bl21 de3 cells  (Thermo Fisher)


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    Structured Review

    Thermo Fisher bl21 de3 cells
    Bl21 De3 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bl21 de3 cells/product/Thermo Fisher
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    bl21 de3 cells - by Bioz Stars, 2020-01
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1
    Article Snippet: Paragraph title: Recombinant protein cloning, expression and purification ... This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15 N ammonium chloride and 3 g/l 13 C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) ).

    Article Title: Cardiomyopathy mutation (F88L) in troponin T abolishes length dependency of myofilament Ca2+ sensitivity
    Article Snippet: Recombinant c-myc –tagged guinea pig TnT (TnTWT and TnTF88L ), guinea pig WT troponin I (TnI), and guinea pig WT troponin C (TnC) were generated and cloned into a pSBETa vector (GenScript USA). .. Recombinant DNA was transformed and expressed in BL21*DE3 cells (Invitrogen).

    Article Title: Reversal of Aging‐Induced Increases in Aortic Stiffness by Targeting Cytoskeletal Protein‐Protein Interfaces
    Article Snippet: Briefly, the EVH1‐TAT construct was cloned in a pTYB1 vector (New England Biolabs). .. The construct was expressed in BL21 (DE3) cells (Invitrogen, Carlsbad, CA), which were grown in terrific broth medium at 37°C until an optical density at 600 nm reached a value of 0.8.

    Article Title: Saturation of recognition elements blocks evolution of new tRNA identities
    Article Snippet: Paragraph title: Enzyme cloning, expression, and purification ... BL21 (DE3) cells (Life Technologies), transformed with pQE70-AlaRS or pQE70-GlyRS, were grown at 37°C up to an OD600 (optical density at 600 nm) of 0.6.

    Article Title: Caenorhabditis elegans PRMT-7 and PRMT-9 Are Evolutionarily Conserved Protein Arginine Methyltransferases with Distinct Substrate Specificities
    Article Snippet: C. elegans PRMT-7 and PRMT-9 cDNA were cloned into a pGEX-6P-1 plasmids and were a kind gift from Dr. Akiyoshi Fukamizu. .. The proteins were expressed in BL21 DE3 cells (Invitrogen); C. elegans GST-PRMT-7 and -9 enzymes were purified using the conditions described in , but with induction for 20 h at 18 °C using 0.13 mM isopropyl D-thiogalactopyranoside (IPTG) for GST-PRMT-7 and 1 mM IPTG for 20 h at 18 °C for both GST-PRMT-9 wild type and A391H mutant.

    Article Title: Engineering a dirhodium artificial metalloenzyme for selective olefin cyclopropanation
    Article Snippet: A codon-optimized gene for POP was obtained from GenScript USA Inc. (Piscataway, NJ) and cloned into the NcoI and XhoI restriction sites of the pET28a plasmid vector. .. E. coli DH5α and BL21 (DE3) cells were purchased from Invitrogen (Carlsbad, CA).

    Article Title: Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire
    Article Snippet: The genes of Isd proteins were directly synthesized by GeneArt (Thermo Fisher Scientific) and cloned into either the pET-20 or the pET-47 expression vector (Novagen). .. Isd proteins and variants (truncations and point mutants) were expressed in BL21 DE3 cells (Thermo Fisher Scientific) under the control of lac operator with an N-term histidine tag and C-term Flag Tag.

    Centrifugation:

    Article Title: 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1
    Article Snippet: This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15 N ammonium chloride and 3 g/l 13 C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) ). .. Re-suspended cells were disrupted using sonication and centrifugation at 13,500 rpm was carried out to separate the cell pellet from the supernatant.

    Article Title: Installation of the Ether Bridge of Lolines by the Iron- and 2-Oxoglutarate-Dependent Oxygenase, LolO: Regio- and Stereochemistry of Sequential Hydroxylation and Oxacyclization Reactions
    Article Snippet: The plasmid was used to transform BL21 (DE3) cells (Invitrogen, Carlsbad, CA) for protein expression. .. Expression was induced by addition of isopropyl β-d -1-thiogalactopyranoside to a final concentration of 0.5 mM, and cells were shaken overnight at 16 °C before being harvested by centrifugation.

    Article Title: Reversal of Aging‐Induced Increases in Aortic Stiffness by Targeting Cytoskeletal Protein‐Protein Interfaces
    Article Snippet: The construct was expressed in BL21 (DE3) cells (Invitrogen, Carlsbad, CA), which were grown in terrific broth medium at 37°C until an optical density at 600 nm reached a value of 0.8. .. Cells were harvested by centrifugation, suspended in a column buffer (20 mmol/L Na‐HEPES, pH8.5, 500 mmol/L NaCl, 1 mmol/L EDTA, 0.1% Triton X‐100, and 20 μmol/L protease inhibitor), and lysed by sonication.

    Amplification:

    Article Title: 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1
    Article Snippet: Restriction digestion was carried out with NcoI and KpnI to create sticky ends for both the amplified R1 TraIC126 gene fragment and the template for subsequent ligation. .. This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15 N ammonium chloride and 3 g/l 13 C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) ).

