shuffle t7 express competent e coli  (New England Biolabs)


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    Name:
    SHuffle T7 Express Competent E coli
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    SHuffle T7 Express Competent E coli 12x0 05 ml
    Catalog Number:
    c3029j
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    12x0 05 ml
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    Competent Bacteria
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    New England Biolabs shuffle t7 express competent e coli
    SHuffle T7 Express Competent E coli
    SHuffle T7 Express Competent E coli 12x0 05 ml
    https://www.bioz.com/result/shuffle t7 express competent e coli/product/New England Biolabs
    Average 95 stars, based on 70 article reviews
    Price from $9.99 to $1999.99
    shuffle t7 express competent e coli - by Bioz Stars, 2020-02
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    1) Product Images from "Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host"

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host

    Journal: BMC Biochemistry

    doi: 10.1186/s12858-018-0101-0

    Large-scale soluble expression of recombinant AaLT-NL zymogen protease grown in TB media at 10 °C (induced with 0.1 mM IPTG). Plasmid construct was transformed into SHuffle® T7 Express Competent E. coli cells (NEB). Samples were collected at the given time points (in hours). The MW ladder is in kilo-Daltons (kDa). Gel analysis of samples collected from the growth of AaLT was first visualized using InVision™ His-Tag In-Gel Stain (Invitrogen), which specifically chelates to and enhances the fluorescence of poly his-tagged proteins (top figure). The His-Tag stain is the positive identification that the bands expressed in the gel below are indeed the expression of soluble AaLT-zymogen (MW ~ 27.6 kDa, red arrows). The growth was extended beyond 24 h due to the 10 °C growth conditions, which helped in solubly expressing the protease, but also to increase bacterial cell density in order to obtain a large quantity of cell paste for purification
    Figure Legend Snippet: Large-scale soluble expression of recombinant AaLT-NL zymogen protease grown in TB media at 10 °C (induced with 0.1 mM IPTG). Plasmid construct was transformed into SHuffle® T7 Express Competent E. coli cells (NEB). Samples were collected at the given time points (in hours). The MW ladder is in kilo-Daltons (kDa). Gel analysis of samples collected from the growth of AaLT was first visualized using InVision™ His-Tag In-Gel Stain (Invitrogen), which specifically chelates to and enhances the fluorescence of poly his-tagged proteins (top figure). The His-Tag stain is the positive identification that the bands expressed in the gel below are indeed the expression of soluble AaLT-zymogen (MW ~ 27.6 kDa, red arrows). The growth was extended beyond 24 h due to the 10 °C growth conditions, which helped in solubly expressing the protease, but also to increase bacterial cell density in order to obtain a large quantity of cell paste for purification

    Techniques Used: Expressing, Recombinant, Plasmid Preparation, Construct, Transformation Assay, Staining, Fluorescence, Purification

    Initial attempt at solubly expressing recombinant midgut proteases in SHuffle® T7 Express Competent E. coli cells (NEB). For each growth experiment, TB media and a 30 °C growth temperature was used. Cells were induced with 0.1 mM IPTG when reaching the log phase (OD 600nm ~ 0.5–1.0). Samples were collected at the given time points (in hours) and prepared for SDS-PAGE analysis. The MW ladder is in kilo-Daltons (kDa). In all cases, the arrow indicates where the expected soluble over-expressed protease should appear. However, all proteases under these conditions were expressed insolubly, only observed in the total samples. a 4–12% BIS-TRIS gel over-expression of AaET grown for a total of 26 h. The MW of the his 6 -tagged AaET-NL zymogen is ~ 27.0 kDa. b 12% BIS-TRIS gel over-expression of AaSPVI grown for a total of 4 h. The MW of the his 6 -tagged AaSPVI-NL zymogen is ~ 28.7 kDa. c 12% BIS-TRIS gel over-expression of AaSPVII grown for a total of 4 h. The MW of the his 6 -tagged AaSPVII-NL zymogen is ~ 28.7 kDa. d 12% BIS-TRIS gel over-expression of AaLT grown for a total of 4 h. The MW of the his 6 -tagged AaLT-NL zymogen is ~ 27.6 kDa
    Figure Legend Snippet: Initial attempt at solubly expressing recombinant midgut proteases in SHuffle® T7 Express Competent E. coli cells (NEB). For each growth experiment, TB media and a 30 °C growth temperature was used. Cells were induced with 0.1 mM IPTG when reaching the log phase (OD 600nm ~ 0.5–1.0). Samples were collected at the given time points (in hours) and prepared for SDS-PAGE analysis. The MW ladder is in kilo-Daltons (kDa). In all cases, the arrow indicates where the expected soluble over-expressed protease should appear. However, all proteases under these conditions were expressed insolubly, only observed in the total samples. a 4–12% BIS-TRIS gel over-expression of AaET grown for a total of 26 h. The MW of the his 6 -tagged AaET-NL zymogen is ~ 27.0 kDa. b 12% BIS-TRIS gel over-expression of AaSPVI grown for a total of 4 h. The MW of the his 6 -tagged AaSPVI-NL zymogen is ~ 28.7 kDa. c 12% BIS-TRIS gel over-expression of AaSPVII grown for a total of 4 h. The MW of the his 6 -tagged AaSPVII-NL zymogen is ~ 28.7 kDa. d 12% BIS-TRIS gel over-expression of AaLT grown for a total of 4 h. The MW of the his 6 -tagged AaLT-NL zymogen is ~ 27.6 kDa

    Techniques Used: Expressing, Recombinant, SDS Page, Over Expression

    Soluble expression of recombinant AaET-NL and AaSPVII-NL zymogen proteases grown in TB media at 23 °C post-induction (induced with 0.1 mM IPTG). Plasmid constructs were transformed into SHuffle® T7 Express Competent E. coli cells (NEB). The MW ladder is in kilo-Daltons (kDa). a Western blot analysis utilizing an AaET-specific antibody of soluble samples collected from the growth and expression of AaET (a total of 4 h post-induction). The zymogen (inactive form of the protease) is observed in the first 2 h (MW ~ 27.0 kDa, red arrow), but a second species hypothesized to be the active mature form begins to appear at the two-hour time-point (MW ~ 22.4 kDa, green arrow) while the zymogen completely disappears by the third hour post-induction. b Large scale expression analysis of AaSPVII-zymogen grown for a total of 5 h post-induction. A single band at ~ 28.7 kDa (orange arrow) is observed to be increasing over time after induction with no observable band present in both the total and soluble pre-induction samples ( t = 0 h)
    Figure Legend Snippet: Soluble expression of recombinant AaET-NL and AaSPVII-NL zymogen proteases grown in TB media at 23 °C post-induction (induced with 0.1 mM IPTG). Plasmid constructs were transformed into SHuffle® T7 Express Competent E. coli cells (NEB). The MW ladder is in kilo-Daltons (kDa). a Western blot analysis utilizing an AaET-specific antibody of soluble samples collected from the growth and expression of AaET (a total of 4 h post-induction). The zymogen (inactive form of the protease) is observed in the first 2 h (MW ~ 27.0 kDa, red arrow), but a second species hypothesized to be the active mature form begins to appear at the two-hour time-point (MW ~ 22.4 kDa, green arrow) while the zymogen completely disappears by the third hour post-induction. b Large scale expression analysis of AaSPVII-zymogen grown for a total of 5 h post-induction. A single band at ~ 28.7 kDa (orange arrow) is observed to be increasing over time after induction with no observable band present in both the total and soluble pre-induction samples ( t = 0 h)

