shuffle t7 express e coli strain c3029  (New England Biolabs)


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    Name:
    SHuffle T7 Express Competent E coli
    Description:
    SHuffle T7 Express Competent E coli 12x0 05 ml
    Catalog Number:
    C3029J
    Price:
    224
    Size:
    12x0 05 ml
    Category:
    Competent Bacteria
    Score:
    85
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    Structured Review

    New England Biolabs shuffle t7 express e coli strain c3029
    SHuffle T7 Express Competent E coli
    SHuffle T7 Express Competent E coli 12x0 05 ml
    https://www.bioz.com/result/shuffle t7 express e coli strain c3029/product/New England Biolabs
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    shuffle t7 express e coli strain c3029 - by Bioz Stars, 2019-10
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    Related Articles

    Transduction:

    Article Title: A water-soluble DsbB variant that catalyzes disulfide bond formation in vivo
    Article Snippet: The following E. coli strains were used in this study: DHB4 (MC1000 ΔphoA (PvuII) phoR ΔmalF3 F’[lac +(lacI Q ) pro ]), SHuffle T7 Express (New England Biolabs), and BL21(DE3) (Novagen). .. DHB4 ΔdsbAB strain was derived from DHB4 by insertional inactivation of the dsbA and dsbB genes with the kanamycin resistance gene as described previously .

    Clone Assay:

    Article Title: A Reexamination of Thioredoxin Reductase from Thermoplasmaacidophilum, a Thermoacidophilic Euryarchaeon, Identifies It as an NADH-Dependent Enzyme
    Article Snippet: Coding sequences for Ta-TrxR and Ta-Trx were amplified from T. acidophilum genomic DNA using Q5 DNA polymerase (NEB, Ipswich, MA), and the respective amplicons were cloned into Nde I and Bam HI sites of pTev5, a T7-based expression vector, resulting pUL207 and pUL208, respectively. .. Both plasmids were transformed into E. coli SHuffle T7 Express competent cells (NEB, Ipswich, MA) containing pRIL plasmid (Stratagene, La Jolla, CA).

    Article Title: A water-soluble DsbB variant that catalyzes disulfide bond formation in vivo
    Article Snippet: The following E. coli strains were used in this study: DHB4 (MC1000 ΔphoA (PvuII) phoR ΔmalF3 F’[lac +(lacI Q ) pro ]), SHuffle T7 Express (New England Biolabs), and BL21(DE3) (Novagen). .. Briefly, this was accomplished by P1 phage transduction using the BW25113 dsbA ::kan and BW25113 dsbB ::kan strains from the Keio collection as donors and plasmid pCP20 to remove the Kan marker as needed.

    Article Title: Nanobody‐mediated resistance to Grapevine fanleaf virus in plants
    Article Snippet: For Nb23:EGFP production, the whole Nb23:EGFP:6‐His coding sequence was cloned into the pET‐22b(+) expression vector (Novagen, Madison) as a NdeI/XhoI fragment subsequent to a PCR amplification using pEAQ‐HT‐DEST3‐Nb23:EGFP as template. .. Expression was performed in E. coli SHuffle T7 Express (New England Biolabs, France) grown in TB medium and induced overnight with 0.1 mm IPTG at 20 °C.

    Article Title: C9orf72, a protein associated with amyotrophic lateral sclerosis (ALS) is a guanine nucleotide exchange factor
    Article Snippet: The C9orf72 gene was initially cloned into a variety of vectors such as pTriEx-4 Ek/LIC (N-terminal His6 Tag), pET-SUMO (N-terminal SUMO-His6 tag), pGEX-6p-1 (N-terminal glutathione S-transferase (GST) tag), as well as a modified version of pMAL-p2X (N-terminal His6 -MBP tag). .. A selection of bacterial expression strains was tried (such as Shuffle T7 express and Nico21(DE3) (from New England Biolabs, Hitchin, UK), Rosetta strains, Rosettagami strains (from Novagen, Watford, UK), SoluBL21 (from Amsbio, Abingdon, UK)), along with a variety of media types and expression trials varying different parameters, but none managed to improve expression levels or produced protein that was stable.

    Article Title: Functional and structural insight into properdin control of complement alternative pathway amplification
    Article Snippet: The human C3 C345c domain (Ala1492‐Asn1641) cDNA was synthesized (GenScript) with an N‐terminal TEV cleavage site and cloned into the pET‐32a(+) expression vector (Novagen) downstream of thioredoxin A (TrxA) and a His‐tag using BamHI and HindIII restriction sites. .. Protein was expressed in the E. coli SHuffle T7 strain (New England Biolabs) in LB Broth containing 2% (w/v) glucose and 100 μg/ml ampicillin.

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
    Article Snippet: The most commonly used bacterial strain for expression of genes cloned into the pET vector system are the BL21(DE3) and E. coli K12 lineage strains [ ]. .. To overcome this, we turned to a BL21(DE3) derivative known as SHuffle® T7 Express Competent E. coli (New England Biolabs #C3029J, Ipswich, MA).

    Centrifugation:

    Article Title: Electrostatically-guided inhibition of Curli amyloid nucleation by the CsgC-like family of chaperones
    Article Snippet: CsgH was expressed in E. coli SHuffle T7 Express cells (NEB) by growth in LB medium at 37 °C until the OD600 reached 0.8 units. .. Expression was induced at 30 °C by addition of 0.5 mM IPTG.

    Article Title: A Reexamination of Thioredoxin Reductase from Thermoplasmaacidophilum, a Thermoacidophilic Euryarchaeon, Identifies It as an NADH-Dependent Enzyme
    Article Snippet: Both plasmids were transformed into E. coli SHuffle T7 Express competent cells (NEB, Ipswich, MA) containing pRIL plasmid (Stratagene, La Jolla, CA). .. When a growing culture reached an optical density (OD600 ) of 0.8, as measured with a DU800 UV–visible spectrophotometer (Beckman Coulter, Inc., Brea, CA), IPTG was added to a final concentration of 0.4 mM and the cultivation was continued for additional 5 h at 37 °C for Ta-TrxR overexpression and for 12 h at 15 °C for Ta-Trx overexpression.

    Article Title: Functional and structural insight into properdin control of complement alternative pathway amplification
    Article Snippet: Protein was expressed in the E. coli SHuffle T7 strain (New England Biolabs) in LB Broth containing 2% (w/v) glucose and 100 μg/ml ampicillin. .. Protein expression was induced at OD600nm = 0.6 by adding IPTG to 1 mM final concentration.

    Amplification:

    Article Title: A Reexamination of Thioredoxin Reductase from Thermoplasmaacidophilum, a Thermoacidophilic Euryarchaeon, Identifies It as an NADH-Dependent Enzyme
    Article Snippet: Coding sequences for Ta-TrxR and Ta-Trx were amplified from T. acidophilum genomic DNA using Q5 DNA polymerase (NEB, Ipswich, MA), and the respective amplicons were cloned into Nde I and Bam HI sites of pTev5, a T7-based expression vector, resulting pUL207 and pUL208, respectively. .. Both plasmids were transformed into E. coli SHuffle T7 Express competent cells (NEB, Ipswich, MA) containing pRIL plasmid (Stratagene, La Jolla, CA).

    Article Title: Nanobody‐mediated resistance to Grapevine fanleaf virus in plants
    Article Snippet: For Nb23:EGFP production, the whole Nb23:EGFP:6‐His coding sequence was cloned into the pET‐22b(+) expression vector (Novagen, Madison) as a NdeI/XhoI fragment subsequent to a PCR amplification using pEAQ‐HT‐DEST3‐Nb23:EGFP as template. .. Expression was performed in E. coli SHuffle T7 Express (New England Biolabs, France) grown in TB medium and induced overnight with 0.1 mm IPTG at 20 °C.

    Article Title: Genetically Enhanced Lysozyme Evades a Pathogen Derived Inhibitory Protein
    Article Snippet: Genes coding for Ivyc and E. coli MliC were amplified from E. coli JM105, and that for Ivyp from P. aeruginosa strain PA01. .. Expression vectors pET26b-Ivyc-his, pET26b-Ivyp-his, and pET26b-MliC-his were constructed and subsequently transformed into Shuffle® T7 Express cells (New England Biolabs, USA) and produced as described previously ( ).