    Article Title: A mutated recombinant subunit vaccine protects mice and guinea pigs against botulinum type A intoxication
    Article Snippet: The HCR template was amplified and subcloned into BamHI and NotI restriction sites of a pGEX4T3 vector (GE Healthcare Life Sciences, Chicago, IL) and tagged with N-terminal glutathione S-transferase. .. BA15 was transformed into BL21 DE3 cells (Invitrogen, Carlsbad, CA) for protein expression.

    Synthesized:

    Article Title: Installation of the Ether Bridge of Lolines by the Iron- and 2-Oxoglutarate-Dependent Oxygenase, LolO: Regio- and Stereochemistry of Sequential Hydroxylation and Oxacyclization Reactions
    Article Snippet: Overexpression of LolO in E. coli A gene encoding the same amino acid sequence as the lolO2 gene from Ep. uncinata e167 (GenBank accession no. AY723750.1) but codon-optimized for expression in Escherichia coli was synthesized and inserted into pET28a between the Nde I and Bam HI restriction sites by GeneArt (Regensburg, Germany). .. The plasmid was used to transform BL21 (DE3) cells (Invitrogen, Carlsbad, CA) for protein expression.

    Article Title: A mutated recombinant subunit vaccine protects mice and guinea pigs against botulinum type A intoxication
    Article Snippet: Construction and purification The downloaded HCR domain of BoNT/A (residues T876 to L1296) was synthesized (CosmoGentech, Seoul, South Korea) with codon optimization for E.coli expression. .. BA15 was transformed into BL21 DE3 cells (Invitrogen, Carlsbad, CA) for protein expression.

    Article Title: Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire
    Article Snippet: The genes of Isd proteins were directly synthesized by GeneArt (Thermo Fisher Scientific) and cloned into either the pET-20 or the pET-47 expression vector (Novagen). .. Isd proteins and variants (truncations and point mutants) were expressed in BL21 DE3 cells (Thermo Fisher Scientific) under the control of lac operator with an N-term histidine tag and C-term Flag Tag.

    Construct:

    Article Title: 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1
    Article Snippet: .. This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15 N ammonium chloride and 3 g/l 13 C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) ). .. In 1 L of culture, the protein expression was induced with 1 mM (final concentration) of Isopropyl-β-D-thiogalactopyranoside (IPTG) at an OD600 nm of 0.8 and growth was continued with shaking at 20 °C for 16 h. The cells were harvested and re-suspended in lysis buffer (50 mM Na2 HPO4 , 10 mM imidazole, 300 mM NaCl pH 8.0).

    Article Title: Reversal of Aging‐Induced Increases in Aortic Stiffness by Targeting Cytoskeletal Protein‐Protein Interfaces
    Article Snippet: .. The construct was expressed in BL21 (DE3) cells (Invitrogen, Carlsbad, CA), which were grown in terrific broth medium at 37°C until an optical density at 600 nm reached a value of 0.8. .. The recombinant protein expression was induced by addition of 0.5 mmol/L Isopropyl β‐D‐thiogalactoside (Invitrogen), and cultivation was continued at 20°C for 16 hours.

    Article Title: Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire
    Article Snippet: Isd proteins and variants (truncations and point mutants) were expressed in BL21 DE3 cells (Thermo Fisher Scientific) under the control of lac operator with an N-term histidine tag and C-term Flag Tag. .. These constructs were purified with Ni Sepharose Excel (GE Healthcare).

    Nuclear Magnetic Resonance:

    Article Title: 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1
    Article Snippet: .. This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15 N ammonium chloride and 3 g/l 13 C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) ). .. In 1 L of culture, the protein expression was induced with 1 mM (final concentration) of Isopropyl-β-D-thiogalactopyranoside (IPTG) at an OD600 nm of 0.8 and growth was continued with shaking at 20 °C for 16 h. The cells were harvested and re-suspended in lysis buffer (50 mM Na2 HPO4 , 10 mM imidazole, 300 mM NaCl pH 8.0).

    Activity Assay:

    Article Title: Saturation of recognition elements blocks evolution of new tRNA identities
    Article Snippet: BL21 (DE3) cells (Life Technologies), transformed with pQE70-AlaRS or pQE70-GlyRS, were grown at 37°C up to an OD600 (optical density at 600 nm) of 0.6. .. Recombinant ARS activity in crude cell extracts was assayed by aminoacylation assay.

    Expressing:

    Article Title: 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1
    Article Snippet: Paragraph title: Recombinant protein cloning, expression and purification ... This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15 N ammonium chloride and 3 g/l 13 C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) ).

    Article Title: Installation of the Ether Bridge of Lolines by the Iron- and 2-Oxoglutarate-Dependent Oxygenase, LolO: Regio- and Stereochemistry of Sequential Hydroxylation and Oxacyclization Reactions
    Article Snippet: .. The plasmid was used to transform BL21 (DE3) cells (Invitrogen, Carlsbad, CA) for protein expression. ..

    Article Title: A mutated recombinant subunit vaccine protects mice and guinea pigs against botulinum type A intoxication
    Article Snippet: .. BA15 was transformed into BL21 DE3 cells (Invitrogen, Carlsbad, CA) for protein expression. ..