    Techniques Used: Expressing, Recombinant, Plasmid Preparation, Construct, Transformation Assay, Western Blot

    2) Product Images from "Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host"

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host

    Journal: BMC Biochemistry

    doi: 10.1186/s12858-018-0101-0

    Large-scale soluble expression of recombinant AaLT-NL zymogen protease grown in TB media at 10 °C (induced with 0.1 mM IPTG). Plasmid construct was transformed into SHuffle® T7 Express Competent E. coli cells (NEB). Samples were collected at the given time points (in hours). The MW ladder is in kilo-Daltons (kDa). Gel analysis of samples collected from the growth of AaLT was first visualized using InVision™ His-Tag In-Gel Stain (Invitrogen), which specifically chelates to and enhances the fluorescence of poly his-tagged proteins (top figure). The His-Tag stain is the positive identification that the bands expressed in the gel below are indeed the expression of soluble AaLT-zymogen (MW ~ 27.6 kDa, red arrows). The growth was extended beyond 24 h due to the 10 °C growth conditions, which helped in solubly expressing the protease, but also to increase bacterial cell density in order to obtain a large quantity of cell paste for purification
    Figure Legend Snippet: Large-scale soluble expression of recombinant AaLT-NL zymogen protease grown in TB media at 10 °C (induced with 0.1 mM IPTG). Plasmid construct was transformed into SHuffle® T7 Express Competent E. coli cells (NEB). Samples were collected at the given time points (in hours). The MW ladder is in kilo-Daltons (kDa). Gel analysis of samples collected from the growth of AaLT was first visualized using InVision™ His-Tag In-Gel Stain (Invitrogen), which specifically chelates to and enhances the fluorescence of poly his-tagged proteins (top figure). The His-Tag stain is the positive identification that the bands expressed in the gel below are indeed the expression of soluble AaLT-zymogen (MW ~ 27.6 kDa, red arrows). The growth was extended beyond 24 h due to the 10 °C growth conditions, which helped in solubly expressing the protease, but also to increase bacterial cell density in order to obtain a large quantity of cell paste for purification

    Techniques Used: Expressing, Recombinant, Plasmid Preparation, Construct, Transformation Assay, Staining, Fluorescence, Purification

    Initial attempt at solubly expressing recombinant midgut proteases in SHuffle® T7 Express Competent E. coli cells (NEB). For each growth experiment, TB media and a 30 °C growth temperature was used. Cells were induced with 0.1 mM IPTG when reaching the log phase (OD 600nm ~ 0.5–1.0). Samples were collected at the given time points (in hours) and prepared for SDS-PAGE analysis. The MW ladder is in kilo-Daltons (kDa). In all cases, the arrow indicates where the expected soluble over-expressed protease should appear. However, all proteases under these conditions were expressed insolubly, only observed in the total samples. a 4–12% BIS-TRIS gel over-expression of AaET grown for a total of 26 h. The MW of the his 6 -tagged AaET-NL zymogen is ~ 27.0 kDa. b 12% BIS-TRIS gel over-expression of AaSPVI grown for a total of 4 h. The MW of the his 6 -tagged AaSPVI-NL zymogen is ~ 28.7 kDa. c 12% BIS-TRIS gel over-expression of AaSPVII grown for a total of 4 h. The MW of the his 6 -tagged AaSPVII-NL zymogen is ~ 28.7 kDa. d 12% BIS-TRIS gel over-expression of AaLT grown for a total of 4 h. The MW of the his 6 -tagged AaLT-NL zymogen is ~ 27.6 kDa
    Figure Legend Snippet: Initial attempt at solubly expressing recombinant midgut proteases in SHuffle® T7 Express Competent E. coli cells (NEB). For each growth experiment, TB media and a 30 °C growth temperature was used. Cells were induced with 0.1 mM IPTG when reaching the log phase (OD 600nm ~ 0.5–1.0). Samples were collected at the given time points (in hours) and prepared for SDS-PAGE analysis. The MW ladder is in kilo-Daltons (kDa). In all cases, the arrow indicates where the expected soluble over-expressed protease should appear. However, all proteases under these conditions were expressed insolubly, only observed in the total samples. a 4–12% BIS-TRIS gel over-expression of AaET grown for a total of 26 h. The MW of the his 6 -tagged AaET-NL zymogen is ~ 27.0 kDa. b 12% BIS-TRIS gel over-expression of AaSPVI grown for a total of 4 h. The MW of the his 6 -tagged AaSPVI-NL zymogen is ~ 28.7 kDa. c 12% BIS-TRIS gel over-expression of AaSPVII grown for a total of 4 h. The MW of the his 6 -tagged AaSPVII-NL zymogen is ~ 28.7 kDa. d 12% BIS-TRIS gel over-expression of AaLT grown for a total of 4 h. The MW of the his 6 -tagged AaLT-NL zymogen is ~ 27.6 kDa

