shuffle t7 express lysy competent e coli cells  (New England Biolabs)


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    Name:
    SHuffle T7 Express lysY Competent E coli
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    SHuffle T7 Express lysY Competent E coli 12x0 05 ml
    Catalog Number:
    c3030j
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    12x0 05 ml
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    Competent Bacteria
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    New England Biolabs shuffle t7 express lysy competent e coli cells
    SHuffle T7 Express lysY Competent E coli
    SHuffle T7 Express lysY Competent E coli 12x0 05 ml
    https://www.bioz.com/result/shuffle t7 express lysy competent e coli cells/product/New England Biolabs
    Average 95 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    shuffle t7 express lysy competent e coli cells - by Bioz Stars, 2020-02
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    Images

    1) Product Images from "Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant Pfs25, a Leading Transmission-Blocking Vaccine Candidate"

    Article Title: Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant Pfs25, a Leading Transmission-Blocking Vaccine Candidate

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00486-17

    Expression of chimeric r Pf s25/8(CΔS) and unfused r Pf s25. (A) Schematic depiction of expression constructs for the production of r Pf s25/8(CΔS) and r Pf s25. Expression plasmids for chimeric r Pf s25/8(CΔS) or unfused r Pf s25 were transformed into E. coli SHuffle T7 Express lysY cells. (B and C) r Pf s25/8(CΔS) lysates harvested before ( T 0 ) or 3 h after ( T 3 ) induction were separated by SDS-PAGE (10% gel) under reducing conditions, followed by Coomassie blue staining (B) or immunoblot analysis (C) using rabbit-anti-r Pf MSP8 IgG. The asterisk highlights r Pf s25/8(CΔS) at the predicted size. (D and E) The expression of unfused r Pf s25 was assessed as described above, on 12% polyacrylamide gels under reducing conditions, followed by Coomassie blue staining (D) or immunoblot analysis (E) using an anti-His MAb. The asterisk highlights r Pf s25 at the predicted size.
    Figure Legend Snippet: Expression of chimeric r Pf s25/8(CΔS) and unfused r Pf s25. (A) Schematic depiction of expression constructs for the production of r Pf s25/8(CΔS) and r Pf s25. Expression plasmids for chimeric r Pf s25/8(CΔS) or unfused r Pf s25 were transformed into E. coli SHuffle T7 Express lysY cells. (B and C) r Pf s25/8(CΔS) lysates harvested before ( T 0 ) or 3 h after ( T 3 ) induction were separated by SDS-PAGE (10% gel) under reducing conditions, followed by Coomassie blue staining (B) or immunoblot analysis (C) using rabbit-anti-r Pf MSP8 IgG. The asterisk highlights r Pf s25/8(CΔS) at the predicted size. (D and E) The expression of unfused r Pf s25 was assessed as described above, on 12% polyacrylamide gels under reducing conditions, followed by Coomassie blue staining (D) or immunoblot analysis (E) using an anti-His MAb. The asterisk highlights r Pf s25 at the predicted size.

    Techniques Used: Expressing, Construct, Transformation Assay, SDS Page, Staining

    2) Product Images from "A high-throughput expression screening platform to optimize the production of antimicrobial peptides"

    Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-017-0637-5

    Expression screening in E. coli . Expression screening was carried out in 96-well microtiter plates to identify high producers for three different target proteins. The promoter, protein fusion partner and E. coli strain were varied. a IMPI, b BR021, c AFP. Rosetta-gami 2 Rosetta-gami 2 (DE3) pLysS, Origami Origami 2, SHuffle SHuffle T7 Express lysY, BL21 BL21 (DE3), C41 OverExpress C41 (DE3) pLysS, C43 OverExpress C43 (DE3) pLysS
    Figure Legend Snippet: Expression screening in E. coli . Expression screening was carried out in 96-well microtiter plates to identify high producers for three different target proteins. The promoter, protein fusion partner and E. coli strain were varied. a IMPI, b BR021, c AFP. Rosetta-gami 2 Rosetta-gami 2 (DE3) pLysS, Origami Origami 2, SHuffle SHuffle T7 Express lysY, BL21 BL21 (DE3), C41 OverExpress C41 (DE3) pLysS, C43 OverExpress C43 (DE3) pLysS

    Techniques Used: Expressing

    3) Product Images from "Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli"

    Article Title: Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli

    Journal: Biotechnology Letters

    doi: 10.1007/s10529-013-1180-z

    Cytoplamic expression of proTHI-TRX fusion proteins in strain C3030. For all fusion proteins, 1 ml of cell culture was pelleted and dissolved in 100 μl sample buffer. 10 μl from this extract and an equivalent amount for total soluble cytoplasmic fractions were separated on Tricine/SDS gels. (M) Protein marker, ( 1 ) un-induced crude extract, ( 2 ) induced crude extract, ( 3 ) total soluble fraction taken after cell lysis by sonication, ( 4 ) insoluble fraction after sonication. Red stars indicate the 25 kDa fusion protein
    Figure Legend Snippet: Cytoplamic expression of proTHI-TRX fusion proteins in strain C3030. For all fusion proteins, 1 ml of cell culture was pelleted and dissolved in 100 μl sample buffer. 10 μl from this extract and an equivalent amount for total soluble cytoplasmic fractions were separated on Tricine/SDS gels. (M) Protein marker, ( 1 ) un-induced crude extract, ( 2 ) induced crude extract, ( 3 ) total soluble fraction taken after cell lysis by sonication, ( 4 ) insoluble fraction after sonication. Red stars indicate the 25 kDa fusion protein

    Techniques Used: Expressing, Cell Culture, Marker, Lysis, Sonication

    Comparison of Ni–NTA purified proTHI-TRX fusion proteins from strain C3030. a Coomassie Brilliant Blue staining. b Western blot with anti-His tag antibody. Each well contained 3 μg protein. (M) Prestained protein marker, ( 1 ) proTHI2.1-TRX, ( 2 ) proTHI2.2-TRX, ( 3 ) proTHI2.3-TRX, ( 4 ) proTHI2.4-TRX
    Figure Legend Snippet: Comparison of Ni–NTA purified proTHI-TRX fusion proteins from strain C3030. a Coomassie Brilliant Blue staining. b Western blot with anti-His tag antibody. Each well contained 3 μg protein. (M) Prestained protein marker, ( 1 ) proTHI2.1-TRX, ( 2 ) proTHI2.2-TRX, ( 3 ) proTHI2.3-TRX, ( 4 ) proTHI2.4-TRX

    Techniques Used: Purification, Staining, Western Blot, Marker

    4) Product Images from "Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant Pfs25, a Leading Transmission-Blocking Vaccine Candidate"

    Article Title: Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant Pfs25, a Leading Transmission-Blocking Vaccine Candidate

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00486-17

    Expression of chimeric r Pf s25/8(CΔS) and unfused r Pf s25. (A) Schematic depiction of expression constructs for the production of r Pf s25/8(CΔS) and r Pf s25. Expression plasmids for chimeric r Pf s25/8(CΔS) or unfused r Pf s25 were transformed into E. coli SHuffle T7 Express lysY cells. (B and C) r Pf s25/8(CΔS) lysates harvested before ( T 0 ) or 3 h after ( T 3 ) induction were separated by SDS-PAGE (10% gel) under reducing conditions, followed by Coomassie blue staining (B) or immunoblot analysis (C) using rabbit-anti-r Pf MSP8 IgG. The asterisk highlights r Pf s25/8(CΔS) at the predicted size. (D and E) The expression of unfused r Pf s25 was assessed as described above, on 12% polyacrylamide gels under reducing conditions, followed by Coomassie blue staining (D) or immunoblot analysis (E) using an anti-His MAb. The asterisk highlights r Pf s25 at the predicted size.
    Figure Legend Snippet: Expression of chimeric r Pf s25/8(CΔS) and unfused r Pf s25. (A) Schematic depiction of expression constructs for the production of r Pf s25/8(CΔS) and r Pf s25. Expression plasmids for chimeric r Pf s25/8(CΔS) or unfused r Pf s25 were transformed into E. coli SHuffle T7 Express lysY cells. (B and C) r Pf s25/8(CΔS) lysates harvested before ( T 0 ) or 3 h after ( T 3 ) induction were separated by SDS-PAGE (10% gel) under reducing conditions, followed by Coomassie blue staining (B) or immunoblot analysis (C) using rabbit-anti-r Pf MSP8 IgG. The asterisk highlights r Pf s25/8(CΔS) at the predicted size. (D and E) The expression of unfused r Pf s25 was assessed as described above, on 12% polyacrylamide gels under reducing conditions, followed by Coomassie blue staining (D) or immunoblot analysis (E) using an anti-His MAb. The asterisk highlights r Pf s25 at the predicted size.