    Article Title: MRSA isolates from the United States hospitals carry dfrG and dfrK resistance genes and succumb to propargyl-linked antifolates
    Article Snippet: Paragraph title: DfrA, DfrB, DfrG and DfrK Protein Expression and Purification ... BL21 (DE3) cells (Invitrogen) were transformed with DfrA and DfrG in pET-41a(+) and DfrK in pET-24-a(+) were transfected into BL21 (DE3) cells (Invitrogen) were transformed with.

    Article Title: Cardiomyopathy mutation (F88L) in troponin T abolishes length dependency of myofilament Ca2+ sensitivity
    Article Snippet: Paragraph title: Expression and purification of recombinant guinea pig cardiac troponin subunits ... Recombinant DNA was transformed and expressed in BL21*DE3 cells (Invitrogen).

    Article Title: Archaeal TFEα/β is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39
    Article Snippet: .. Co-purification assays For co-purification assays, pET-21a(+) and pRSF-1b-based vectors for the expression of Sso TFEα and Sso TFEβ or their mutant versions were co-transformed into BL21* (DE3) cells (Life Technologies). ..

    Article Title: Reversal of Aging‐Induced Increases in Aortic Stiffness by Targeting Cytoskeletal Protein‐Protein Interfaces
    Article Snippet: Paragraph title: Expression and Purification of VASP EVH1‐TAT Recombinant Protein ... The construct was expressed in BL21 (DE3) cells (Invitrogen, Carlsbad, CA), which were grown in terrific broth medium at 37°C until an optical density at 600 nm reached a value of 0.8.

    Article Title: Saturation of recognition elements blocks evolution of new tRNA identities
    Article Snippet: Paragraph title: Enzyme cloning, expression, and purification ... BL21 (DE3) cells (Life Technologies), transformed with pQE70-AlaRS or pQE70-GlyRS, were grown at 37°C up to an OD600 (optical density at 600 nm) of 0.6.

    Article Title: Caenorhabditis elegans PRMT-7 and PRMT-9 Are Evolutionarily Conserved Protein Arginine Methyltransferases with Distinct Substrate Specificities
    Article Snippet: Paragraph title: Bacterial Protein Expression and Purification ... The proteins were expressed in BL21 DE3 cells (Invitrogen); C. elegans GST-PRMT-7 and -9 enzymes were purified using the conditions described in , but with induction for 20 h at 18 °C using 0.13 mM isopropyl D-thiogalactopyranoside (IPTG) for GST-PRMT-7 and 1 mM IPTG for 20 h at 18 °C for both GST-PRMT-9 wild type and A391H mutant.

    Article Title: Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire
    Article Snippet: Paragraph title: Expression and purification of mAbs and protein reagents ... Isd proteins and variants (truncations and point mutants) were expressed in BL21 DE3 cells (Thermo Fisher Scientific) under the control of lac operator with an N-term histidine tag and C-term Flag Tag.

    Transformation Assay:

    Article Title: 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1
    Article Snippet: .. This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15 N ammonium chloride and 3 g/l 13 C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) ). .. In 1 L of culture, the protein expression was induced with 1 mM (final concentration) of Isopropyl-β-D-thiogalactopyranoside (IPTG) at an OD600 nm of 0.8 and growth was continued with shaking at 20 °C for 16 h. The cells were harvested and re-suspended in lysis buffer (50 mM Na2 HPO4 , 10 mM imidazole, 300 mM NaCl pH 8.0).

    Article Title: A mutated recombinant subunit vaccine protects mice and guinea pigs against botulinum type A intoxication
    Article Snippet: .. BA15 was transformed into BL21 DE3 cells (Invitrogen, Carlsbad, CA) for protein expression. ..

    Article Title: MRSA isolates from the United States hospitals carry dfrG and dfrK resistance genes and succumb to propargyl-linked antifolates
    Article Snippet: .. BL21 (DE3) cells (Invitrogen) were transformed with DfrA and DfrG in pET-41a(+) and DfrK in pET-24-a(+) were transfected into BL21 (DE3) cells (Invitrogen) were transformed with. ..

    Article Title: Cardiomyopathy mutation (F88L) in troponin T abolishes length dependency of myofilament Ca2+ sensitivity
    Article Snippet: .. Recombinant DNA was transformed and expressed in BL21*DE3 cells (Invitrogen). .. In brief, TnTWT and TnTF88L were purified by ion-exchange chromatography on a DEAE-Fast Sepharose column (GE Healthcare Biosciences).

    Article Title: Saturation of recognition elements blocks evolution of new tRNA identities
    Article Snippet: .. BL21 (DE3) cells (Life Technologies), transformed with pQE70-AlaRS or pQE70-GlyRS, were grown at 37°C up to an OD600 (optical density at 600 nm) of 0.6. .. Protein expression was then induced with 1 mM isopropyl-β-d -thiogalactopyranoside (IPTG) for 3 hours.

    Over Expression:

    Article Title: Installation of the Ether Bridge of Lolines by the Iron- and 2-Oxoglutarate-Dependent Oxygenase, LolO: Regio- and Stereochemistry of Sequential Hydroxylation and Oxacyclization Reactions
    Article Snippet: Paragraph title: Overexpression of LolO in E. coli ... The plasmid was used to transform BL21 (DE3) cells (Invitrogen, Carlsbad, CA) for protein expression.