    Techniques Used: Expressing, Recombinant, SDS Page, Over Expression

    Soluble expression of recombinant AaET-NL and AaSPVII-NL zymogen proteases grown in TB media at 23 °C post-induction (induced with 0.1 mM IPTG). Plasmid constructs were transformed into SHuffle® T7 Express Competent E. coli cells (NEB). The MW ladder is in kilo-Daltons (kDa). a Western blot analysis utilizing an AaET-specific antibody of soluble samples collected from the growth and expression of AaET (a total of 4 h post-induction). The zymogen (inactive form of the protease) is observed in the first 2 h (MW ~ 27.0 kDa, red arrow), but a second species hypothesized to be the active mature form begins to appear at the two-hour time-point (MW ~ 22.4 kDa, green arrow) while the zymogen completely disappears by the third hour post-induction. b Large scale expression analysis of AaSPVII-zymogen grown for a total of 5 h post-induction. A single band at ~ 28.7 kDa (orange arrow) is observed to be increasing over time after induction with no observable band present in both the total and soluble pre-induction samples ( t = 0 h)
    Figure Legend Snippet: Soluble expression of recombinant AaET-NL and AaSPVII-NL zymogen proteases grown in TB media at 23 °C post-induction (induced with 0.1 mM IPTG). Plasmid constructs were transformed into SHuffle® T7 Express Competent E. coli cells (NEB). The MW ladder is in kilo-Daltons (kDa). a Western blot analysis utilizing an AaET-specific antibody of soluble samples collected from the growth and expression of AaET (a total of 4 h post-induction). The zymogen (inactive form of the protease) is observed in the first 2 h (MW ~ 27.0 kDa, red arrow), but a second species hypothesized to be the active mature form begins to appear at the two-hour time-point (MW ~ 22.4 kDa, green arrow) while the zymogen completely disappears by the third hour post-induction. b Large scale expression analysis of AaSPVII-zymogen grown for a total of 5 h post-induction. A single band at ~ 28.7 kDa (orange arrow) is observed to be increasing over time after induction with no observable band present in both the total and soluble pre-induction samples ( t = 0 h)

    Techniques Used: Expressing, Recombinant, Plasmid Preparation, Construct, Transformation Assay, Western Blot

    Related Articles

    Transduction:

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    Article Snippet: Strains and plasmids E. coli strain MC4100 (F− araD139 Δ(argF-lac )U169 flbB5301 deoC1 ptsF25 relA1 rbsR22 rpsL150 thiA ), SHuffle T7 Express (New England Biolabs), and Origami2(DE3) (Novagen) were used for heterologous expression of different eukaryotic GnTs. .. This strain was created by introducing sequential mutations using P1vir phage transduction with the respective strains from the Keio collection as donors.

    Article Title: A water-soluble DsbB variant that catalyzes disulfide bond formation in vivo
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    Clone Assay:

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    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
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    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
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    Centrifugation:

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    Article Title: MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP
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    Amplification:

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    Stable Transfection:

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    Construct:

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    Incubation:

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    Solubility:

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    Expressing:

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    Article Title: Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application
    Article Snippet: Paragraph title: Protein Expression and Purification ... Recombinant disulfide mutants were expressed from SHuffle T7 Express E coli (New England BioLabs, Ipswich, MA) and Luria broth media.

    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: Fusion to Tetrahymena thermophila granule lattice protein 1 confers solubility to sexual stage malaria antigens in Escherichia coli
    Article Snippet: Paragraph title: 2.3. Expression and protein purification ... The E. coli SHuffle strain (New England Biolabs, catalog #C3029), which has been engineered for improved production of disulfide-bonded proteins in the cytoplasm [ ], was transformed with individual plasmids and grown at 30 °C unless otherwise stated.

    Article Title: MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP
    Article Snippet: Paragraph title: Protein expression and purification ... Full-length mouse MANF (wild-type and mutants; UK2006, UK2209, UK2210, UK2212, UK2280) and SAP domain (126-169; UK2079) were expressed in the Origami B (DE3) E. coli strain (New England BioLabs, cat. # C3029).

    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: 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: Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation
    Article Snippet: .. This pCOLADuet-1 vector was transformed into the of E. coli SHuffle T7 Express strain (New England Biolabs) for expression. .. To make disulfide-cross-linked trimers, a gene encoding untagged A349C HslU was cloned into MCS1 of pCOLADuet-1 between the NcoI and BamHI sites, and a gene encoding His6 -ENLYFQS-E47CT361C HslU was cloned into MCS2 between the NdeI and XhoI sites.

    Article Title: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform
    Article Snippet: .. For prokaryotic expression of the α-EGFR TM the E. coli strain SHuffle® T7 (New England BioLabs GmbH, Frankfurt am Main, Germany) was transformed with the vector pET-28a_α-EGFR TM using standard heat shock method. .. One single clone of genetically modified bacteria was grown over night at 37°C in 10 mL SOC medium containing 2% [w/v] Bacto™ Trypton (BD Biosciences), 0.5% [w/v] Bacto™ Yeast (BD Biosciences), 10 mM NaCl (VWR International GmbH, Darmstadt, Germany), 2.5 mM KCl (Fluka Chemie AG, Neu-Ulm, Germany), 10 mM MgCl2 (VWR International GmbH), 10 mM MgSO4 (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), 20 mM glucose (Carl Roth GmbH & Co. KG, Karlsruhe, Deutschland), and 10 μg/mL kanamycin (Carl Roth GmbH & Co. KG).

    Transformation Assay:

    Article Title: MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked
    Article Snippet: .. E. coli Shuffle T7 (NEB, C3026H) cells transformed with a TFMI-3 variant construct were grown in LB medium at 30 °C. .. The expression was induced at OD600 = 0.8 by 0.4 mM IPTG and was continued O/N at 18 °C.

    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: Fusion to Tetrahymena thermophila granule lattice protein 1 confers solubility to sexual stage malaria antigens in Escherichia coli
    Article Snippet: .. The E. coli SHuffle strain (New England Biolabs, catalog #C3029), which has been engineered for improved production of disulfide-bonded proteins in the cytoplasm [ ], was transformed with individual plasmids and grown at 30 °C unless otherwise stated. ..

    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). ..

    Article Title: Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation
    Article Snippet: .. This pCOLADuet-1 vector was transformed into the of E. coli SHuffle T7 Express strain (New England Biolabs) for expression. .. To make disulfide-cross-linked trimers, a gene encoding untagged A349C HslU was cloned into MCS1 of pCOLADuet-1 between the NcoI and BamHI sites, and a gene encoding His6 -ENLYFQS-E47CT361C HslU was cloned into MCS2 between the NdeI and XhoI sites.

    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). .. 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.

    Genetically Modified:

    Article Title: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform
    Article Snippet: For prokaryotic expression of the α-EGFR TM the E. coli strain SHuffle® T7 (New England BioLabs GmbH, Frankfurt am Main, Germany) was transformed with the vector pET-28a_α-EGFR TM using standard heat shock method. .. One single clone of genetically modified bacteria was grown over night at 37°C in 10 mL SOC medium containing 2% [w/v] Bacto™ Trypton (BD Biosciences), 0.5% [w/v] Bacto™ Yeast (BD Biosciences), 10 mM NaCl (VWR International GmbH, Darmstadt, Germany), 2.5 mM KCl (Fluka Chemie AG, Neu-Ulm, Germany), 10 mM MgCl2 (VWR International GmbH), 10 mM MgSO4 (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), 20 mM glucose (Carl Roth GmbH & Co. KG, Karlsruhe, Deutschland), and 10 μg/mL kanamycin (Carl Roth GmbH & Co. KG).