    Techniques Used: Expressing, Construct, Transformation Assay, SDS Page, Staining

    5) Product Images from "Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli"

    Article Title: Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli

    Journal: Biotechnology Letters

    doi: 10.1007/s10529-013-1180-z

    Cytoplamic expression of proTHI-TRX fusion proteins in strain C3030. For all fusion proteins, 1 ml of cell culture was pelleted and dissolved in 100 μl sample buffer. 10 μl from this extract and an equivalent amount for total soluble cytoplasmic fractions were separated on Tricine/SDS gels. (M) Protein marker, ( 1 ) un-induced crude extract, ( 2 ) induced crude extract, ( 3 ) total soluble fraction taken after cell lysis by sonication, ( 4 ) insoluble fraction after sonication. Red stars indicate the 25 kDa fusion protein
    Figure Legend Snippet: Cytoplamic expression of proTHI-TRX fusion proteins in strain C3030. For all fusion proteins, 1 ml of cell culture was pelleted and dissolved in 100 μl sample buffer. 10 μl from this extract and an equivalent amount for total soluble cytoplasmic fractions were separated on Tricine/SDS gels. (M) Protein marker, ( 1 ) un-induced crude extract, ( 2 ) induced crude extract, ( 3 ) total soluble fraction taken after cell lysis by sonication, ( 4 ) insoluble fraction after sonication. Red stars indicate the 25 kDa fusion protein

    Techniques Used: Expressing, Cell Culture, Marker, Lysis, Sonication

    Comparison of Ni–NTA purified proTHI-TRX fusion proteins from strain C3030. a Coomassie Brilliant Blue staining. b Western blot with anti-His tag antibody. Each well contained 3 μg protein. (M) Prestained protein marker, ( 1 ) proTHI2.1-TRX, ( 2 ) proTHI2.2-TRX, ( 3 ) proTHI2.3-TRX, ( 4 ) proTHI2.4-TRX
    Figure Legend Snippet: Comparison of Ni–NTA purified proTHI-TRX fusion proteins from strain C3030. a Coomassie Brilliant Blue staining. b Western blot with anti-His tag antibody. Each well contained 3 μg protein. (M) Prestained protein marker, ( 1 ) proTHI2.1-TRX, ( 2 ) proTHI2.2-TRX, ( 3 ) proTHI2.3-TRX, ( 4 ) proTHI2.4-TRX

    Techniques Used: Purification, Staining, Western Blot, Marker

    Related Articles

    Clone Assay:

    Article Title: The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity
    Article Snippet: Two additional constructs containing mutations in cysteine 69 (C69A and C69Y) were also synthesized and cloned into the Bam HI and Xho I sites of pET21a(+) by GenScript Biotech (Piscataway, NJ, USA). .. Each expression construct was transformed into SHuffle® T7 Express lysY Escherichia coli (C3030; New England Biolabs, USA), which allows for expression of cytosolic proteins with disulfide bonds.

    Article Title: Structure of the Lectin Mannose 6-Phosphate Receptor Homology (MRH) Domain of Glucosidase II, an Enzyme That Regulates Glycoprotein Folding Quality Control in the Endoplasmic Reticulum *
    Article Snippet: Escherichia coli DH5α was used for cloning purposes. .. Recombinant GIIβ expression was performed in SHuffle T7 Express lysY competent E. coli cells (BL21 C3030, New England Biolabs).

    Article Title: Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli
    Article Snippet: .. We therefore cloned the expression plasmids for all four fusion proteins into the SHuffle strain C3030 (New England Biolabs) in which the lethal phenotype of lacking the gor and trxB reductases is suppressed by a mutation in the peroxidase ahpC (Faulkner et al. ) thus providing an oxidising environment in the cytoplasm. .. Additionally, expression of periplasmic disulfide bond isomerase (DsbC) in the cytoplasm of these strains is supposed to enhance proper disulfide bond formation (Maskos et al. ).

    Article Title: Evaluation of the Immunogenicity and Vaccine Potential of Recombinant Plasmodium falciparum Merozoite Surface Protein 8
    Article Snippet: The codon-harmonized r Pf MSP8 and r Pf MSP8(ΔAsn/Asp) constructs were commercially synthesized, cloned into pBR322 and pUCminusMCS cloning vectors, respectively, and sequenced (Blue Heron Biotechnology Inc., Bothell, WA). .. The resulting expression vectors were transformed into SHuffle T7 Express lysY -competent E. coli cells (New England BioLabs, Ipswich, MA).

    Article Title: The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity
    Article Snippet: Two additional constructs containing mutations in cysteine 69 (C69A and C69Y) were also synthesized and cloned into the Bam HI and Xho I sites of pET21a(+) by GenScript Biotech (Piscataway, NJ, USA). .. Each expression construct was transformed into SHuffle® T7 Express lysY Escherichia coli (C3030; New England Biolabs, USA), which allows for expression of cytosolic proteins with disulfide bonds.

    Article Title: Comprehensive analysis of Fc-mediated IgM binding to the Plasmodium falciparum erythrocyte membrane protein 1 family in three parasite clones
    Article Snippet: Recombinant protein expression Recombinant proteins with N-terminal his-tags and representing various PfEMP1 domains were produced in Escherichia coli Shuffle C3030 cells (New England Biolabs). .. Constructs were amplified from parasite genomic DNA, using specific primer sets (Table ) and cloned into the pET15b modified plasmid .

    Centrifugation:

    Article Title: Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli
    Article Snippet: We therefore cloned the expression plasmids for all four fusion proteins into the SHuffle strain C3030 (New England Biolabs) in which the lethal phenotype of lacking the gor and trxB reductases is suppressed by a mutation in the peroxidase ahpC (Faulkner et al. ) thus providing an oxidising environment in the cytoplasm. .. We induced the expression with IPTG and separated the crude protein extract into soluble and insoluble proteins by centrifugation (Fig. ).

    Amplification:

    Article Title: The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity
    Article Snippet: Protein expression and purification The predicted mature BrLTP2.1 (amino acids 27–98, minus signal peptide) was PCR amplified from genomic DNA and cloned into the Bam HI and Xho I restriction sites of the protein expression vector pET21a(+) in frame with the N-terminal T7-tag and C-terminal His6 tag, to form pCH1, which was verified by Sanger sequencing at the University of Minnesota Genomics Center. .. Each expression construct was transformed into SHuffle® T7 Express lysY Escherichia coli (C3030; New England Biolabs, USA), which allows for expression of cytosolic proteins with disulfide bonds.

    Article Title: The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus
    Article Snippet: The gene fragment encoding ΔN-TcpB (residues 25–423) was PCR amplified with primers TcpB25-vc-fpcr-nde1 and TcpB25-vc-rpcr-BamH1 from V . cholerae WT strain O395 genomic DNA, followed by digestion with restriction enzymes NdeI and BamHI. .. The construct was transformed into SHuffle T7 Express lysY Competent E . coli (New England Biolabs) by heat shock.

    Article Title: The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity
    Article Snippet: The predicted mature BrLTP2.1 (amino acids 27–98, minus signal peptide) was PCR amplified from genomic DNA and cloned into the Bam HI and Xho I restriction sites of the protein expression vector pET21a(+) in frame with the N-terminal T7-tag and C-terminal His6 tag, to form pCH1, which was verified by Sanger sequencing at the University of Minnesota Genomics Center. .. Each expression construct was transformed into SHuffle® T7 Express lysY Escherichia coli (C3030; New England Biolabs, USA), which allows for expression of cytosolic proteins with disulfide bonds.

