expression vector pet 15b  (Millipore)


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    Structured Review

    Millipore expression vector pet 15b
    (a) E. coli -expressed ScFv in renaturating buffer applied directly to microplates was indicated by γ chain- and Fab-specific antibodies. (b) Analysis of the inhibition of MAb ZX10 binding to rNS3 by NS3-ScFv-pET or <t>pET-15b</t> vector without insert, both expressed and purified from E. coli and diluted in renaturating buffer. Residual binding of ZX10 was indicated by goat anti-mouse immunoglobulin G. OD, optical density.
    Expression Vector Pet 15b, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/expression vector pet 15b/product/Millipore
    Average 98 stars, based on 44 article reviews
    Price from $9.99 to $1999.99
    expression vector pet 15b - by Bioz Stars, 2020-04
    98/100 stars

    Images

    1) Product Images from "Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein"

    Article Title: Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein

    Journal: Clinical and Diagnostic Laboratory Immunology

    doi:

    (a) E. coli -expressed ScFv in renaturating buffer applied directly to microplates was indicated by γ chain- and Fab-specific antibodies. (b) Analysis of the inhibition of MAb ZX10 binding to rNS3 by NS3-ScFv-pET or pET-15b vector without insert, both expressed and purified from E. coli and diluted in renaturating buffer. Residual binding of ZX10 was indicated by goat anti-mouse immunoglobulin G. OD, optical density.
    Figure Legend Snippet: (a) E. coli -expressed ScFv in renaturating buffer applied directly to microplates was indicated by γ chain- and Fab-specific antibodies. (b) Analysis of the inhibition of MAb ZX10 binding to rNS3 by NS3-ScFv-pET or pET-15b vector without insert, both expressed and purified from E. coli and diluted in renaturating buffer. Residual binding of ZX10 was indicated by goat anti-mouse immunoglobulin G. OD, optical density.

    Techniques Used: Inhibition, Binding Assay, Positron Emission Tomography, Plasmid Preparation, Purification

    Analysis of NS3-ScFv-pET expressed in E. coli by use of a silver-stained SDS minigel (right) and by Western blotting (left). Total E. coli lysate of the pET-15b vector without insert (lanes 1 and 7) and lysate of the E. coli pellet containing NS3-ScFv-pET (lanes 2 and 8) or containing the NS3-ScFv-pET eluted from the Ni-NTA column by Tris buffer with 8 M urea at pH 5.0 (lanes 3 and 9) or pH 4.0 (lanes 5 and 11) or with 0.1 M EDTA (lanes 6 and 12) are shown. Lanes 4 and 10, molecular size markers. The Western blot was developed with an Ni-NTA conjugate and the insoluble substrate BCIP-NBT.
    Figure Legend Snippet: Analysis of NS3-ScFv-pET expressed in E. coli by use of a silver-stained SDS minigel (right) and by Western blotting (left). Total E. coli lysate of the pET-15b vector without insert (lanes 1 and 7) and lysate of the E. coli pellet containing NS3-ScFv-pET (lanes 2 and 8) or containing the NS3-ScFv-pET eluted from the Ni-NTA column by Tris buffer with 8 M urea at pH 5.0 (lanes 3 and 9) or pH 4.0 (lanes 5 and 11) or with 0.1 M EDTA (lanes 6 and 12) are shown. Lanes 4 and 10, molecular size markers. The Western blot was developed with an Ni-NTA conjugate and the insoluble substrate BCIP-NBT.

    Techniques Used: Positron Emission Tomography, Staining, Western Blot, Plasmid Preparation

    Related Articles

    Clone Assay:

    Article Title: Overexpression, crystallization and preliminary X--ray crystallographic analysis of the C-terminal cytosolic domain of mouse anoctamin 1
    Article Snippet: .. Protein expression and purification mANO1-CTD was cloned into the expression vector pET-15b(+) (Novagen), adding a hexahistidine-containing 20-residue tag to the N-terminus. .. The recombinant protein was overexpressed in Escherichia coli Rosetta2(DE3)pLysS cells using Luria broth culture medium.