    Derivative Assay:

    Article Title: A water-soluble DsbB variant that catalyzes disulfide bond formation in vivo
    Article Snippet: Bacterial strains and plasmids 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 .

    Chromatography:

    Article Title: MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked
    Article Snippet: The construct enabled high level expression of the fusion proteins in E. coli Shuffle T7 (NEB, C3026H) cells, purification through Ni-NTA chromatography, and liberation of TFMI-3 through TEV protease processing as follows. .. E. coli Shuffle T7 (NEB, C3026H) cells transformed with a TFMI-3 variant construct were grown in LB medium at 30 °C.

    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 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).

    Introduce:

    Article Title: Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application
    Article Snippet: The QuickChange™ site-directed mutagenesis protocol (Agilent Technologies, Santa Clara, CA) was used to introduce all FGF mutations and were confirmed by DNA sequencing (Biomolecular Analysis Synthesis and Sequencing Laboratory, Florida State University). .. Recombinant disulfide mutants were expressed from SHuffle T7 Express E coli (New England BioLabs, Ipswich, MA) and Luria broth media.

    Generated:

    Article Title: A library of chemically defined human N-glycans synthesized from microbial oligosaccharide precursors
    Article Snippet: Strains and plasmids E. coli strain MC4100 (F− araD139 Δ(argF-lac )U169 flbB5301 deoC1 ptsF25 relA1 rbsR22 rpsL150 thiA ), SHuffle T7 Express (New England Biolabs), and Origami2(DE3) (Novagen) were used for heterologous expression of different eukaryotic GnTs. .. Briefly, to delete the nanA gene a lysate was generated from donor cells (JW31194-1) containing the ΔnanA 735::kan mutation.

    Article Title: A water-soluble DsbB variant that catalyzes disulfide bond formation in vivo
    Article Snippet: Bacterial strains and plasmids 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 DHB4(DE3) and DHB4(DE3) ΔdsbAB strains were generated using a λDE3 lysogenization kit (Novagen) according to manufacturer’s instructions and positive clones identified based on the ability to produce active GFP from plasmid pET-GFP .

    other:

    Article Title: An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism
    Article Snippet: The bispecific proteins Fv2 and Fv2 (WQL) were expressed in the cytoplasm of SHuffle T7 Express cells from pET28- Fv2 and pET28-Fv2 (WQL), respectively.

    DNA Sequencing:

    Article Title: Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application
    Article Snippet: The QuickChange™ site-directed mutagenesis protocol (Agilent Technologies, Santa Clara, CA) was used to introduce all FGF mutations and were confirmed by DNA sequencing (Biomolecular Analysis Synthesis and Sequencing Laboratory, Florida State University). .. Recombinant disulfide mutants were expressed from SHuffle T7 Express E coli (New England BioLabs, Ipswich, MA) and Luria broth media.

    Sequencing:

    Article Title: Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application
    Article Snippet: The QuickChange™ site-directed mutagenesis protocol (Agilent Technologies, Santa Clara, CA) was used to introduce all FGF mutations and were confirmed by DNA sequencing (Biomolecular Analysis Synthesis and Sequencing Laboratory, Florida State University). .. Recombinant disulfide mutants were expressed from SHuffle T7 Express E coli (New England BioLabs, Ipswich, MA) and Luria broth media.

    Article Title: Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation
    Article Snippet: To make disulfide-cross-linked dimers, a gene encoding untagged E47C HslU was cloned into the first multiple cloning site (MCS1) of the pCOLADuet-1 (Novagen) vector between the NcoI and BamHI sites and a gene encoding His6- ENLYFQS-A349C HslU was cloned into MCS2 between the NdeI and XhoI sites, where His6 is the hexahistidine tag and ENLYFQS is the sequence recognized and cleaved by the tobacco etch virus protease. .. This pCOLADuet-1 vector was transformed into the of E. coli SHuffle T7 Express strain (New England Biolabs) for expression.

    Sonication:

    Article Title: MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked
    Article Snippet: E. coli Shuffle T7 (NEB, C3026H) cells transformed with a TFMI-3 variant construct were grown in LB medium at 30 °C. .. Cells were harvested by centrifugation (5 min, 7500g), resuspended in 1/10 culture volume 50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0 buffer (chromatography buffer), and disrupted by 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 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).

    Recombinant:

    Article Title: Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application
    Article Snippet: .. Recombinant disulfide mutants were expressed from SHuffle T7 Express E coli (New England BioLabs, Ipswich, MA) and Luria broth media. .. The E coli culture was incubated at 30°C until OD600 = 0.6, at which point the temperature was shifted to 20°C and 1 mM isopropyl-β-D-thio-galactoside was added to induce protein expression with overnight incubation.

    Article Title: MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP
    Article Snippet: Full-length mouse MANF (wild-type and mutants; UK2006, UK2209, UK2210, UK2212, UK2280) and SAP domain (126-169; UK2079) were expressed in the Origami B (DE3) E. coli strain (New England BioLabs, cat. # C3029). .. The bacterial cultures were grown at 37 °C to an optical density (OD600 ) of 0.8 in 2 × TY medium supplemented with 100 μg/ml ampicillin, and expression of recombinant protein was induced at 22 °C for 16 h by the addition of 0.5 mM isopropylthio β-D-1-galactopyranoside (IPTG).

    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: 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: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform
    Article Snippet: Paragraph title: Expression and purification of recombinant antibodies ... For prokaryotic expression of the α-EGFR TM the E. coli strain SHuffle® T7 (New England BioLabs GmbH, Frankfurt am Main, Germany) was transformed with the vector pET-28a_α-EGFR TM using standard heat shock method.

    Mutagenesis:

    Article Title: A library of chemically defined human N-glycans synthesized from microbial oligosaccharide precursors
    Article Snippet: Strains and plasmids E. coli strain MC4100 (F− araD139 Δ(argF-lac )U169 flbB5301 deoC1 ptsF25 relA1 rbsR22 rpsL150 thiA ), SHuffle T7 Express (New England Biolabs), and Origami2(DE3) (Novagen) were used for heterologous expression of different eukaryotic GnTs. .. Briefly, to delete the nanA gene a lysate was generated from donor cells (JW31194-1) containing the ΔnanA 735::kan mutation.

    Article Title: MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked
    Article Snippet: A P1 Glu mutant of the HA-tagged inhibitor (TFMI-3_K135E_HA) was created using overlap extension PCR. .. E. coli Shuffle T7 (NEB, C3026H) cells transformed with a TFMI-3 variant construct were grown in LB medium at 30 °C.