    Synthesized:

    Article Title: Functional and structural insight into properdin control of complement alternative pathway amplification
    Article Snippet: The human C3 C345c domain (Ala1492‐Asn1641) cDNA was synthesized (GenScript) with an N‐terminal TEV cleavage site and cloned into the pET‐32a(+) expression vector (Novagen) downstream of thioredoxin A (TrxA) and a His‐tag using BamHI and HindIII restriction sites. .. Protein was expressed in the E. coli SHuffle T7 strain (New England Biolabs) in LB Broth containing 2% (w/v) glucose and 100 μg/ml ampicillin.

    Construct:

    Article Title: Electrostatically-guided inhibition of Curli amyloid nucleation by the CsgC-like family of chaperones
    Article Snippet: From sequence/structural comparison with CsgC, we expressed a CsgH construct starting at residue 10. .. CsgH was expressed in E. coli SHuffle T7 Express cells (NEB) by growth in LB medium at 37 °C until the OD600 reached 0.8 units.

    Article Title: Expression of Functional Human Sialyltransferases ST3Gal1 and ST6Gal1 in Escherichia coli
    Article Snippet: The pLgals1 construct is the fusion of an N -terminal human galectin-1 with hST3gal1 and was selected with 100 μg/ml ampicillin. .. Plasmids were transformed for expression in E . coli BL21 (New England BioLabs), SHuffle T7 Express (New England BioLabs) and Origami 2 DE3 (Novagen, Darmstadt, Germany) and plated onto LB agar plates containing the appropriate antibiotic.

    Article Title: A water-soluble DsbB variant that catalyzes disulfide bond formation in vivo
    Article Snippet: The following E. coli strains were used in this study: DHB4 (MC1000 ΔphoA (PvuII) phoR ΔmalF3 F’[lac +(lacI Q ) pro ]), SHuffle T7 Express (New England Biolabs), and BL21(DE3) (Novagen). .. The following E. coli strains were used in this study: DHB4 (MC1000 ΔphoA (PvuII) phoR ΔmalF3 F’[lac +(lacI Q ) pro ]), SHuffle T7 Express (New England Biolabs), and BL21(DE3) (Novagen).

    Article Title: Genetically Enhanced Lysozyme Evades a Pathogen Derived Inhibitory Protein
    Article Snippet: The centromeric expression vector p4GM-LYZ was constructed as previously described ( ). .. E. coli T7 Shuffle Express was purchased from New England Biolabs.

    Article Title: Structural basis of mammalian glycan targeting by Vibrio cholerae cytolysin and biofilm proteins
    Article Snippet: Paragraph title: Expression and purification of β-prism lectin and VCC full-length constructs ... VCC full-length protein was expressed in Shuffle T7 E . coli cells (New England Biolabs) for 4 hours at 30°C and the VCC β-prism lectin domain was expressed for 8 hours at 37°C in T7 Express E . coli cells.

    Article Title: C9orf72, a protein associated with amyotrophic lateral sclerosis (ALS) is a guanine nucleotide exchange factor
    Article Snippet: The SUMO-His6 tagged C9orf72 expressed as inclusion bodies and the construct with the minimal tag (His6 ) did not express at all in E. coli . .. A selection of bacterial expression strains was tried (such as Shuffle T7 express and Nico21(DE3) (from New England Biolabs, Hitchin, UK), Rosetta strains, Rosettagami strains (from Novagen, Watford, UK), SoluBL21 (from Amsbio, Abingdon, UK)), along with a variety of media types and expression trials varying different parameters, but none managed to improve expression levels or produced protein that was stable.

    Article Title: Genetically Enhanced Lysozyme Evades a Pathogen Derived Inhibitory Protein
    Article Snippet: Genes coding for Ivyc and E. coli MliC were amplified from E. coli JM105, and that for Ivyp from P. aeruginosa strain PA01. .. Expression vectors pET26b-Ivyc-his, pET26b-Ivyp-his, and pET26b-MliC-his were constructed and subsequently transformed into Shuffle® T7 Express cells (New England Biolabs, USA) and produced as described previously ( ). .. GMD were produced as described in detail elsewhere , with the exception that M. luteus , instead of Staphylococcus aureus , was employed as the target bacteria.

    Article Title: Novel, Anti-hTNF-α Variable New Antigen Receptor Formats with Enhanced Neutralizing Potency and Multifunctionality, Generated for Therapeutic Development
    Article Snippet: Expression in E. coli HB2151 cells was induced with 1 mM isopropyl β-d -1-thiogalactopyranoside (IPTG), and soluble VNAR protein was extracted from the periplasm (E. coli HB2151 cells) ( ). .. All multivalent non-Fc VNAR constructs were expressed as cytoplasmic protein in IPTG-induced E. coli SHuffle® T7 Express cells (NEB) using pET28b(+) expression vector. .. Extraction of cytoplasmic VNAR protein was achieved using the BugBuster™ protein extraction reagent plus Benzonase® (Novagen).

    Incubation:

    Article Title: Immunizing Adult Female Mice with a TcpA-A2-CTB Chimera Provides a High Level of Protection for Their Pups in the Infant Mouse Model of Cholera
    Article Snippet: 6His-TcpA(29–199) was produced in SHuffle™ T7 Express E. coli (NEB, Ipswich, MA) in half liter cultures of TCYM media pH 7.5 (tryptone 1%, NaCl 0.5%, yeast extract 0.5%, casamino acids 0.1%, MgSO4 0.2%) and 100 µg/ml kanamycin at 37°C, 250 rpm until cultures reached an OD600 of ∼3.0. .. 6His-TcpA(29–199) was produced in SHuffle™ T7 Express E. coli (NEB, Ipswich, MA) in half liter cultures of TCYM media pH 7.5 (tryptone 1%, NaCl 0.5%, yeast extract 0.5%, casamino acids 0.1%, MgSO4 0.2%) and 100 µg/ml kanamycin at 37°C, 250 rpm until cultures reached an OD600 of ∼3.0.

    Article Title: The endoplasmic reticulum HSP40 co‐chaperone ERdj3/ DNAJB11 assembles and functions as a tetramer
    Article Snippet: The fractions were collected with interval of 1 ml and analyzed by SDS–PAGE and Western blot. .. 6xHis Smt3 tagged ERdj3 mutants were transformed into SHuffle® T7 Express competent E. coli (NEB) and incubated at 30°C. .. The overnight culture grown at 30°C in LB media was inoculated in LB media at ratio of 1:1,000 to grow at 30°C till O.D. reaches ~0.6, and then, 0.1 mM IPTG was used to induce overnight expression at 25°C.

    Article Title: Structural basis of mammalian glycan targeting by Vibrio cholerae cytolysin and biofilm proteins
    Article Snippet: To remove the GFP tag, the fusion proteins were incubated with 1:100 (wt/wt) human α-thrombin (Haematologic Technologies) for 4 hours at room temperature or 1:500 (wt/wt) trypsin (Sigma Aldrich) for 1 hour at room temperature and the reaction stopped with 20 mM EDTA and 1 mM AEBSF. .. VCC full-length protein was expressed in Shuffle T7 E . coli cells (New England Biolabs) for 4 hours at 30°C and the VCC β-prism lectin domain was expressed for 8 hours at 37°C in T7 Express E . coli cells.

    Expressing:

    Article Title: Electrostatically-guided inhibition of Curli amyloid nucleation by the CsgC-like family of chaperones
    Article Snippet: Paragraph title: CsgH expression and purification ... CsgH was expressed in E. coli SHuffle T7 Express cells (NEB) by growth in LB medium at 37 °C until the OD600 reached 0.8 units.

    Article Title: Expression of Functional Human Sialyltransferases ST3Gal1 and ST6Gal1 in Escherichia coli
    Article Snippet: The pLgals1 construct is the fusion of an N -terminal human galectin-1 with hST3gal1 and was selected with 100 μg/ml ampicillin. .. Plasmids were transformed for expression in E . coli BL21 (New England BioLabs), SHuffle T7 Express (New England BioLabs) and Origami 2 DE3 (Novagen, Darmstadt, Germany) and plated onto LB agar plates containing the appropriate antibiotic. .. Tetracycline (12.5 μg/ml final concentration) was also included for selection of the gor mutation in Origami.

