEN, Hilden, Germany) expression vector with an N-terminal six-histidine tag and expressed in the SHuffle strain E. coli (New England Biolabs) transformed with pREP4 (QIAGEN). .. Cell pellets were collected by centrifugation and lysed by ultrasonication in 50 mM Tris (pH 8.0), 100 mM sodium chloride, 0.1% Triton X-100.

    Article Title: Structural basis of ribosomal peptide macrocyclization in plants
    Article Snippet: DNA sequence encoding residues 28–491 of HaAEP1 (accession code: ), Ricinus communis AEP (RcAEP1) residues 58–497 (accession code: ) and Arabidopsis thaliana AEP (AtAEP2) residues 47–486 (accession code: ) were cloned into a pQE30 (QIAGEN, Hilden, Germany) expression vector with an N-terminal six-histidine tag and expressed in the SHuffle strain E. coli (New England Biolabs) transformed with pREP4 (QIAGEN). .. Cell pellets were collected by centrifugation and lysed by ultrasonication in 50 mM Tris (pH 8.0), 100 mM sodium chloride, 0.1% Triton X-100.

    Amplification:

    Article Title: Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae
    Article Snippet: .. The amplified DNA was purified, digested, and cloned into the BamHI site of pTrcHisA (Invitrogen Life Technologies), resulting in pTrcHisA-pPhlyP, which was transformed into Shuffle Express competent E. coli cells (New England BioLabs [NEB]). ..

    Filtration:

    Article Title: Computationally designed libraries for rapid enzyme stabilization
    Article Snippet: Only variants with single disulfide bonds were expressed in E.coli NEB Shuffle Express (New England Biolabs, USA). .. If needed, further purification was carried out by gel filtration (Supradex 75, Life technologies, UK).

    Cytometry:

    Article Title: Constrained combinatorial libraries of Gp2 proteins enhance discovery of PD-L1 binders
    Article Snippet: When using the NEB SHuffle E. coli strain, four were able to be produced and purified from the soluble fraction. .. Flow cytometry verified all four PD-L1 binding Gp2 variants bound to CHO cells transfected to overexpress hPD-L1 but did not bind untransfected CHO ( ).

    Construct:

    Article Title: Evidence for Ancient Origins of Bowman-Birk Inhibitors from Selaginella moellendorffii
    Article Snippet: .. The pQE30- BBI3 construct and the suppressor plasmid pREP4 (Qiagen) were cotransformed into the E. coli strain Shuffle Express (New England Biolabs; catalog no. C3028H) for protein production. .. To test the predicted P1 inhibitory residues of BBI3, a series of mutant sequences were generated by site-directed mutagenesis ( ).

    Incubation:

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: Similarly, nanobodies were expressed in E. coli SHuffle Express bacteria (New England Biolabs) at 25 °C overnight, 120 rpm. .. After incubation on ice for 20 minutes, bacteria were lysed by sonication with a Branson DigitalSonifier (Branson Ultrasonics, S. Louis, MO, USA) applying five times five pulses at 95% power.

    Article Title: Structural basis of ribosomal peptide macrocyclization in plants
    Article Snippet: Protein expression and purification DNA sequence encoding residues 28–491 of HaAEP1 (accession code: KJ147147), Ricinus communis AEP (RcAEP1) residues 58–497 (accession code: D17401) and Arabidopsis thaliana AEP (AtAEP2) residues 47–486 (accession code: Q39044) were cloned into a pQE30 (QIAGEN, Hilden, Germany) expression vector with an N-terminal six-histidine tag and expressed in the SHuffle strain E. coli (New England Biolabs) transformed with pREP4 (QIAGEN). .. The soluble fraction was then harvested by centrifugation and the supernatant was incubated (batch wise) with Ni-NTA resin overnight at 4°C.

    Article Title: Structural basis of ribosomal peptide macrocyclization in plants
    Article Snippet: DNA sequence encoding residues 28–491 of HaAEP1 (accession code: ), Ricinus communis AEP (RcAEP1) residues 58–497 (accession code: ) and Arabidopsis thaliana AEP (AtAEP2) residues 47–486 (accession code: ) were cloned into a pQE30 (QIAGEN, Hilden, Germany) expression vector with an N-terminal six-histidine tag and expressed in the SHuffle strain E. coli (New England Biolabs) transformed with pREP4 (QIAGEN). .. The soluble fraction was then harvested by centrifugation and the supernatant was incubated (batch wise) with Ni-NTA resin overnight at 4°C.

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: One representative member of each family was sub-cloned into SHuffle Express E. coli (New England Biolabs) for nanobody expression as described above. .. The crude lysate of each clone was incubated on 4% PFA fixed COS-7 cells transfected with the antigen of interest fused to EGFP.

    Activity Assay:

    Article Title: Mining the biomass deconstructing capabilities of rice yellow stem borer symbionts
    Article Snippet: The encoding gene was cloned (without the signal peptide sequence) into the expression vector pET30a (Fig. b) and recombinant protein expressed in E. coli strain shuffle (NEB), purified by metal affinity chromatography (Fig. c). .. The purified protein was active towards beechwood xylan and we found that the recombinant xylanase showed maximum activity at 60 °C, a pH optimum of 7.0 (Fig. d, e) and V max and K M values were found to be 72.2 µmol/min/mg and 2.859 mg/mL, respectively.

    Cell Culture:

    Article Title: Structural basis of ribosomal peptide macrocyclization in plants
    Article Snippet: Protein expression and purification DNA sequence encoding residues 28–491 of HaAEP1 (accession code: KJ147147), Ricinus communis AEP (RcAEP1) residues 58–497 (accession code: D17401) and Arabidopsis thaliana AEP (AtAEP2) residues 47–486 (accession code: Q39044) were cloned into a pQE30 (QIAGEN, Hilden, Germany) expression vector with an N-terminal six-histidine tag and expressed in the SHuffle strain E. coli (New England Biolabs) transformed with pREP4 (QIAGEN). .. Briefly, cultures were grown at 30°C to an OD600 of 0.8–1.0 in Luria Broth medium containing 100 μg/mL ampicillin and 35 μg/mL kanamycin with expression induced at 16°C with 0.1 mM isopropyl β-D-1-thiogalactopyranoside then cells cultured overnight.