    Article Title: Quantitative methods for measuring DNA flexibility in vitro and in vivo
    Article Snippet: .. The S. cerevisiae Nhp6A coding region is cloned between the Nco I and Bam HI sites of bacterial expression vector pET-15b (Novagen). .. The resulting plasmid (pJ1400) encodes untagged Nhp6A protein (molecular weight 10,801) with the following amino acid sequence: MVTPREPK2 RT2 RK3 DPNAPKRALSAYMF2 ANENRDIVRSENPDITFGQVGK2 LGE-KWKALTPE2 KQPYEAKAQADK2 RYESEKELYNATLA A 6-mL overnight LB culture of E. coli strain BL21(DE3) containing carbenicillin (Cb) (50 µg/mL) is grown from a single colony, pelleted by centrifugation, washed with 1 mL fresh LB medium to remove secreted β-lactamase, and resuspended in 1 ml of medium.

    Article Title: Deciphering the regulatory and catalytic mechanisms of an unusual SAM-dependent enzyme
    Article Snippet: .. Gene cloning, protein expression, and purification A codon-optimized gene encoding LepI, originated from A. flavus , was integrated into the expression vector pET-15b (Novagen, Madison, WI, USA) between the NdeI and XhoI sites with an N-terminal histidine tag. .. DNA sequencing and transformation into Escherichia coli BL21 (DE3) cells for expression verified the constructed plasmid pET-15b-LepI.

    Article Title: hnRNP H Is a Component of a Splicing Enhancer Complex That Activates a c-src Alternative Exon in Neuronal Cells
    Article Snippet: .. Both PCR products were digested with Xho I and Bam HI and then cloned into the expression vector pET 15b (Novagen) at the same sites. .. The recombinant N-terminal histidine-tagged proteins were expressed in Escherichia coli BL21(DE3).

    Article Title: Solution structure of the cytohesin-1 (B2-1) Sec7 domain and its interaction with the GTPase ADP ribosylation factor 1
    Article Snippet: .. The gene was cloned into the Nde I– Xho I restriction sites of the expression vector pET-15b (Novagen). .. Residues 18–181 (Δ17Arf-1) were subcloned into the Nde I– Xho I restriction sites of pET-20b. (Novagen).

    Article Title: Expression, purification and preliminary X-ray crystallographic analysis of Arf1-GDP in complex with dimeric p23 peptide
    Article Snippet: Paragraph title: 2.1. Cloning, expression and purification  ... The PCR products were digested with Nde I and Bam HI restriction enzymes (New England Biolabs) and then inserted into expression vector pET-15b (Novagen).

    Article Title: Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein
    Article Snippet: The NS3-ScFv DNA was reamplified to introduce the cloning sites and to omit the leader sequence. .. The digested fragment was inserted into the bacterial expression vector pET-15b (Novagen, Madison, Wis.) to give NS3-ScFv-pET.

    Article Title: Human and yeast Rad52 proteins promote DNA strand exchange
    Article Snippet: The complete coding sequence of the HsRad52 gene was amplified by PCR from plasmid pEG2 ( ) and inserted into bacterial expression vector pET 15b (Novagen) between Nde I and Bam HI sites in frame with a 5′-end sequence coding for a series of six histidine residues, which function as a metal-binding domain in the translated fusion protein. .. The sequence of the cloned cDNA for HsRad52 was identical to the sequence published by Muris et al. ( ).

    Article Title: Biochemical characterization of an unclassified glutathione S-transferase of Plutella xylostella
    Article Snippet: The pxgstu1 cDNA was cloned into the pGEM-T Easy Vector, as described above. .. After digestion of the PCR product with Nde I and Bam HI, the obtained fragment was subcloned into the Nde I– Bam HI site of expression vector pET-15b (Novagen; EMD Biosciences, Inc., Darmstadt, Germany).

    Article Title: Potential Alu Function: Regulation of the Activity of Double-Stranded RNA-Activated Kinase PKR
    Article Snippet: .. PKR was cloned into expression vector pET-15b (Novagen) and bacterially expressed as a hexahistidine-tagged fusion protein as described previously ( ). .. Cell culture, cell lysis and extraction, and protein purification by nickel-agarose affinity column chromatography and by gel filtration fast-performance liquid chromatography were carried out as previously described ( ) with the following modifications.

    Centrifugation:

    Article Title: Quantitative methods for measuring DNA flexibility in vitro and in vivo
    Article Snippet: The S. cerevisiae Nhp6A coding region is cloned between the Nco I and Bam HI sites of bacterial expression vector pET-15b (Novagen). .. The resulting plasmid (pJ1400) encodes untagged Nhp6A protein (molecular weight 10,801) with the following amino acid sequence: MVTPREPK2 RT2 RK3 DPNAPKRALSAYMF2 ANENRDIVRSENPDITFGQVGK2 LGE-KWKALTPE2 KQPYEAKAQADK2 RYESEKELYNATLA A 6-mL overnight LB culture of E. coli strain BL21(DE3) containing carbenicillin (Cb) (50 µg/mL) is grown from a single colony, pelleted by centrifugation, washed with 1 mL fresh LB medium to remove secreted β-lactamase, and resuspended in 1 ml of medium.