    Crystallization Assay:

    Article Title: Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire
    Article Snippet: Isd proteins and variants (truncations and point mutants) were expressed in BL21 DE3 cells (Thermo Fisher Scientific) under the control of lac operator with an N-term histidine tag and C-term Flag Tag. .. The NEAT1 and NEAT2 domains utilized in crystallization were expressed with an N-terminal histidine tag followed by a HRV 3C site using the pET-47b vector (Novagen).

    Transfection:

    Article Title: MRSA isolates from the United States hospitals carry dfrG and dfrK resistance genes and succumb to propargyl-linked antifolates
    Article Snippet: .. BL21 (DE3) cells (Invitrogen) were transformed with DfrA and DfrG in pET-41a(+) and DfrK in pET-24-a(+) were transfected into BL21 (DE3) cells (Invitrogen) were transformed with. ..

    Ligation:

    Article Title: 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1
    Article Snippet: Restriction digestion was carried out with NcoI and KpnI to create sticky ends for both the amplified R1 TraIC126 gene fragment and the template for subsequent ligation. .. This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15 N ammonium chloride and 3 g/l 13 C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) ).

    Protease Inhibitor:

    Article Title: MRSA isolates from the United States hospitals carry dfrG and dfrK resistance genes and succumb to propargyl-linked antifolates
    Article Snippet: BL21 (DE3) cells (Invitrogen) were transformed with DfrA and DfrG in pET-41a(+) and DfrK in pET-24-a(+) were transfected into BL21 (DE3) cells (Invitrogen) were transformed with. .. DfrG was resuspended to 30 mL using a buffer containing 0.5 M KCl, 50 mM Tris, pH 8.0, 5 mM beta-mercaptoethanol, 5% glycerol, 0.8 mg/mL lysozyme, 5 mM imidazole, DNase, RNase and a protease inhibitor tablet (Life Technologies) and lysed via French Press.

    Article Title: Reversal of Aging‐Induced Increases in Aortic Stiffness by Targeting Cytoskeletal Protein‐Protein Interfaces
    Article Snippet: The construct was expressed in BL21 (DE3) cells (Invitrogen, Carlsbad, CA), which were grown in terrific broth medium at 37°C until an optical density at 600 nm reached a value of 0.8. .. Cells were harvested by centrifugation, suspended in a column buffer (20 mmol/L Na‐HEPES, pH8.5, 500 mmol/L NaCl, 1 mmol/L EDTA, 0.1% Triton X‐100, and 20 μmol/L protease inhibitor), and lysed by sonication.

    Generated:

    Article Title: Cardiomyopathy mutation (F88L) in troponin T abolishes length dependency of myofilament Ca2+ sensitivity
    Article Snippet: Recombinant c-myc –tagged guinea pig TnT (TnTWT and TnTF88L ), guinea pig WT troponin I (TnI), and guinea pig WT troponin C (TnC) were generated and cloned into a pSBETa vector (GenScript USA). .. Recombinant DNA was transformed and expressed in BL21*DE3 cells (Invitrogen).

    Sequencing:

    Article Title: Installation of the Ether Bridge of Lolines by the Iron- and 2-Oxoglutarate-Dependent Oxygenase, LolO: Regio- and Stereochemistry of Sequential Hydroxylation and Oxacyclization Reactions
    Article Snippet: Overexpression of LolO in E. coli A gene encoding the same amino acid sequence as the lolO2 gene from Ep. uncinata e167 (GenBank accession no. AY723750.1) but codon-optimized for expression in Escherichia coli was synthesized and inserted into pET28a between the Nde I and Bam HI restriction sites by GeneArt (Regensburg, Germany). .. The plasmid was used to transform BL21 (DE3) cells (Invitrogen, Carlsbad, CA) for protein expression.

    Article Title: Saturation of recognition elements blocks evolution of new tRNA identities
    Article Snippet: The correct sequence of the cDNA was confirmed by sequencing. .. BL21 (DE3) cells (Life Technologies), transformed with pQE70-AlaRS or pQE70-GlyRS, were grown at 37°C up to an OD600 (optical density at 600 nm) of 0.6.

    Sonication:

    Article Title: 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1
    Article Snippet: This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15 N ammonium chloride and 3 g/l 13 C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) ). .. Re-suspended cells were disrupted using sonication and centrifugation at 13,500 rpm was carried out to separate the cell pellet from the supernatant.

    Article Title: A mutated recombinant subunit vaccine protects mice and guinea pigs against botulinum type A intoxication
    Article Snippet: BA15 was transformed into BL21 DE3 cells (Invitrogen, Carlsbad, CA) for protein expression. .. Cell pellets were resuspended in ice-cold 20 mMTris pH 8.0, 200 mMNaCl lysis buffer and then disrupted via sonication (Qsonica, Newtown, CT).