    Article Title: A Reexamination of Thioredoxin Reductase from Thermoplasmaacidophilum, a Thermoacidophilic Euryarchaeon, Identifies It as an NADH-Dependent Enzyme
    Article Snippet: Paragraph title: Heterologous Expression of TrxR and Trx of T.acidophilum (Ta-TrxR and Ta-Trx) in E. coli SHuffle T7 Express ... Both plasmids were transformed into E. coli SHuffle T7 Express competent cells (NEB, Ipswich, MA) containing pRIL plasmid (Stratagene, La Jolla, CA).

    Article Title: Genetically Enhanced Lysozyme Evades a Pathogen Derived Inhibitory Protein
    Article Snippet: The centromeric expression vector p4GM-LYZ was constructed as previously described ( ). .. E. coli T7 Shuffle Express was purchased from New England Biolabs.

    Article Title: Nanobody‐mediated resistance to Grapevine fanleaf virus in plants
    Article Snippet: For Nb23:EGFP production, the whole Nb23:EGFP:6‐His coding sequence was cloned into the pET‐22b(+) expression vector (Novagen, Madison) as a NdeI/XhoI fragment subsequent to a PCR amplification using pEAQ‐HT‐DEST3‐Nb23:EGFP as template. .. Expression was performed in E. coli SHuffle T7 Express (New England Biolabs, France) grown in TB medium and induced overnight with 0.1 mm IPTG at 20 °C. .. Pelleted cells resuspended in PB‐NaCl buffer (10 mm phosphate buffer, 300 mm NaCl, pH 7.4) were lysed by sonication (80% amplitude for 2 min with 13 mm diameter probe, Vibra‐Cell VCX 500 (Sonics & Materials Inc) before purification of cytoplasmic extract.

    Article Title: Structural basis of mammalian glycan targeting by Vibrio cholerae cytolysin and biofilm proteins
    Article Snippet: Paragraph title: Expression and purification of β-prism lectin and VCC full-length constructs ... VCC full-length protein was expressed in Shuffle T7 E . coli cells (New England Biolabs) for 4 hours at 30°C and the VCC β-prism lectin domain was expressed for 8 hours at 37°C in T7 Express E . coli cells.

    Article Title: C9orf72, a protein associated with amyotrophic lateral sclerosis (ALS) is a guanine nucleotide exchange factor
    Article Snippet: The SUMO-His6 tagged C9orf72 expressed as inclusion bodies and the construct with the minimal tag (His6 ) did not express at all in E. coli . .. A selection of bacterial expression strains was tried (such as Shuffle T7 express and Nico21(DE3) (from New England Biolabs, Hitchin, UK), Rosetta strains, Rosettagami strains (from Novagen, Watford, UK), SoluBL21 (from Amsbio, Abingdon, UK)), along with a variety of media types and expression trials varying different parameters, but none managed to improve expression levels or produced protein that was stable. .. We also prepared constructs where maltose-binding protein (MBP) and GST tags were moved to the C-terminal end of the protein but obtained similar results as above.

    Article Title: Functional and structural insight into properdin control of complement alternative pathway amplification
    Article Snippet: The human C3 C345c domain (Ala1492‐Asn1641) cDNA was synthesized (GenScript) with an N‐terminal TEV cleavage site and cloned into the pET‐32a(+) expression vector (Novagen) downstream of thioredoxin A (TrxA) and a His‐tag using BamHI and HindIII restriction sites. .. Protein was expressed in the E. coli SHuffle T7 strain (New England Biolabs) in LB Broth containing 2% (w/v) glucose and 100 μg/ml ampicillin.

    Article Title: A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach
    Article Snippet: B. thailandensis and P. fluorescens strains used in this study were derived from the sequenced strains E264 and Pf-5, respectively ( ; ). .. E. coli strains included in this study included DH5α for plasmid maintenance, BL21 pLysS for expression of effectors for toxicity assays, SM10 for conjugal transfer of plasmids into P. fluorescens , and Shuffle T7 pLysS Express (New England Biolabs) for purification of effectors. .. Growth conditions for all strains and plasmid and strain construction details are described in .

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
    Article Snippet: Paragraph title: Expression of recombinant proteases using SHuffle® (NEB) E. coli cells and Bis-Tris gel analysis ... To overcome this, we turned to a BL21(DE3) derivative known as SHuffle® T7 Express Competent E. coli (New England Biolabs #C3029J, Ipswich, MA).

    Article Title: Phosphatidylserine transport by Ups2–Mdm35 in respiration-active mitochondria
    Article Snippet: Paragraph title: Protein expression and purification ... The target proteins were expressed in E. coli SHuffle T7 cells (New England Biolabs, Inc.) cultured in LB medium.

    Article Title: Genetically Enhanced Lysozyme Evades a Pathogen Derived Inhibitory Protein
    Article Snippet: Genes coding for Ivyc and E. coli MliC were amplified from E. coli JM105, and that for Ivyp from P. aeruginosa strain PA01. .. Expression vectors pET26b-Ivyc-his, pET26b-Ivyp-his, and pET26b-MliC-his were constructed and subsequently transformed into Shuffle® T7 Express cells (New England Biolabs, USA) and produced as described previously ( ). .. GMD were produced as described in detail elsewhere , with the exception that M. luteus , instead of Staphylococcus aureus , was employed as the target bacteria.

    Article Title: Structural and mechanistic insights into phospholipid transfer by Ups1–Mdm35 in mitochondria
    Article Snippet: Paragraph title: Protein expression and purification ... The target proteins were expressed in E. coli SHuffle T7 cells (NEB) cultured in LB medium.

    Article Title: Novel, Anti-hTNF-α Variable New Antigen Receptor Formats with Enhanced Neutralizing Potency and Multifunctionality, Generated for Therapeutic Development
    Article Snippet: Expression in E. coli HB2151 cells was induced with 1 mM isopropyl β-d -1-thiogalactopyranoside (IPTG), and soluble VNAR protein was extracted from the periplasm (E. coli HB2151 cells) ( ). .. All multivalent non-Fc VNAR constructs were expressed as cytoplasmic protein in IPTG-induced E. coli SHuffle® T7 Express cells (NEB) using pET28b(+) expression vector. .. Extraction of cytoplasmic VNAR protein was achieved using the BugBuster™ protein extraction reagent plus Benzonase® (Novagen).

    Article Title: Effect of N- and C-Terminal Amino Acids on the Interfacial Binding Properties of Phospholipase D from Vibrio parahaemolyticus
    Article Snippet: The expression vectors pET21a and pET28a were purchased from Stratagene (La Jolla, CA, USA). .. Escherichia coli Shuffle T7 Express Competent cells were purchased from New England BioLabs (Beijing, China).

    BIA-KA:

    Article Title: Effect of N- and C-Terminal Amino Acids on the Interfacial Binding Properties of Phospholipase D from Vibrio parahaemolyticus
    Article Snippet: Escherichia coli Shuffle T7 Express Competent cells were purchased from New England BioLabs (Beijing, China). .. Ni2+ -NTA Sepharose fast-flow and anion exchange chromatography (Q-Sepharose XL) columns were purchased from GE Healthcare (Boston, MA, USA).

    Modification:

    Article Title: C9orf72, a protein associated with amyotrophic lateral sclerosis (ALS) is a guanine nucleotide exchange factor
    Article Snippet: The C9orf72 gene was initially cloned into a variety of vectors such as pTriEx-4 Ek/LIC (N-terminal His6 Tag), pET-SUMO (N-terminal SUMO-His6 tag), pGEX-6p-1 (N-terminal glutathione S-transferase (GST) tag), as well as a modified version of pMAL-p2X (N-terminal His6 -MBP tag). .. A selection of bacterial expression strains was tried (such as Shuffle T7 express and Nico21(DE3) (from New England Biolabs, Hitchin, UK), Rosetta strains, Rosettagami strains (from Novagen, Watford, UK), SoluBL21 (from Amsbio, Abingdon, UK)), along with a variety of media types and expression trials varying different parameters, but none managed to improve expression levels or produced protein that was stable.

