e coli bl21  (Millipore)


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    Structured Review

    Millipore e coli bl21
    E Coli Bl21, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 912 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli bl21/product/Millipore
    Average 99 stars, based on 912 article reviews
    Price from $9.99 to $1999.99
    e coli bl21 - by Bioz Stars, 2020-04
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Molecular characterization and expression analysis of iron superoxide dismutase gene from Pseudochlorella pringsheimii (Trebouxiophyceae, Chlorophyta)
    Article Snippet: The previous product (644 bp, Fig. a) was gel-purified, and cloned into the pET28c expression vector (Novagen, USA). .. The FeSOD nucleotide sequences in the expression vector were sequenced again for validation, and then the vector was transformed into E. coli BL21(DE3)pLysS cells (Novagen, USA).

    Article Title: NS5B induces up-regulation of the BH3-only protein, BIK, essential for the hepatitis C virus RNA replication and viral release.
    Article Snippet: Generation of anti-NS5B monoclonal antibody The cDNA encoding for the N-terminal fragment (residues 1 to 385) of NS5B of JFH-1 HCV was cloned into the pGEX-6P1 vector (GE Healthcare, Uppsala, Sweden). .. Glutathione S-transferease (GST)-fusion NS5B protein was then expressed in Escherichia coli BL21(DE3) (Novagen, EMD Chemicals, Inc., Madison, WI) and purified as previously described .

    Article Title: Comparative Structural Analysis of Different Mycobacteriophage-Derived Mycolylarabinogalactan Esterases (Lysin B).
    Article Snippet: Paragraph title: 2.4. Cloning of LysB-His6 Enzymes ... After ligation with T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA, USA), the ligation mixture was transformed into E. coli BL21(DE3) (Novagen, Madison, WI, USA), plated on LB agar (Saveen and Werner AB, Limhamn, Sweden) supplemented with 100 µg/mL ampicillin (Sigma-Aldrich, St Louis, MO, USA), and grown overnight at 37 ◦C.

    Centrifugation:

    Article Title: Differential biochemical properties of three canonical Dps proteins from the cyanobacterium Nostoc punctiforme suggest distinct cellular functions
    Article Snippet: E. coli BL21(DE3) (Novagen) was used for heterologous protein expression. .. Cells were harvested by centrifugation at 4,000 × g for 20 min at 4 °C, and stored overnight at −20 °C.

    Article Title: Peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus.
    Article Snippet: GST fusion proteins were expressed in Escherichia coli BL21 (Novagen) as described in Wootton et al. (1998) . .. One hundred milliliters of log phase bacterial culture was induced with 1 mM IPTG, and after 3 h bacteria were pelleted by centrifugation, resuspended in 5 ml of PBS, and sonicated on ice three times for 30 s. Triton X-100 was added to a final concentration of 1%, and proteins were disassociated for 30 min at 4°C with constant agitation.

    MTT Assay:

    Article Title: 6-Hydroxypseudooxynicotine Dehydrogenase Delivers Electrons to Electron Transfer Flavoprotein during Nicotine Degradation by Agrobacterium tumefaciens S33
    Article Snippet: E. coli BL21(DE3) [F− ompT hsdS (rB − mB − ) gal dcm lacY1 (DE3); Novagen], E. coli C43(DE3) [F− ompT hsdS (rB − mB − ) gal dcm (DE3), harboring pCodonPlus (chloramphenicol resistant {Cmr }) and pRKISC (tetracycline resistant {Tcr }); gifted by Yasuhiro Takahashi from Osaka University], and E. coli S17-1 ( recA pro hsdR17 RP4-2 -Tcr ::Mu-Kmr ::Tn 7 integrated into the chromosome) ( ) were cultured in LB medium at 37°C. .. DCPIP, PMS, MTT, TTC, FAD, ADP, AMP, ferene [3-(2-pyridyl)-5,6-bis(5-sulfo-2-furyl)-1,2,4-triazinedisodium trihydrate], and coenzyme Q1 were purchased from Sigma-Aldrich (St. Louis, MO).

    Construct:

    Article Title: Dystrophin As A Molecular Shock Absorber
    Article Snippet: Paragraph title: Plasmids constructs and protein expression. ... Each of the resulting plasmids was co-transformed with a BirA plasmid and expressed in Escherichia coli BL21 (DE3) cultured in LB-media with D-Biotin (Sigma Aldrich), and affinity purified through His-tag.

    Incubation:

    Article Title: Molecular characterization and expression analysis of iron superoxide dismutase gene from Pseudochlorella pringsheimii (Trebouxiophyceae, Chlorophyta)
    Article Snippet: The FeSOD nucleotide sequences in the expression vector were sequenced again for validation, and then the vector was transformed into E. coli BL21(DE3)pLysS cells (Novagen, USA). .. After the E. coli cells reached a cell-density of OD600  = 0.6, the expression of the FeSOD protein was induced by the addition of 25 μM IPTG (isopropyl-β-D-thiogalactopyranoside), followed by incubation at 28 °C for 15 h. Purification of the expressed protein was done via affinity chromatography on a HiTrap HP column (GE Healthcare, USA), quantified by A280 and Bradford ( ) method (Biorad, USA) and the purity was validated by 12% SDS-PAGE (SDS–polyacrylamide gel electrophoresis; Laemmli , Fig. b).

    Article Title: 6-Hydroxypseudooxynicotine Dehydrogenase Delivers Electrons to Electron Transfer Flavoprotein during Nicotine Degradation by Agrobacterium tumefaciens S33
    Article Snippet: It was incubated aerobically in nicotine or HSP medium at 30°C, as described previously , where nicotine and HSP were used as the sole source of carbon and nitrogen. .. E. coli BL21(DE3) [F− ompT hsdS (rB − mB − ) gal dcm lacY1 (DE3); Novagen], E. coli C43(DE3) [F− ompT hsdS (rB − mB − ) gal dcm (DE3), harboring pCodonPlus (chloramphenicol resistant {Cmr }) and pRKISC (tetracycline resistant {Tcr }); gifted by Yasuhiro Takahashi from Osaka University], and E. coli S17-1 ( recA pro hsdR17 RP4-2 -Tcr ::Mu-Kmr ::Tn 7 integrated into the chromosome) ( ) were cultured in LB medium at 37°C.

    Article Title: Differential biochemical properties of three canonical Dps proteins from the cyanobacterium Nostoc punctiforme suggest distinct cellular functions
    Article Snippet: E. coli BL21(DE3) (Novagen) was used for heterologous protein expression. .. E. coli BL21(DE3) cells harboring the recombinant plasmids were incubated at 37 °C with shaking (250 rpm) in 600 ml of lysogeny broth (LB) culture medium containing 50 μg ml−1 of ampicillin and 0.1 g liter−1 of FeCl3 and grown to an optical density at 600 nm of 0.6.

    Article Title: Peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus.
    Article Snippet: GST fusion proteins were expressed in Escherichia coli BL21 (Novagen) as described in Wootton et al. (1998) . .. The insoluble fraction was removed by centrifugation, and the supernatant, containing the GST fusion proteins, was incubated with 100 l of a 50% slurry of glutathione Sepharose 4B beads (Pharmacia) overnight at 4°C.