    Article Title: MRSA isolates from the United States hospitals carry dfrG and dfrK resistance genes and succumb to propargyl-linked antifolates
    Article Snippet: BL21 (DE3) cells (Invitrogen) were transformed with DfrA and DfrG in pET-41a(+) and DfrK in pET-24-a(+) were transfected into BL21 (DE3) cells (Invitrogen) were transformed with. .. Cells were pelleted and resuspended to 30 mL using a buffer containing 0.4 M KCl, 25 mM Tris, pH 8.0, 5 mM beta-mercaptoethanol, 5% glycerol, 100 μg/mL lysozyme, 5 mM imidazole and DNase (Fisher Scientific) and lysed via sonication.

    Article Title: Archaeal TFEα/β is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39
    Article Snippet: Co-purification assays For co-purification assays, pET-21a(+) and pRSF-1b-based vectors for the expression of Sso TFEα and Sso TFEβ or their mutant versions were co-transformed into BL21* (DE3) cells (Life Technologies). .. Cells from 400 ml culture were resuspended in 4 ml TK(500) buffer and disrupted by sonication.

    Article Title: Reversal of Aging‐Induced Increases in Aortic Stiffness by Targeting Cytoskeletal Protein‐Protein Interfaces
    Article Snippet: The construct was expressed in BL21 (DE3) cells (Invitrogen, Carlsbad, CA), which were grown in terrific broth medium at 37°C until an optical density at 600 nm reached a value of 0.8. .. Cells were harvested by centrifugation, suspended in a column buffer (20 mmol/L Na‐HEPES, pH8.5, 500 mmol/L NaCl, 1 mmol/L EDTA, 0.1% Triton X‐100, and 20 μmol/L protease inhibitor), and lysed by sonication.

    Affinity Purification:

    Article Title: Reversal of Aging‐Induced Increases in Aortic Stiffness by Targeting Cytoskeletal Protein‐Protein Interfaces
    Article Snippet: This vector consists of a chitin‐binding domain (for affinity purification) and an intein tag (for self‐cleavage after purification). .. The construct was expressed in BL21 (DE3) cells (Invitrogen, Carlsbad, CA), which were grown in terrific broth medium at 37°C until an optical density at 600 nm reached a value of 0.8.

    Binding Assay:

    Article Title: 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1
    Article Snippet: The two primers: R1 TraIC126-F 5′-AGT CCATGG GCAATAGCGTCCAGCCG-3′ and R1 TraIC126-R 5′-TTT GGTACC CTAATCGCCGCCCAA-3′, were used to create the R1 TraIC126 encoding insert by PCR amplification. pET Z2 contains a 6xHis, IgG binding domain (Z-domain), TEV cleavage site, followed by a multi-cloning site and carries a kanamycin resistance gene. .. This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15 N ammonium chloride and 3 g/l 13 C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) ).

    Ion Exchange Chromatography:

    Article Title: Cardiomyopathy mutation (F88L) in troponin T abolishes length dependency of myofilament Ca2+ sensitivity
    Article Snippet: Recombinant DNA was transformed and expressed in BL21*DE3 cells (Invitrogen). .. In brief, TnTWT and TnTF88L were purified by ion-exchange chromatography on a DEAE-Fast Sepharose column (GE Healthcare Biosciences).

    Mutagenesis:

    Article Title: A mutated recombinant subunit vaccine protects mice and guinea pigs against botulinum type A intoxication
    Article Snippet: HCR/A mutants were created via overlap PCR mutagenesis. .. BA15 was transformed into BL21 DE3 cells (Invitrogen, Carlsbad, CA) for protein expression.

    Article Title: Archaeal TFEα/β is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39
    Article Snippet: .. Co-purification assays For co-purification assays, pET-21a(+) and pRSF-1b-based vectors for the expression of Sso TFEα and Sso TFEβ or their mutant versions were co-transformed into BL21* (DE3) cells (Life Technologies). ..

    Article Title: Caenorhabditis elegans PRMT-7 and PRMT-9 Are Evolutionarily Conserved Protein Arginine Methyltransferases with Distinct Substrate Specificities
    Article Snippet: .. The proteins were expressed in BL21 DE3 cells (Invitrogen); C. elegans GST-PRMT-7 and -9 enzymes were purified using the conditions described in , but with induction for 20 h at 18 °C using 0.13 mM isopropyl D-thiogalactopyranoside (IPTG) for GST-PRMT-7 and 1 mM IPTG for 20 h at 18 °C for both GST-PRMT-9 wild type and A391H mutant. .. The enzymes were purified as described and dialyzed overnight into 100 mM Tris-Cl, 100 mM NaCl, pH 7.5.

    Isolation:

    Article Title: Engineering a dirhodium artificial metalloenzyme for selective olefin cyclopropanation
    Article Snippet: E. coli DH5α and BL21 (DE3) cells were purchased from Invitrogen (Carlsbad, CA). .. Gel extraction (Cat# 28706) and plasmid isolation (Cat# 27106) kits were purchased from QIAGEN Inc. (Valencia, CA) and used according to the manufacturer's instructions.

    Lysis:

    Article Title: 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1
    Article Snippet: This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15 N ammonium chloride and 3 g/l 13 C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) ). .. In 1 L of culture, the protein expression was induced with 1 mM (final concentration) of Isopropyl-β-D-thiogalactopyranoside (IPTG) at an OD600 nm of 0.8 and growth was continued with shaking at 20 °C for 16 h. The cells were harvested and re-suspended in lysis buffer (50 mM Na2 HPO4 , 10 mM imidazole, 300 mM NaCl pH 8.0).