    Transformation Assay:

    Article Title: Expression of Functional Human Sialyltransferases ST3Gal1 and ST6Gal1 in Escherichia coli
    Article Snippet: The pLgals1 construct is the fusion of an N -terminal human galectin-1 with hST3gal1 and was selected with 100 μg/ml ampicillin. .. Plasmids were transformed for expression in E . coli BL21 (New England BioLabs), SHuffle T7 Express (New England BioLabs) and Origami 2 DE3 (Novagen, Darmstadt, Germany) and plated onto LB agar plates containing the appropriate antibiotic. .. Tetracycline (12.5 μg/ml final concentration) was also included for selection of the gor mutation in Origami.

    Article Title: A Reexamination of Thioredoxin Reductase from Thermoplasmaacidophilum, a Thermoacidophilic Euryarchaeon, Identifies It as an NADH-Dependent Enzyme
    Article Snippet: These plasmids were designed to express recombinant proteins with an NH2 -terminal His6 -tag, followed by a TEV protease recognition site. .. Both plasmids were transformed into E. coli SHuffle T7 Express competent cells (NEB, Ipswich, MA) containing pRIL plasmid (Stratagene, La Jolla, CA). .. The resulting strains were grown at 37 °C in Luria Bertani media containing 100 and 34 μg/mL ampicillin and chloramphenicol, respectively.

    Article Title: The endoplasmic reticulum HSP40 co‐chaperone ERdj3/ DNAJB11 assembles and functions as a tetramer
    Article Snippet: The fractions were collected with interval of 1 ml and analyzed by SDS–PAGE and Western blot. .. 6xHis Smt3 tagged ERdj3 mutants were transformed into SHuffle® T7 Express competent E. coli (NEB) and incubated at 30°C. .. The overnight culture grown at 30°C in LB media was inoculated in LB media at ratio of 1:1,000 to grow at 30°C till O.D. reaches ~0.6, and then, 0.1 mM IPTG was used to induce overnight expression at 25°C.

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
    Article Snippet: To overcome this, we turned to a BL21(DE3) derivative known as SHuffle® T7 Express Competent E. coli (New England Biolabs #C3029J, Ipswich, MA). .. To overcome this, we turned to a BL21(DE3) derivative known as SHuffle® T7 Express Competent E. coli (New England Biolabs #C3029J, Ipswich, MA).

    Article Title: Genetically Enhanced Lysozyme Evades a Pathogen Derived Inhibitory Protein
    Article Snippet: Genes coding for Ivyc and E. coli MliC were amplified from E. coli JM105, and that for Ivyp from P. aeruginosa strain PA01. .. Expression vectors pET26b-Ivyc-his, pET26b-Ivyp-his, and pET26b-MliC-his were constructed and subsequently transformed into Shuffle® T7 Express cells (New England Biolabs, USA) and produced as described previously ( ). .. GMD were produced as described in detail elsewhere , with the exception that M. luteus , instead of Staphylococcus aureus , was employed as the target bacteria.

    Over Expression:

    Article Title: A Reexamination of Thioredoxin Reductase from Thermoplasmaacidophilum, a Thermoacidophilic Euryarchaeon, Identifies It as an NADH-Dependent Enzyme
    Article Snippet: Both plasmids were transformed into E. coli SHuffle T7 Express competent cells (NEB, Ipswich, MA) containing pRIL plasmid (Stratagene, La Jolla, CA). .. For the expression of Ta-Trx, the LB media was supplemented with DTT at a final concentration of 0.2 mM.

    Crystallization Assay:

    Article Title: Structural and mechanistic insights into phospholipid transfer by Ups1–Mdm35 in mitochondria
    Article Snippet: The target proteins were expressed in E. coli SHuffle T7 cells (NEB) cultured in LB medium. .. GST–Mdm35 derivatives were affinity purified by a glutathione-Sepharose 4B (GS4B) column (GE Healthcare).

    Derivative Assay:

    Article Title: A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach
    Article Snippet: B. thailandensis and P. fluorescens strains used in this study were derived from the sequenced strains E264 and Pf-5, respectively ( ; ). .. E. coli strains included in this study included DH5α for plasmid maintenance, BL21 pLysS for expression of effectors for toxicity assays, SM10 for conjugal transfer of plasmids into P. fluorescens , and Shuffle T7 pLysS Express (New England Biolabs) for purification of effectors.

    Transfection:

    Article Title: Novel, Anti-hTNF-α Variable New Antigen Receptor Formats with Enhanced Neutralizing Potency and Multifunctionality, Generated for Therapeutic Development
    Article Snippet: All multivalent non-Fc VNAR constructs were expressed as cytoplasmic protein in IPTG-induced E. coli SHuffle® T7 Express cells (NEB) using pET28b(+) expression vector. .. All multivalent non-Fc VNAR constructs were expressed as cytoplasmic protein in IPTG-induced E. coli SHuffle® T7 Express cells (NEB) using pET28b(+) expression vector.

    Chromatography:

    Article Title: Electrostatically-guided inhibition of Curli amyloid nucleation by the CsgC-like family of chaperones
    Article Snippet: CsgH was expressed in E. coli SHuffle T7 Express cells (NEB) by growth in LB medium at 37 °C until the OD600 reached 0.8 units. .. Expression was induced at 30 °C by addition of 0.5 mM IPTG.

    Article Title: Effect of N- and C-Terminal Amino Acids on the Interfacial Binding Properties of Phospholipase D from Vibrio parahaemolyticus
    Article Snippet: Escherichia coli Shuffle T7 Express Competent cells were purchased from New England BioLabs (Beijing, China). .. Escherichia coli Shuffle T7 Express Competent cells were purchased from New England BioLabs (Beijing, China).

    Protease Inhibitor:

    Article Title: The endoplasmic reticulum HSP40 co‐chaperone ERdj3/ DNAJB11 assembles and functions as a tetramer
    Article Snippet: 6xHis Smt3 tagged ERdj3 mutants were transformed into SHuffle® T7 Express competent E. coli (NEB) and incubated at 30°C. .. The overnight culture grown at 30°C in LB media was inoculated in LB media at ratio of 1:1,000 to grow at 30°C till O.D. reaches ~0.6, and then, 0.1 mM IPTG was used to induce overnight expression at 25°C.

    Cell Culture:

    Article Title: Phosphatidylserine transport by Ups2–Mdm35 in respiration-active mitochondria
    Article Snippet: NBD signals were detected and quantified with an imaging analyzer (FLA-5000; Fujifilm) and MultiGauge software (Fujifilm). .. The target proteins were expressed in E. coli SHuffle T7 cells (New England Biolabs, Inc.) cultured in LB medium. .. After addition of 0.1 mM isopropyl-d -thiogalactoside, the cells were cultured at 20°C for 20 h, and were disrupted by sonication.

    Article Title: Structural and mechanistic insights into phospholipid transfer by Ups1–Mdm35 in mitochondria
    Article Snippet: We refer to Ups1ΔC5 and Mdm35ΔC5 as Ups1 and Mdm35 below. .. The target proteins were expressed in E. coli SHuffle T7 cells (NEB) cultured in LB medium. .. After addition of 0.1 mM isopropyl-D -thiogalactoside, the cells were cultured at 20 °C for 20 h, and were disrupted by sonication.

    Generated:

    Article Title: A water-soluble DsbB variant that catalyzes disulfide bond formation in vivo
    Article Snippet: The following E. coli strains were used in this study: DHB4 (MC1000 ΔphoA (PvuII) phoR ΔmalF3 F’[lac +(lacI Q ) pro ]), SHuffle T7 Express (New England Biolabs), and BL21(DE3) (Novagen). .. Briefly, this was accomplished by P1 phage transduction using the BW25113 dsbA ::kan and BW25113 dsbB ::kan strains from the Keio collection as donors and plasmid pCP20 to remove the Kan marker as needed.