    Article Title: Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application
    Article Snippet: The QuickChange™ site-directed mutagenesis protocol (Agilent Technologies, Santa Clara, CA) was used to introduce all FGF mutations and were confirmed by DNA sequencing (Biomolecular Analysis Synthesis and Sequencing Laboratory, Florida State University). .. Recombinant disulfide mutants were expressed from SHuffle T7 Express E coli (New England BioLabs, Ipswich, MA) and Luria broth media.

    Article Title: Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation
    Article Snippet: HslU mutants used to make disulfide-cross-linked pseudohexamers were constructed in the cysteine-free C262A/C288S HslU background with or without the E257Q mutation. .. This pCOLADuet-1 vector was transformed into the of E. coli SHuffle T7 Express strain (New England Biolabs) for expression.

    Purification:

    Article Title: MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked
    Article Snippet: Paragraph title: Construction, expression and purification of TFMI-3 variants ... E. coli Shuffle T7 (NEB, C3026H) cells transformed with a TFMI-3 variant construct were grown in LB medium at 30 °C.

    Article Title: Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application
    Article Snippet: Paragraph title: Protein Expression and Purification ... Recombinant disulfide mutants were expressed from SHuffle T7 Express E coli (New England BioLabs, Ipswich, MA) and Luria broth media.

    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: Fusion to Tetrahymena thermophila granule lattice protein 1 confers solubility to sexual stage malaria antigens in Escherichia coli
    Article Snippet: The E. coli SHuffle strain (New England Biolabs, catalog #C3029), which has been engineered for improved production of disulfide-bonded proteins in the cytoplasm [ ], was transformed with individual plasmids and grown at 30 °C unless otherwise stated. .. Bacterial cells were collected by centrifugation and pellets were stored at −80 °C until purification (for proteins induced at 30 °C, except Pfs25 induced at 16 °C) or solubility analysis (for proteins induced at 30 °C and 16 °C).

    Article Title: MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP
    Article Snippet: Paragraph title: Protein expression and purification ... Full-length mouse MANF (wild-type and mutants; UK2006, UK2209, UK2210, UK2212, UK2280) and SAP domain (126-169; UK2079) were expressed in the Origami B (DE3) E. coli strain (New England BioLabs, cat. # C3029).

    Article Title: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform
    Article Snippet: Paragraph title: Expression and purification of recombinant antibodies ... For prokaryotic expression of the α-EGFR TM the E. coli strain SHuffle® T7 (New England BioLabs GmbH, Frankfurt am Main, Germany) was transformed with the vector pET-28a_α-EGFR TM using standard heat shock method.

    Protein Purification:

    Article Title: Fusion to Tetrahymena thermophila granule lattice protein 1 confers solubility to sexual stage malaria antigens in Escherichia coli
    Article Snippet: Paragraph title: 2.3. Expression and protein purification ... The E. coli SHuffle strain (New England Biolabs, catalog #C3029), which has been engineered for improved production of disulfide-bonded proteins in the cytoplasm [ ], was transformed with individual plasmids and grown at 30 °C unless otherwise stated.

    Article Title: Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation
    Article Snippet: Paragraph title: Cloning, expression, and protein purification ... This pCOLADuet-1 vector was transformed into the of E. coli SHuffle T7 Express strain (New England Biolabs) for expression.

    Polymerase Chain Reaction:

    Article Title: MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked
    Article Snippet: A P1 Glu mutant of the HA-tagged inhibitor (TFMI-3_K135E_HA) was created using overlap extension PCR. .. E. coli Shuffle T7 (NEB, C3026H) cells transformed with a TFMI-3 variant construct were grown in LB medium at 30 °C.

    Cell Culture:

    Article Title: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform
    Article Snippet: Purification of the his-tagged recombinant proteins from cell culture supernatant was performed by affinity chromatography on Ni-NTA columns (Qiagen, Hilden, Germany) according to previously published protocols. .. For prokaryotic expression of the α-EGFR TM the E. coli strain SHuffle® T7 (New England BioLabs GmbH, Frankfurt am Main, Germany) was transformed with the vector pET-28a_α-EGFR TM using standard heat shock method.

    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 lysate was centrifuged and then filtered through 0.45 μm filter before loading onto HisTrap FF column (GE Healthcare) using ÄKTA FPLC system (GE Healthcare).

    SDS Page:

    Article Title: Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application
    Article Snippet: Recombinant disulfide mutants were expressed from SHuffle T7 Express E coli (New England BioLabs, Ipswich, MA) and Luria broth media. .. Protein purity was evaluated by gel densitometry of Coomassie blue–stained SDS-PAGE.

    Plasmid Preparation:

    Article Title: MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked
    Article Snippet: TFMI-3 variant genes were cloned into this vector using BamHI and XhoI ( ). .. E. coli Shuffle T7 (NEB, C3026H) cells transformed with a TFMI-3 variant construct were grown in LB medium at 30 °C.

    Article Title: A water-soluble DsbB variant that catalyzes disulfide bond formation in vivo
    Article Snippet: Bacterial strains and plasmids 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: Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application
    Article Snippet: Recombinant protein expression used a pET21a(+) expression vector (EMD Millipore, Billerica, MA) with a codon-optimized synthetic gene encoding the 140 amino acid “mature” form of human WT FGF-1 and with an N-terminal 6× His tag. .. Recombinant disulfide mutants were expressed from SHuffle T7 Express E coli (New England BioLabs, Ipswich, MA) and Luria broth media.

    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: 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). ..

    Article Title: Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation
    Article Snippet: .. This pCOLADuet-1 vector was transformed into the of E. coli SHuffle T7 Express strain (New England Biolabs) for expression. .. To make disulfide-cross-linked trimers, a gene encoding untagged A349C HslU was cloned into MCS1 of pCOLADuet-1 between the NcoI and BamHI sites, and a gene encoding His6 -ENLYFQS-E47CT361C HslU was cloned into MCS2 between the NdeI and XhoI sites.

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
    Article Snippet: Expression of recombinant proteases using SHuffle® (NEB) E. coli cells and Bis-Tris gel analysis 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: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform
    Article Snippet: .. For prokaryotic expression of the α-EGFR TM the E. coli strain SHuffle® T7 (New England BioLabs GmbH, Frankfurt am Main, Germany) was transformed with the vector pET-28a_α-EGFR TM using standard heat shock method. .. One single clone of genetically modified bacteria was grown over night at 37°C in 10 mL SOC medium containing 2% [w/v] Bacto™ Trypton (BD Biosciences), 0.5% [w/v] Bacto™ Yeast (BD Biosciences), 10 mM NaCl (VWR International GmbH, Darmstadt, Germany), 2.5 mM KCl (Fluka Chemie AG, Neu-Ulm, Germany), 10 mM MgCl2 (VWR International GmbH), 10 mM MgSO4 (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), 20 mM glucose (Carl Roth GmbH & Co. KG, Karlsruhe, Deutschland), and 10 μg/mL kanamycin (Carl Roth GmbH & Co. KG).