    Article Title: Deciphering the regulatory and catalytic mechanisms of an unusual SAM-dependent enzyme
    Article Snippet: Gene cloning, protein expression, and purification A codon-optimized gene encoding LepI, originated from A. flavus , was integrated into the expression vector pET-15b (Novagen, Madison, WI, USA) between the NdeI and XhoI sites with an N-terminal histidine tag. .. When the cell density reached an OD 600 nm of 0.6, the culture temperature was decreased from 37 °C to 15 °C, and the culture was supplemented with 0.3 mmol/L isopropyl-d-1-thiogalactopyranoside to induce the expression of LepI for 18 h. Cell pellets harvested by 4000 rpm centrifugation for 15 min were resuspended in 300 mL lysis buffer (25 mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride) and disrupted using a low-temperature high-pressure cell disruptor.

    Article Title: hnRNP H Is a Component of a Splicing Enhancer Complex That Activates a c-src Alternative Exon in Neuronal Cells
    Article Snippet: Both PCR products were digested with Xho I and Bam HI and then cloned into the expression vector pET 15b (Novagen) at the same sites. .. The bacterial cells were grown in LB medium containing ampicillin (100 μg/ml) at 37°C to an optical density of 0.7 at 600 nm before induction with 1 mM isopropyl-β- d -thiogalactopyranoside (IPTG) for 3 h. For the purification of hnRNPs H and F, 1 liter each of the IPTG-induced cells was pelleted by centrifugation and washed with 50 mM Tris-HCl (pH 8.0) containing 0.1 M NaCl.

    Article Title: Biochemical characterization of an unclassified glutathione S-transferase of Plutella xylostella
    Article Snippet: After digestion of the PCR product with Nde I and Bam HI, the obtained fragment was subcloned into the Nde I– Bam HI site of expression vector pET-15b (Novagen; EMD Biosciences, Inc., Darmstadt, Germany). .. After further incubation overnight at 30°C, cells were harvested by centrifugation; homogenized in 20 mM Tris-HCl buffer (pH 8.0) that contained 0.5 M NaCl, 4 mg/mL of lysozyme, and 2×103 U Cryonase Cold-active Nuclease (Takara, Tokyo, Japan); and disrupted by sonication.

    Amplification:

    Article Title: Solution structure of the cytohesin-1 (B2-1) Sec7 domain and its interaction with the GTPase ADP ribosylation factor 1
    Article Snippet: The Arf-1 cDNA was isolated by PCR amplification from a human spleen cell cDNA library ( ) by using primers based on the Arf-1 sequence ( ). .. The gene was cloned into the Nde I– Xho I restriction sites of the expression vector pET-15b (Novagen).

    Article Title: Expression, purification and preliminary X-ray crystallographic analysis of Arf1-GDP in complex with dimeric p23 peptide
    Article Snippet: The gene encoding human Arf1 (residues 18–181; UniProt accession No. ) was amplified from a human brain cDNA library (Stratagene) using upstream primer 5′-GGAATTCCATATGATGCGCATCCTCATGGTGGGCCTGG-3′ and downstream primer 5′-CGCGGATCCTCACTTCTGGTTCCGGAGCTGATTGGACAG-3′ which contained Nde I and Bam HI restriction sites. .. The PCR products were digested with Nde I and Bam HI restriction enzymes (New England Biolabs) and then inserted into expression vector pET-15b (Novagen).

    Article Title: DipA, a Pore-Forming Protein in the Outer Membrane of Lyme Disease Spirochetes Exhibits Specificity for the Permeation of Dicarboxylates
    Article Snippet: Overexpression of a recombinant fragment of DipA A fragment of DipA representing the 90 C-terminal amino acids was produced in E. coli Rosetta™ 2 (DE3) (Novagen), using expression vector pET 15b (Novagen) containing an N-terminal His6 -Tag. .. The gene fragment of 285 bp was amplified by PCR using following oligonucleotides: rbb0418_f ( 5′-CTG CATATG GAAGGAAAAACACAAATTGG-3′ ) containing NdeI restriction site and rbb0418_r ( 5′-GACTTTA GGATCC TTAAGTTATAGACATTCC-3′ ) containing BamHI restriction site.