    Sequencing:

    Article Title: Electrostatically-guided inhibition of Curli amyloid nucleation by the CsgC-like family of chaperones
    Article Snippet: From sequence/structural comparison with CsgC, we expressed a CsgH construct starting at residue 10. .. CsgH was expressed in E. coli SHuffle T7 Express cells (NEB) by growth in LB medium at 37 °C until the OD600 reached 0.8 units.

    Article Title: Nanobody‐mediated resistance to Grapevine fanleaf virus in plants
    Article Snippet: For Nb23:EGFP production, the whole Nb23:EGFP:6‐His coding sequence was cloned into the pET‐22b(+) expression vector (Novagen, Madison) as a NdeI/XhoI fragment subsequent to a PCR amplification using pEAQ‐HT‐DEST3‐Nb23:EGFP as template. .. Expression was performed in E. coli SHuffle T7 Express (New England Biolabs, France) grown in TB medium and induced overnight with 0.1 mm IPTG at 20 °C.

    Article Title: C9orf72, a protein associated with amyotrophic lateral sclerosis (ALS) is a guanine nucleotide exchange factor
    Article Snippet: A selection of bacterial expression strains was tried (such as Shuffle T7 express and Nico21(DE3) (from New England Biolabs, Hitchin, UK), Rosetta strains, Rosettagami strains (from Novagen, Watford, UK), SoluBL21 (from Amsbio, Abingdon, UK)), along with a variety of media types and expression trials varying different parameters, but none managed to improve expression levels or produced protein that was stable. .. Producing a functional mammalian protein often necessitates the use of a eukaryotic expression host to ensure appropriate posttranslational modifications (e.g., disulphide bonds, glycosylation).

    Sonication:

    Article Title: The endoplasmic reticulum HSP40 co‐chaperone ERdj3/ DNAJB11 assembles and functions as a tetramer
    Article Snippet: 6xHis Smt3 tagged ERdj3 mutants were transformed into SHuffle® T7 Express competent E. coli (NEB) and incubated at 30°C. .. The overnight culture grown at 30°C in LB media was inoculated in LB media at ratio of 1:1,000 to grow at 30°C till O.D. reaches ~0.6, and then, 0.1 mM IPTG was used to induce overnight expression at 25°C.

    Article Title: Functional and structural insight into properdin control of complement alternative pathway amplification
    Article Snippet: Protein was expressed in the E. coli SHuffle T7 strain (New England Biolabs) in LB Broth containing 2% (w/v) glucose and 100 μg/ml ampicillin. .. Protein expression was induced at OD600nm = 0.6 by adding IPTG to 1 mM final concentration.

    Affinity Purification:

    Article Title: Phosphatidylserine transport by Ups2–Mdm35 in respiration-active mitochondria
    Article Snippet: The target proteins were expressed in E. coli SHuffle T7 cells (New England Biolabs, Inc.) cultured in LB medium. .. After addition of 0.1 mM isopropyl-d -thiogalactoside, the cells were cultured at 20°C for 20 h, and were disrupted by sonication.

    Article Title: Structural and mechanistic insights into phospholipid transfer by Ups1–Mdm35 in mitochondria
    Article Snippet: The target proteins were expressed in E. coli SHuffle T7 cells (NEB) cultured in LB medium. .. After addition of 0.1 mM isopropyl-D -thiogalactoside, the cells were cultured at 20 °C for 20 h, and were disrupted by sonication.

    Binding Assay:

    Article Title: Functional and structural insight into properdin control of complement alternative pathway amplification
    Article Snippet: Protein was expressed in the E. coli SHuffle T7 strain (New England Biolabs) in LB Broth containing 2% (w/v) glucose and 100 μg/ml ampicillin. .. Protein expression was induced at OD600nm = 0.6 by adding IPTG to 1 mM final concentration.

    CtB Assay:

    Article Title: Immunizing Adult Female Mice with a TcpA-A2-CTB Chimera Provides a High Level of Protection for Their Pups in the Infant Mouse Model of Cholera
    Article Snippet: 6His-TcpA(29–199) was produced in SHuffle™ T7 Express E. coli (NEB, Ipswich, MA) in half liter cultures of TCYM media pH 7.5 (tryptone 1%, NaCl 0.5%, yeast extract 0.5%, casamino acids 0.1%, MgSO4 0.2%) and 100 µg/ml kanamycin at 37°C, 250 rpm until cultures reached an OD600 of ∼3.0. .. After acclimating to 30°C with shaking at 250 rpm for 30 minutes, the cultures were then induced with 0.5 mM IPTG and incubated for 4 hrs.

    Size-exclusion Chromatography:

    Article Title: SMOC Binds to Pro-EGF, but Does Not Induce Erk Phosphorylation via the EGFR
    Article Snippet: Full length Xenopus X SMOC-1, X SMOC-1ΔEC (25 to K309) lacking the EC domain, and X SMOC-1EC containing the EC domain only (K309 to end) in the pET- 28b(+) vector (Novagen) were expressed in the Shuffle® T7 Express E.coli strain C3029 (New England Biolabs). .. Full length Xenopus X SMOC-1, X SMOC-1ΔEC (25 to K309) lacking the EC domain, and X SMOC-1EC containing the EC domain only (K309 to end) in the pET- 28b(+) vector (Novagen) were expressed in the Shuffle® T7 Express E.coli strain C3029 (New England Biolabs).

    Purification:

    Article Title: Immunizing Adult Female Mice with a TcpA-A2-CTB Chimera Provides a High Level of Protection for Their Pups in the Infant Mouse Model of Cholera
    Article Snippet: Paragraph title: Recombinant protein production and purification ... 6His-TcpA(29–199) was produced in SHuffle™ T7 Express E. coli (NEB, Ipswich, MA) in half liter cultures of TCYM media pH 7.5 (tryptone 1%, NaCl 0.5%, yeast extract 0.5%, casamino acids 0.1%, MgSO4 0.2%) and 100 µg/ml kanamycin at 37°C, 250 rpm until cultures reached an OD600 of ∼3.0.

    Article Title: Electrostatically-guided inhibition of Curli amyloid nucleation by the CsgC-like family of chaperones
    Article Snippet: Paragraph title: CsgH expression and purification ... CsgH was expressed in E. coli SHuffle T7 Express cells (NEB) by growth in LB medium at 37 °C until the OD600 reached 0.8 units.

    Article Title: The endoplasmic reticulum HSP40 co‐chaperone ERdj3/ DNAJB11 assembles and functions as a tetramer
    Article Snippet: Paragraph title: R ERdj3 purification and characterization ... 6xHis Smt3 tagged ERdj3 mutants were transformed into SHuffle® T7 Express competent E. coli (NEB) and incubated at 30°C.

    Article Title: Nanobody‐mediated resistance to Grapevine fanleaf virus in plants
    Article Snippet: Paragraph title: Nb23:EGFP purification ... Expression was performed in E. coli SHuffle T7 Express (New England Biolabs, France) grown in TB medium and induced overnight with 0.1 mm IPTG at 20 °C.

    Article Title: Structural basis of mammalian glycan targeting by Vibrio cholerae cytolysin and biofilm proteins
    Article Snippet: Paragraph title: Expression and purification of β-prism lectin and VCC full-length constructs ... VCC full-length protein was expressed in Shuffle T7 E . coli cells (New England Biolabs) for 4 hours at 30°C and the VCC β-prism lectin domain was expressed for 8 hours at 37°C in T7 Express E . coli cells.

    Article Title: An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism
    Article Snippet: After purification, eluted MBP fusions were further desalted using a Zeba™ Spin Desalting Column (Fisher) into 20 mM Tris/Cl(pH.4). .. The gpD and scFv-D10 proteins were expressed from pET28-gpD and pET28-scFv-D10, respectively, in BL21(DE3) cells, while scFv-GCN4 and scFv-GCN4(WQL) were expressed in the cytoplasm of SHuffle T7 Express cells (New England Biolabs) from pET28-scFv-GCN4 and pET28-GCN4(WQL), respectively.

    Article Title: A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach
    Article Snippet: B. thailandensis and P. fluorescens strains used in this study were derived from the sequenced strains E264 and Pf-5, respectively ( ; ). .. E. coli strains included in this study included DH5α for plasmid maintenance, BL21 pLysS for expression of effectors for toxicity assays, SM10 for conjugal transfer of plasmids into P. fluorescens , and Shuffle T7 pLysS Express (New England Biolabs) for purification of effectors. .. Growth conditions for all strains and plasmid and strain construction details are described in .