    Article Title: Structural basis of ribosomal peptide macrocyclization in plants
    Article Snippet: DNA sequence encoding residues 28–491 of HaAEP1 (accession code: ), Ricinus communis AEP (RcAEP1) residues 58–497 (accession code: ) and Arabidopsis thaliana AEP (AtAEP2) residues 47–486 (accession code: ) were cloned into a pQE30 (QIAGEN, Hilden, Germany) expression vector with an N-terminal six-histidine tag and expressed in the SHuffle strain E. coli (New England Biolabs) transformed with pREP4 (QIAGEN). .. Briefly, cultures were grown at 30°C to an OD600 of 0.8–1.0 in Luria Broth medium containing 100 μg/mL ampicillin and 35 μg/mL kanamycin with expression induced at 16°C with 0.1 mM isopropyl β-D-1-thiogalactopyranoside then cells cultured overnight.

    Expressing:

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: Paragraph title: Protein expression and purification ... Similarly, nanobodies were expressed in E. coli SHuffle Express bacteria (New England Biolabs) at 25 °C overnight, 120 rpm.

    Article Title: Structural basis of ribosomal peptide macrocyclization in plants
    Article Snippet: .. Protein expression and purification DNA sequence encoding residues 28–491 of HaAEP1 (accession code: KJ147147), Ricinus communis AEP (RcAEP1) residues 58–497 (accession code: D17401) and Arabidopsis thaliana AEP (AtAEP2) residues 47–486 (accession code: Q39044) were cloned into a pQE30 (QIAGEN, Hilden, Germany) expression vector with an N-terminal six-histidine tag and expressed in the SHuffle strain E. coli (New England Biolabs) transformed with pREP4 (QIAGEN). .. Briefly, cultures were grown at 30°C to an OD600 of 0.8–1.0 in Luria Broth medium containing 100 μg/mL ampicillin and 35 μg/mL kanamycin with expression induced at 16°C with 0.1 mM isopropyl β-D-1-thiogalactopyranoside then cells cultured overnight.

    Article Title: Production and characterization of neurosecretory protein GM using Escherichia coli and Chinese Hamster Ovary cells
    Article Snippet: .. 2.3 Expression of recombinant His6 -TF tagged NPGM-Gly The pCold-NPGM-Gly plasmid was transformed into E. coli BL21 strain (GE Healthcare, Little Chalfont, UK) or E. coli SHuffle strain (New England Biolabs, Ipswich, MA, USA). ..

    Article Title: Structural basis of ribosomal peptide macrocyclization in plants
    Article Snippet: .. DNA sequence encoding residues 28–491 of HaAEP1 (accession code: ), Ricinus communis AEP (RcAEP1) residues 58–497 (accession code: ) and Arabidopsis thaliana AEP (AtAEP2) residues 47–486 (accession code: ) were cloned into a pQE30 (QIAGEN, Hilden, Germany) expression vector with an N-terminal six-histidine tag and expressed in the SHuffle strain E. coli (New England Biolabs) transformed with pREP4 (QIAGEN). .. Briefly, cultures were grown at 30°C to an OD600 of 0.8–1.0 in Luria Broth medium containing 100 μg/mL ampicillin and 35 μg/mL kanamycin with expression induced at 16°C with 0.1 mM isopropyl β-D-1-thiogalactopyranoside then cells cultured overnight.

    Article Title: Exploring Codon Optimization and Response Surface Methodology to Express Biologically Active Transmembrane RANKL in E. coli
    Article Snippet: .. For preliminary screening experiments, E. coli BL21 (DE3) (Stratagene, USA), E. coli BL21 (Stratagene, USA), TOP10F'E. coli (Invitrogen, USA) and SHuffle Express E.coli (New England Biolabs, UK) cells were tested for expression of proteins. .. To optimize the production of both forms of RANKL, SHuffle Express E. coli was used as expression host.

    Article Title: A Dual-Intein Autoprocessing Domain that Directs Synchronized Protein Co-Expression in Both Prokaryotes and Eukaryotes
    Article Snippet: .. In addition to E. coli BL21(DE3), protein expression was performed in SHuffle E. coli strain (New England Biolab, Ipswich, MA) lacking the trxB and gor reductases along with an additional suppressor mutation (ahpC) which restores viability . ..

    Article Title: Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae
    Article Snippet: Paragraph title: Cloning, expression, and purification of toxins. ... The amplified DNA was purified, digested, and cloned into the BamHI site of pTrcHisA (Invitrogen Life Technologies), resulting in pTrcHisA-pPhlyP, which was transformed into Shuffle Express competent E. coli cells (New England BioLabs [NEB]).

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: .. One representative member of each family was sub-cloned into SHuffle Express E. coli (New England Biolabs) for nanobody expression as described above. .. The crude lysate of each clone was incubated on 4% PFA fixed COS-7 cells transfected with the antigen of interest fused to EGFP.

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
    Article Snippet: Initial attempts at recombinantly expressing mosquito midgut proteases using Escherichia coli led to insoluble expression (inclusion bodies), which were isolated and purified using a denaturation/renaturation strategy [ ]. .. With engineering of the SHuffle E. coli strain (New England Biolabs), with a more oxidizing cytoplasm, the four most abundant proteases were successfully solubly expressed in a bacterial system.

    Article Title: Mining the biomass deconstructing capabilities of rice yellow stem borer symbionts
    Article Snippet: .. The encoding gene was cloned (without the signal peptide sequence) into the expression vector pET30a (Fig. b) and recombinant protein expressed in E. coli strain shuffle (NEB), purified by metal affinity chromatography (Fig. c). .. The purified protein was active towards beechwood xylan and we found that the recombinant xylanase showed maximum activity at 60 °C, a pH optimum of 7.0 (Fig. d, e) and V max and K M values were found to be 72.2 µmol/min/mg and 2.859 mg/mL, respectively.

    Article Title: Evidence for Ancient Origins of Bowman-Birk Inhibitors from Selaginella moellendorffii
    Article Snippet: Paragraph title: Recombinant Protein Expression, Purification, and Mutagenesis ... The pQE30- BBI3 construct and the suppressor plasmid pREP4 (Qiagen) were cotransformed into the E. coli strain Shuffle Express (New England Biolabs; catalog no. C3028H) for protein production.

    Western Blot:

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA). .. Purification was assessed by SDS-PAGE and western blotting using anti-His-tag mAb-HRP-DirecT antibody (Code number: D291-7, Medical & Biological Laboratories, Japan) at 1:4000 (v/v) dilution with ImmunoStar zeta chemiluminescence reagent (Wako, Japan) (Supplementary Fig. ).

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA). .. Purification was assessed by SDS-PAGE and western blotting using anti-His-tag mAb-HRP-DirecT antibody (Code number: D291-7, Medical & Biological Laboratories, Japan) at 1:4000 (v/v) dilution with ImmunoStar zeta chemiluminescence reagent (Wako, Japan) (Supplementary Fig. ).