    Article Title: Comprehensive analysis of Fc-mediated IgM binding to the Plasmodium falciparum erythrocyte membrane protein 1 family in three parasite clones
    Article Snippet: Recombinant protein expression Recombinant proteins with N-terminal his-tags and representing various PfEMP1 domains were produced in Escherichia coli Shuffle C3030 cells (New England Biolabs). .. Constructs were amplified from parasite genomic DNA, using specific primer sets (Table ) and cloned into the pET15b modified plasmid .

    Article Title: Natural and Vaccine-Induced Acquisition of Cross-Reactive IgG-Inhibiting ICAM-1-Specific Binding of a Plasmodium falciparum PfEMP1 Subtype Associated Specifically with Cerebral Malaria
    Article Snippet: The genes encoding the DBLβ domains used were amplified from genomic DNA or produced as synthetic genes ( http://eurofins.dk ) ( ). .. Amplicons were subcloned into a modified pET15b vector and expressed as His-tagged proteins in Escherichia coli Shuffle C3030 cells (New England BioLabs) as described previously ( ).

    Synthesized:

    Article Title: The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity
    Article Snippet: Two additional constructs containing mutations in cysteine 69 (C69A and C69Y) were also synthesized and cloned into the Bam HI and Xho I sites of pET21a(+) by GenScript Biotech (Piscataway, NJ, USA). .. Each expression construct was transformed into SHuffle® T7 Express lysY Escherichia coli (C3030; New England Biolabs, USA), which allows for expression of cytosolic proteins with disulfide bonds.

    Article Title: Evaluation of the Immunogenicity and Vaccine Potential of Recombinant Plasmodium falciparum Merozoite Surface Protein 8
    Article Snippet: The codon-harmonized r Pf MSP8 and r Pf MSP8(ΔAsn/Asp) constructs were commercially synthesized, cloned into pBR322 and pUCminusMCS cloning vectors, respectively, and sequenced (Blue Heron Biotechnology Inc., Bothell, WA). .. The resulting expression vectors were transformed into SHuffle T7 Express lysY -competent E. coli cells (New England BioLabs, Ipswich, MA).

    Article Title: The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity
    Article Snippet: Two additional constructs containing mutations in cysteine 69 (C69A and C69Y) were also synthesized and cloned into the Bam HI and Xho I sites of pET21a(+) by GenScript Biotech (Piscataway, NJ, USA). .. Each expression construct was transformed into SHuffle® T7 Express lysY Escherichia coli (C3030; New England Biolabs, USA), which allows for expression of cytosolic proteins with disulfide bonds.

    Construct:

    Article Title: The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity
    Article Snippet: .. Each expression construct was transformed into SHuffle® T7 Express lysY Escherichia coli (C3030; New England Biolabs, USA), which allows for expression of cytosolic proteins with disulfide bonds. .. Escherichia coli cultures for protein expression were grown at 30 °C until log phase was reached (OD600 of ~0.6) and induced for expression of T7-BrLTP2.1-His6 with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG).

    Article Title: Acquisition of IgG to ICAM-1-Binding DBLβ Domains in the Plasmodium falciparum Erythrocyte Membrane Protein 1 Antigen Family Varies between Groups A, B, and C
    Article Snippet: .. His-tagged DBLβ domains (see Table S1 in the supplemental material) were expressed in Escherichia coli Shuffle C3030 cells (New England Biolabs) from synthetic genes ( https://eurofins.dk ) or from DNA constructs generated by PCR from genomic DNA using specific primers (Table S2). .. The domains were purified by immobilized metal affinity chromatography ( , , ).

    Article Title: The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus
    Article Snippet: .. The construct was transformed into SHuffle T7 Express lysY Competent E . coli (New England Biolabs) by heat shock. .. The cells were grown in LB broth supplemented with ampicillin (100 μg/ml) to OD600 of 0.1 in a shaking incubator 37°C, and protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG, final concentration to 0.1 mM).

    Article Title: Evaluation of the Immunogenicity and Vaccine Potential of Recombinant Plasmodium falciparum Merozoite Surface Protein 8
    Article Snippet: Paragraph title: r Pf MSP8 and r Pf MSP8(ΔAsn/Asp) expression constructs. ... The resulting expression vectors were transformed into SHuffle T7 Express lysY -competent E. coli cells (New England BioLabs, Ipswich, MA).

    Article Title: The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity
    Article Snippet: .. Each expression construct was transformed into SHuffle® T7 Express lysY Escherichia coli (C3030; New England Biolabs, USA), which allows for expression of cytosolic proteins with disulfide bonds. .. Escherichia coli cultures for protein expression were grown at 30 °C until log phase was reached (OD600 of ~0.6) and induced for expression of T7-BrLTP2.1-His6 with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG).

    Article Title: Comprehensive analysis of Fc-mediated IgM binding to the Plasmodium falciparum erythrocyte membrane protein 1 family in three parasite clones
    Article Snippet: Recombinant protein expression Recombinant proteins with N-terminal his-tags and representing various PfEMP1 domains were produced in Escherichia coli Shuffle C3030 cells (New England Biolabs). .. Constructs were amplified from parasite genomic DNA, using specific primer sets (Table ) and cloned into the pET15b modified plasmid .

    Article Title: Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant Pfs25, a Leading Transmission-Blocking Vaccine Candidate
    Article Snippet: .. The r Pf s25 expression construct was transformed into SHuffle T7 Express lysY competent E. coli cells (New England BioLabs). .. For expression, 3-liter bacterial cultures were grown by using a BioFlo115 benchtop bioreactor (New Brunswick Scientific).

    Expressing:

    Article Title: The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity
    Article Snippet: .. Each expression construct was transformed into SHuffle® T7 Express lysY Escherichia coli (C3030; New England Biolabs, USA), which allows for expression of cytosolic proteins with disulfide bonds. .. Escherichia coli cultures for protein expression were grown at 30 °C until log phase was reached (OD600 of ~0.6) and induced for expression of T7-BrLTP2.1-His6 with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG).

    Article Title: Structure of the Lectin Mannose 6-Phosphate Receptor Homology (MRH) Domain of Glucosidase II, an Enzyme That Regulates Glycoprotein Folding Quality Control in the Endoplasmic Reticulum *
    Article Snippet: .. Recombinant GIIβ expression was performed in SHuffle T7 Express lysY competent E. coli cells (BL21 C3030, New England Biolabs). ..

    Article Title: Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli
    Article Snippet: .. We therefore cloned the expression plasmids for all four fusion proteins into the SHuffle strain C3030 (New England Biolabs) in which the lethal phenotype of lacking the gor and trxB reductases is suppressed by a mutation in the peroxidase ahpC (Faulkner et al. ) thus providing an oxidising environment in the cytoplasm. .. Additionally, expression of periplasmic disulfide bond isomerase (DsbC) in the cytoplasm of these strains is supposed to enhance proper disulfide bond formation (Maskos et al. ).

    Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides
    Article Snippet: .. Transformation of E. coli expression strains Six different E. coli strains were used for expression screening: Rosetta-gami 2 (DE3) pLysS (Merck Millipore), Origami 2 (Merck Millipore), BL21 (DE3) (NEB), SHuffle T7 Express lysY (NEB), OverExpress C41 (DE3) pLysS (Sigma-Aldrich), OverExpress C43 (DE3) pLysS (Sigma-Aldrich). .. E. coli expression strains were transformed with the expression plasmids by heat shock.

    Article Title: The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus
    Article Snippet: Paragraph title: Expression and purification of ΔN-TcpA and ΔN-TcpB ... The construct was transformed into SHuffle T7 Express lysY Competent E . coli (New England Biolabs) by heat shock.