    GPC Assay:

    Article Title: The RNase YbeY Is Vital for Ribosome Maturation, Stress Resistance, and Virulence of the Natural Genetic Engineer Agrobacterium tumefaciens
    Article Snippet: Hfq6×His and YbeY6×His fusion proteins were expressed in E. coli BL21(DE3) cells, using pET-plasmid-based expression from a T7 promoter (Novagen, Madison, WI). .. To assess the oligomeric state, we performed gel permeation chromatography.

    Expressing:

    Article Title: Molecular characterization and expression analysis of iron superoxide dismutase gene from Pseudochlorella pringsheimii (Trebouxiophyceae, Chlorophyta)
    Article Snippet: .. The FeSOD nucleotide sequences in the expression vector were sequenced again for validation, and then the vector was transformed into E. coli BL21(DE3)pLysS cells (Novagen, USA). .. After the E. coli cells reached a cell-density of OD600  = 0.6, the expression of the FeSOD protein was induced by the addition of 25 μM IPTG (isopropyl-β-D-thiogalactopyranoside), followed by incubation at 28 °C for 15 h. Purification of the expressed protein was done via affinity chromatography on a HiTrap HP column (GE Healthcare, USA), quantified by A280 and Bradford ( ) method (Biorad, USA) and the purity was validated by 12% SDS-PAGE (SDS–polyacrylamide gel electrophoresis; Laemmli , Fig. b).

    Article Title: The RNase YbeY Is Vital for Ribosome Maturation, Stress Resistance, and Virulence of the Natural Genetic Engineer Agrobacterium tumefaciens
    Article Snippet: .. Hfq6×His and YbeY6×His fusion proteins were expressed in E. coli BL21(DE3) cells, using pET-plasmid-based expression from a T7 promoter (Novagen, Madison, WI). .. To assess the oligomeric state, we performed gel permeation chromatography.

    Article Title: Complete reconstitution of the diverse pathways of gentamicin B biosynthesis
    Article Snippet: .. For expression of the recombinant proteins (GenM1, GenD2, GenS2, GenN, GenD1, GenQ, GenB1, GenJ, and GenK2), the E. coli BL21(DE3) (Novagen), E. coli BL21(DE3)pLysS (Novagen), E. coli Rosetta gamiTM2 (DE3) (Novagen) and E. coli ArcticExpress(DE3) (Agilent technologies, Santa Clara, CA, USA) were used as heterologous hosts. ..

    Article Title: Binding characterization of determinants in porcine aminopeptidase N, the cellular receptor for transmissible gastroenteritis virus.
    Article Snippet: .. Expression and purification of pAPN in E. coli The above-mentioned recombinant plasmids were transformed into E. coli BL21(DE3)pLysS (Novagen, Germany) and the transformed cells were cultured in Luria bertani (LB) medium containing Ampicillin (50 g/ml) at 37 • C with shaking until the optical density (OD) of the culture at 600 nm reached 0.5. .. Isopropyl ␤d-thiogalactoside (IPTG) was then added into the culture to a final concentration of 1 mM to induce pAPN expression at 37 • C for 6 h. The empty vector-transformed culture was used as control.

    Article Title: Mucosal and systemic immune responses elicited by recombinant Lactococcus lactis expressing a fusion protein composed of pertussis toxin and filamentous hemagglutinin from Bordetella pertussis.
    Article Snippet: Bacterial strains, plasmids, growth condition and animals L. lactis NZ3900 (LL) and L. lactis food-grade expression vector pNZ8149 were from NIZO food research (Kernhemseweg, Netherlands). .. E. coli BL21 (DE3; Novagen, USA) was grown in Luria-Bertani (LB) medium at 37°C with shaking at 220 rpm.

    Article Title: Structural analysis of the STAT1:STAT2 heterodimer revealed the mechanism of Sendai virus C protein–mediated blockade of type 1 interferon signaling
    Article Snippet: .. Expression of wild-type Y3, Y3W125A , or ProS2-Y3W125A was carried out in E. coli BL21(DE3) CodonPlus RIL (Novagen) at 15 °C for 24 h by induction with 0.2 m m isopropyl 1-thio-β- d -galactopyranoside, and expression of the STAT1ND:STAT2ND fusion protein was carried out in E. coli BL21(DE3)pLysS (Novagen) in a similar manner. .. All proteins were N terminally histidine-tagged and purified by nickel affinity chromatography using His-Bind Resin (Novagen) according to the supplier's instruction manual.

    Article Title: Differential biochemical properties of three canonical Dps proteins from the cyanobacterium Nostoc punctiforme suggest distinct cellular functions
    Article Snippet: .. E. coli BL21(DE3) (Novagen) was used for heterologous protein expression. .. E. coli BL21(DE3) cells harboring the recombinant plasmids were incubated at 37 °C with shaking (250 rpm) in 600 ml of lysogeny broth (LB) culture medium containing 50 μg ml−1 of ampicillin and 0.1 g liter−1 of FeCl3 and grown to an optical density at 600 nm of 0.6.

    Article Title: Dystrophin As A Molecular Shock Absorber
    Article Snippet: Paragraph title: Plasmids constructs and protein expression. ... Each of the resulting plasmids was co-transformed with a BirA plasmid and expressed in Escherichia coli BL21 (DE3) cultured in LB-media with D-Biotin (Sigma Aldrich), and affinity purified through His-tag.

    Article Title: Comparative Structural Analysis of Different Mycobacteriophage-Derived Mycolylarabinogalactan Esterases (Lysin B).
    Article Snippet: LysB-His6 gBlocks were cloned in EcoRI and NdeI restriction sites of pET22b (+) expression vector (Novagen, Madison, WI, USA). .. After ligation with T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA, USA), the ligation mixture was transformed into E. coli BL21(DE3) (Novagen, Madison, WI, USA), plated on LB agar (Saveen and Werner AB, Limhamn, Sweden) supplemented with 100 µg/mL ampicillin (Sigma-Aldrich, St Louis, MO, USA), and grown overnight at 37 ◦C.

    Transformation Assay:

    Article Title: Molecular characterization and expression analysis of iron superoxide dismutase gene from Pseudochlorella pringsheimii (Trebouxiophyceae, Chlorophyta)
    Article Snippet: .. The FeSOD nucleotide sequences in the expression vector were sequenced again for validation, and then the vector was transformed into E. coli BL21(DE3)pLysS cells (Novagen, USA). .. After the E. coli cells reached a cell-density of OD600  = 0.6, the expression of the FeSOD protein was induced by the addition of 25 μM IPTG (isopropyl-β-D-thiogalactopyranoside), followed by incubation at 28 °C for 15 h. Purification of the expressed protein was done via affinity chromatography on a HiTrap HP column (GE Healthcare, USA), quantified by A280 and Bradford ( ) method (Biorad, USA) and the purity was validated by 12% SDS-PAGE (SDS–polyacrylamide gel electrophoresis; Laemmli , Fig. b).

    Article Title: Binding characterization of determinants in porcine aminopeptidase N, the cellular receptor for transmissible gastroenteritis virus.
    Article Snippet: .. Expression and purification of pAPN in E. coli The above-mentioned recombinant plasmids were transformed into E. coli BL21(DE3)pLysS (Novagen, Germany) and the transformed cells were cultured in Luria bertani (LB) medium containing Ampicillin (50 g/ml) at 37 • C with shaking until the optical density (OD) of the culture at 600 nm reached 0.5. .. Isopropyl ␤d-thiogalactoside (IPTG) was then added into the culture to a final concentration of 1 mM to induce pAPN expression at 37 • C for 6 h. The empty vector-transformed culture was used as control.