    Article Title: Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae
    Article Snippet: The amplified DNA was purified, digested, and cloned into the BamHI site of pTrcHisA (Invitrogen Life Technologies), resulting in pTrcHisA-pPhlyP, which was transformed into Shuffle Express competent E. coli cells (New England BioLabs [NEB]). .. The purity of the material was estimated to be at least 90%, as estimated by densitometric analysis of Coomassie-stained SDS-PAGE gels, and the concentration was estimated to be ∼20 μg/ml as assessed by an anti-His6× Western blot with His6× -tagged pVCC serving as a standard.

    Transformation Assay:

    Article Title: Structural basis of ribosomal peptide macrocyclization in plants
    Article Snippet: .. Protein expression and purification DNA sequence encoding residues 28–491 of HaAEP1 (accession code: KJ147147), Ricinus communis AEP (RcAEP1) residues 58–497 (accession code: D17401) and Arabidopsis thaliana AEP (AtAEP2) residues 47–486 (accession code: Q39044) were cloned into a pQE30 (QIAGEN, Hilden, Germany) expression vector with an N-terminal six-histidine tag and expressed in the SHuffle strain E. coli (New England Biolabs) transformed with pREP4 (QIAGEN). .. Briefly, cultures were grown at 30°C to an OD600 of 0.8–1.0 in Luria Broth medium containing 100 μg/mL ampicillin and 35 μg/mL kanamycin with expression induced at 16°C with 0.1 mM isopropyl β-D-1-thiogalactopyranoside then cells cultured overnight.

    Article Title: Production and characterization of neurosecretory protein GM using Escherichia coli and Chinese Hamster Ovary cells
    Article Snippet: .. 2.3 Expression of recombinant His6 -TF tagged NPGM-Gly The pCold-NPGM-Gly plasmid was transformed into E. coli BL21 strain (GE Healthcare, Little Chalfont, UK) or E. coli SHuffle strain (New England Biolabs, Ipswich, MA, USA). ..

    Article Title: Structural basis of ribosomal peptide macrocyclization in plants
    Article Snippet: .. DNA sequence encoding residues 28–491 of HaAEP1 (accession code: ), Ricinus communis AEP (RcAEP1) residues 58–497 (accession code: ) and Arabidopsis thaliana AEP (AtAEP2) residues 47–486 (accession code: ) were cloned into a pQE30 (QIAGEN, Hilden, Germany) expression vector with an N-terminal six-histidine tag and expressed in the SHuffle strain E. coli (New England Biolabs) transformed with pREP4 (QIAGEN). .. Briefly, cultures were grown at 30°C to an OD600 of 0.8–1.0 in Luria Broth medium containing 100 μg/mL ampicillin and 35 μg/mL kanamycin with expression induced at 16°C with 0.1 mM isopropyl β-D-1-thiogalactopyranoside then cells cultured overnight.

    Article Title: A Dual-Intein Autoprocessing Domain that Directs Synchronized Protein Co-Expression in Both Prokaryotes and Eukaryotes
    Article Snippet: Expression in Escherichia coli The pET vectors encoding DI-polyprotein sequences were transformed into E. coli BL21(DE3). .. In addition to E. coli BL21(DE3), protein expression was performed in SHuffle E. coli strain (New England Biolab, Ipswich, MA) lacking the trxB and gor reductases along with an additional suppressor mutation (ahpC) which restores viability .

    Article Title: Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae
    Article Snippet: .. The amplified DNA was purified, digested, and cloned into the BamHI site of pTrcHisA (Invitrogen Life Technologies), resulting in pTrcHisA-pPhlyP, which was transformed into Shuffle Express competent E. coli cells (New England BioLabs [NEB]). ..

    Derivative Assay:

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: The expression vector was derived from the LacO-pQLinkN-construct containing a N-terminal Histidine-tag and a bdSUMO-domain fused to the protein of interest to increase protein solubility and allow cleavage with bdSUMO protease. .. Similarly, nanobodies were expressed in E. coli SHuffle Express bacteria (New England Biolabs) at 25 °C overnight, 120 rpm.

    Flow Cytometry:

    Article Title: Constrained combinatorial libraries of Gp2 proteins enhance discovery of PD-L1 binders
    Article Snippet: When using the NEB SHuffle E. coli strain, four were able to be produced and purified from the soluble fraction. .. Flow cytometry verified all four PD-L1 binding Gp2 variants bound to CHO cells transfected to overexpress hPD-L1 but did not bind untransfected CHO ( ).

    Concentration Assay:

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: Protein expression was induced by adding IPTG to a final concentration of 1 mM. .. Similarly, nanobodies were expressed in E. coli SHuffle Express bacteria (New England Biolabs) at 25 °C overnight, 120 rpm.

    Article Title: Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae
    Article Snippet: The preparation was ∼99% pure as estimated by densitometric analysis of a Coomassie-stained SDS-PAGE gel, and it had a concentration of 1 mg/ml as determined by using the Bio-Rad protein assay. .. The amplified DNA was purified, digested, and cloned into the BamHI site of pTrcHisA (Invitrogen Life Technologies), resulting in pTrcHisA-pPhlyP, which was transformed into Shuffle Express competent E. coli cells (New England BioLabs [NEB]).

    Solubility:

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: The expression vector was derived from the LacO-pQLinkN-construct containing a N-terminal Histidine-tag and a bdSUMO-domain fused to the protein of interest to increase protein solubility and allow cleavage with bdSUMO protease. .. Similarly, nanobodies were expressed in E. coli SHuffle Express bacteria (New England Biolabs) at 25 °C overnight, 120 rpm.

    Generated:

    Article Title: Evidence for Ancient Origins of Bowman-Birk Inhibitors from Selaginella moellendorffii
    Article Snippet: The pQE30- BBI3 construct and the suppressor plasmid pREP4 (Qiagen) were cotransformed into the E. coli strain Shuffle Express (New England Biolabs; catalog no. C3028H) for protein production. .. To test the predicted P1 inhibitory residues of BBI3, a series of mutant sequences were generated by site-directed mutagenesis ( ).

    Imaging:

    Article Title: Constrained combinatorial libraries of Gp2 proteins enhance discovery of PD-L1 binders
    Article Snippet: High affinity PD-L1 ligands are of interest in the clinic as PET imaging agents to stratify patients and predict therapeutic response. .. When using the NEB SHuffle E. coli strain, four were able to be produced and purified from the soluble fraction.