    Article Title: Phosphatidylserine transport by Ups2–Mdm35 in respiration-active mitochondria
    Article Snippet: Paragraph title: Protein expression and purification ... The target proteins were expressed in E. coli SHuffle T7 cells (New England Biolabs, Inc.) cultured in LB medium.

    Article Title: Genetically Enhanced Lysozyme Evades a Pathogen Derived Inhibitory Protein
    Article Snippet: Paragraph title: Purification of lysozyme inhibitors ... Expression vectors pET26b-Ivyc-his, pET26b-Ivyp-his, and pET26b-MliC-his were constructed and subsequently transformed into Shuffle® T7 Express cells (New England Biolabs, USA) and produced as described previously ( ).

    Article Title: Structural and mechanistic insights into phospholipid transfer by Ups1–Mdm35 in mitochondria
    Article Snippet: Paragraph title: Protein expression and purification ... The target proteins were expressed in E. coli SHuffle T7 cells (NEB) cultured in LB medium.

    Article Title: Novel, Anti-hTNF-α Variable New Antigen Receptor Formats with Enhanced Neutralizing Potency and Multifunctionality, Generated for Therapeutic Development
    Article Snippet: Paragraph title: Soluble VNAR Protein Expression and Purification ... All multivalent non-Fc VNAR constructs were expressed as cytoplasmic protein in IPTG-induced E. coli SHuffle® T7 Express cells (NEB) using pET28b(+) expression vector.

    Protein Purification:

    Article Title: An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism
    Article Snippet: Paragraph title: Protein purification ... The gpD and scFv-D10 proteins were expressed from pET28-gpD and pET28-scFv-D10, respectively, in BL21(DE3) cells, while scFv-GCN4 and scFv-GCN4(WQL) were expressed in the cytoplasm of SHuffle T7 Express cells (New England Biolabs) from pET28-scFv-GCN4 and pET28-GCN4(WQL), respectively.

    Polymerase Chain Reaction:

    Article Title: Nanobody‐mediated resistance to Grapevine fanleaf virus in plants
    Article Snippet: For Nb23:EGFP production, the whole Nb23:EGFP:6‐His coding sequence was cloned into the pET‐22b(+) expression vector (Novagen, Madison) as a NdeI/XhoI fragment subsequent to a PCR amplification using pEAQ‐HT‐DEST3‐Nb23:EGFP as template. .. Expression was performed in E. coli SHuffle T7 Express (New England Biolabs, France) grown in TB medium and induced overnight with 0.1 mm IPTG at 20 °C.

    Selection:

    Article Title: C9orf72, a protein associated with amyotrophic lateral sclerosis (ALS) is a guanine nucleotide exchange factor
    Article Snippet: The SUMO-His6 tagged C9orf72 expressed as inclusion bodies and the construct with the minimal tag (His6 ) did not express at all in E. coli . .. A selection of bacterial expression strains was tried (such as Shuffle T7 express and Nico21(DE3) (from New England Biolabs, Hitchin, UK), Rosetta strains, Rosettagami strains (from Novagen, Watford, UK), SoluBL21 (from Amsbio, Abingdon, UK)), along with a variety of media types and expression trials varying different parameters, but none managed to improve expression levels or produced protein that was stable. .. We also prepared constructs where maltose-binding protein (MBP) and GST tags were moved to the C-terminal end of the protein but obtained similar results as above.

    Fast Protein Liquid Chromatography:

    Article Title: The endoplasmic reticulum HSP40 co‐chaperone ERdj3/ DNAJB11 assembles and functions as a tetramer
    Article Snippet: 6xHis Smt3 tagged ERdj3 mutants were transformed into SHuffle® T7 Express competent E. coli (NEB) and incubated at 30°C. .. The harvested cells were sonicated in Ni A buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 20 mM imidazole) with cOmplete™ Ultra EDTA free protease inhibitor (Roche).

    Article Title: An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism
    Article Snippet: The gpD and scFv-D10 proteins were expressed from pET28-gpD and pET28-scFv-D10, respectively, in BL21(DE3) cells, while scFv-GCN4 and scFv-GCN4(WQL) were expressed in the cytoplasm of SHuffle T7 Express cells (New England Biolabs) from pET28-scFv-GCN4 and pET28-GCN4(WQL), respectively. .. The gpD and scFv-D10 proteins were expressed from pET28-gpD and pET28-scFv-D10, respectively, in BL21(DE3) cells, while scFv-GCN4 and scFv-GCN4(WQL) were expressed in the cytoplasm of SHuffle T7 Express cells (New England Biolabs) from pET28-scFv-GCN4 and pET28-GCN4(WQL), respectively.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Nanobody‐mediated resistance to Grapevine fanleaf virus in plants
    Article Snippet: Expression was performed in E. coli SHuffle T7 Express (New England Biolabs, France) grown in TB medium and induced overnight with 0.1 mm IPTG at 20 °C. .. Pelleted cells resuspended in PB‐NaCl buffer (10 mm phosphate buffer, 300 mm NaCl, pH 7.4) were lysed by sonication (80% amplitude for 2 min with 13 mm diameter probe, Vibra‐Cell VCX 500 (Sonics & Materials Inc) before purification of cytoplasmic extract.

    Plasmid Preparation:

    Article Title: A Reexamination of Thioredoxin Reductase from Thermoplasmaacidophilum, a Thermoacidophilic Euryarchaeon, Identifies It as an NADH-Dependent Enzyme
    Article Snippet: These plasmids were designed to express recombinant proteins with an NH2 -terminal His6 -tag, followed by a TEV protease recognition site. .. Both plasmids were transformed into E. coli SHuffle T7 Express competent cells (NEB, Ipswich, MA) containing pRIL plasmid (Stratagene, La Jolla, CA). .. The resulting strains were grown at 37 °C in Luria Bertani media containing 100 and 34 μg/mL ampicillin and chloramphenicol, respectively.

    Article Title: A water-soluble DsbB variant that catalyzes disulfide bond formation in vivo
    Article Snippet: The following E. coli strains were used in this study: DHB4 (MC1000 ΔphoA (PvuII) phoR ΔmalF3 F’[lac +(lacI Q ) pro ]), SHuffle T7 Express (New England Biolabs), and BL21(DE3) (Novagen). .. DHB4 ΔdsbAB strain was derived from DHB4 by insertional inactivation of the dsbA and dsbB genes with the kanamycin resistance gene as described previously .

    Article Title: Genetically Enhanced Lysozyme Evades a Pathogen Derived Inhibitory Protein
    Article Snippet: The centromeric expression vector p4GM-LYZ was constructed as previously described ( ). .. E. coli T7 Shuffle Express was purchased from New England Biolabs.

    Article Title: Nanobody‐mediated resistance to Grapevine fanleaf virus in plants
    Article Snippet: For Nb23:EGFP production, the whole Nb23:EGFP:6‐His coding sequence was cloned into the pET‐22b(+) expression vector (Novagen, Madison) as a NdeI/XhoI fragment subsequent to a PCR amplification using pEAQ‐HT‐DEST3‐Nb23:EGFP as template. .. Expression was performed in E. coli SHuffle T7 Express (New England Biolabs, France) grown in TB medium and induced overnight with 0.1 mm IPTG at 20 °C.

    Article Title: Functional and structural insight into properdin control of complement alternative pathway amplification
    Article Snippet: The human C3 C345c domain (Ala1492‐Asn1641) cDNA was synthesized (GenScript) with an N‐terminal TEV cleavage site and cloned into the pET‐32a(+) expression vector (Novagen) downstream of thioredoxin A (TrxA) and a His‐tag using BamHI and HindIII restriction sites. .. Protein was expressed in the E. coli SHuffle T7 strain (New England Biolabs) in LB Broth containing 2% (w/v) glucose and 100 μg/ml ampicillin.