    Article Title: A mutated recombinant subunit vaccine protects mice and guinea pigs against botulinum type A intoxication
    Article Snippet: BA15 was transformed into BL21 DE3 cells (Invitrogen, Carlsbad, CA) for protein expression. .. Cell pellets were resuspended in ice-cold 20 mMTris pH 8.0, 200 mMNaCl lysis buffer and then disrupted via sonication (Qsonica, Newtown, CT).

    Purification:

    Article Title: 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1
    Article Snippet: Paragraph title: Recombinant protein cloning, expression and purification ... This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15 N ammonium chloride and 3 g/l 13 C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) ).

    Article Title: A mutated recombinant subunit vaccine protects mice and guinea pigs against botulinum type A intoxication
    Article Snippet: Paragraph title: Construction and purification ... BA15 was transformed into BL21 DE3 cells (Invitrogen, Carlsbad, CA) for protein expression.

    Article Title: MRSA isolates from the United States hospitals carry dfrG and dfrK resistance genes and succumb to propargyl-linked antifolates
    Article Snippet: Paragraph title: DfrA, DfrB, DfrG and DfrK Protein Expression and Purification ... BL21 (DE3) cells (Invitrogen) were transformed with DfrA and DfrG in pET-41a(+) and DfrK in pET-24-a(+) were transfected into BL21 (DE3) cells (Invitrogen) were transformed with.

    Article Title: Cardiomyopathy mutation (F88L) in troponin T abolishes length dependency of myofilament Ca2+ sensitivity
    Article Snippet: Paragraph title: Expression and purification of recombinant guinea pig cardiac troponin subunits ... Recombinant DNA was transformed and expressed in BL21*DE3 cells (Invitrogen).

    Article Title: Reversal of Aging‐Induced Increases in Aortic Stiffness by Targeting Cytoskeletal Protein‐Protein Interfaces
    Article Snippet: Paragraph title: Expression and Purification of VASP EVH1‐TAT Recombinant Protein ... The construct was expressed in BL21 (DE3) cells (Invitrogen, Carlsbad, CA), which were grown in terrific broth medium at 37°C until an optical density at 600 nm reached a value of 0.8.

    Article Title: Saturation of recognition elements blocks evolution of new tRNA identities
    Article Snippet: Paragraph title: Enzyme cloning, expression, and purification ... BL21 (DE3) cells (Life Technologies), transformed with pQE70-AlaRS or pQE70-GlyRS, were grown at 37°C up to an OD600 (optical density at 600 nm) of 0.6.

    Article Title: Caenorhabditis elegans PRMT-7 and PRMT-9 Are Evolutionarily Conserved Protein Arginine Methyltransferases with Distinct Substrate Specificities
    Article Snippet: .. The proteins were expressed in BL21 DE3 cells (Invitrogen); C. elegans GST-PRMT-7 and -9 enzymes were purified using the conditions described in , but with induction for 20 h at 18 °C using 0.13 mM isopropyl D-thiogalactopyranoside (IPTG) for GST-PRMT-7 and 1 mM IPTG for 20 h at 18 °C for both GST-PRMT-9 wild type and A391H mutant. .. The enzymes were purified as described and dialyzed overnight into 100 mM Tris-Cl, 100 mM NaCl, pH 7.5.

    Article Title: Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire
    Article Snippet: Paragraph title: Expression and purification of mAbs and protein reagents ... Isd proteins and variants (truncations and point mutants) were expressed in BL21 DE3 cells (Thermo Fisher Scientific) under the control of lac operator with an N-term histidine tag and C-term Flag Tag.

    Protein Purification:

    Article Title: 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1
    Article Snippet: This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15 N ammonium chloride and 3 g/l 13 C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) ). .. The supernatant was filtered through a 0.45 µm filter and protein purification was performed using Ni–NTA affinity chromatography.

    Article Title: Cardiomyopathy mutation (F88L) in troponin T abolishes length dependency of myofilament Ca2+ sensitivity
    Article Snippet: Recombinant DNA was transformed and expressed in BL21*DE3 cells (Invitrogen). .. Details on protein purification can be found in the Supplemental materials and methods.

    Polymerase Chain Reaction:

    Article Title: 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1
    Article Snippet: The two primers: R1 TraIC126-F 5′-AGT CCATGG GCAATAGCGTCCAGCCG-3′ and R1 TraIC126-R 5′-TTT GGTACC CTAATCGCCGCCCAA-3′, were used to create the R1 TraIC126 encoding insert by PCR amplification. pET Z2 contains a 6xHis, IgG binding domain (Z-domain), TEV cleavage site, followed by a multi-cloning site and carries a kanamycin resistance gene. .. This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15 N ammonium chloride and 3 g/l 13 C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) ).

    Article Title: A mutated recombinant subunit vaccine protects mice and guinea pigs against botulinum type A intoxication
    Article Snippet: HCR/A mutants were created via overlap PCR mutagenesis. .. BA15 was transformed into BL21 DE3 cells (Invitrogen, Carlsbad, CA) for protein expression.