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: Paragraph title: Imaging validation of first candidates ... One representative member of each family was sub-cloned into SHuffle Express E. coli (New England Biolabs) for nanobody expression as described above.

    Transmission Assay:

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
    Article Snippet: Mosquito control is key in slowing the spread of mosquito-borne diseases, especially in areas with high pathogen transmission. .. With engineering of the SHuffle E. coli strain (New England Biolabs), with a more oxidizing cytoplasm, the four most abundant proteases were successfully solubly expressed in a bacterial system.

    Polymerase Chain Reaction:

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: PCR products were inserted in frame between the Factor Xa recognition site and the stop codon of pCold I vector (Takara Bio, Japan) by In-Fusion cloning (Clontech, USA). .. Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA).

    Article Title: Exploring Codon Optimization and Response Surface Methodology to Express Biologically Active Transmembrane RANKL in E. coli
    Article Snippet: For preliminary screening experiments, E. coli BL21 (DE3) (Stratagene, USA), E. coli BL21 (Stratagene, USA), TOP10F'E. coli (Invitrogen, USA) and SHuffle Express E.coli (New England Biolabs, UK) cells were tested for expression of proteins. .. While pGEM-T Easy vector (Promega, USA) was used for the cloning of PCR products, pMAL-c5X vector (New England Biolabs, UK), pET32a (+) vector (Novagen, USA), and pGEX-5X-1 (GE Healthcare, UK) were used as expression vectors.

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: PCR products were inserted in frame between the Factor Xa recognition site and the stop codon of pCold I vector (Takara Bio, Japan) by In-Fusion cloning (Clontech, USA). .. Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA).

    Article Title: Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae
    Article Snippet: For the production of recombinant pro-PhlyP (pPhlyP), the gene encoding pPhlyP was amplified by PCR from P. damselae subsp. damselae AR57 by using high-fidelity Kapa Taq DNA polymerase (Kapa) and primers PhlyP-Forward (5′-AACTATTCTACACCTGCAGA) and PhlyP-Reverse (5′-TTAACCCCAAATGAGCTAAT). .. The amplified DNA was purified, digested, and cloned into the BamHI site of pTrcHisA (Invitrogen Life Technologies), resulting in pTrcHisA-pPhlyP, which was transformed into Shuffle Express competent E. coli cells (New England BioLabs [NEB]).

    Article Title: Evidence for Ancient Origins of Bowman-Birk Inhibitors from Selaginella moellendorffii
    Article Snippet: The pQE30- BBI3 construct and the suppressor plasmid pREP4 (Qiagen) were cotransformed into the E. coli strain Shuffle Express (New England Biolabs; catalog no. C3028H) for protein production. .. In all three cases, the putative P1 Lys was mutated to Ala by site-directed PCR mutagenesis using primers listed in .

    Sonication:

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: Similarly, nanobodies were expressed in E. coli SHuffle Express bacteria (New England Biolabs) at 25 °C overnight, 120 rpm. .. After incubation on ice for 20 minutes, bacteria were lysed by sonication with a Branson DigitalSonifier (Branson Ultrasonics, S. Louis, MO, USA) applying five times five pulses at 95% power.

    Affinity Purification:

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: A Twin-Strep-Tag (IBA GmbH, Göttingen, Germany) was fused to the C-terminus of the protein for affinity purification. .. Similarly, nanobodies were expressed in E. coli SHuffle Express bacteria (New England Biolabs) at 25 °C overnight, 120 rpm.

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: .. Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA). .. Purification was assessed by SDS-PAGE and western blotting using anti-His-tag mAb-HRP-DirecT antibody (Code number: D291-7, Medical & Biological Laboratories, Japan) at 1:4000 (v/v) dilution with ImmunoStar zeta chemiluminescence reagent (Wako, Japan) (Supplementary Fig. ).

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: .. Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA). .. Purification was assessed by SDS-PAGE and western blotting using anti-His-tag mAb-HRP-DirecT antibody (Code number: D291-7, Medical & Biological Laboratories, Japan) at 1:4000 (v/v) dilution with ImmunoStar zeta chemiluminescence reagent (Wako, Japan) (Supplementary Fig. ).

    Article Title: Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae
    Article Snippet: The amplified DNA was purified, digested, and cloned into the BamHI site of pTrcHisA (Invitrogen Life Technologies), resulting in pTrcHisA-pPhlyP, which was transformed into Shuffle Express competent E. coli cells (New England BioLabs [NEB]). .. N-terminally His6× -tagged pPhlyP was affinity purified from E. coli cell lysates by using Ni-NTA agarose (Qiagen) following Qiagen protocols.

    Binding Assay:

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: Paragraph title: Binding assay of recombinant VCBP-C ... Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA).

    Article Title: Constrained combinatorial libraries of Gp2 proteins enhance discovery of PD-L1 binders
    Article Snippet: The six most abundant variants sequenced from the PD-L1 binding population were transferred to a production vector and produced in E. coli. .. When using the NEB SHuffle E. coli strain, four were able to be produced and purified from the soluble fraction.

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: One representative member of each family was sub-cloned into SHuffle Express E. coli (New England Biolabs) for nanobody expression as described above. .. A colocalization of the fluorescent antibody signal with EGFP without significant background binding was considered to indicate a specifically bound nanobody ( ).

    Mutagenesis:

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: The full-length transcript of VCBP-C, which encodes 349 amino acid residues, was used as a PCR template to amplify DNA fragments encoding mature VCBP-C (Wt), a deletion mutant of C-terminal CBD (ΔC) or that of N-terminal two Ig-V domains (ΔV), which covers residues 22-349, 22–280, or 278–349, respectively. .. Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA).

    Article Title: A Dual-Intein Autoprocessing Domain that Directs Synchronized Protein Co-Expression in Both Prokaryotes and Eukaryotes
    Article Snippet: .. In addition to E. coli BL21(DE3), protein expression was performed in SHuffle E. coli strain (New England Biolab, Ipswich, MA) lacking the trxB and gor reductases along with an additional suppressor mutation (ahpC) which restores viability . ..

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: Binding assay of recombinant VCBP-C The full-length transcript of VCBP-C, which encodes 349 amino acid residues, was used as a PCR template to amplify DNA fragments encoding mature VCBP-C (Wt), a deletion mutant of C-terminal CBD (ΔC) or that of N-terminal two Ig-V domains (ΔV), which covers residues 22-349, 22–280, or 278–349, respectively. .. Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA).