    Article Title: Evaluation of the Immunogenicity and Vaccine Potential of Recombinant Plasmodium falciparum Merozoite Surface Protein 8
    Article Snippet: .. The resulting expression vectors were transformed into SHuffle T7 Express lysY -competent E. coli cells (New England BioLabs, Ipswich, MA). ..

    Article Title: The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity
    Article Snippet: .. Each expression construct was transformed into SHuffle® T7 Express lysY Escherichia coli (C3030; New England Biolabs, USA), which allows for expression of cytosolic proteins with disulfide bonds. .. Escherichia coli cultures for protein expression were grown at 30 °C until log phase was reached (OD600 of ~0.6) and induced for expression of T7-BrLTP2.1-His6 with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG).

    Article Title: Comprehensive analysis of Fc-mediated IgM binding to the Plasmodium falciparum erythrocyte membrane protein 1 family in three parasite clones
    Article Snippet: .. Recombinant protein expression Recombinant proteins with N-terminal his-tags and representing various PfEMP1 domains were produced in Escherichia coli Shuffle C3030 cells (New England Biolabs). .. Constructs were amplified from parasite genomic DNA, using specific primer sets (Table ) and cloned into the pET15b modified plasmid .

    Article Title: Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant Pfs25, a Leading Transmission-Blocking Vaccine Candidate
    Article Snippet: .. The r Pf s25 expression construct was transformed into SHuffle T7 Express lysY competent E. coli cells (New England BioLabs). .. For expression, 3-liter bacterial cultures were grown by using a BioFlo115 benchtop bioreactor (New Brunswick Scientific).

    Modification:

    Article Title: Comprehensive analysis of Fc-mediated IgM binding to the Plasmodium falciparum erythrocyte membrane protein 1 family in three parasite clones
    Article Snippet: Recombinant protein expression Recombinant proteins with N-terminal his-tags and representing various PfEMP1 domains were produced in Escherichia coli Shuffle C3030 cells (New England Biolabs). .. Constructs were amplified from parasite genomic DNA, using specific primer sets (Table ) and cloned into the pET15b modified plasmid .

    Article Title: Natural and Vaccine-Induced Acquisition of Cross-Reactive IgG-Inhibiting ICAM-1-Specific Binding of a Plasmodium falciparum PfEMP1 Subtype Associated Specifically with Cerebral Malaria
    Article Snippet: .. Amplicons were subcloned into a modified pET15b vector and expressed as His-tagged proteins in Escherichia coli Shuffle C3030 cells (New England BioLabs) as described previously ( ). .. All the proteins were purified (see Fig. S1 in the supplemental material) by immobilized metal ion affinity chromatography using HisTrap HP 1-ml columns (GE Healthcare) and are referred to by the designations listed in . ) as described previously ( ).

    Western Blot:

    Article Title: Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli
    Article Snippet: Western blotting with the anti-His-tag antibody also detected a large band in the proTHI2.1 fraction at around 60 kDa and a smaller (20 kDa) and larger band (30 kDa) in the proTHI2.3 fraction. .. We therefore cloned the expression plasmids for all four fusion proteins into the SHuffle strain C3030 (New England Biolabs) in which the lethal phenotype of lacking the gor and trxB reductases is suppressed by a mutation in the peroxidase ahpC (Faulkner et al. ) thus providing an oxidising environment in the cytoplasm.

    Transformation Assay:

    Article Title: The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity
    Article Snippet: .. Each expression construct was transformed into SHuffle® T7 Express lysY Escherichia coli (C3030; New England Biolabs, USA), which allows for expression of cytosolic proteins with disulfide bonds. .. Escherichia coli cultures for protein expression were grown at 30 °C until log phase was reached (OD600 of ~0.6) and induced for expression of T7-BrLTP2.1-His6 with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG).

    Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides
    Article Snippet: .. Transformation of E. coli expression strains Six different E. coli strains were used for expression screening: Rosetta-gami 2 (DE3) pLysS (Merck Millipore), Origami 2 (Merck Millipore), BL21 (DE3) (NEB), SHuffle T7 Express lysY (NEB), OverExpress C41 (DE3) pLysS (Sigma-Aldrich), OverExpress C43 (DE3) pLysS (Sigma-Aldrich). .. E. coli expression strains were transformed with the expression plasmids by heat shock.

    Article Title: The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus
    Article Snippet: .. The construct was transformed into SHuffle T7 Express lysY Competent E . coli (New England Biolabs) by heat shock. .. The cells were grown in LB broth supplemented with ampicillin (100 μg/ml) to OD600 of 0.1 in a shaking incubator 37°C, and protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG, final concentration to 0.1 mM).

    Article Title: Evaluation of the Immunogenicity and Vaccine Potential of Recombinant Plasmodium falciparum Merozoite Surface Protein 8
    Article Snippet: .. The resulting expression vectors were transformed into SHuffle T7 Express lysY -competent E. coli cells (New England BioLabs, Ipswich, MA). ..

    Article Title: The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity
    Article Snippet: .. Each expression construct was transformed into SHuffle® T7 Express lysY Escherichia coli (C3030; New England Biolabs, USA), which allows for expression of cytosolic proteins with disulfide bonds. .. Escherichia coli cultures for protein expression were grown at 30 °C until log phase was reached (OD600 of ~0.6) and induced for expression of T7-BrLTP2.1-His6 with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG).

    Article Title: Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant Pfs25, a Leading Transmission-Blocking Vaccine Candidate
    Article Snippet: .. The r Pf s25 expression construct was transformed into SHuffle T7 Express lysY competent E. coli cells (New England BioLabs). .. For expression, 3-liter bacterial cultures were grown by using a BioFlo115 benchtop bioreactor (New Brunswick Scientific).

    Protease Inhibitor:

    Article Title: The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus
    Article Snippet: The construct was transformed into SHuffle T7 Express lysY Competent E . coli (New England Biolabs) by heat shock. .. The culture supernatant was discarded and the cell pellet was resuspended in lysis buffer (50 mM Na2 HPO4 /NaH2 PO4 pH 7.4, 20 mM Tris HCl pH 7.4, 100 mM NaCl, EDTA-free protease inhibitor cocktail [Roche], and 1 mg/ml lysozyme) and gently stirred at room temperature for an hour.

    Generated:

    Article Title: Acquisition of IgG to ICAM-1-Binding DBLβ Domains in the Plasmodium falciparum Erythrocyte Membrane Protein 1 Antigen Family Varies between Groups A, B, and C
    Article Snippet: .. His-tagged DBLβ domains (see Table S1 in the supplemental material) were expressed in Escherichia coli Shuffle C3030 cells (New England Biolabs) from synthetic genes ( https://eurofins.dk ) or from DNA constructs generated by PCR from genomic DNA using specific primers (Table S2). .. The domains were purified by immobilized metal affinity chromatography ( , , ).

    Protein Concentration:

    Article Title: Thermophilic phosphoribosyltransferases Thermus thermophilus HB27 in nucleotide synthesis
    Article Snippet: Bacterial strains: a) E. coli C3030 [MiniF lysY (CamR ) / fhuA2 lacZ::T7 gene1 [lon] ompT ahpC gal λatt ::pNEB3-r1- cDsbC (SpecR , lacIq ) ΔtrxB sulA11 R(mcr-73::miniTn1 0--TetS )2 [dcm] R(zgb-210::Tn10 --TetS ) endA1 Δgor ∆(mcrC-mrr)114::IS10 ] New England Biolabs (USA, MA), b) E.coli BL21(DE3) fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS λ DE3 = λ sBamHIo ΔEcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 Δnin5. .. The protein concentration was determined by the Bradford method [ ], using BSA as a standard.