    Positron Emission Tomography:

    Article Title: A water-soluble DsbB variant that catalyzes disulfide bond formation in vivo
    Article Snippet: Bacterial strains and plasmids 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 DHB4(DE3) and DHB4(DE3) ΔdsbAB strains were generated using a λDE3 lysogenization kit (Novagen) according to manufacturer’s instructions and positive clones identified based on the ability to produce active GFP from plasmid pET-GFP .

    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: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host
    Article Snippet: Expression of recombinant proteases using SHuffle® (NEB) E. coli cells and Bis-Tris gel analysis 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: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform
    Article Snippet: .. For prokaryotic expression of the α-EGFR TM the E. coli strain SHuffle® T7 (New England BioLabs GmbH, Frankfurt am Main, Germany) was transformed with the vector pET-28a_α-EGFR TM using standard heat shock method. .. One single clone of genetically modified bacteria was grown over night at 37°C in 10 mL SOC medium containing 2% [w/v] Bacto™ Trypton (BD Biosciences), 0.5% [w/v] Bacto™ Yeast (BD Biosciences), 10 mM NaCl (VWR International GmbH, Darmstadt, Germany), 2.5 mM KCl (Fluka Chemie AG, Neu-Ulm, Germany), 10 mM MgCl2 (VWR International GmbH), 10 mM MgSO4 (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), 20 mM glucose (Carl Roth GmbH & Co. KG, Karlsruhe, Deutschland), and 10 μg/mL kanamycin (Carl Roth GmbH & Co. KG).

    Column Chromatography:

    Article Title: Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application
    Article Snippet: Recombinant disulfide mutants were expressed from SHuffle T7 Express E coli (New England BioLabs, Ipswich, MA) and Luria broth media. .. The expressed protein was purified using sequential column chromatography on Ni-nitrilotriacetic acid affinity resin (Qiagen, Valencia, CA) and heparin Sepharose resin (GE Life Sciences, Pittsburgh, PA).

    Spectrophotometry:

    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). .. For small or large-scale growth expression, a single colony from transformed overnight plates was selected to set overnight liquid cultures in LB media supplemented with kanamycin (30 μg/ml) and set in a 30 °C shaker (250 rpm) for 16 to 18 h. The optical density at 600 nm (OD600nm ) of the overnight cultures was determined using a spectrophotometer and used as the starter culture to initiate growth at a starting OD600nm ~ 0.05 in fresh, autoclaved media.

    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: 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). .. For small or large-scale growth expression, a single colony from transformed overnight plates was selected to set overnight liquid cultures in LB media supplemented with kanamycin (30 μg/ml) and set in a 30 °C shaker (250 rpm) for 16 to 18 h. The optical density at 600 nm (OD600nm ) of the overnight cultures was determined using a spectrophotometer and used as the starter culture to initiate growth at a starting OD600nm ~ 0.05 in fresh, autoclaved media.

    Affinity Chromatography:

    Article Title: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform
    Article Snippet: Purification of the his-tagged recombinant proteins from cell culture supernatant was performed by affinity chromatography on Ni-NTA columns (Qiagen, Hilden, Germany) according to previously published protocols. .. For prokaryotic expression of the α-EGFR TM the E. coli strain SHuffle® T7 (New England BioLabs GmbH, Frankfurt am Main, Germany) was transformed with the vector pET-28a_α-EGFR TM using standard heat shock method.

    Concentration Assay:

    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: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform
    Article Snippet: For prokaryotic expression of the α-EGFR TM the E. coli strain SHuffle® T7 (New England BioLabs GmbH, Frankfurt am Main, Germany) was transformed with the vector pET-28a_α-EGFR TM using standard heat shock method. .. Subsequently, TM expression was induced by adding isopropyl-ß-D-1-thiogalactopyranoside (Gerbu Biotechnik GmbH, Wieblingen, Germany) at a final concentration of 1 mM.

    Marker:

    Article Title: A water-soluble DsbB variant that catalyzes disulfide bond formation in vivo
    Article Snippet: Bacterial strains and plasmids 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.

    Variant Assay:

    Article Title: MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked
    Article Snippet: .. E. coli Shuffle T7 (NEB, C3026H) cells transformed with a TFMI-3 variant construct were grown in LB medium at 30 °C. .. The expression was induced at OD600 = 0.8 by 0.4 mM IPTG and was continued O/N at 18 °C.

    Article Title: Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation
    Article Snippet: This pCOLADuet-1 vector was transformed into the of E. coli SHuffle T7 Express strain (New England Biolabs) for expression. .. This vector was co-transformed with a pet12b (Novagen) vector encoding the untagged Q39C HslU variant into the SHuffle T7 Express strain.

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    New England Biolabs shuffle t7 express competent e coli
    Large-scale soluble expression of recombinant AaLT-NL zymogen protease grown in TB media at 10 °C (induced with 0.1 mM IPTG). Plasmid construct was transformed into SHuffle® <t>T7</t> Express Competent E. coli cells (NEB). Samples were collected at the given time points (in hours). The MW ladder is in kilo-Daltons (kDa). Gel analysis of samples collected from the growth of AaLT was first visualized using InVision™ His-Tag In-Gel Stain (Invitrogen), which specifically chelates to and enhances the fluorescence of poly his-tagged proteins (top figure). The His-Tag stain is the positive identification that the bands expressed in the gel below are indeed the expression of soluble AaLT-zymogen (MW ~ 27.6 kDa, red arrows). The growth was extended beyond 24 h due to the 10 °C growth conditions, which helped in solubly expressing the protease, but also to increase bacterial cell density in order to obtain a large quantity of cell paste for purification
    Shuffle T7 Express Competent E Coli, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Large-scale soluble expression of recombinant AaLT-NL zymogen protease grown in TB media at 10 °C (induced with 0.1 mM IPTG). Plasmid construct was transformed into SHuffle® T7 Express Competent E. coli cells (NEB). Samples were collected at the given time points (in hours). The MW ladder is in kilo-Daltons (kDa). Gel analysis of samples collected from the growth of AaLT was first visualized using InVision™ His-Tag In-Gel Stain (Invitrogen), which specifically chelates to and enhances the fluorescence of poly his-tagged proteins (top figure). The His-Tag stain is the positive identification that the bands expressed in the gel below are indeed the expression of soluble AaLT-zymogen (MW ~ 27.6 kDa, red arrows). The growth was extended beyond 24 h due to the 10 °C growth conditions, which helped in solubly expressing the protease, but also to increase bacterial cell density in order to obtain a large quantity of cell paste for purification