    Article Title: Computationally designed libraries for rapid enzyme stabilization
    Article Snippet: Mutants of LEH were created by QuikChange mutagenesis (Agilent, USA) in a 96-well plate. .. Only variants with single disulfide bonds were expressed in E.coli NEB Shuffle Express (New England Biolabs, USA).

    Article Title: Evidence for Ancient Origins of Bowman-Birk Inhibitors from Selaginella moellendorffii
    Article Snippet: Paragraph title: Recombinant Protein Expression, Purification, and Mutagenesis ... The pQE30- BBI3 construct and the suppressor plasmid pREP4 (Qiagen) were cotransformed into the E. coli strain Shuffle Express (New England Biolabs; catalog no. C3028H) for protein production.

    Isolation:

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
    Article Snippet: Initial attempts at recombinantly expressing mosquito midgut proteases using Escherichia coli led to insoluble expression (inclusion bodies), which were isolated and purified using a denaturation/renaturation strategy [ ]. .. With engineering of the SHuffle E. coli strain (New England Biolabs), with a more oxidizing cytoplasm, the four most abundant proteases were successfully solubly expressed in a bacterial system.

    Transfection:

    Article Title: Constrained combinatorial libraries of Gp2 proteins enhance discovery of PD-L1 binders
    Article Snippet: When using the NEB SHuffle E. coli strain, four were able to be produced and purified from the soluble fraction. .. Flow cytometry verified all four PD-L1 binding Gp2 variants bound to CHO cells transfected to overexpress hPD-L1 but did not bind untransfected CHO ( ).

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: One representative member of each family was sub-cloned into SHuffle Express E. coli (New England Biolabs) for nanobody expression as described above. .. The crude lysate of each clone was incubated on 4% PFA fixed COS-7 cells transfected with the antigen of interest fused to EGFP.

    Purification:

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: Paragraph title: Protein expression and purification ... Similarly, nanobodies were expressed in E. coli SHuffle Express bacteria (New England Biolabs) at 25 °C overnight, 120 rpm.

    Article Title: Structural basis of ribosomal peptide macrocyclization in plants
    Article Snippet: .. Protein expression and purification DNA sequence encoding residues 28–491 of HaAEP1 (accession code: KJ147147), Ricinus communis AEP (RcAEP1) residues 58–497 (accession code: D17401) and Arabidopsis thaliana AEP (AtAEP2) residues 47–486 (accession code: Q39044) were cloned into a pQE30 (QIAGEN, Hilden, Germany) expression vector with an N-terminal six-histidine tag and expressed in the SHuffle strain E. coli (New England Biolabs) transformed with pREP4 (QIAGEN). .. Briefly, cultures were grown at 30°C to an OD600 of 0.8–1.0 in Luria Broth medium containing 100 μg/mL ampicillin and 35 μg/mL kanamycin with expression induced at 16°C with 0.1 mM isopropyl β-D-1-thiogalactopyranoside then cells cultured overnight.

    Article Title: Structural basis of ribosomal peptide macrocyclization in plants
    Article Snippet: Paragraph title: Protein expression and purification ... DNA sequence encoding residues 28–491 of HaAEP1 (accession code: ), Ricinus communis AEP (RcAEP1) residues 58–497 (accession code: ) and Arabidopsis thaliana AEP (AtAEP2) residues 47–486 (accession code: ) were cloned into a pQE30 (QIAGEN, Hilden, Germany) expression vector with an N-terminal six-histidine tag and expressed in the SHuffle strain E. coli (New England Biolabs) transformed with pREP4 (QIAGEN).

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA). .. Purification was assessed by SDS-PAGE and western blotting using anti-His-tag mAb-HRP-DirecT antibody (Code number: D291-7, Medical & Biological Laboratories, Japan) at 1:4000 (v/v) dilution with ImmunoStar zeta chemiluminescence reagent (Wako, Japan) (Supplementary Fig. ).

    Article Title: Constrained combinatorial libraries of Gp2 proteins enhance discovery of PD-L1 binders
    Article Snippet: .. When using the NEB SHuffle E. coli strain, four were able to be produced and purified from the soluble fraction. .. Flow cytometry verified all four PD-L1 binding Gp2 variants bound to CHO cells transfected to overexpress hPD-L1 but did not bind untransfected CHO ( ).

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA). .. Purification was assessed by SDS-PAGE and western blotting using anti-His-tag mAb-HRP-DirecT antibody (Code number: D291-7, Medical & Biological Laboratories, Japan) at 1:4000 (v/v) dilution with ImmunoStar zeta chemiluminescence reagent (Wako, Japan) (Supplementary Fig. ).

    Article Title: Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae
    Article Snippet: .. The amplified DNA was purified, digested, and cloned into the BamHI site of pTrcHisA (Invitrogen Life Technologies), resulting in pTrcHisA-pPhlyP, which was transformed into Shuffle Express competent E. coli cells (New England BioLabs [NEB]). ..

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
    Article Snippet: Initial attempts at recombinantly expressing mosquito midgut proteases using Escherichia coli led to insoluble expression (inclusion bodies), which were isolated and purified using a denaturation/renaturation strategy [ ]. .. With engineering of the SHuffle E. coli strain (New England Biolabs), with a more oxidizing cytoplasm, the four most abundant proteases were successfully solubly expressed in a bacterial system.

    Article Title: Mining the biomass deconstructing capabilities of rice yellow stem borer symbionts
    Article Snippet: .. The encoding gene was cloned (without the signal peptide sequence) into the expression vector pET30a (Fig. b) and recombinant protein expressed in E. coli strain shuffle (NEB), purified by metal affinity chromatography (Fig. c). .. The purified protein was active towards beechwood xylan and we found that the recombinant xylanase showed maximum activity at 60 °C, a pH optimum of 7.0 (Fig. d, e) and V max and K M values were found to be 72.2 µmol/min/mg and 2.859 mg/mL, respectively.

    Article Title: Computationally designed libraries for rapid enzyme stabilization
    Article Snippet: Only variants with single disulfide bonds were expressed in E.coli NEB Shuffle Express (New England Biolabs, USA). .. Purification was carried out on Ni–NTA columns with a C-terminal hexa-histidine tag using cell lysate prepared from cells grown in 1 l of Terrific Broth.

    Article Title: Evidence for Ancient Origins of Bowman-Birk Inhibitors from Selaginella moellendorffii
    Article Snippet: Paragraph title: Recombinant Protein Expression, Purification, and Mutagenesis ... The pQE30- BBI3 construct and the suppressor plasmid pREP4 (Qiagen) were cotransformed into the E. coli strain Shuffle Express (New England Biolabs; catalog no. C3028H) for protein production.