    Article Title: Thermophilic phosphoribosyltransferases Thermus thermophilus HB27 in nucleotide synthesis
    Article Snippet: Bacterial strains: a) E. coli C3030 [MiniF lysY (CamR ) / fhuA2 lacZ::T7 gene1 [lon] ompT ahpC gal λatt ::pNEB3-r1-cDsbC (SpecR , lacIq ) ΔtrxB sulA11 R(mcr-73::miniTn1 0--TetS )2 [dcm] R(zgb-210::Tn10 --TetS ) endA1 Δgor ∆(mcrC-mrr)114::IS10 ] New England Biolabs (USA, MA), b) E.coli BL21(DE3) fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS λ DE3 = λ sBamHIo ΔEcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 Δnin5. .. The protein concentration was determined by the Bradford method [ ], using BSA as a standard.

    Sequencing:

    Article Title: The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity
    Article Snippet: Protein expression and purification The predicted mature BrLTP2.1 (amino acids 27–98, minus signal peptide) was PCR amplified from genomic DNA and cloned into the Bam HI and Xho I restriction sites of the protein expression vector pET21a(+) in frame with the N-terminal T7-tag and C-terminal His6 tag, to form pCH1, which was verified by Sanger sequencing at the University of Minnesota Genomics Center. .. Each expression construct was transformed into SHuffle® T7 Express lysY Escherichia coli (C3030; New England Biolabs, USA), which allows for expression of cytosolic proteins with disulfide bonds.

    Article Title: Evaluation of the Immunogenicity and Vaccine Potential of Recombinant Plasmodium falciparum Merozoite Surface Protein 8
    Article Snippet: Two stop codons (TGA and TAA) were incorporated at the 3′ end of each sequence. .. The resulting expression vectors were transformed into SHuffle T7 Express lysY -competent E. coli cells (New England BioLabs, Ipswich, MA).

    Article Title: The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity
    Article Snippet: The predicted mature BrLTP2.1 (amino acids 27–98, minus signal peptide) was PCR amplified from genomic DNA and cloned into the Bam HI and Xho I restriction sites of the protein expression vector pET21a(+) in frame with the N-terminal T7-tag and C-terminal His6 tag, to form pCH1, which was verified by Sanger sequencing at the University of Minnesota Genomics Center. .. Each expression construct was transformed into SHuffle® T7 Express lysY Escherichia coli (C3030; New England Biolabs, USA), which allows for expression of cytosolic proteins with disulfide bonds.

    Recombinant:

    Article Title: Structure of the Lectin Mannose 6-Phosphate Receptor Homology (MRH) Domain of Glucosidase II, an Enzyme That Regulates Glycoprotein Folding Quality Control in the Endoplasmic Reticulum *
    Article Snippet: .. Recombinant GIIβ expression was performed in SHuffle T7 Express lysY competent E. coli cells (BL21 C3030, New England Biolabs). ..

    Article Title: Acquisition of IgG to ICAM-1-Binding DBLβ Domains in the Plasmodium falciparum Erythrocyte Membrane Protein 1 Antigen Family Varies between Groups A, B, and C
    Article Snippet: Paragraph title: Production of recombinant proteins. ... His-tagged DBLβ domains (see Table S1 in the supplemental material) were expressed in Escherichia coli Shuffle C3030 cells (New England Biolabs) from synthetic genes ( https://eurofins.dk ) or from DNA constructs generated by PCR from genomic DNA using specific primers (Table S2).

    Article Title: Evaluation of the Immunogenicity and Vaccine Potential of Recombinant Plasmodium falciparum Merozoite Surface Protein 8
    Article Snippet: The resulting expression vectors were transformed into SHuffle T7 Express lysY -competent E. coli cells (New England BioLabs, Ipswich, MA). .. Expression of the recombinant proteins was accomplished using a BioFLo115 benchtop bioreactor (New Brunswick Scientific, Edison, NJ).

    Article Title: Comprehensive analysis of Fc-mediated IgM binding to the Plasmodium falciparum erythrocyte membrane protein 1 family in three parasite clones
    Article Snippet: .. Recombinant protein expression Recombinant proteins with N-terminal his-tags and representing various PfEMP1 domains were produced in Escherichia coli Shuffle C3030 cells (New England Biolabs). .. Constructs were amplified from parasite genomic DNA, using specific primer sets (Table ) and cloned into the pET15b modified plasmid .

    Article Title: Natural and Vaccine-Induced Acquisition of Cross-Reactive IgG-Inhibiting ICAM-1-Specific Binding of a Plasmodium falciparum PfEMP1 Subtype Associated Specifically with Cerebral Malaria
    Article Snippet: Paragraph title: Recombinant proteins. ... Amplicons were subcloned into a modified pET15b vector and expressed as His-tagged proteins in Escherichia coli Shuffle C3030 cells (New England BioLabs) as described previously ( ).

    Mutagenesis:

    Article Title: Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli
    Article Snippet: .. We therefore cloned the expression plasmids for all four fusion proteins into the SHuffle strain C3030 (New England Biolabs) in which the lethal phenotype of lacking the gor and trxB reductases is suppressed by a mutation in the peroxidase ahpC (Faulkner et al. ) thus providing an oxidising environment in the cytoplasm. .. Additionally, expression of periplasmic disulfide bond isomerase (DsbC) in the cytoplasm of these strains is supposed to enhance proper disulfide bond formation (Maskos et al. ).

    Subcloning:

    Article Title: Evaluation of the Immunogenicity and Vaccine Potential of Recombinant Plasmodium falciparum Merozoite Surface Protein 8
    Article Snippet: To facilitate subcloning, NcoI and NotI restriction sites were added to the 5′ and 3′ ends, respectively. .. The resulting expression vectors were transformed into SHuffle T7 Express lysY -competent E. coli cells (New England BioLabs, Ipswich, MA).

    Labeling:

    Article Title: Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli
    Article Snippet: These were also labeled with the anti-His-tag antibody and seemed to be degradation products. .. We therefore cloned the expression plasmids for all four fusion proteins into the SHuffle strain C3030 (New England Biolabs) in which the lethal phenotype of lacking the gor and trxB reductases is suppressed by a mutation in the peroxidase ahpC (Faulkner et al. ) thus providing an oxidising environment in the cytoplasm.

    Purification:

    Article Title: The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity
    Article Snippet: Paragraph title: Protein expression and purification ... Each expression construct was transformed into SHuffle® T7 Express lysY Escherichia coli (C3030; New England Biolabs, USA), which allows for expression of cytosolic proteins with disulfide bonds.

    Article Title: Acquisition of IgG to ICAM-1-Binding DBLβ Domains in the Plasmodium falciparum Erythrocyte Membrane Protein 1 Antigen Family Varies between Groups A, B, and C
    Article Snippet: His-tagged DBLβ domains (see Table S1 in the supplemental material) were expressed in Escherichia coli Shuffle C3030 cells (New England Biolabs) from synthetic genes ( https://eurofins.dk ) or from DNA constructs generated by PCR from genomic DNA using specific primers (Table S2). .. The domains were purified by immobilized metal affinity chromatography ( , , ).

    Article Title: The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus
    Article Snippet: Paragraph title: Expression and purification of ΔN-TcpA and ΔN-TcpB ... The construct was transformed into SHuffle T7 Express lysY Competent E . coli (New England Biolabs) by heat shock.

    Article Title: Evaluation of the Immunogenicity and Vaccine Potential of Recombinant Plasmodium falciparum Merozoite Surface Protein 8
    Article Snippet: A leader sequence that includes a 6-histidine tag and a glycine-serine linker (MAHHHHHHPGGSGSGT) was inserted onto the 5′ end of both codon-harmonized coding sequences to allow for downstream purification. .. The resulting expression vectors were transformed into SHuffle T7 Express lysY -competent E. coli cells (New England BioLabs, Ipswich, MA).

    Article Title: Comprehensive analysis of Fc-mediated IgM binding to the Plasmodium falciparum erythrocyte membrane protein 1 family in three parasite clones
    Article Snippet: Recombinant protein expression Recombinant proteins with N-terminal his-tags and representing various PfEMP1 domains were produced in Escherichia coli Shuffle C3030 cells (New England Biolabs). .. All expressed proteins were purified by immobilized metal ion affinity chromatography using His-Trap High Performance columns (GE Healthcare).