    Article Title: Comparative Structural Analysis of Different Mycobacteriophage-Derived Mycolylarabinogalactan Esterases (Lysin B).
    Article Snippet: .. After ligation with T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA, USA), the ligation mixture was transformed into E. coli BL21(DE3) (Novagen, Madison, WI, USA), plated on LB agar (Saveen and Werner AB, Limhamn, Sweden) supplemented with 100 µg/mL ampicillin (Sigma-Aldrich, St Louis, MO, USA), and grown overnight at 37 ◦C. .. Plasmids (pET22b(+)-LysB-His6) were extracted from the transformant colonies and sequenced (GATC Biotech AB, Solna, Sweden); the one with the correct sequence was used for protein expression in E. coli BL21(DE3).

    Chromatography:

    Article Title: The RNase YbeY Is Vital for Ribosome Maturation, Stress Resistance, and Virulence of the Natural Genetic Engineer Agrobacterium tumefaciens
    Article Snippet: Hfq6×His and YbeY6×His fusion proteins were expressed in E. coli BL21(DE3) cells, using pET-plasmid-based expression from a T7 promoter (Novagen, Madison, WI). .. After Ni-NTA chromatography, purified His-tagged fusion proteins were separated by size via a Superdex 200 10/300 GL column (HEPES buffer [pH 7.5]) on an ÄKTA system (GE Healthcare).

    Ligation:

    Article Title: Comparative Structural Analysis of Different Mycobacteriophage-Derived Mycolylarabinogalactan Esterases (Lysin B).
    Article Snippet: .. After ligation with T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA, USA), the ligation mixture was transformed into E. coli BL21(DE3) (Novagen, Madison, WI, USA), plated on LB agar (Saveen and Werner AB, Limhamn, Sweden) supplemented with 100 µg/mL ampicillin (Sigma-Aldrich, St Louis, MO, USA), and grown overnight at 37 ◦C. .. Plasmids (pET22b(+)-LysB-His6) were extracted from the transformant colonies and sequenced (GATC Biotech AB, Solna, Sweden); the one with the correct sequence was used for protein expression in E. coli BL21(DE3).

    Synthesized:

    Article Title: Dystrophin As A Molecular Shock Absorber
    Article Snippet: Four fragments of the dystrophin central rod domain ( i.e ., SR01–05: 338th -938th ; SR06–10: 939th -1466th ; SR11–17: 1464th -2210th ; SR18–24: 2209th -3044th ) were synthesized by PCR using plasmid template containing the full-length human dystrophin sequence. .. Each of the resulting plasmids was co-transformed with a BirA plasmid and expressed in Escherichia coli BL21 (DE3) cultured in LB-media with D-Biotin (Sigma Aldrich), and affinity purified through His-tag.

    Cell Culture:

    Article Title: 6-Hydroxypseudooxynicotine Dehydrogenase Delivers Electrons to Electron Transfer Flavoprotein during Nicotine Degradation by Agrobacterium tumefaciens S33
    Article Snippet: .. E. coli BL21(DE3) [F− ompT hsdS (rB − mB − ) gal dcm lacY1 (DE3); Novagen], E. coli C43(DE3) [F− ompT hsdS (rB − mB − ) gal dcm (DE3), harboring pCodonPlus (chloramphenicol resistant {Cmr }) and pRKISC (tetracycline resistant {Tcr }); gifted by Yasuhiro Takahashi from Osaka University], and E. coli S17-1 ( recA pro hsdR17 RP4-2 -Tcr ::Mu-Kmr ::Tn 7 integrated into the chromosome) ( ) were cultured in LB medium at 37°C. .. DCPIP, PMS, MTT, TTC, FAD, ADP, AMP, ferene [3-(2-pyridyl)-5,6-bis(5-sulfo-2-furyl)-1,2,4-triazinedisodium trihydrate], and coenzyme Q1 were purchased from Sigma-Aldrich (St. Louis, MO).

    Article Title: Binding characterization of determinants in porcine aminopeptidase N, the cellular receptor for transmissible gastroenteritis virus.
    Article Snippet: .. Expression and purification of pAPN in E. coli The above-mentioned recombinant plasmids were transformed into E. coli BL21(DE3)pLysS (Novagen, Germany) and the transformed cells were cultured in Luria bertani (LB) medium containing Ampicillin (50 g/ml) at 37 • C with shaking until the optical density (OD) of the culture at 600 nm reached 0.5. .. Isopropyl ␤d-thiogalactoside (IPTG) was then added into the culture to a final concentration of 1 mM to induce pAPN expression at 37 • C for 6 h. The empty vector-transformed culture was used as control.

    Article Title: Dystrophin As A Molecular Shock Absorber
    Article Snippet: .. Each of the resulting plasmids was co-transformed with a BirA plasmid and expressed in Escherichia coli BL21 (DE3) cultured in LB-media with D-Biotin (Sigma Aldrich), and affinity purified through His-tag. .. A vertical magnetic tweezers setup, , was combined with a disturbance-free, rapid solution-exchange flow channel for conducting in vitro protein stretching experiments.

    Sequencing:

    Article Title: Molecular characterization and expression analysis of iron superoxide dismutase gene from Pseudochlorella pringsheimii (Trebouxiophyceae, Chlorophyta)
    Article Snippet: The FeSOD nucleotide sequences in the expression vector were sequenced again for validation, and then the vector was transformed into E. coli BL21(DE3)pLysS cells (Novagen, USA). .. The nucleotide sequence of the FeSOD of P. pringsheimii was deposited at the NCBI GenBank database under accession number .

    Article Title: Dystrophin As A Molecular Shock Absorber
    Article Snippet: Four fragments of the dystrophin central rod domain ( i.e ., SR01–05: 338th -938th ; SR06–10: 939th -1466th ; SR11–17: 1464th -2210th ; SR18–24: 2209th -3044th ) were synthesized by PCR using plasmid template containing the full-length human dystrophin sequence. .. Each of the resulting plasmids was co-transformed with a BirA plasmid and expressed in Escherichia coli BL21 (DE3) cultured in LB-media with D-Biotin (Sigma Aldrich), and affinity purified through His-tag.

    Article Title: Comparative Structural Analysis of Different Mycobacteriophage-Derived Mycolylarabinogalactan Esterases (Lysin B).
    Article Snippet: After ligation with T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA, USA), the ligation mixture was transformed into E. coli BL21(DE3) (Novagen, Madison, WI, USA), plated on LB agar (Saveen and Werner AB, Limhamn, Sweden) supplemented with 100 µg/mL ampicillin (Sigma-Aldrich, St Louis, MO, USA), and grown overnight at 37 ◦C. .. Plasmids (pET22b(+)-LysB-His6) were extracted from the transformant colonies and sequenced (GATC Biotech AB, Solna, Sweden); the one with the correct sequence was used for protein expression in E. coli BL21(DE3).

    Sonication:

    Article Title: Peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus.
    Article Snippet: GST fusion proteins were expressed in Escherichia coli BL21 (Novagen) as described in Wootton et al. (1998) . .. One hundred milliliters of log phase bacterial culture was induced with 1 mM IPTG, and after 3 h bacteria were pelleted by centrifugation, resuspended in 5 ml of PBS, and sonicated on ice three times for 30 s. Triton X-100 was added to a final concentration of 1%, and proteins were disassociated for 30 min at 4°C with constant agitation.