    Sequencing:

    Article Title: Structural basis of ribosomal peptide macrocyclization in plants
    Article Snippet: .. Protein expression and purification DNA sequence encoding residues 28–491 of HaAEP1 (accession code: KJ147147), Ricinus communis AEP (RcAEP1) residues 58–497 (accession code: D17401) and Arabidopsis thaliana AEP (AtAEP2) residues 47–486 (accession code: Q39044) were cloned into a pQE30 (QIAGEN, Hilden, Germany) expression vector with an N-terminal six-histidine tag and expressed in the SHuffle strain E. coli (New England Biolabs) transformed with pREP4 (QIAGEN). .. Briefly, cultures were grown at 30°C to an OD600 of 0.8–1.0 in Luria Broth medium containing 100 μg/mL ampicillin and 35 μg/mL kanamycin with expression induced at 16°C with 0.1 mM isopropyl β-D-1-thiogalactopyranoside then cells cultured overnight.

    Article Title: Structural basis of ribosomal peptide macrocyclization in plants
    Article Snippet: .. DNA sequence encoding residues 28–491 of HaAEP1 (accession code: ), Ricinus communis AEP (RcAEP1) residues 58–497 (accession code: ) and Arabidopsis thaliana AEP (AtAEP2) residues 47–486 (accession code: ) were cloned into a pQE30 (QIAGEN, Hilden, Germany) expression vector with an N-terminal six-histidine tag and expressed in the SHuffle strain E. coli (New England Biolabs) transformed with pREP4 (QIAGEN). .. Briefly, cultures were grown at 30°C to an OD600 of 0.8–1.0 in Luria Broth medium containing 100 μg/mL ampicillin and 35 μg/mL kanamycin with expression induced at 16°C with 0.1 mM isopropyl β-D-1-thiogalactopyranoside then cells cultured overnight.

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
    Article Snippet: With engineering of the SHuffle E. coli strain (New England Biolabs), with a more oxidizing cytoplasm, the four most abundant proteases were successfully solubly expressed in a bacterial system. .. Therefore, here we describe the cloning and successful recombinant soluble expression of wild type zymogen (inactive no leader sequence) forms of the most abundant Ae. aegypti mosquito midgut proteases using SHuffle E. coli cells.

    Article Title: Mining the biomass deconstructing capabilities of rice yellow stem borer symbionts
    Article Snippet: .. The encoding gene was cloned (without the signal peptide sequence) into the expression vector pET30a (Fig. b) and recombinant protein expressed in E. coli strain shuffle (NEB), purified by metal affinity chromatography (Fig. c). .. The purified protein was active towards beechwood xylan and we found that the recombinant xylanase showed maximum activity at 60 °C, a pH optimum of 7.0 (Fig. d, e) and V max and K M values were found to be 72.2 µmol/min/mg and 2.859 mg/mL, respectively.

    Article Title: Evidence for Ancient Origins of Bowman-Birk Inhibitors from Selaginella moellendorffii
    Article Snippet: The sequence was subcloned into the Bam HI and Sal I sites of pQE30 (Qiagen). .. The pQE30- BBI3 construct and the suppressor plasmid pREP4 (Qiagen) were cotransformed into the E. coli strain Shuffle Express (New England Biolabs; catalog no. C3028H) for protein production.

    Protein Extraction:

    Article Title: A Dual-Intein Autoprocessing Domain that Directs Synchronized Protein Co-Expression in Both Prokaryotes and Eukaryotes
    Article Snippet: In addition to E. coli BL21(DE3), protein expression was performed in SHuffle E. coli strain (New England Biolab, Ipswich, MA) lacking the trxB and gor reductases along with an additional suppressor mutation (ahpC) which restores viability . .. Washed cell pellets were disrupted by ultrasonication at 5 Watt for 5 min in PBS protein extraction buffer (20 mM sodium phosphate pH 7.5, 150 mM sodium chloride, 1 mM phenylmethylsulfonyl fluoride, 200 μM lysozyme, 1 μg/ml leupeptin, 100 ng/ml pepstatin).

    Affinity Chromatography:

    Article Title: Mining the biomass deconstructing capabilities of rice yellow stem borer symbionts
    Article Snippet: .. The encoding gene was cloned (without the signal peptide sequence) into the expression vector pET30a (Fig. b) and recombinant protein expressed in E. coli strain shuffle (NEB), purified by metal affinity chromatography (Fig. c). .. The purified protein was active towards beechwood xylan and we found that the recombinant xylanase showed maximum activity at 60 °C, a pH optimum of 7.0 (Fig. d, e) and V max and K M values were found to be 72.2 µmol/min/mg and 2.859 mg/mL, respectively.

    Lysis:

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: Similarly, nanobodies were expressed in E. coli SHuffle Express bacteria (New England Biolabs) at 25 °C overnight, 120 rpm. .. The next day, bacteria were harvested by centrifugation for 20 minutes at 3200 x g. The pellet was resuspended in lysis buffer containing 50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 500 µg/ml lysozyme and 100 µg/ml DNaseI (all from Sigma-Aldrich) at pH 8.0.

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: .. Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA). .. Purification was assessed by SDS-PAGE and western blotting using anti-His-tag mAb-HRP-DirecT antibody (Code number: D291-7, Medical & Biological Laboratories, Japan) at 1:4000 (v/v) dilution with ImmunoStar zeta chemiluminescence reagent (Wako, Japan) (Supplementary Fig. ).

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: .. Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA). .. Purification was assessed by SDS-PAGE and western blotting using anti-His-tag mAb-HRP-DirecT antibody (Code number: D291-7, Medical & Biological Laboratories, Japan) at 1:4000 (v/v) dilution with ImmunoStar zeta chemiluminescence reagent (Wako, Japan) (Supplementary Fig. ).

    SDS Page:

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA). .. Purification was assessed by SDS-PAGE and western blotting using anti-His-tag mAb-HRP-DirecT antibody (Code number: D291-7, Medical & Biological Laboratories, Japan) at 1:4000 (v/v) dilution with ImmunoStar zeta chemiluminescence reagent (Wako, Japan) (Supplementary Fig. ).

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA). .. Purification was assessed by SDS-PAGE and western blotting using anti-His-tag mAb-HRP-DirecT antibody (Code number: D291-7, Medical & Biological Laboratories, Japan) at 1:4000 (v/v) dilution with ImmunoStar zeta chemiluminescence reagent (Wako, Japan) (Supplementary Fig. ).