    Journal: BMC Biochemistry

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host

    doi: 10.1186/s12858-018-0101-0

    Figure Lengend Snippet: Large-scale soluble expression of recombinant AaLT-NL zymogen protease grown in TB media at 10 °C (induced with 0.1 mM IPTG). Plasmid construct was transformed into SHuffle® T7 Express Competent E. coli cells (NEB). Samples were collected at the given time points (in hours). The MW ladder is in kilo-Daltons (kDa). Gel analysis of samples collected from the growth of AaLT was first visualized using InVision™ His-Tag In-Gel Stain (Invitrogen), which specifically chelates to and enhances the fluorescence of poly his-tagged proteins (top figure). The His-Tag stain is the positive identification that the bands expressed in the gel below are indeed the expression of soluble AaLT-zymogen (MW ~ 27.6 kDa, red arrows). The growth was extended beyond 24 h due to the 10 °C growth conditions, which helped in solubly expressing the protease, but also to increase bacterial cell density in order to obtain a large quantity of cell paste for purification

    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).

    Techniques: Expressing, Recombinant, Plasmid Preparation, Construct, Transformation Assay, Staining, Fluorescence, Purification

    Initial attempt at solubly expressing recombinant midgut proteases in SHuffle® T7 Express Competent E. coli cells (NEB). For each growth experiment, TB media and a 30 °C growth temperature was used. Cells were induced with 0.1 mM IPTG when reaching the log phase (OD 600nm ~ 0.5–1.0). Samples were collected at the given time points (in hours) and prepared for SDS-PAGE analysis. The MW ladder is in kilo-Daltons (kDa). In all cases, the arrow indicates where the expected soluble over-expressed protease should appear. However, all proteases under these conditions were expressed insolubly, only observed in the total samples. a 4–12% BIS-TRIS gel over-expression of AaET grown for a total of 26 h. The MW of the his 6 -tagged AaET-NL zymogen is ~ 27.0 kDa. b 12% BIS-TRIS gel over-expression of AaSPVI grown for a total of 4 h. The MW of the his 6 -tagged AaSPVI-NL zymogen is ~ 28.7 kDa. c 12% BIS-TRIS gel over-expression of AaSPVII grown for a total of 4 h. The MW of the his 6 -tagged AaSPVII-NL zymogen is ~ 28.7 kDa. d 12% BIS-TRIS gel over-expression of AaLT grown for a total of 4 h. The MW of the his 6 -tagged AaLT-NL zymogen is ~ 27.6 kDa

    Journal: BMC Biochemistry

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host

    doi: 10.1186/s12858-018-0101-0

    Figure Lengend Snippet: Initial attempt at solubly expressing recombinant midgut proteases in SHuffle® T7 Express Competent E. coli cells (NEB). For each growth experiment, TB media and a 30 °C growth temperature was used. Cells were induced with 0.1 mM IPTG when reaching the log phase (OD 600nm ~ 0.5–1.0). Samples were collected at the given time points (in hours) and prepared for SDS-PAGE analysis. The MW ladder is in kilo-Daltons (kDa). In all cases, the arrow indicates where the expected soluble over-expressed protease should appear. However, all proteases under these conditions were expressed insolubly, only observed in the total samples. a 4–12% BIS-TRIS gel over-expression of AaET grown for a total of 26 h. The MW of the his 6 -tagged AaET-NL zymogen is ~ 27.0 kDa. b 12% BIS-TRIS gel over-expression of AaSPVI grown for a total of 4 h. The MW of the his 6 -tagged AaSPVI-NL zymogen is ~ 28.7 kDa. c 12% BIS-TRIS gel over-expression of AaSPVII grown for a total of 4 h. The MW of the his 6 -tagged AaSPVII-NL zymogen is ~ 28.7 kDa. d 12% BIS-TRIS gel over-expression of AaLT grown for a total of 4 h. The MW of the his 6 -tagged AaLT-NL zymogen is ~ 27.6 kDa

    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).

    Techniques: Expressing, Recombinant, SDS Page, Over Expression

    Soluble expression of recombinant AaET-NL and AaSPVII-NL zymogen proteases grown in TB media at 23 °C post-induction (induced with 0.1 mM IPTG). Plasmid constructs were transformed into SHuffle® T7 Express Competent E. coli cells (NEB). The MW ladder is in kilo-Daltons (kDa). a Western blot analysis utilizing an AaET-specific antibody of soluble samples collected from the growth and expression of AaET (a total of 4 h post-induction). The zymogen (inactive form of the protease) is observed in the first 2 h (MW ~ 27.0 kDa, red arrow), but a second species hypothesized to be the active mature form begins to appear at the two-hour time-point (MW ~ 22.4 kDa, green arrow) while the zymogen completely disappears by the third hour post-induction. b Large scale expression analysis of AaSPVII-zymogen grown for a total of 5 h post-induction. A single band at ~ 28.7 kDa (orange arrow) is observed to be increasing over time after induction with no observable band present in both the total and soluble pre-induction samples ( t = 0 h)

    Journal: BMC Biochemistry

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host

    doi: 10.1186/s12858-018-0101-0

    Figure Lengend Snippet: Soluble expression of recombinant AaET-NL and AaSPVII-NL zymogen proteases grown in TB media at 23 °C post-induction (induced with 0.1 mM IPTG). Plasmid constructs were transformed into SHuffle® T7 Express Competent E. coli cells (NEB). The MW ladder is in kilo-Daltons (kDa). a Western blot analysis utilizing an AaET-specific antibody of soluble samples collected from the growth and expression of AaET (a total of 4 h post-induction). The zymogen (inactive form of the protease) is observed in the first 2 h (MW ~ 27.0 kDa, red arrow), but a second species hypothesized to be the active mature form begins to appear at the two-hour time-point (MW ~ 22.4 kDa, green arrow) while the zymogen completely disappears by the third hour post-induction. b Large scale expression analysis of AaSPVII-zymogen grown for a total of 5 h post-induction. A single band at ~ 28.7 kDa (orange arrow) is observed to be increasing over time after induction with no observable band present in both the total and soluble pre-induction samples ( t = 0 h)

    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).