    Affinity Purification:

    Article Title: Dystrophin As A Molecular Shock Absorber
    Article Snippet: .. Each of the resulting plasmids was co-transformed with a BirA plasmid and expressed in Escherichia coli BL21 (DE3) cultured in LB-media with D-Biotin (Sigma Aldrich), and affinity purified through His-tag. .. A vertical magnetic tweezers setup, , was combined with a disturbance-free, rapid solution-exchange flow channel for conducting in vitro protein stretching experiments.

    Recombinant:

    Article Title: Complete reconstitution of the diverse pathways of gentamicin B biosynthesis
    Article Snippet: .. For expression of the recombinant proteins (GenM1, GenD2, GenS2, GenN, GenD1, GenQ, GenB1, GenJ, and GenK2), the E. coli BL21(DE3) (Novagen), E. coli BL21(DE3)pLysS (Novagen), E. coli Rosetta gamiTM2 (DE3) (Novagen) and E. coli ArcticExpress(DE3) (Agilent technologies, Santa Clara, CA, USA) were used as heterologous hosts. ..

    Article Title: Binding characterization of determinants in porcine aminopeptidase N, the cellular receptor for transmissible gastroenteritis virus.
    Article Snippet: .. Expression and purification of pAPN in E. coli The above-mentioned recombinant plasmids were transformed into E. coli BL21(DE3)pLysS (Novagen, Germany) and the transformed cells were cultured in Luria bertani (LB) medium containing Ampicillin (50 g/ml) at 37 • C with shaking until the optical density (OD) of the culture at 600 nm reached 0.5. .. Isopropyl ␤d-thiogalactoside (IPTG) was then added into the culture to a final concentration of 1 mM to induce pAPN expression at 37 • C for 6 h. The empty vector-transformed culture was used as control.

    Article Title: Differential biochemical properties of three canonical Dps proteins from the cyanobacterium Nostoc punctiforme suggest distinct cellular functions
    Article Snippet: Paragraph title: Bacterial strains, growth conditions, and expression of recombinant protein ... E. coli BL21(DE3) (Novagen) was used for heterologous protein expression.

    Article Title: Comparative Structural Analysis of Different Mycobacteriophage-Derived Mycolylarabinogalactan Esterases (Lysin B).
    Article Snippet: After ligation with T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA, USA), the ligation mixture was transformed into E. coli BL21(DE3) (Novagen, Madison, WI, USA), plated on LB agar (Saveen and Werner AB, Limhamn, Sweden) supplemented with 100 µg/mL ampicillin (Sigma-Aldrich, St Louis, MO, USA), and grown overnight at 37 ◦C. .. Cells were grown overnight at 37 ◦C, 200 rpm in LB supplemented with 100 µg/mL ampicillin, and glycerol stocks of the recombinant cells were prepared and stored at −80 ◦C.

    DNA Extraction:

    Article Title: Complete reconstitution of the diverse pathways of gentamicin B biosynthesis
    Article Snippet: For genomic DNA isolation and cultivation, M. echinospora was grown for 5 d at 28 °C in ATCC 172 medium (1% glucose, 0.5% yeast extract, 2% soluble starch, 0.5% N-Z amine, and 0.2% CaCO3 ). .. For expression of the recombinant proteins (GenM1, GenD2, GenS2, GenN, GenD1, GenQ, GenB1, GenJ, and GenK2), the E. coli BL21(DE3) (Novagen), E. coli BL21(DE3)pLysS (Novagen), E. coli Rosetta gamiTM2 (DE3) (Novagen) and E. coli ArcticExpress(DE3) (Agilent technologies, Santa Clara, CA, USA) were used as heterologous hosts.

    Nucleic Acid Electrophoresis:

    Article Title: Molecular characterization and expression analysis of iron superoxide dismutase gene from Pseudochlorella pringsheimii (Trebouxiophyceae, Chlorophyta)
    Article Snippet: The FeSOD nucleotide sequences in the expression vector were sequenced again for validation, and then the vector was transformed into E. coli BL21(DE3)pLysS cells (Novagen, USA). .. After the E. coli cells reached a cell-density of OD600  = 0.6, the expression of the FeSOD protein was induced by the addition of 25 μM IPTG (isopropyl-β-D-thiogalactopyranoside), followed by incubation at 28 °C for 15 h. Purification of the expressed protein was done via affinity chromatography on a HiTrap HP column (GE Healthcare, USA), quantified by A280 and Bradford ( ) method (Biorad, USA) and the purity was validated by 12% SDS-PAGE (SDS–polyacrylamide gel electrophoresis; Laemmli , Fig. b).

    Pull Down Assay:

    Article Title: Peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus.
    Article Snippet: Paragraph title: GST-bead pull-down assay ... GST fusion proteins were expressed in Escherichia coli BL21 (Novagen) as described in Wootton et al. (1998) .

    Subcloning:

    Article Title: Complete reconstitution of the diverse pathways of gentamicin B biosynthesis
    Article Snippet: E. coli DH5α and plasmid pGEM-T Easy Vector (Promega, Madison, WI, USA) were used for routine subcloning. .. For expression of the recombinant proteins (GenM1, GenD2, GenS2, GenN, GenD1, GenQ, GenB1, GenJ, and GenK2), the E. coli BL21(DE3) (Novagen), E. coli BL21(DE3)pLysS (Novagen), E. coli Rosetta gamiTM2 (DE3) (Novagen) and E. coli ArcticExpress(DE3) (Agilent technologies, Santa Clara, CA, USA) were used as heterologous hosts.

    Purification:

    Article Title: Molecular characterization and expression analysis of iron superoxide dismutase gene from Pseudochlorella pringsheimii (Trebouxiophyceae, Chlorophyta)
    Article Snippet: The FeSOD nucleotide sequences in the expression vector were sequenced again for validation, and then the vector was transformed into E. coli BL21(DE3)pLysS cells (Novagen, USA). .. After the E. coli cells reached a cell-density of OD600  = 0.6, the expression of the FeSOD protein was induced by the addition of 25 μM IPTG (isopropyl-β-D-thiogalactopyranoside), followed by incubation at 28 °C for 15 h. Purification of the expressed protein was done via affinity chromatography on a HiTrap HP column (GE Healthcare, USA), quantified by A280 and Bradford ( ) method (Biorad, USA) and the purity was validated by 12% SDS-PAGE (SDS–polyacrylamide gel electrophoresis; Laemmli , Fig. b).

    Article Title: The RNase YbeY Is Vital for Ribosome Maturation, Stress Resistance, and Virulence of the Natural Genetic Engineer Agrobacterium tumefaciens
    Article Snippet: Hfq6×His and YbeY6×His fusion proteins were expressed in E. coli BL21(DE3) cells, using pET-plasmid-based expression from a T7 promoter (Novagen, Madison, WI). .. After Ni-NTA chromatography, purified His-tagged fusion proteins were separated by size via a Superdex 200 10/300 GL column (HEPES buffer [pH 7.5]) on an ÄKTA system (GE Healthcare).

    Article Title: Binding characterization of determinants in porcine aminopeptidase N, the cellular receptor for transmissible gastroenteritis virus.
    Article Snippet: .. Expression and purification of pAPN in E. coli The above-mentioned recombinant plasmids were transformed into E. coli BL21(DE3)pLysS (Novagen, Germany) and the transformed cells were cultured in Luria bertani (LB) medium containing Ampicillin (50 g/ml) at 37 • C with shaking until the optical density (OD) of the culture at 600 nm reached 0.5. .. Isopropyl ␤d-thiogalactoside (IPTG) was then added into the culture to a final concentration of 1 mM to induce pAPN expression at 37 • C for 6 h. The empty vector-transformed culture was used as control.