    Positron Emission Tomography:

    Article Title: 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1
    Article Snippet: .. This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15 N ammonium chloride and 3 g/l 13 C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) ). .. In 1 L of culture, the protein expression was induced with 1 mM (final concentration) of Isopropyl-β-D-thiogalactopyranoside (IPTG) at an OD600 nm of 0.8 and growth was continued with shaking at 20 °C for 16 h. The cells were harvested and re-suspended in lysis buffer (50 mM Na2 HPO4 , 10 mM imidazole, 300 mM NaCl pH 8.0).

    Article Title: MRSA isolates from the United States hospitals carry dfrG and dfrK resistance genes and succumb to propargyl-linked antifolates
    Article Snippet: .. BL21 (DE3) cells (Invitrogen) were transformed with DfrA and DfrG in pET-41a(+) and DfrK in pET-24-a(+) were transfected into BL21 (DE3) cells (Invitrogen) were transformed with. ..

    Article Title: Archaeal TFEα/β is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39
    Article Snippet: .. Co-purification assays For co-purification assays, pET-21a(+) and pRSF-1b-based vectors for the expression of Sso TFEα and Sso TFEβ or their mutant versions were co-transformed into BL21* (DE3) cells (Life Technologies). ..

    Article Title: Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire
    Article Snippet: The genes of Isd proteins were directly synthesized by GeneArt (Thermo Fisher Scientific) and cloned into either the pET-20 or the pET-47 expression vector (Novagen). .. Isd proteins and variants (truncations and point mutants) were expressed in BL21 DE3 cells (Thermo Fisher Scientific) under the control of lac operator with an N-term histidine tag and C-term Flag Tag.

    Recombinant:

    Article Title: 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1
    Article Snippet: Paragraph title: Recombinant protein cloning, expression and purification ... This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15 N ammonium chloride and 3 g/l 13 C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) ).

    Article Title: Cardiomyopathy mutation (F88L) in troponin T abolishes length dependency of myofilament Ca2+ sensitivity
    Article Snippet: .. Recombinant DNA was transformed and expressed in BL21*DE3 cells (Invitrogen). .. In brief, TnTWT and TnTF88L were purified by ion-exchange chromatography on a DEAE-Fast Sepharose column (GE Healthcare Biosciences).

    Article Title: Reversal of Aging‐Induced Increases in Aortic Stiffness by Targeting Cytoskeletal Protein‐Protein Interfaces
    Article Snippet: Paragraph title: Expression and Purification of VASP EVH1‐TAT Recombinant Protein ... The construct was expressed in BL21 (DE3) cells (Invitrogen, Carlsbad, CA), which were grown in terrific broth medium at 37°C until an optical density at 600 nm reached a value of 0.8.

    Article Title: Saturation of recognition elements blocks evolution of new tRNA identities
    Article Snippet: BL21 (DE3) cells (Life Technologies), transformed with pQE70-AlaRS or pQE70-GlyRS, were grown at 37°C up to an OD600 (optical density at 600 nm) of 0.6. .. Recombinant ARS activity in crude cell extracts was assayed by aminoacylation assay.

    Aminoacylation Assay:

    Article Title: Saturation of recognition elements blocks evolution of new tRNA identities
    Article Snippet: BL21 (DE3) cells (Life Technologies), transformed with pQE70-AlaRS or pQE70-GlyRS, were grown at 37°C up to an OD600 (optical density at 600 nm) of 0.6. .. Recombinant ARS activity in crude cell extracts was assayed by aminoacylation assay.

    Plasmid Preparation:

    Article Title: 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1
    Article Snippet: Recombinant protein cloning, expression and purification R1 TraIC126 was cloned into pET Z2 vector (EMBL Heidelberg) (Bogomolovas et al. ) by classical cloning methods where full length R1 TraI was used as a template. .. This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15 N ammonium chloride and 3 g/l 13 C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) ).

    Article Title: Installation of the Ether Bridge of Lolines by the Iron- and 2-Oxoglutarate-Dependent Oxygenase, LolO: Regio- and Stereochemistry of Sequential Hydroxylation and Oxacyclization Reactions
    Article Snippet: .. The plasmid was used to transform BL21 (DE3) cells (Invitrogen, Carlsbad, CA) for protein expression. ..

    Article Title: A mutated recombinant subunit vaccine protects mice and guinea pigs against botulinum type A intoxication
    Article Snippet: The HCR template was amplified and subcloned into BamHI and NotI restriction sites of a pGEX4T3 vector (GE Healthcare Life Sciences, Chicago, IL) and tagged with N-terminal glutathione S-transferase. .. BA15 was transformed into BL21 DE3 cells (Invitrogen, Carlsbad, CA) for protein expression.

    Article Title: Cardiomyopathy mutation (F88L) in troponin T abolishes length dependency of myofilament Ca2+ sensitivity
    Article Snippet: Recombinant c-myc –tagged guinea pig TnT (TnTWT and TnTF88L ), guinea pig WT troponin I (TnI), and guinea pig WT troponin C (TnC) were generated and cloned into a pSBETa vector (GenScript USA). .. Recombinant DNA was transformed and expressed in BL21*DE3 cells (Invitrogen).