    Article Title: SMOC Binds to Pro-EGF, but Does Not Induce Erk Phosphorylation via the EGFR
    Article Snippet: In an attempt to identify potential cell surface receptor(s) that are activated by SMOC we conducted a phosphoproteomic screen of HEK293 cells following exposure to X SMOC-1 protein. .. Full length Xenopus X SMOC-1, X SMOC-1ΔEC (25 to K309) lacking the EC domain, and X SMOC-1EC containing the EC domain only (K309 to end) in the pET- 28b(+) vector (Novagen) were expressed in the Shuffle® T7 Express E.coli strain C3029 (New England Biolabs). .. Following bacterial cell lysis solubilized inclusion bodies were applied to and eluted from Ni-NTA agarose (Qiagen).

    Article Title: A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach
    Article Snippet: B. thailandensis and P. fluorescens strains used in this study were derived from the sequenced strains E264 and Pf-5, respectively ( ; ). .. E. coli strains included in this study included DH5α for plasmid maintenance, BL21 pLysS for expression of effectors for toxicity assays, SM10 for conjugal transfer of plasmids into P. fluorescens , and Shuffle T7 pLysS Express (New England Biolabs) for purification of effectors. .. Growth conditions for all strains and plasmid and strain construction details are described in .

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
    Article Snippet: The most commonly used bacterial strain for expression of genes cloned into the pET vector system are the BL21(DE3) and E. coli K12 lineage strains [ ]. .. To overcome this, we turned to a BL21(DE3) derivative known as SHuffle® T7 Express Competent E. coli (New England Biolabs #C3029J, Ipswich, MA).

    Article Title: Novel, Anti-hTNF-α Variable New Antigen Receptor Formats with Enhanced Neutralizing Potency and Multifunctionality, Generated for Therapeutic Development
    Article Snippet: Expression in E. coli HB2151 cells was induced with 1 mM isopropyl β-d -1-thiogalactopyranoside (IPTG), and soluble VNAR protein was extracted from the periplasm (E. coli HB2151 cells) ( ). .. All multivalent non-Fc VNAR constructs were expressed as cytoplasmic protein in IPTG-induced E. coli SHuffle® T7 Express cells (NEB) using pET28b(+) expression vector. .. Extraction of cytoplasmic VNAR protein was achieved using the BugBuster™ protein extraction reagent plus Benzonase® (Novagen).

    Functional Assay:

    Article Title: C9orf72, a protein associated with amyotrophic lateral sclerosis (ALS) is a guanine nucleotide exchange factor
    Article Snippet: A selection of bacterial expression strains was tried (such as Shuffle T7 express and Nico21(DE3) (from New England Biolabs, Hitchin, UK), Rosetta strains, Rosettagami strains (from Novagen, Watford, UK), SoluBL21 (from Amsbio, Abingdon, UK)), along with a variety of media types and expression trials varying different parameters, but none managed to improve expression levels or produced protein that was stable. .. We also prepared constructs where maltose-binding protein (MBP) and GST tags were moved to the C-terminal end of the protein but obtained similar results as above.

    Recombinant:

    Article Title: Immunizing Adult Female Mice with a TcpA-A2-CTB Chimera Provides a High Level of Protection for Their Pups in the Infant Mouse Model of Cholera
    Article Snippet: Paragraph title: Recombinant protein production and purification ... 6His-TcpA(29–199) was produced in SHuffle™ T7 Express E. coli (NEB, Ipswich, MA) in half liter cultures of TCYM media pH 7.5 (tryptone 1%, NaCl 0.5%, yeast extract 0.5%, casamino acids 0.1%, MgSO4 0.2%) and 100 µg/ml kanamycin at 37°C, 250 rpm until cultures reached an OD600 of ∼3.0.

    Article Title: A Reexamination of Thioredoxin Reductase from Thermoplasmaacidophilum, a Thermoacidophilic Euryarchaeon, Identifies It as an NADH-Dependent Enzyme
    Article Snippet: These plasmids were designed to express recombinant proteins with an NH2 -terminal His6 -tag, followed by a TEV protease recognition site. .. Both plasmids were transformed into E. coli SHuffle T7 Express competent cells (NEB, Ipswich, MA) containing pRIL plasmid (Stratagene, La Jolla, CA).

    Article Title: SMOC Binds to Pro-EGF, but Does Not Induce Erk Phosphorylation via the EGFR
    Article Snippet: Paragraph title: Recombinant SMOC Proteins ... Full length Xenopus X SMOC-1, X SMOC-1ΔEC (25 to K309) lacking the EC domain, and X SMOC-1EC containing the EC domain only (K309 to end) in the pET- 28b(+) vector (Novagen) were expressed in the Shuffle® T7 Express E.coli strain C3029 (New England Biolabs).

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
    Article Snippet: Paragraph title: Expression of recombinant proteases using SHuffle® (NEB) E. coli cells and Bis-Tris gel analysis ... To overcome this, we turned to a BL21(DE3) derivative known as SHuffle® T7 Express Competent E. coli (New England Biolabs #C3029J, Ipswich, MA).

    Laser Capture Microdissection:

    Article Title: Genetically Enhanced Lysozyme Evades a Pathogen Derived Inhibitory Protein
    Article Snippet: E. coli strains BW25113 (F-, DE(araD-araB)567, lacZ4787(del)::rrnB-3, LAM-, rph-1, DE(rhaD-rhaB)568, hsdR514 ) and JW1631-1 (F-, DE(araD-araB)567, lacZ4787(del)::rrnB-3, LAM-, rph-1, DE(rhaD-rhaB)568, hsdR514, ΔydhA788::kan) were purchased from The Coli Genetic Stock Center at Yale University, CT, USA. ydhA was previously renamed mliC ( ). .. E. coli T7 Shuffle Express was purchased from New England Biolabs.

    Spectrophotometry:

    Article Title: A Reexamination of Thioredoxin Reductase from Thermoplasmaacidophilum, a Thermoacidophilic Euryarchaeon, Identifies It as an NADH-Dependent Enzyme
    Article Snippet: Both plasmids were transformed into E. coli SHuffle T7 Express competent cells (NEB, Ipswich, MA) containing pRIL plasmid (Stratagene, La Jolla, CA). .. For the expression of Ta-Trx, the LB media was supplemented with DTT at a final concentration of 0.2 mM.

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
    Article Snippet: To overcome this, we turned to a BL21(DE3) derivative known as SHuffle® T7 Express Competent E. coli (New England Biolabs #C3029J, Ipswich, MA). .. To overcome this, we turned to a BL21(DE3) derivative known as SHuffle® T7 Express Competent E. coli (New England Biolabs #C3029J, Ipswich, MA).

    Affinity Chromatography:

    Article Title: An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism
    Article Snippet: The resulting MBP fusions were each purified using amylose affinity chromatography. .. The gpD and scFv-D10 proteins were expressed from pET28-gpD and pET28-scFv-D10, respectively, in BL21(DE3) cells, while scFv-GCN4 and scFv-GCN4(WQL) were expressed in the cytoplasm of SHuffle T7 Express cells (New England Biolabs) from pET28-scFv-GCN4 and pET28-GCN4(WQL), respectively.

    Produced:

    Article Title: Immunizing Adult Female Mice with a TcpA-A2-CTB Chimera Provides a High Level of Protection for Their Pups in the Infant Mouse Model of Cholera
    Article Snippet: The cultures were then placed at 16°C and 250 rpm for 30 minutes to acclimate to the new temperature then induced with 0.2 mM IPTG and grown overnight at 16°C for ∼16–18 hrs. .. 6His-TcpA(29–199) was produced in SHuffle™ T7 Express E. coli (NEB, Ipswich, MA) in half liter cultures of TCYM media pH 7.5 (tryptone 1%, NaCl 0.5%, yeast extract 0.5%, casamino acids 0.1%, MgSO4 0.2%) and 100 µg/ml kanamycin at 37°C, 250 rpm until cultures reached an OD600 of ∼3.0. .. The cultures were then placed at 16°C, 250 rpm for 30 minutes, and then induced with 0.1 mM IPTG and grown overnight as above.