    Article Title: Natural and Vaccine-Induced Acquisition of Cross-Reactive IgG-Inhibiting ICAM-1-Specific Binding of a Plasmodium falciparum PfEMP1 Subtype Associated Specifically with Cerebral Malaria
    Article Snippet: Amplicons were subcloned into a modified pET15b vector and expressed as His-tagged proteins in Escherichia coli Shuffle C3030 cells (New England BioLabs) as described previously ( ). .. All the proteins were purified (see Fig. S1 in the supplemental material) by immobilized metal ion affinity chromatography using HisTrap HP 1-ml columns (GE Healthcare) and are referred to by the designations listed in . ) as described previously ( ).

    Polymerase Chain Reaction:

    Article Title: The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity
    Article Snippet: Protein expression and purification The predicted mature BrLTP2.1 (amino acids 27–98, minus signal peptide) was PCR amplified from genomic DNA and cloned into the Bam HI and Xho I restriction sites of the protein expression vector pET21a(+) in frame with the N-terminal T7-tag and C-terminal His6 tag, to form pCH1, which was verified by Sanger sequencing at the University of Minnesota Genomics Center. .. Each expression construct was transformed into SHuffle® T7 Express lysY Escherichia coli (C3030; New England Biolabs, USA), which allows for expression of cytosolic proteins with disulfide bonds.

    Article Title: Acquisition of IgG to ICAM-1-Binding DBLβ Domains in the Plasmodium falciparum Erythrocyte Membrane Protein 1 Antigen Family Varies between Groups A, B, and C
    Article Snippet: .. His-tagged DBLβ domains (see Table S1 in the supplemental material) were expressed in Escherichia coli Shuffle C3030 cells (New England Biolabs) from synthetic genes ( https://eurofins.dk ) or from DNA constructs generated by PCR from genomic DNA using specific primers (Table S2). .. The domains were purified by immobilized metal affinity chromatography ( , , ).

    Article Title: The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus
    Article Snippet: The gene fragment encoding ΔN-TcpB (residues 25–423) was PCR amplified with primers TcpB25-vc-fpcr-nde1 and TcpB25-vc-rpcr-BamH1 from V . cholerae WT strain O395 genomic DNA, followed by digestion with restriction enzymes NdeI and BamHI. .. The construct was transformed into SHuffle T7 Express lysY Competent E . coli (New England Biolabs) by heat shock.

    Article Title: The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity
    Article Snippet: The predicted mature BrLTP2.1 (amino acids 27–98, minus signal peptide) was PCR amplified from genomic DNA and cloned into the Bam HI and Xho I restriction sites of the protein expression vector pET21a(+) in frame with the N-terminal T7-tag and C-terminal His6 tag, to form pCH1, which was verified by Sanger sequencing at the University of Minnesota Genomics Center. .. Each expression construct was transformed into SHuffle® T7 Express lysY Escherichia coli (C3030; New England Biolabs, USA), which allows for expression of cytosolic proteins with disulfide bonds.

    Affinity Chromatography:

    Article Title: Acquisition of IgG to ICAM-1-Binding DBLβ Domains in the Plasmodium falciparum Erythrocyte Membrane Protein 1 Antigen Family Varies between Groups A, B, and C
    Article Snippet: His-tagged DBLβ domains (see Table S1 in the supplemental material) were expressed in Escherichia coli Shuffle C3030 cells (New England Biolabs) from synthetic genes ( https://eurofins.dk ) or from DNA constructs generated by PCR from genomic DNA using specific primers (Table S2). .. The domains were purified by immobilized metal affinity chromatography ( , , ).

    Article Title: Comprehensive analysis of Fc-mediated IgM binding to the Plasmodium falciparum erythrocyte membrane protein 1 family in three parasite clones
    Article Snippet: Recombinant protein expression Recombinant proteins with N-terminal his-tags and representing various PfEMP1 domains were produced in Escherichia coli Shuffle C3030 cells (New England Biolabs). .. All expressed proteins were purified by immobilized metal ion affinity chromatography using His-Trap High Performance columns (GE Healthcare).

    Article Title: Natural and Vaccine-Induced Acquisition of Cross-Reactive IgG-Inhibiting ICAM-1-Specific Binding of a Plasmodium falciparum PfEMP1 Subtype Associated Specifically with Cerebral Malaria
    Article Snippet: Amplicons were subcloned into a modified pET15b vector and expressed as His-tagged proteins in Escherichia coli Shuffle C3030 cells (New England BioLabs) as described previously ( ). .. All the proteins were purified (see Fig. S1 in the supplemental material) by immobilized metal ion affinity chromatography using HisTrap HP 1-ml columns (GE Healthcare) and are referred to by the designations listed in . ) as described previously ( ).

    Polyacrylamide Gel Electrophoresis:

    Article Title: Comprehensive analysis of Fc-mediated IgM binding to the Plasmodium falciparum erythrocyte membrane protein 1 family in three parasite clones
    Article Snippet: Recombinant protein expression Recombinant proteins with N-terminal his-tags and representing various PfEMP1 domains were produced in Escherichia coli Shuffle C3030 cells (New England Biolabs). .. The purity of each protein was analyzed by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis, followed by Coomassie staining with InstantBlue (Expedeon) following the manufacturer’s instructions.

    Lysis:

    Article Title: The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus
    Article Snippet: The construct was transformed into SHuffle T7 Express lysY Competent E . coli (New England Biolabs) by heat shock. .. The culture supernatant was discarded and the cell pellet was resuspended in lysis buffer (50 mM Na2 HPO4 /NaH2 PO4 pH 7.4, 20 mM Tris HCl pH 7.4, 100 mM NaCl, EDTA-free protease inhibitor cocktail [Roche], and 1 mg/ml lysozyme) and gently stirred at room temperature for an hour.

    Plasmid Preparation:

    Article Title: The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity
    Article Snippet: Protein expression and purification The predicted mature BrLTP2.1 (amino acids 27–98, minus signal peptide) was PCR amplified from genomic DNA and cloned into the Bam HI and Xho I restriction sites of the protein expression vector pET21a(+) in frame with the N-terminal T7-tag and C-terminal His6 tag, to form pCH1, which was verified by Sanger sequencing at the University of Minnesota Genomics Center. .. Each expression construct was transformed into SHuffle® T7 Express lysY Escherichia coli (C3030; New England Biolabs, USA), which allows for expression of cytosolic proteins with disulfide bonds.

    Article Title: Thermophilic phosphoribosyltransferases Thermus thermophilus HB27 in nucleotide synthesis
    Article Snippet: Bacterial strains: a) E. coli C3030 [MiniF lysY (CamR ) / fhuA2 lacZ::T7 gene1 [lon] ompT ahpC gal λatt ::pNEB3-r1- cDsbC (SpecR , lacIq ) ΔtrxB sulA11 R(mcr-73::miniTn1 0--TetS )2 [dcm] R(zgb-210::Tn10 --TetS ) endA1 Δgor ∆(mcrC-mrr)114::IS10 ] New England Biolabs (USA, MA), b) E.coli BL21(DE3) fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS λ DE3 = λ sBamHIo ΔEcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 Δnin5. .. Plasmid vector: pET 23a+, pET 23d+ (Merck Millipore, USA, MA).

    Article Title: Thermophilic phosphoribosyltransferases Thermus thermophilus HB27 in nucleotide synthesis
    Article Snippet: Bacterial strains: a) E. coli C3030 [MiniF lysY (CamR ) / fhuA2 lacZ::T7 gene1 [lon] ompT ahpC gal λatt ::pNEB3-r1-cDsbC (SpecR , lacIq ) ΔtrxB sulA11 R(mcr-73::miniTn1 0--TetS )2 [dcm] R(zgb-210::Tn10 --TetS ) endA1 Δgor ∆(mcrC-mrr)114::IS10 ] New England Biolabs (USA, MA), b) E.coli BL21(DE3) fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS λ DE3 = λ sBamHIo ΔEcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 Δnin5. .. Plasmid vector: pET 23a+, pET 23d+ (Merck Millipore, USA, MA).