    Article Title: Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae
    Article Snippet: The preparation was ∼99% pure as estimated by densitometric analysis of a Coomassie-stained SDS-PAGE gel, and it had a concentration of 1 mg/ml as determined by using the Bio-Rad protein assay. .. The amplified DNA was purified, digested, and cloned into the BamHI site of pTrcHisA (Invitrogen Life Technologies), resulting in pTrcHisA-pPhlyP, which was transformed into Shuffle Express competent E. coli cells (New England BioLabs [NEB]).

    Plasmid Preparation:

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: The expression vector was derived from the LacO-pQLinkN-construct containing a N-terminal Histidine-tag and a bdSUMO-domain fused to the protein of interest to increase protein solubility and allow cleavage with bdSUMO protease. .. Similarly, nanobodies were expressed in E. coli SHuffle Express bacteria (New England Biolabs) at 25 °C overnight, 120 rpm.

    Article Title: Structural basis of ribosomal peptide macrocyclization in plants
    Article Snippet: .. Protein expression and purification DNA sequence encoding residues 28–491 of HaAEP1 (accession code: KJ147147), Ricinus communis AEP (RcAEP1) residues 58–497 (accession code: D17401) and Arabidopsis thaliana AEP (AtAEP2) residues 47–486 (accession code: Q39044) were cloned into a pQE30 (QIAGEN, Hilden, Germany) expression vector with an N-terminal six-histidine tag and expressed in the SHuffle strain E. coli (New England Biolabs) transformed with pREP4 (QIAGEN). .. Briefly, cultures were grown at 30°C to an OD600 of 0.8–1.0 in Luria Broth medium containing 100 μg/mL ampicillin and 35 μg/mL kanamycin with expression induced at 16°C with 0.1 mM isopropyl β-D-1-thiogalactopyranoside then cells cultured overnight.

    Article Title: Production and characterization of neurosecretory protein GM using Escherichia coli and Chinese Hamster Ovary cells
    Article Snippet: .. 2.3 Expression of recombinant His6 -TF tagged NPGM-Gly The pCold-NPGM-Gly plasmid was transformed into E. coli BL21 strain (GE Healthcare, Little Chalfont, UK) or E. coli SHuffle strain (New England Biolabs, Ipswich, MA, USA). ..

    Article Title: Structural basis of ribosomal peptide macrocyclization in plants
    Article Snippet: .. DNA sequence encoding residues 28–491 of HaAEP1 (accession code: ), Ricinus communis AEP (RcAEP1) residues 58–497 (accession code: ) and Arabidopsis thaliana AEP (AtAEP2) residues 47–486 (accession code: ) were cloned into a pQE30 (QIAGEN, Hilden, Germany) expression vector with an N-terminal six-histidine tag and expressed in the SHuffle strain E. coli (New England Biolabs) transformed with pREP4 (QIAGEN). .. Briefly, cultures were grown at 30°C to an OD600 of 0.8–1.0 in Luria Broth medium containing 100 μg/mL ampicillin and 35 μg/mL kanamycin with expression induced at 16°C with 0.1 mM isopropyl β-D-1-thiogalactopyranoside then cells cultured overnight.

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: PCR products were inserted in frame between the Factor Xa recognition site and the stop codon of pCold I vector (Takara Bio, Japan) by In-Fusion cloning (Clontech, USA). .. Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA).

    Article Title: Exploring Codon Optimization and Response Surface Methodology to Express Biologically Active Transmembrane RANKL in E. coli
    Article Snippet: For preliminary screening experiments, E. coli BL21 (DE3) (Stratagene, USA), E. coli BL21 (Stratagene, USA), TOP10F'E. coli (Invitrogen, USA) and SHuffle Express E.coli (New England Biolabs, UK) cells were tested for expression of proteins. .. While pGEM-T Easy vector (Promega, USA) was used for the cloning of PCR products, pMAL-c5X vector (New England Biolabs, UK), pET32a (+) vector (Novagen, USA), and pGEX-5X-1 (GE Healthcare, UK) were used as expression vectors.

    Article Title: Constrained combinatorial libraries of Gp2 proteins enhance discovery of PD-L1 binders
    Article Snippet: The six most abundant variants sequenced from the PD-L1 binding population were transferred to a production vector and produced in E. coli. .. When using the NEB SHuffle E. coli strain, four were able to be produced and purified from the soluble fraction.

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: PCR products were inserted in frame between the Factor Xa recognition site and the stop codon of pCold I vector (Takara Bio, Japan) by In-Fusion cloning (Clontech, USA). .. Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA).

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
    Article Snippet: However, before validating midgut proteases as a potential vector control strategy, we must first fully understand how individual midgut proteases digest blood meal proteins. .. With engineering of the SHuffle E. coli strain (New England Biolabs), with a more oxidizing cytoplasm, the four most abundant proteases were successfully solubly expressed in a bacterial system.

    Article Title: Mining the biomass deconstructing capabilities of rice yellow stem borer symbionts
    Article Snippet: .. The encoding gene was cloned (without the signal peptide sequence) into the expression vector pET30a (Fig. b) and recombinant protein expressed in E. coli strain shuffle (NEB), purified by metal affinity chromatography (Fig. c). .. The purified protein was active towards beechwood xylan and we found that the recombinant xylanase showed maximum activity at 60 °C, a pH optimum of 7.0 (Fig. d, e) and V max and K M values were found to be 72.2 µmol/min/mg and 2.859 mg/mL, respectively.

    Article Title: Computationally designed libraries for rapid enzyme stabilization
    Article Snippet: A plasmid containing the gene for the LEH was kindly provided by Prof. Dr. M. Arand (University of Zurich). .. Only variants with single disulfide bonds were expressed in E.coli NEB Shuffle Express (New England Biolabs, USA).

    Article Title: Evidence for Ancient Origins of Bowman-Birk Inhibitors from Selaginella moellendorffii
    Article Snippet: .. The pQE30- BBI3 construct and the suppressor plasmid pREP4 (Qiagen) were cotransformed into the E. coli strain Shuffle Express (New England Biolabs; catalog no. C3028H) for protein production. .. To test the predicted P1 inhibitory residues of BBI3, a series of mutant sequences were generated by site-directed mutagenesis ( ).

    Functional Assay:

    Article Title: Mining the biomass deconstructing capabilities of rice yellow stem borer symbionts
    Article Snippet: Paragraph title: Recombinant expression and functional validation of a xylanase from the GH10 family ... The encoding gene was cloned (without the signal peptide sequence) into the expression vector pET30a (Fig. b) and recombinant protein expressed in E. coli strain shuffle (NEB), purified by metal affinity chromatography (Fig. c).