    Article Title: C9orf72, a protein associated with amyotrophic lateral sclerosis (ALS) is a guanine nucleotide exchange factor
    Article Snippet: The SUMO-His6 tagged C9orf72 expressed as inclusion bodies and the construct with the minimal tag (His6 ) did not express at all in E. coli . .. A selection of bacterial expression strains was tried (such as Shuffle T7 express and Nico21(DE3) (from New England Biolabs, Hitchin, UK), Rosetta strains, Rosettagami strains (from Novagen, Watford, UK), SoluBL21 (from Amsbio, Abingdon, UK)), along with a variety of media types and expression trials varying different parameters, but none managed to improve expression levels or produced protein that was stable. .. We also prepared constructs where maltose-binding protein (MBP) and GST tags were moved to the C-terminal end of the protein but obtained similar results as above.

    Article Title: Genetically Enhanced Lysozyme Evades a Pathogen Derived Inhibitory Protein
    Article Snippet: Genes coding for Ivyc and E. coli MliC were amplified from E. coli JM105, and that for Ivyp from P. aeruginosa strain PA01. .. Expression vectors pET26b-Ivyc-his, pET26b-Ivyp-his, and pET26b-MliC-his were constructed and subsequently transformed into Shuffle® T7 Express cells (New England Biolabs, USA) and produced as described previously ( ). .. GMD were produced as described in detail elsewhere , with the exception that M. luteus , instead of Staphylococcus aureus , was employed as the target bacteria.

    Concentration Assay:

    Article Title: Expression of Functional Human Sialyltransferases ST3Gal1 and ST6Gal1 in Escherichia coli
    Article Snippet: pMAL constructs were expressed as N -terminus MBP fusion proteins carrying a C -terminal His tag and selected on medium containing ampicillin (200 μg/ml final concentration). pET28b+ constructs were expressed as N -terminus His tagged enzymes and selected on kanamycin (30 μg/ml final concentration). .. Plasmids were transformed for expression in E . coli BL21 (New England BioLabs), SHuffle T7 Express (New England BioLabs) and Origami 2 DE3 (Novagen, Darmstadt, Germany) and plated onto LB agar plates containing the appropriate antibiotic.

    Article Title: A Reexamination of Thioredoxin Reductase from Thermoplasmaacidophilum, a Thermoacidophilic Euryarchaeon, Identifies It as an NADH-Dependent Enzyme
    Article Snippet: Both plasmids were transformed into E. coli SHuffle T7 Express competent cells (NEB, Ipswich, MA) containing pRIL plasmid (Stratagene, La Jolla, CA). .. Both plasmids were transformed into E. coli SHuffle T7 Express competent cells (NEB, Ipswich, MA) containing pRIL plasmid (Stratagene, La Jolla, CA).

    Marker:

    Article Title: A water-soluble DsbB variant that catalyzes disulfide bond formation in vivo
    Article Snippet: The following E. coli strains were used in this study: DHB4 (MC1000 ΔphoA (PvuII) phoR ΔmalF3 F’[lac +(lacI Q ) pro ]), SHuffle T7 Express (New England Biolabs), and BL21(DE3) (Novagen). .. DHB4 ΔdsbAB strain was derived from DHB4 by insertional inactivation of the dsbA and dsbB genes with the kanamycin resistance gene as described previously .

    Staining:

    Article Title: Nanobody‐mediated resistance to Grapevine fanleaf virus in plants
    Article Snippet: Expression was performed in E. coli SHuffle T7 Express (New England Biolabs, France) grown in TB medium and induced overnight with 0.1 mm IPTG at 20 °C. .. Pelleted cells resuspended in PB‐NaCl buffer (10 mm phosphate buffer, 300 mm NaCl, pH 7.4) were lysed by sonication (80% amplitude for 2 min with 13 mm diameter probe, Vibra‐Cell VCX 500 (Sonics & Materials Inc) before purification of cytoplasmic extract.

    Positron Emission Tomography:

    Article Title: A water-soluble DsbB variant that catalyzes disulfide bond formation in vivo
    Article Snippet: The following E. coli strains were used in this study: DHB4 (MC1000 ΔphoA (PvuII) phoR ΔmalF3 F’[lac +(lacI Q ) pro ]), SHuffle T7 Express (New England Biolabs), and BL21(DE3) (Novagen). .. Briefly, this was accomplished by P1 phage transduction using the BW25113 dsbA ::kan and BW25113 dsbB ::kan strains from the Keio collection as donors and plasmid pCP20 to remove the Kan marker as needed.

    Article Title: Nanobody‐mediated resistance to Grapevine fanleaf virus in plants
    Article Snippet: For Nb23:EGFP production, the whole Nb23:EGFP:6‐His coding sequence was cloned into the pET‐22b(+) expression vector (Novagen, Madison) as a NdeI/XhoI fragment subsequent to a PCR amplification using pEAQ‐HT‐DEST3‐Nb23:EGFP as template. .. Expression was performed in E. coli SHuffle T7 Express (New England Biolabs, France) grown in TB medium and induced overnight with 0.1 mm IPTG at 20 °C.

    Article Title: C9orf72, a protein associated with amyotrophic lateral sclerosis (ALS) is a guanine nucleotide exchange factor
    Article Snippet: The C9orf72 gene was initially cloned into a variety of vectors such as pTriEx-4 Ek/LIC (N-terminal His6 Tag), pET-SUMO (N-terminal SUMO-His6 tag), pGEX-6p-1 (N-terminal glutathione S-transferase (GST) tag), as well as a modified version of pMAL-p2X (N-terminal His6 -MBP tag). .. A selection of bacterial expression strains was tried (such as Shuffle T7 express and Nico21(DE3) (from New England Biolabs, Hitchin, UK), Rosetta strains, Rosettagami strains (from Novagen, Watford, UK), SoluBL21 (from Amsbio, Abingdon, UK)), along with a variety of media types and expression trials varying different parameters, but none managed to improve expression levels or produced protein that was stable.

    Article Title: Functional and structural insight into properdin control of complement alternative pathway amplification
    Article Snippet: The human C3 C345c domain (Ala1492‐Asn1641) cDNA was synthesized (GenScript) with an N‐terminal TEV cleavage site and cloned into the pET‐32a(+) expression vector (Novagen) downstream of thioredoxin A (TrxA) and a His‐tag using BamHI and HindIII restriction sites. .. Protein was expressed in the E. coli SHuffle T7 strain (New England Biolabs) in LB Broth containing 2% (w/v) glucose and 100 μg/ml ampicillin.

    Article Title: An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism
    Article Snippet: Gcn4 and JunLZ were expressed as MBP fusions from pET28-MBP-Gcn4 and pET-MBP-JunLZ, respectively, in the cytoplasm of E. coli BL21(DE3) cells. .. The gpD and scFv-D10 proteins were expressed from pET28-gpD and pET28-scFv-D10, respectively, in BL21(DE3) cells, while scFv-GCN4 and scFv-GCN4(WQL) were expressed in the cytoplasm of SHuffle T7 Express cells (New England Biolabs) from pET28-scFv-GCN4 and pET28-GCN4(WQL), respectively.

    Article Title: SMOC Binds to Pro-EGF, but Does Not Induce Erk Phosphorylation via the EGFR
    Article Snippet: In an attempt to identify potential cell surface receptor(s) that are activated by SMOC we conducted a phosphoproteomic screen of HEK293 cells following exposure to X SMOC-1 protein. .. Full length Xenopus X SMOC-1, X SMOC-1ΔEC (25 to K309) lacking the EC domain, and X SMOC-1EC containing the EC domain only (K309 to end) in the pET- 28b(+) vector (Novagen) were expressed in the Shuffle® T7 Express E.coli strain C3029 (New England Biolabs). .. Following bacterial cell lysis solubilized inclusion bodies were applied to and eluted from Ni-NTA agarose (Qiagen).

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
    Article Snippet: The most commonly used bacterial strain for expression of genes cloned into the pET vector system are the BL21(DE3) and E. coli K12 lineage strains [ ]. .. To overcome this, we turned to a BL21(DE3) derivative known as SHuffle® T7 Express Competent E. coli (New England Biolabs #C3029J, Ipswich, MA).

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    New England Biolabs shuffle t7 express e coli strain c3029
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