    Article Title: Reversal of Aging‐Induced Increases in Aortic Stiffness by Targeting Cytoskeletal Protein‐Protein Interfaces
    Article Snippet: This vector consists of a chitin‐binding domain (for affinity purification) and an intein tag (for self‐cleavage after purification). .. The construct was expressed in BL21 (DE3) cells (Invitrogen, Carlsbad, CA), which were grown in terrific broth medium at 37°C until an optical density at 600 nm reached a value of 0.8.

    Article Title: Saturation of recognition elements blocks evolution of new tRNA identities
    Article Snippet: Enzyme cloning, expression, and purification Human cytoplasmic alanyl-tRNA synthetase (AlaRS) and GlyRS complementary DNAs (cDNAs) (NM_001605 and NM_002047, respectively) were cloned into a pQE-70 vector (Qiagen) for bacterial expression of a C-terminal His6 -tagged protein. .. BL21 (DE3) cells (Life Technologies), transformed with pQE70-AlaRS or pQE70-GlyRS, were grown at 37°C up to an OD600 (optical density at 600 nm) of 0.6.

    Article Title: Engineering a dirhodium artificial metalloenzyme for selective olefin cyclopropanation
    Article Snippet: A codon-optimized gene for POP was obtained from GenScript USA Inc. (Piscataway, NJ) and cloned into the NcoI and XhoI restriction sites of the pET28a plasmid vector. .. E. coli DH5α and BL21 (DE3) cells were purchased from Invitrogen (Carlsbad, CA).

    Article Title: Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire
    Article Snippet: The genes of Isd proteins were directly synthesized by GeneArt (Thermo Fisher Scientific) and cloned into either the pET-20 or the pET-47 expression vector (Novagen). .. Isd proteins and variants (truncations and point mutants) were expressed in BL21 DE3 cells (Thermo Fisher Scientific) under the control of lac operator with an N-term histidine tag and C-term Flag Tag.

    Copurification:

    Article Title: Archaeal TFEα/β is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39
    Article Snippet: .. Co-purification assays For co-purification assays, pET-21a(+) and pRSF-1b-based vectors for the expression of Sso TFEα and Sso TFEβ or their mutant versions were co-transformed into BL21* (DE3) cells (Life Technologies). ..

    Incubation:

    Article Title: Archaeal TFEα/β is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39
    Article Snippet: Co-purification assays For co-purification assays, pET-21a(+) and pRSF-1b-based vectors for the expression of Sso TFEα and Sso TFEβ or their mutant versions were co-transformed into BL21* (DE3) cells (Life Technologies). .. The heat-stable supernatant was mixed with 0.2 ml pre-equilibrated His-Select resin (Sigma–Aldrich) and incubated for 10 min at 4°C.

    Affinity Chromatography:

    Article Title: 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1
    Article Snippet: This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15 N ammonium chloride and 3 g/l 13 C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) ). .. The supernatant was filtered through a 0.45 µm filter and protein purification was performed using Ni–NTA affinity chromatography.

    Concentration Assay:

    Article Title: 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1
    Article Snippet: This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15 N ammonium chloride and 3 g/l 13 C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) ). .. In 1 L of culture, the protein expression was induced with 1 mM (final concentration) of Isopropyl-β-D-thiogalactopyranoside (IPTG) at an OD600 nm of 0.8 and growth was continued with shaking at 20 °C for 16 h. The cells were harvested and re-suspended in lysis buffer (50 mM Na2 HPO4 , 10 mM imidazole, 300 mM NaCl pH 8.0).

    Article Title: Installation of the Ether Bridge of Lolines by the Iron- and 2-Oxoglutarate-Dependent Oxygenase, LolO: Regio- and Stereochemistry of Sequential Hydroxylation and Oxacyclization Reactions
    Article Snippet: The plasmid was used to transform BL21 (DE3) cells (Invitrogen, Carlsbad, CA) for protein expression. .. Expression was induced by addition of isopropyl β-d -1-thiogalactopyranoside to a final concentration of 0.5 mM, and cells were shaken overnight at 16 °C before being harvested by centrifugation.

    Article Title: A mutated recombinant subunit vaccine protects mice and guinea pigs against botulinum type A intoxication
    Article Snippet: BA15 was transformed into BL21 DE3 cells (Invitrogen, Carlsbad, CA) for protein expression. .. Expression was induced by adding isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM with a cell density of 0.4–0.5 OD600 in an overnight culture at 18°C.

    FLAG-tag:

    Article Title: Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire
    Article Snippet: .. Isd proteins and variants (truncations and point mutants) were expressed in BL21 DE3 cells (Thermo Fisher Scientific) under the control of lac operator with an N-term histidine tag and C-term Flag Tag. .. These constructs were purified with Ni Sepharose Excel (GE Healthcare).

    Gel Extraction:

    Article Title: Engineering a dirhodium artificial metalloenzyme for selective olefin cyclopropanation
    Article Snippet: E. coli DH5α and BL21 (DE3) cells were purchased from Invitrogen (Carlsbad, CA). .. Gel extraction (Cat# 28706) and plasmid isolation (Cat# 27106) kits were purchased from QIAGEN Inc. (Valencia, CA) and used according to the manufacturer's instructions.

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