    Article Title: NS5B induces up-regulation of the BH3-only protein, BIK, essential for the hepatitis C virus RNA replication and viral release.
    Article Snippet: .. Glutathione S-transferease (GST)-fusion NS5B protein was then expressed in Escherichia coli BL21(DE3) (Novagen, EMD Chemicals, Inc., Madison, WI) and purified as previously described . .. The GST-fusion protein was then used to immunize mice and generate hybridomas as previously described (Oh et al., 2010) .

    Article Title: Structural analysis of the STAT1:STAT2 heterodimer revealed the mechanism of Sendai virus C protein–mediated blockade of type 1 interferon signaling
    Article Snippet: Expression of wild-type Y3, Y3W125A , or ProS2-Y3W125A was carried out in E. coli BL21(DE3) CodonPlus RIL (Novagen) at 15 °C for 24 h by induction with 0.2 m m isopropyl 1-thio-β- d -galactopyranoside, and expression of the STAT1ND:STAT2ND fusion protein was carried out in E. coli BL21(DE3)pLysS (Novagen) in a similar manner. .. All proteins were N terminally histidine-tagged and purified by nickel affinity chromatography using His-Bind Resin (Novagen) according to the supplier's instruction manual.

    Article Title: Comparative Structural Analysis of Different Mycobacteriophage-Derived Mycolylarabinogalactan Esterases (Lysin B).
    Article Snippet: After ligation with T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA, USA), the ligation mixture was transformed into E. coli BL21(DE3) (Novagen, Madison, WI, USA), plated on LB agar (Saveen and Werner AB, Limhamn, Sweden) supplemented with 100 µg/mL ampicillin (Sigma-Aldrich, St Louis, MO, USA), and grown overnight at 37 ◦C. .. Production and Purification of LysB-His6 Enzymes 2.5.1.

    Protein Purification:

    Article Title: The RNase YbeY Is Vital for Ribosome Maturation, Stress Resistance, and Virulence of the Natural Genetic Engineer Agrobacterium tumefaciens
    Article Snippet: Paragraph title: Protein purification and GPC. ... Hfq6×His and YbeY6×His fusion proteins were expressed in E. coli BL21(DE3) cells, using pET-plasmid-based expression from a T7 promoter (Novagen, Madison, WI).

    Polymerase Chain Reaction:

    Article Title: Dystrophin As A Molecular Shock Absorber
    Article Snippet: Four fragments of the dystrophin central rod domain ( i.e ., SR01–05: 338th -938th ; SR06–10: 939th -1466th ; SR11–17: 1464th -2210th ; SR18–24: 2209th -3044th ) were synthesized by PCR using plasmid template containing the full-length human dystrophin sequence. .. Each of the resulting plasmids was co-transformed with a BirA plasmid and expressed in Escherichia coli BL21 (DE3) cultured in LB-media with D-Biotin (Sigma Aldrich), and affinity purified through His-tag.

    Affinity Chromatography:

    Article Title: Molecular characterization and expression analysis of iron superoxide dismutase gene from Pseudochlorella pringsheimii (Trebouxiophyceae, Chlorophyta)
    Article Snippet: The FeSOD nucleotide sequences in the expression vector were sequenced again for validation, and then the vector was transformed into E. coli BL21(DE3)pLysS cells (Novagen, USA). .. After the E. coli cells reached a cell-density of OD600  = 0.6, the expression of the FeSOD protein was induced by the addition of 25 μM IPTG (isopropyl-β-D-thiogalactopyranoside), followed by incubation at 28 °C for 15 h. Purification of the expressed protein was done via affinity chromatography on a HiTrap HP column (GE Healthcare, USA), quantified by A280 and Bradford ( ) method (Biorad, USA) and the purity was validated by 12% SDS-PAGE (SDS–polyacrylamide gel electrophoresis; Laemmli , Fig. b).

    Article Title: Structural analysis of the STAT1:STAT2 heterodimer revealed the mechanism of Sendai virus C protein–mediated blockade of type 1 interferon signaling
    Article Snippet: Expression of wild-type Y3, Y3W125A , or ProS2-Y3W125A was carried out in E. coli BL21(DE3) CodonPlus RIL (Novagen) at 15 °C for 24 h by induction with 0.2 m m isopropyl 1-thio-β- d -galactopyranoside, and expression of the STAT1ND:STAT2ND fusion protein was carried out in E. coli BL21(DE3)pLysS (Novagen) in a similar manner. .. All proteins were N terminally histidine-tagged and purified by nickel affinity chromatography using His-Bind Resin (Novagen) according to the supplier's instruction manual.

    Mouse Assay:

    Article Title: NS5B induces up-regulation of the BH3-only protein, BIK, essential for the hepatitis C virus RNA replication and viral release.
    Article Snippet: Glutathione S-transferease (GST)-fusion NS5B protein was then expressed in Escherichia coli BL21(DE3) (Novagen, EMD Chemicals, Inc., Madison, WI) and purified as previously described . .. The GST-fusion protein was then used to immunize mice and generate hybridomas as previously described (Oh et al., 2010) .

    Article Title: Mucosal and systemic immune responses elicited by recombinant Lactococcus lactis expressing a fusion protein composed of pertussis toxin and filamentous hemagglutinin from Bordetella pertussis.
    Article Snippet: E. coli BL21 (DE3; Novagen, USA) was grown in Luria-Bertani (LB) medium at 37°C with shaking at 220 rpm. .. Female BALB/c mice (4-6 weeks old) were purchased from the animal facility of Production Complex of Pasteur Institute of Iran (Karaj, Iran).

    SDS Page:

    Article Title: Molecular characterization and expression analysis of iron superoxide dismutase gene from Pseudochlorella pringsheimii (Trebouxiophyceae, Chlorophyta)
    Article Snippet: The FeSOD nucleotide sequences in the expression vector were sequenced again for validation, and then the vector was transformed into E. coli BL21(DE3)pLysS cells (Novagen, USA). .. After the E. coli cells reached a cell-density of OD600  = 0.6, the expression of the FeSOD protein was induced by the addition of 25 μM IPTG (isopropyl-β-D-thiogalactopyranoside), followed by incubation at 28 °C for 15 h. Purification of the expressed protein was done via affinity chromatography on a HiTrap HP column (GE Healthcare, USA), quantified by A280 and Bradford ( ) method (Biorad, USA) and the purity was validated by 12% SDS-PAGE (SDS–polyacrylamide gel electrophoresis; Laemmli , Fig. b).

    Plasmid Preparation:

    Article Title: Molecular characterization and expression analysis of iron superoxide dismutase gene from Pseudochlorella pringsheimii (Trebouxiophyceae, Chlorophyta)
    Article Snippet: .. The FeSOD nucleotide sequences in the expression vector were sequenced again for validation, and then the vector was transformed into E. coli BL21(DE3)pLysS cells (Novagen, USA). .. After the E. coli cells reached a cell-density of OD600  = 0.6, the expression of the FeSOD protein was induced by the addition of 25 μM IPTG (isopropyl-β-D-thiogalactopyranoside), followed by incubation at 28 °C for 15 h. Purification of the expressed protein was done via affinity chromatography on a HiTrap HP column (GE Healthcare, USA), quantified by A280 and Bradford ( ) method (Biorad, USA) and the purity was validated by 12% SDS-PAGE (SDS–polyacrylamide gel electrophoresis; Laemmli , Fig. b).