    Positron Emission Tomography:

    Article Title: Constrained combinatorial libraries of Gp2 proteins enhance discovery of PD-L1 binders
    Article Snippet: High affinity PD-L1 ligands are of interest in the clinic as PET imaging agents to stratify patients and predict therapeutic response. .. When using the NEB SHuffle E. coli strain, four were able to be produced and purified from the soluble fraction.

    Article Title: A Dual-Intein Autoprocessing Domain that Directs Synchronized Protein Co-Expression in Both Prokaryotes and Eukaryotes
    Article Snippet: Expression in Escherichia coli The pET vectors encoding DI-polyprotein sequences were transformed into E. coli BL21(DE3). .. In addition to E. coli BL21(DE3), protein expression was performed in SHuffle E. coli strain (New England Biolab, Ipswich, MA) lacking the trxB and gor reductases along with an additional suppressor mutation (ahpC) which restores viability .

    In Vitro:

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
    Article Snippet: This approach did lead to active recombinant midgut proteases, but rather than produce recombinant proteases with the native (wild type) propeptide region, a heterologous enterokinase cleavage site was engineered to allow for the activation of the proteases in vitro. .. With engineering of the SHuffle E. coli strain (New England Biolabs), with a more oxidizing cytoplasm, the four most abundant proteases were successfully solubly expressed in a bacterial system.

    Produced:

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: Protein expression and purification Full antigens and their truncated form ( and ) were produced by recombinant expression in E. coli NEB Express strain (New England Biolabs, Ipswich, MA, USA). .. Similarly, nanobodies were expressed in E. coli SHuffle Express bacteria (New England Biolabs) at 25 °C overnight, 120 rpm.

    Article Title: Constrained combinatorial libraries of Gp2 proteins enhance discovery of PD-L1 binders
    Article Snippet: .. When using the NEB SHuffle E. coli strain, four were able to be produced and purified from the soluble fraction. .. Flow cytometry verified all four PD-L1 binding Gp2 variants bound to CHO cells transfected to overexpress hPD-L1 but did not bind untransfected CHO ( ).

    Activation Assay:

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
    Article Snippet: This approach did lead to active recombinant midgut proteases, but rather than produce recombinant proteases with the native (wild type) propeptide region, a heterologous enterokinase cleavage site was engineered to allow for the activation of the proteases in vitro. .. With engineering of the SHuffle E. coli strain (New England Biolabs), with a more oxidizing cytoplasm, the four most abundant proteases were successfully solubly expressed in a bacterial system.

    Recombinant:

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: Protein expression and purification Full antigens and their truncated form ( and ) were produced by recombinant expression in E. coli NEB Express strain (New England Biolabs, Ipswich, MA, USA). .. Similarly, nanobodies were expressed in E. coli SHuffle Express bacteria (New England Biolabs) at 25 °C overnight, 120 rpm.

    Article Title: Production and characterization of neurosecretory protein GM using Escherichia coli and Chinese Hamster Ovary cells
    Article Snippet: .. 2.3 Expression of recombinant His6 -TF tagged NPGM-Gly The pCold-NPGM-Gly plasmid was transformed into E. coli BL21 strain (GE Healthcare, Little Chalfont, UK) or E. coli SHuffle strain (New England Biolabs, Ipswich, MA, USA). ..

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: .. Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA). .. Purification was assessed by SDS-PAGE and western blotting using anti-His-tag mAb-HRP-DirecT antibody (Code number: D291-7, Medical & Biological Laboratories, Japan) at 1:4000 (v/v) dilution with ImmunoStar zeta chemiluminescence reagent (Wako, Japan) (Supplementary Fig. ).

    Article Title: Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota
    Article Snippet: .. Recombinant proteins were expressed in SHuffle Express competent Escherichia coli (New England BioLabs, USA), followed by cell lysis with B-Per reagent (Thermo Fisher Scientific, USA) and affinity purification with Talon resin (Clontech, USA). .. Purification was assessed by SDS-PAGE and western blotting using anti-His-tag mAb-HRP-DirecT antibody (Code number: D291-7, Medical & Biological Laboratories, Japan) at 1:4000 (v/v) dilution with ImmunoStar zeta chemiluminescence reagent (Wako, Japan) (Supplementary Fig. ).

    Article Title: Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae
    Article Snippet: For the production of recombinant pro-PhlyP (pPhlyP), the gene encoding pPhlyP was amplified by PCR from P. damselae subsp. damselae AR57 by using high-fidelity Kapa Taq DNA polymerase (Kapa) and primers PhlyP-Forward (5′-AACTATTCTACACCTGCAGA) and PhlyP-Reverse (5′-TTAACCCCAAATGAGCTAAT). .. The amplified DNA was purified, digested, and cloned into the BamHI site of pTrcHisA (Invitrogen Life Technologies), resulting in pTrcHisA-pPhlyP, which was transformed into Shuffle Express competent E. coli cells (New England BioLabs [NEB]).

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
    Article Snippet: This approach did lead to active recombinant midgut proteases, but rather than produce recombinant proteases with the native (wild type) propeptide region, a heterologous enterokinase cleavage site was engineered to allow for the activation of the proteases in vitro. .. With engineering of the SHuffle E. coli strain (New England Biolabs), with a more oxidizing cytoplasm, the four most abundant proteases were successfully solubly expressed in a bacterial system.

    Article Title: Mining the biomass deconstructing capabilities of rice yellow stem borer symbionts
    Article Snippet: .. The encoding gene was cloned (without the signal peptide sequence) into the expression vector pET30a (Fig. b) and recombinant protein expressed in E. coli strain shuffle (NEB), purified by metal affinity chromatography (Fig. c). .. The purified protein was active towards beechwood xylan and we found that the recombinant xylanase showed maximum activity at 60 °C, a pH optimum of 7.0 (Fig. d, e) and V max and K M values were found to be 72.2 µmol/min/mg and 2.859 mg/mL, respectively.

    Article Title: Evidence for Ancient Origins of Bowman-Birk Inhibitors from Selaginella moellendorffii
    Article Snippet: Paragraph title: Recombinant Protein Expression, Purification, and Mutagenesis ... The pQE30- BBI3 construct and the suppressor plasmid pREP4 (Qiagen) were cotransformed into the E. coli strain Shuffle Express (New England Biolabs; catalog no. C3028H) for protein production.

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    New England Biolabs shuffle express competent escherichia coli
    Shuffle Express Competent Escherichia Coli, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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