    Article Title: The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus
    Article Snippet: The product was ligated into expression vector pET15b (Novagen), which encodes a His-tag and linker with a thrombin cleavage site. .. The construct was transformed into SHuffle T7 Express lysY Competent E . coli (New England Biolabs) by heat shock.

    Article Title: Evaluation of the Immunogenicity and Vaccine Potential of Recombinant Plasmodium falciparum Merozoite Surface Protein 8
    Article Snippet: The synthetic gene inserts were excised and ligated into the NcoI and NotI sites of the pET-28 expression vector (EMD Biosciences, San Diego, CA). .. The resulting expression vectors were transformed into SHuffle T7 Express lysY -competent E. coli cells (New England BioLabs, Ipswich, MA).

    Article Title: The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity
    Article Snippet: The predicted mature BrLTP2.1 (amino acids 27–98, minus signal peptide) was PCR amplified from genomic DNA and cloned into the Bam HI and Xho I restriction sites of the protein expression vector pET21a(+) in frame with the N-terminal T7-tag and C-terminal His6 tag, to form pCH1, which was verified by Sanger sequencing at the University of Minnesota Genomics Center. .. Each expression construct was transformed into SHuffle® T7 Express lysY Escherichia coli (C3030; New England Biolabs, USA), which allows for expression of cytosolic proteins with disulfide bonds.

    Article Title: Comprehensive analysis of Fc-mediated IgM binding to the Plasmodium falciparum erythrocyte membrane protein 1 family in three parasite clones
    Article Snippet: Recombinant protein expression Recombinant proteins with N-terminal his-tags and representing various PfEMP1 domains were produced in Escherichia coli Shuffle C3030 cells (New England Biolabs). .. Constructs were amplified from parasite genomic DNA, using specific primer sets (Table ) and cloned into the pET15b modified plasmid .

    Article Title: Natural and Vaccine-Induced Acquisition of Cross-Reactive IgG-Inhibiting ICAM-1-Specific Binding of a Plasmodium falciparum PfEMP1 Subtype Associated Specifically with Cerebral Malaria
    Article Snippet: .. Amplicons were subcloned into a modified pET15b vector and expressed as His-tagged proteins in Escherichia coli Shuffle C3030 cells (New England BioLabs) as described previously ( ). .. All the proteins were purified (see Fig. S1 in the supplemental material) by immobilized metal ion affinity chromatography using HisTrap HP 1-ml columns (GE Healthcare) and are referred to by the designations listed in . ) as described previously ( ).

    Functional Assay:

    Article Title: Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli
    Article Snippet: However, E. coli strains with an oxidizing cytoplasm have been reported that result in high levels of functional proteins with disulfide bridges. .. We therefore cloned the expression plasmids for all four fusion proteins into the SHuffle strain C3030 (New England Biolabs) in which the lethal phenotype of lacking the gor and trxB reductases is suppressed by a mutation in the peroxidase ahpC (Faulkner et al. ) thus providing an oxidising environment in the cytoplasm.

    Positron Emission Tomography:

    Article Title: Thermophilic phosphoribosyltransferases Thermus thermophilus HB27 in nucleotide synthesis
    Article Snippet: Bacterial strains: a) E. coli C3030 [MiniF lysY (CamR ) / fhuA2 lacZ::T7 gene1 [lon] ompT ahpC gal λatt ::pNEB3-r1- cDsbC (SpecR , lacIq ) ΔtrxB sulA11 R(mcr-73::miniTn1 0--TetS )2 [dcm] R(zgb-210::Tn10 --TetS ) endA1 Δgor ∆(mcrC-mrr)114::IS10 ] New England Biolabs (USA, MA), b) E.coli BL21(DE3) fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS λ DE3 = λ sBamHIo ΔEcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 Δnin5. .. Plasmid vector: pET 23a+, pET 23d+ (Merck Millipore, USA, MA).

    Article Title: Thermophilic phosphoribosyltransferases Thermus thermophilus HB27 in nucleotide synthesis
    Article Snippet: Bacterial strains: a) E. coli C3030 [MiniF lysY (CamR ) / fhuA2 lacZ::T7 gene1 [lon] ompT ahpC gal λatt ::pNEB3-r1-cDsbC (SpecR , lacIq ) ΔtrxB sulA11 R(mcr-73::miniTn1 0--TetS )2 [dcm] R(zgb-210::Tn10 --TetS ) endA1 Δgor ∆(mcrC-mrr)114::IS10 ] New England Biolabs (USA, MA), b) E.coli BL21(DE3) fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS λ DE3 = λ sBamHIo ΔEcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 Δnin5. .. Plasmid vector: pET 23a+, pET 23d+ (Merck Millipore, USA, MA).

    Article Title: Evaluation of the Immunogenicity and Vaccine Potential of Recombinant Plasmodium falciparum Merozoite Surface Protein 8
    Article Snippet: The synthetic gene inserts were excised and ligated into the NcoI and NotI sites of the pET-28 expression vector (EMD Biosciences, San Diego, CA). .. The resulting expression vectors were transformed into SHuffle T7 Express lysY -competent E. coli cells (New England BioLabs, Ipswich, MA).

    Produced:

    Article Title: Comprehensive analysis of Fc-mediated IgM binding to the Plasmodium falciparum erythrocyte membrane protein 1 family in three parasite clones
    Article Snippet: .. Recombinant protein expression Recombinant proteins with N-terminal his-tags and representing various PfEMP1 domains were produced in Escherichia coli Shuffle C3030 cells (New England Biolabs). .. Constructs were amplified from parasite genomic DNA, using specific primer sets (Table ) and cloned into the pET15b modified plasmid .

    Article Title: Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli
    Article Snippet: .. The amount of fusion protein that could be produced from the SHuffle strain C3030 is shown in Table and was higher than obtained with Rosetta(DE3)pLysS. .. Western blotting of the purified fusion proteins (Fig. ) showed less contaminating proteins than with the Rosetta(DE3)pLysS strain (Supplementary Fig. 6).

    Article Title: Natural and Vaccine-Induced Acquisition of Cross-Reactive IgG-Inhibiting ICAM-1-Specific Binding of a Plasmodium falciparum PfEMP1 Subtype Associated Specifically with Cerebral Malaria
    Article Snippet: The genes encoding the DBLβ domains used were amplified from genomic DNA or produced as synthetic genes ( http://eurofins.dk ) ( ). .. Amplicons were subcloned into a modified pET15b vector and expressed as His-tagged proteins in Escherichia coli Shuffle C3030 cells (New England BioLabs) as described previously ( ).

    Concentration Assay:

    Article Title: The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus
    Article Snippet: The construct was transformed into SHuffle T7 Express lysY Competent E . coli (New England Biolabs) by heat shock. .. The cells were grown in LB broth supplemented with ampicillin (100 μg/ml) to OD600 of 0.1 in a shaking incubator 37°C, and protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG, final concentration to 0.1 mM).

    Article Title: Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant Pfs25, a Leading Transmission-Blocking Vaccine Candidate
    Article Snippet: The r Pf s25 expression construct was transformed into SHuffle T7 Express lysY competent E. coli cells (New England BioLabs). .. At an optical density at 600 nm (OD600 ) of ∼4.0, the expression of r Pf s25 was induced by the addition of IPTG (ThermoFisher Scientific) to a final concentration of 1 mM.

    Staining:

    Article Title: Comprehensive analysis of Fc-mediated IgM binding to the Plasmodium falciparum erythrocyte membrane protein 1 family in three parasite clones
    Article Snippet: Recombinant protein expression Recombinant proteins with N-terminal his-tags and representing various PfEMP1 domains were produced in Escherichia coli Shuffle C3030 cells (New England Biolabs). .. The purity of each protein was analyzed by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis, followed by Coomassie staining with InstantBlue (Expedeon) following the manufacturer’s instructions.