    Article Title: Complete reconstitution of the diverse pathways of gentamicin B biosynthesis
    Article Snippet: E. coli DH5α and plasmid pGEM-T Easy Vector (Promega, Madison, WI, USA) were used for routine subcloning. .. For expression of the recombinant proteins (GenM1, GenD2, GenS2, GenN, GenD1, GenQ, GenB1, GenJ, and GenK2), the E. coli BL21(DE3) (Novagen), E. coli BL21(DE3)pLysS (Novagen), E. coli Rosetta gamiTM2 (DE3) (Novagen) and E. coli ArcticExpress(DE3) (Agilent technologies, Santa Clara, CA, USA) were used as heterologous hosts.

    Article Title: Binding characterization of determinants in porcine aminopeptidase N, the cellular receptor for transmissible gastroenteritis virus.
    Article Snippet: Expression and purification of pAPN in E. coli The above-mentioned recombinant plasmids were transformed into E. coli BL21(DE3)pLysS (Novagen, Germany) and the transformed cells were cultured in Luria bertani (LB) medium containing Ampicillin (50 g/ml) at 37 • C with shaking until the optical density (OD) of the culture at 600 nm reached 0.5. .. Isopropyl ␤d-thiogalactoside (IPTG) was then added into the culture to a final concentration of 1 mM to induce pAPN expression at 37 • C for 6 h. The empty vector-transformed culture was used as control.

    Article Title: NS5B induces up-regulation of the BH3-only protein, BIK, essential for the hepatitis C virus RNA replication and viral release.
    Article Snippet: Generation of anti-NS5B monoclonal antibody The cDNA encoding for the N-terminal fragment (residues 1 to 385) of NS5B of JFH-1 HCV was cloned into the pGEX-6P1 vector (GE Healthcare, Uppsala, Sweden). .. Glutathione S-transferease (GST)-fusion NS5B protein was then expressed in Escherichia coli BL21(DE3) (Novagen, EMD Chemicals, Inc., Madison, WI) and purified as previously described .

    Article Title: Mucosal and systemic immune responses elicited by recombinant Lactococcus lactis expressing a fusion protein composed of pertussis toxin and filamentous hemagglutinin from Bordetella pertussis.
    Article Snippet: Bacterial strains, plasmids, growth condition and animals L. lactis NZ3900 (LL) and L. lactis food-grade expression vector pNZ8149 were from NIZO food research (Kernhemseweg, Netherlands). .. E. coli BL21 (DE3; Novagen, USA) was grown in Luria-Bertani (LB) medium at 37°C with shaking at 220 rpm.

    Article Title: Dystrophin As A Molecular Shock Absorber
    Article Snippet: .. Each of the resulting plasmids was co-transformed with a BirA plasmid and expressed in Escherichia coli BL21 (DE3) cultured in LB-media with D-Biotin (Sigma Aldrich), and affinity purified through His-tag. .. A vertical magnetic tweezers setup, , was combined with a disturbance-free, rapid solution-exchange flow channel for conducting in vitro protein stretching experiments.

    Article Title: Comparative Structural Analysis of Different Mycobacteriophage-Derived Mycolylarabinogalactan Esterases (Lysin B).
    Article Snippet: LysB-His6 gBlocks were cloned in EcoRI and NdeI restriction sites of pET22b (+) expression vector (Novagen, Madison, WI, USA). .. After ligation with T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA, USA), the ligation mixture was transformed into E. coli BL21(DE3) (Novagen, Madison, WI, USA), plated on LB agar (Saveen and Werner AB, Limhamn, Sweden) supplemented with 100 µg/mL ampicillin (Sigma-Aldrich, St Louis, MO, USA), and grown overnight at 37 ◦C.

    Positron Emission Tomography:

    Article Title: The RNase YbeY Is Vital for Ribosome Maturation, Stress Resistance, and Virulence of the Natural Genetic Engineer Agrobacterium tumefaciens
    Article Snippet: .. Hfq6×His and YbeY6×His fusion proteins were expressed in E. coli BL21(DE3) cells, using pET-plasmid-based expression from a T7 promoter (Novagen, Madison, WI). .. To assess the oligomeric state, we performed gel permeation chromatography.

    Article Title: Complete reconstitution of the diverse pathways of gentamicin B biosynthesis
    Article Snippet: The pET expression vectors, such as pET15b and pET28a, were purchased from Novagen (Madison, WI, USA). .. For expression of the recombinant proteins (GenM1, GenD2, GenS2, GenN, GenD1, GenQ, GenB1, GenJ, and GenK2), the E. coli BL21(DE3) (Novagen), E. coli BL21(DE3)pLysS (Novagen), E. coli Rosetta gamiTM2 (DE3) (Novagen) and E. coli ArcticExpress(DE3) (Agilent technologies, Santa Clara, CA, USA) were used as heterologous hosts.

    Gel Permeation Chromatography:

    Article Title: The RNase YbeY Is Vital for Ribosome Maturation, Stress Resistance, and Virulence of the Natural Genetic Engineer Agrobacterium tumefaciens
    Article Snippet: Paragraph title: Protein purification and GPC. ... Hfq6×His and YbeY6×His fusion proteins were expressed in E. coli BL21(DE3) cells, using pET-plasmid-based expression from a T7 promoter (Novagen, Madison, WI).

    Concentration Assay:

    Article Title: Binding characterization of determinants in porcine aminopeptidase N, the cellular receptor for transmissible gastroenteritis virus.
    Article Snippet: Expression and purification of pAPN in E. coli The above-mentioned recombinant plasmids were transformed into E. coli BL21(DE3)pLysS (Novagen, Germany) and the transformed cells were cultured in Luria bertani (LB) medium containing Ampicillin (50 g/ml) at 37 • C with shaking until the optical density (OD) of the culture at 600 nm reached 0.5. .. Isopropyl ␤d-thiogalactoside (IPTG) was then added into the culture to a final concentration of 1 mM to induce pAPN expression at 37 • C for 6 h. The empty vector-transformed culture was used as control.

    Article Title: Peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus.
    Article Snippet: GST fusion proteins were expressed in Escherichia coli BL21 (Novagen) as described in Wootton et al. (1998) . .. One hundred milliliters of log phase bacterial culture was induced with 1 mM IPTG, and after 3 h bacteria were pelleted by centrifugation, resuspended in 5 ml of PBS, and sonicated on ice three times for 30 s. Triton X-100 was added to a final concentration of 1%, and proteins were disassociated for 30 min at 4°C with constant agitation.

    Marker:

    Article Title: Complete reconstitution of the diverse pathways of gentamicin B biosynthesis
    Article Snippet: For expression of the recombinant proteins (GenM1, GenD2, GenS2, GenN, GenD1, GenQ, GenB1, GenJ, and GenK2), the E. coli BL21(DE3) (Novagen), E. coli BL21(DE3)pLysS (Novagen), E. coli Rosetta gamiTM2 (DE3) (Novagen) and E. coli ArcticExpress(DE3) (Agilent technologies, Santa Clara, CA, USA) were used as heterologous hosts. .. For the recombinant GenM2, the high-copy-number E. coli -Streptomyces shuttle vector pSE34 containing the constitutive ermE * promoter plus a thiostrepton resistance marker was used for expression .