    Techniques: Expressing, Recombinant, Plasmid Preparation, Construct, Transformation Assay, Western Blot

    Large-scale soluble expression of recombinant AaLT-NL zymogen protease grown in TB media at 10 °C (induced with 0.1 mM IPTG). Plasmid construct was transformed into SHuffle® T7 Express Competent E. coli cells (NEB). Samples were collected at the given time points (in hours). The MW ladder is in kilo-Daltons (kDa). Gel analysis of samples collected from the growth of AaLT was first visualized using InVision™ His-Tag In-Gel Stain (Invitrogen), which specifically chelates to and enhances the fluorescence of poly his-tagged proteins (top figure). The His-Tag stain is the positive identification that the bands expressed in the gel below are indeed the expression of soluble AaLT-zymogen (MW ~ 27.6 kDa, red arrows). The growth was extended beyond 24 h due to the 10 °C growth conditions, which helped in solubly expressing the protease, but also to increase bacterial cell density in order to obtain a large quantity of cell paste for purification

    Journal: BMC Biochemistry

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host

    doi: 10.1186/s12858-018-0101-0

    Figure Lengend Snippet: Large-scale soluble expression of recombinant AaLT-NL zymogen protease grown in TB media at 10 °C (induced with 0.1 mM IPTG). Plasmid construct was transformed into SHuffle® T7 Express Competent E. coli cells (NEB). Samples were collected at the given time points (in hours). The MW ladder is in kilo-Daltons (kDa). Gel analysis of samples collected from the growth of AaLT was first visualized using InVision™ His-Tag In-Gel Stain (Invitrogen), which specifically chelates to and enhances the fluorescence of poly his-tagged proteins (top figure). The His-Tag stain is the positive identification that the bands expressed in the gel below are indeed the expression of soluble AaLT-zymogen (MW ~ 27.6 kDa, red arrows). The growth was extended beyond 24 h due to the 10 °C growth conditions, which helped in solubly expressing the protease, but also to increase bacterial cell density in order to obtain a large quantity of cell paste for purification

    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).

    Techniques: Expressing, Recombinant, Plasmid Preparation, Construct, Transformation Assay, Staining, Fluorescence, Purification

    Initial attempt at solubly expressing recombinant midgut proteases in SHuffle® T7 Express Competent E. coli cells (NEB). For each growth experiment, TB media and a 30 °C growth temperature was used. Cells were induced with 0.1 mM IPTG when reaching the log phase (OD 600nm ~ 0.5–1.0). Samples were collected at the given time points (in hours) and prepared for SDS-PAGE analysis. The MW ladder is in kilo-Daltons (kDa). In all cases, the arrow indicates where the expected soluble over-expressed protease should appear. However, all proteases under these conditions were expressed insolubly, only observed in the total samples. a 4–12% BIS-TRIS gel over-expression of AaET grown for a total of 26 h. The MW of the his 6 -tagged AaET-NL zymogen is ~ 27.0 kDa. b 12% BIS-TRIS gel over-expression of AaSPVI grown for a total of 4 h. The MW of the his 6 -tagged AaSPVI-NL zymogen is ~ 28.7 kDa. c 12% BIS-TRIS gel over-expression of AaSPVII grown for a total of 4 h. The MW of the his 6 -tagged AaSPVII-NL zymogen is ~ 28.7 kDa. d 12% BIS-TRIS gel over-expression of AaLT grown for a total of 4 h. The MW of the his 6 -tagged AaLT-NL zymogen is ~ 27.6 kDa

    Journal: BMC Biochemistry

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host

    doi: 10.1186/s12858-018-0101-0

    Figure Lengend Snippet: Initial attempt at solubly expressing recombinant midgut proteases in SHuffle® T7 Express Competent E. coli cells (NEB). For each growth experiment, TB media and a 30 °C growth temperature was used. Cells were induced with 0.1 mM IPTG when reaching the log phase (OD 600nm ~ 0.5–1.0). Samples were collected at the given time points (in hours) and prepared for SDS-PAGE analysis. The MW ladder is in kilo-Daltons (kDa). In all cases, the arrow indicates where the expected soluble over-expressed protease should appear. However, all proteases under these conditions were expressed insolubly, only observed in the total samples. a 4–12% BIS-TRIS gel over-expression of AaET grown for a total of 26 h. The MW of the his 6 -tagged AaET-NL zymogen is ~ 27.0 kDa. b 12% BIS-TRIS gel over-expression of AaSPVI grown for a total of 4 h. The MW of the his 6 -tagged AaSPVI-NL zymogen is ~ 28.7 kDa. c 12% BIS-TRIS gel over-expression of AaSPVII grown for a total of 4 h. The MW of the his 6 -tagged AaSPVII-NL zymogen is ~ 28.7 kDa. d 12% BIS-TRIS gel over-expression of AaLT grown for a total of 4 h. The MW of the his 6 -tagged AaLT-NL zymogen is ~ 27.6 kDa

    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).

    Techniques: Expressing, Recombinant, SDS Page, Over Expression

    Soluble expression of recombinant AaET-NL and AaSPVII-NL zymogen proteases grown in TB media at 23 °C post-induction (induced with 0.1 mM IPTG). Plasmid constructs were transformed into SHuffle® T7 Express Competent E. coli cells (NEB). The MW ladder is in kilo-Daltons (kDa). a Western blot analysis utilizing an AaET-specific antibody of soluble samples collected from the growth and expression of AaET (a total of 4 h post-induction). The zymogen (inactive form of the protease) is observed in the first 2 h (MW ~ 27.0 kDa, red arrow), but a second species hypothesized to be the active mature form begins to appear at the two-hour time-point (MW ~ 22.4 kDa, green arrow) while the zymogen completely disappears by the third hour post-induction. b Large scale expression analysis of AaSPVII-zymogen grown for a total of 5 h post-induction. A single band at ~ 28.7 kDa (orange arrow) is observed to be increasing over time after induction with no observable band present in both the total and soluble pre-induction samples ( t = 0 h)

    Journal: BMC Biochemistry

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host

    doi: 10.1186/s12858-018-0101-0

    Figure Lengend Snippet: Soluble expression of recombinant AaET-NL and AaSPVII-NL zymogen proteases grown in TB media at 23 °C post-induction (induced with 0.1 mM IPTG). Plasmid constructs were transformed into SHuffle® T7 Express Competent E. coli cells (NEB). The MW ladder is in kilo-Daltons (kDa). a Western blot analysis utilizing an AaET-specific antibody of soluble samples collected from the growth and expression of AaET (a total of 4 h post-induction). The zymogen (inactive form of the protease) is observed in the first 2 h (MW ~ 27.0 kDa, red arrow), but a second species hypothesized to be the active mature form begins to appear at the two-hour time-point (MW ~ 22.4 kDa, green arrow) while the zymogen completely disappears by the third hour post-induction. b Large scale expression analysis of AaSPVII-zymogen grown for a total of 5 h post-induction. A single band at ~ 28.7 kDa (orange arrow) is observed to be increasing over time after induction with no observable band present in both the total and soluble pre-induction samples ( t = 0 h)

    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).

    Techniques: Expressing, Recombinant, Plasmid Preparation, Construct, Transformation Assay, Western Blot