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    New England Biolabs shuffle t7 express lysy competent e coli cells
    Expression of chimeric r Pf s25/8(CΔS) and unfused r Pf s25. (A) Schematic depiction of expression constructs for the production of r Pf s25/8(CΔS) and r Pf s25. Expression plasmids for chimeric r Pf s25/8(CΔS) or unfused r Pf s25 were transformed into E. coli SHuffle <t>T7</t> Express <t>lysY</t> cells. (B and C) r Pf s25/8(CΔS) lysates harvested before ( T 0 ) or 3 h after ( T 3 ) induction were separated by SDS-PAGE (10% gel) under reducing conditions, followed by Coomassie blue staining (B) or immunoblot analysis (C) using rabbit-anti-r Pf MSP8 IgG. The asterisk highlights r Pf s25/8(CΔS) at the predicted size. (D and E) The expression of unfused r Pf s25 was assessed as described above, on 12% polyacrylamide gels under reducing conditions, followed by Coomassie blue staining (D) or immunoblot analysis (E) using an anti-His MAb. The asterisk highlights r Pf s25 at the predicted size.
    Shuffle T7 Express Lysy Competent E Coli Cells, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Expression of chimeric r Pf s25/8(CΔS) and unfused r Pf s25. (A) Schematic depiction of expression constructs for the production of r Pf s25/8(CΔS) and r Pf s25. Expression plasmids for chimeric r Pf s25/8(CΔS) or unfused r Pf s25 were transformed into E. coli SHuffle T7 Express lysY cells. (B and C) r Pf s25/8(CΔS) lysates harvested before ( T 0 ) or 3 h after ( T 3 ) induction were separated by SDS-PAGE (10% gel) under reducing conditions, followed by Coomassie blue staining (B) or immunoblot analysis (C) using rabbit-anti-r Pf MSP8 IgG. The asterisk highlights r Pf s25/8(CΔS) at the predicted size. (D and E) The expression of unfused r Pf s25 was assessed as described above, on 12% polyacrylamide gels under reducing conditions, followed by Coomassie blue staining (D) or immunoblot analysis (E) using an anti-His MAb. The asterisk highlights r Pf s25 at the predicted size.

    Journal: Infection and Immunity

    Article Title: Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant Pfs25, a Leading Transmission-Blocking Vaccine Candidate

    doi: 10.1128/IAI.00486-17

    Figure Lengend Snippet: Expression of chimeric r Pf s25/8(CΔS) and unfused r Pf s25. (A) Schematic depiction of expression constructs for the production of r Pf s25/8(CΔS) and r Pf s25. Expression plasmids for chimeric r Pf s25/8(CΔS) or unfused r Pf s25 were transformed into E. coli SHuffle T7 Express lysY cells. (B and C) r Pf s25/8(CΔS) lysates harvested before ( T 0 ) or 3 h after ( T 3 ) induction were separated by SDS-PAGE (10% gel) under reducing conditions, followed by Coomassie blue staining (B) or immunoblot analysis (C) using rabbit-anti-r Pf MSP8 IgG. The asterisk highlights r Pf s25/8(CΔS) at the predicted size. (D and E) The expression of unfused r Pf s25 was assessed as described above, on 12% polyacrylamide gels under reducing conditions, followed by Coomassie blue staining (D) or immunoblot analysis (E) using an anti-His MAb. The asterisk highlights r Pf s25 at the predicted size.

    Article Snippet: The r Pf s25 expression construct was transformed into SHuffle T7 Express lysY competent E. coli cells (New England BioLabs).

    Techniques: Expressing, Construct, Transformation Assay, SDS Page, Staining

    Expression screening in E. coli . Expression screening was carried out in 96-well microtiter plates to identify high producers for three different target proteins. The promoter, protein fusion partner and E. coli strain were varied. a IMPI, b BR021, c AFP. Rosetta-gami 2 Rosetta-gami 2 (DE3) pLysS, Origami Origami 2, SHuffle SHuffle T7 Express lysY, BL21 BL21 (DE3), C41 OverExpress C41 (DE3) pLysS, C43 OverExpress C43 (DE3) pLysS

    Journal: Microbial Cell Factories

    Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides

    doi: 10.1186/s12934-017-0637-5

    Figure Lengend Snippet: Expression screening in E. coli . Expression screening was carried out in 96-well microtiter plates to identify high producers for three different target proteins. The promoter, protein fusion partner and E. coli strain were varied. a IMPI, b BR021, c AFP. Rosetta-gami 2 Rosetta-gami 2 (DE3) pLysS, Origami Origami 2, SHuffle SHuffle T7 Express lysY, BL21 BL21 (DE3), C41 OverExpress C41 (DE3) pLysS, C43 OverExpress C43 (DE3) pLysS

    Article Snippet: Transformation of E. coli expression strains Six different E. coli strains were used for expression screening: Rosetta-gami 2 (DE3) pLysS (Merck Millipore), Origami 2 (Merck Millipore), BL21 (DE3) (NEB), SHuffle T7 Express lysY (NEB), OverExpress C41 (DE3) pLysS (Sigma-Aldrich), OverExpress C43 (DE3) pLysS (Sigma-Aldrich).

    Techniques: Expressing

    Cytoplamic expression of proTHI-TRX fusion proteins in strain C3030. For all fusion proteins, 1 ml of cell culture was pelleted and dissolved in 100 μl sample buffer. 10 μl from this extract and an equivalent amount for total soluble cytoplasmic fractions were separated on Tricine/SDS gels. (M) Protein marker, ( 1 ) un-induced crude extract, ( 2 ) induced crude extract, ( 3 ) total soluble fraction taken after cell lysis by sonication, ( 4 ) insoluble fraction after sonication. Red stars indicate the 25 kDa fusion protein

    Journal: Biotechnology Letters

    Article Title: Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli

    doi: 10.1007/s10529-013-1180-z

    Figure Lengend Snippet: Cytoplamic expression of proTHI-TRX fusion proteins in strain C3030. For all fusion proteins, 1 ml of cell culture was pelleted and dissolved in 100 μl sample buffer. 10 μl from this extract and an equivalent amount for total soluble cytoplasmic fractions were separated on Tricine/SDS gels. (M) Protein marker, ( 1 ) un-induced crude extract, ( 2 ) induced crude extract, ( 3 ) total soluble fraction taken after cell lysis by sonication, ( 4 ) insoluble fraction after sonication. Red stars indicate the 25 kDa fusion protein

    Article Snippet: We therefore cloned the expression plasmids for all four fusion proteins into the SHuffle strain C3030 (New England Biolabs) in which the lethal phenotype of lacking the gor and trxB reductases is suppressed by a mutation in the peroxidase ahpC (Faulkner et al. ) thus providing an oxidising environment in the cytoplasm.

    Techniques: Expressing, Cell Culture, Marker, Lysis, Sonication

    Comparison of Ni–NTA purified proTHI-TRX fusion proteins from strain C3030. a Coomassie Brilliant Blue staining. b Western blot with anti-His tag antibody. Each well contained 3 μg protein. (M) Prestained protein marker, ( 1 ) proTHI2.1-TRX, ( 2 ) proTHI2.2-TRX, ( 3 ) proTHI2.3-TRX, ( 4 ) proTHI2.4-TRX

    Journal: Biotechnology Letters

    Article Title: Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli

    doi: 10.1007/s10529-013-1180-z

    Figure Lengend Snippet: Comparison of Ni–NTA purified proTHI-TRX fusion proteins from strain C3030. a Coomassie Brilliant Blue staining. b Western blot with anti-His tag antibody. Each well contained 3 μg protein. (M) Prestained protein marker, ( 1 ) proTHI2.1-TRX, ( 2 ) proTHI2.2-TRX, ( 3 ) proTHI2.3-TRX, ( 4 ) proTHI2.4-TRX

    Article Snippet: We therefore cloned the expression plasmids for all four fusion proteins into the SHuffle strain C3030 (New England Biolabs) in which the lethal phenotype of lacking the gor and trxB reductases is suppressed by a mutation in the peroxidase ahpC (Faulkner et al. ) thus providing an oxidising environment in the cytoplasm.

    Techniques: Purification, Staining, Western Blot, Marker