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  • 99
    Millipore gst prescission protease
    Accessible hydrophobic residues in the predicted coiled coil are critical for Rab binding. (A) Helical wheel projection of a coiled coil predicted for GCC185 residues 1579−1606. Residues in registers “a-f” were predicted by the Paircoil program. Residues at positions “a” and “d” lie in the dimer interface. Boxed residues are candidates for binding interactions with Rab GTPases. (B, C) Effect of alanine substitutions on Rab binding. Reactions contained wild type or mutant <t>GST-C-110</t> (B; 3 μM, C; 2 μM) and 35 S-GTPγS-preloaded GTPases (B; 170 pmol Rab9-His, C; 190 pmol His-Rab6). Data are mean ± SD. (D) Mass determination of untagged <t>RBD-87</t> I1588A/L1595A by multiple angle static light scattering. The gel filtration elution profile of the protein (black line) and molecular mass (grey line) are shown. Polydispersity of the peak was 1.001.
    Gst Prescission Protease, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore lsrb bl21 ∆ luxs protein
    Binding of <t>LsrB</t> to endogenous AI-2. Purified LsrB (BL21) and LsrB <t>(BL21∆luxS)</t> proteins (5 mg/ml) were incubated for 10 min at 37, 50 and 60 °C to release endogenous AI-2, respectively. After incubation, the LsrB proteins were removed by ultrafiltration (10,000-Da cut-off; EMD Millipore), and the filtered reaction products were tested for AI-2 activity using a V. harveyi BB170 bioassay. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C, respectively ( a ). However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS) ( b ). Moreover, the recombinant LuxS protein, which was expressed in strain BL21 (pColdTF-lsrB) as a negative control, also showed no AI-2 binding activity ( c ). AI-2 (10 μM) was used as a positive control
    Lsrb Bl21 ∆ Luxs Protein, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore bl21 ∆ luxs
    Construction and identification of luxS mutant strain <t>BL21∆luxS</t>
    Bl21 ∆ Luxs, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Accessible hydrophobic residues in the predicted coiled coil are critical for Rab binding. (A) Helical wheel projection of a coiled coil predicted for GCC185 residues 1579−1606. Residues in registers “a-f” were predicted by the Paircoil program. Residues at positions “a” and “d” lie in the dimer interface. Boxed residues are candidates for binding interactions with Rab GTPases. (B, C) Effect of alanine substitutions on Rab binding. Reactions contained wild type or mutant GST-C-110 (B; 3 μM, C; 2 μM) and 35 S-GTPγS-preloaded GTPases (B; 170 pmol Rab9-His, C; 190 pmol His-Rab6). Data are mean ± SD. (D) Mass determination of untagged RBD-87 I1588A/L1595A by multiple angle static light scattering. The gel filtration elution profile of the protein (black line) and molecular mass (grey line) are shown. Polydispersity of the peak was 1.001.

    Journal: Cell

    Article Title: Dual GTPase regulation of the GCC185 Golgin: Communication between adjacent Rab6 and Arl1 binding sites

    doi: 10.1016/j.cell.2007.11.048

    Figure Lengend Snippet: Accessible hydrophobic residues in the predicted coiled coil are critical for Rab binding. (A) Helical wheel projection of a coiled coil predicted for GCC185 residues 1579−1606. Residues in registers “a-f” were predicted by the Paircoil program. Residues at positions “a” and “d” lie in the dimer interface. Boxed residues are candidates for binding interactions with Rab GTPases. (B, C) Effect of alanine substitutions on Rab binding. Reactions contained wild type or mutant GST-C-110 (B; 3 μM, C; 2 μM) and 35 S-GTPγS-preloaded GTPases (B; 170 pmol Rab9-His, C; 190 pmol His-Rab6). Data are mean ± SD. (D) Mass determination of untagged RBD-87 I1588A/L1595A by multiple angle static light scattering. The gel filtration elution profile of the protein (black line) and molecular mass (grey line) are shown. Polydispersity of the peak was 1.001.

    Article Snippet: RBD-87 was then separated from GST, GST-Prescission Protease and glutathione beads over a solid support and concentrated (Amicon Ultra, 5,000 MWCO, Millipore).

    Techniques: Binding Assay, Mutagenesis, Filtration

    Identification of a GCC185 Rab-binding domain. (A) Constructs used to map Rab-GCC185 interactions; numbers represent amino acid residues. The GRIP domain and a Rab binding domain (RBD) are shown. At right: summary of binding to Rab6 or Rab9. (B) GCC185 preferentially binds Rab6-GTP via residues upstream of the GRIP domain. Reactions contained 50 pmol His-Rab6 (left) or 1.2 nmol His-Rab6, or Rab9-His (right) using 35 S-GTPγS or 3 H-GDP-preloaded GTPase and either GST-C110 or GST-RBD-87 (2.8 μM). (C) The GRIP domain is not sufficient for Rab binding. GST-C-110, C-110 Y/A , or C-72 (2 μM) was incubated with 35 S-GTPγS-preloaded GTPases (∼500 pmol) (as in B. except Rab1 and Rab9 were untagged and or ArlQ71L-His was used). Untagged Rab9/1 and His-Rab6 1−174 were employed. (D) Rab6 specifically competes with Rab9 for GCC185 binding. GST-C-110:Rab9 complexes (pair of lanes in the center) were incubated for 3 min with ten fold excess competitor. Rab9-His was detected by immunoblot using a monoclonal anti-Rab9 antibody that did not cross react with Rab6 (see pair of lanes at far left). Lower panel, same as upper panel using indicated amounts of competitor Rab9. Data are mean ± SD.

    Journal: Cell

    Article Title: Dual GTPase regulation of the GCC185 Golgin: Communication between adjacent Rab6 and Arl1 binding sites

    doi: 10.1016/j.cell.2007.11.048

    Figure Lengend Snippet: Identification of a GCC185 Rab-binding domain. (A) Constructs used to map Rab-GCC185 interactions; numbers represent amino acid residues. The GRIP domain and a Rab binding domain (RBD) are shown. At right: summary of binding to Rab6 or Rab9. (B) GCC185 preferentially binds Rab6-GTP via residues upstream of the GRIP domain. Reactions contained 50 pmol His-Rab6 (left) or 1.2 nmol His-Rab6, or Rab9-His (right) using 35 S-GTPγS or 3 H-GDP-preloaded GTPase and either GST-C110 or GST-RBD-87 (2.8 μM). (C) The GRIP domain is not sufficient for Rab binding. GST-C-110, C-110 Y/A , or C-72 (2 μM) was incubated with 35 S-GTPγS-preloaded GTPases (∼500 pmol) (as in B. except Rab1 and Rab9 were untagged and or ArlQ71L-His was used). Untagged Rab9/1 and His-Rab6 1−174 were employed. (D) Rab6 specifically competes with Rab9 for GCC185 binding. GST-C-110:Rab9 complexes (pair of lanes in the center) were incubated for 3 min with ten fold excess competitor. Rab9-His was detected by immunoblot using a monoclonal anti-Rab9 antibody that did not cross react with Rab6 (see pair of lanes at far left). Lower panel, same as upper panel using indicated amounts of competitor Rab9. Data are mean ± SD.

    Article Snippet: RBD-87 was then separated from GST, GST-Prescission Protease and glutathione beads over a solid support and concentrated (Amicon Ultra, 5,000 MWCO, Millipore).

    Techniques: Binding Assay, Construct, Incubation

    Binding of LsrB to endogenous AI-2. Purified LsrB (BL21) and LsrB (BL21∆luxS) proteins (5 mg/ml) were incubated for 10 min at 37, 50 and 60 °C to release endogenous AI-2, respectively. After incubation, the LsrB proteins were removed by ultrafiltration (10,000-Da cut-off; EMD Millipore), and the filtered reaction products were tested for AI-2 activity using a V. harveyi BB170 bioassay. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C, respectively ( a ). However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS) ( b ). Moreover, the recombinant LuxS protein, which was expressed in strain BL21 (pColdTF-lsrB) as a negative control, also showed no AI-2 binding activity ( c ). AI-2 (10 μM) was used as a positive control

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: Binding of LsrB to endogenous AI-2. Purified LsrB (BL21) and LsrB (BL21∆luxS) proteins (5 mg/ml) were incubated for 10 min at 37, 50 and 60 °C to release endogenous AI-2, respectively. After incubation, the LsrB proteins were removed by ultrafiltration (10,000-Da cut-off; EMD Millipore), and the filtered reaction products were tested for AI-2 activity using a V. harveyi BB170 bioassay. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C, respectively ( a ). However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS) ( b ). Moreover, the recombinant LuxS protein, which was expressed in strain BL21 (pColdTF-lsrB) as a negative control, also showed no AI-2 binding activity ( c ). AI-2 (10 μM) was used as a positive control

    Article Snippet: After incubation, the LsrB (BL21∆luxS) protein was removed by ultrafiltration (10,000-Da cut-off, EMD Millipore) and the filtered reaction products were tested for AI-2 activity by investigating the binding activity of LsrB (BL21∆luxS) to exogenous AI-2.

    Techniques: Binding Assay, Purification, Incubation, Activity Assay, Recombinant, Produced, Mutagenesis, Negative Control, Positive Control

    Schematic chart of strategy for binding of LsrB to endogenous AI-2 or exogenous AI-2. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C. However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS). The combination of endogenous AI-2 produced by BL21 (DE3), which is known to interfere with the binding of AI-2 to LsrB (BL21), resulted in the loss in the ability of LsrB (BL21) to bind to exogenous AI-2. Hence, the BL21 (DE3) luxS mutant, which was incapable of producing endogenous AI-2, but could bind to exogenous AI-2

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: Schematic chart of strategy for binding of LsrB to endogenous AI-2 or exogenous AI-2. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C. However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS). The combination of endogenous AI-2 produced by BL21 (DE3), which is known to interfere with the binding of AI-2 to LsrB (BL21), resulted in the loss in the ability of LsrB (BL21) to bind to exogenous AI-2. Hence, the BL21 (DE3) luxS mutant, which was incapable of producing endogenous AI-2, but could bind to exogenous AI-2

    Article Snippet: After incubation, the LsrB (BL21∆luxS) protein was removed by ultrafiltration (10,000-Da cut-off, EMD Millipore) and the filtered reaction products were tested for AI-2 activity by investigating the binding activity of LsrB (BL21∆luxS) to exogenous AI-2.

    Techniques: Binding Assay, Recombinant, Produced, Mutagenesis

    SDS-PAGE analysis of total cellular proteins and purified fusion proteins from BL21∆luxS cells. Lane 1 and lane 5: SDS-PAGE analysis of total cellular proteins from BL21∆luxS containing pCold TF (serve as negative control). Lane 2 and lane 6: SDS-PAGE analysis of total cellular proteins containing expression plasmids pCold-TF-lsrB ( a ) and pCold-TF-luxS ( b ), respectively. Lane 3 and lane 7: SDS-PAGE analysis of total precipitation proteins containing expression plasmids pCold-TF-lsrB and pCold-TF-luxS, respectively. Lane 4 and lane 8: elution of the LsrB and LuxS purified fusion protein from the affinity column, respectively

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: SDS-PAGE analysis of total cellular proteins and purified fusion proteins from BL21∆luxS cells. Lane 1 and lane 5: SDS-PAGE analysis of total cellular proteins from BL21∆luxS containing pCold TF (serve as negative control). Lane 2 and lane 6: SDS-PAGE analysis of total cellular proteins containing expression plasmids pCold-TF-lsrB ( a ) and pCold-TF-luxS ( b ), respectively. Lane 3 and lane 7: SDS-PAGE analysis of total precipitation proteins containing expression plasmids pCold-TF-lsrB and pCold-TF-luxS, respectively. Lane 4 and lane 8: elution of the LsrB and LuxS purified fusion protein from the affinity column, respectively

    Article Snippet: After incubation, the LsrB (BL21∆luxS) protein was removed by ultrafiltration (10,000-Da cut-off, EMD Millipore) and the filtered reaction products were tested for AI-2 activity by investigating the binding activity of LsrB (BL21∆luxS) to exogenous AI-2.

    Techniques: SDS Page, Purification, Negative Control, Expressing, Affinity Column

    Construction and identification of luxS mutant strain BL21∆luxS

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: Construction and identification of luxS mutant strain BL21∆luxS

    Article Snippet: Cell-free culture fluid (CF) was prepared as follows: E. coli strains BL21 (DE3), BL21∆luxS and DH5a were grown in LB at 37 °C, and then pelleted by centrifugation at 12,000 g at 4 °C for 10 min. Then, the resulting supernatants were filtered through a 0.22-μm filter (EMD Millipore, Bedford, MA, USA) to obtain CF samples.

    Techniques: Mutagenesis

    The AI-2 activity in BL21∆luxS. The wild strain BL21 secretes AI-2-like molecules, and can induce V . harveyi BB170 bioluminescence, whereas no bioluminescence induction was observed for the mutant BL21∆ luxS . V . harveyi BB152 served as

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: The AI-2 activity in BL21∆luxS. The wild strain BL21 secretes AI-2-like molecules, and can induce V . harveyi BB170 bioluminescence, whereas no bioluminescence induction was observed for the mutant BL21∆ luxS . V . harveyi BB152 served as

    Article Snippet: Cell-free culture fluid (CF) was prepared as follows: E. coli strains BL21 (DE3), BL21∆luxS and DH5a were grown in LB at 37 °C, and then pelleted by centrifugation at 12,000 g at 4 °C for 10 min. Then, the resulting supernatants were filtered through a 0.22-μm filter (EMD Millipore, Bedford, MA, USA) to obtain CF samples.

    Techniques: Activity Assay, Mutagenesis

    SDS-PAGE analysis of total cellular proteins and purified fusion proteins from BL21∆luxS cells. Lane 1 and lane 5: SDS-PAGE analysis of total cellular proteins from BL21∆luxS containing pCold TF (serve as negative control). Lane 2 and

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: SDS-PAGE analysis of total cellular proteins and purified fusion proteins from BL21∆luxS cells. Lane 1 and lane 5: SDS-PAGE analysis of total cellular proteins from BL21∆luxS containing pCold TF (serve as negative control). Lane 2 and

    Article Snippet: Cell-free culture fluid (CF) was prepared as follows: E. coli strains BL21 (DE3), BL21∆luxS and DH5a were grown in LB at 37 °C, and then pelleted by centrifugation at 12,000 g at 4 °C for 10 min. Then, the resulting supernatants were filtered through a 0.22-μm filter (EMD Millipore, Bedford, MA, USA) to obtain CF samples.

    Techniques: SDS Page, Purification, Negative Control