    Article Title: Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein
    Article Snippet: The VH and VL of NS3 MAb ZX10 were amplified from cDNA by PCR with a recombinant phase antibody system (Pharmacia Biotech, Uppsala, Sweden). .. The digested fragment was inserted into the bacterial expression vector pET-15b (Novagen, Madison, Wis.) to give NS3-ScFv-pET.

    Article Title: Human and yeast Rad52 proteins promote DNA strand exchange
    Article Snippet: .. The complete coding sequence of the HsRad52 gene was amplified by PCR from plasmid pEG2 ( ) and inserted into bacterial expression vector pET 15b (Novagen) between Nde I and Bam HI sites in frame with a 5′-end sequence coding for a series of six histidine residues, which function as a metal-binding domain in the translated fusion protein. .. The resulting plasmid was designated pEG161H1.

    Filtration:

    Article Title: Potential Alu Function: Regulation of the Activity of Double-Stranded RNA-Activated Kinase PKR
    Article Snippet: PKR was cloned into expression vector pET-15b (Novagen) and bacterially expressed as a hexahistidine-tagged fusion protein as described previously ( ). .. Cell culture, cell lysis and extraction, and protein purification by nickel-agarose affinity column chromatography and by gel filtration fast-performance liquid chromatography were carried out as previously described ( ) with the following modifications.

    TA Cloning:

    Article Title: Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein
    Article Snippet: The assembled NS3-ScFv cDNA fragment was directly ligated to the TA cloning vector pCR 2.1 (Invitrogen, San Diego, Calif.). .. The digested fragment was inserted into the bacterial expression vector pET-15b (Novagen, Madison, Wis.) to give NS3-ScFv-pET.

    Construct:

    Article Title: Solution structure of the complex of VEK-30 and plasminogen kringle 2
    Article Snippet: Briefly, the DNA fragment containing the VEK-30 cDNA was inserted into the bacterial expression vector pET-15b (Novagen, Gibbstown, NJ)/GB1 [ ], and expressed in E. coli BL21 (DE3). .. The final construct contained, sequentially, an ATG initiation codon, a purification (His)6 tag, the GB1 fusion partner for increased solubility, a 9-residue linker, and a thrombin cleavage site, LVPR’GS (‘ representing the site of thrombin-catalyzed cleavage), all sequentially inserted into the parent plasmid [ ].

    Article Title: Deciphering the regulatory and catalytic mechanisms of an unusual SAM-dependent enzyme
    Article Snippet: Gene cloning, protein expression, and purification A codon-optimized gene encoding LepI, originated from A. flavus , was integrated into the expression vector pET-15b (Novagen, Madison, WI, USA) between the NdeI and XhoI sites with an N-terminal histidine tag. .. DNA sequencing and transformation into Escherichia coli BL21 (DE3) cells for expression verified the constructed plasmid pET-15b-LepI.

    Incubation:

    Article Title: Overexpression, crystallization and preliminary X--ray crystallographic analysis of the C-terminal cytosolic domain of mouse anoctamin 1
    Article Snippet: Protein expression and purification mANO1-CTD was cloned into the expression vector pET-15b(+) (Novagen), adding a hexahistidine-containing 20-residue tag to the N-terminus. .. Protein expression was induced by 0.5 mM isopropyl β-d -1-thiogalactopyranoside and the cells were incubated for 16 h at 293 K following growth to mid-log phase at 310 K. The cells were lysed by sonication in lysis buffer (20 mM Tris–HCl pH 7.5, 500 mM NaCl, 35 mM imidazole and 1 mM phenylmethanesulfonylfluoride).

    Article Title: Biochemical characterization of an unclassified glutathione S-transferase of Plutella xylostella
    Article Snippet: After digestion of the PCR product with Nde I and Bam HI, the obtained fragment was subcloned into the Nde I– Bam HI site of expression vector pET-15b (Novagen; EMD Biosciences, Inc., Darmstadt, Germany). .. After further incubation overnight at 30°C, cells were harvested by centrifugation; homogenized in 20 mM Tris-HCl buffer (pH 8.0) that contained 0.5 M NaCl, 4 mg/mL of lysozyme, and 2×103 U Cryonase Cold-active Nuclease (Takara, Tokyo, Japan); and disrupted by sonication.

    Solubility:

    Article Title: Solution structure of the complex of VEK-30 and plasminogen kringle 2
    Article Snippet: Briefly, the DNA fragment containing the VEK-30 cDNA was inserted into the bacterial expression vector pET-15b (Novagen, Gibbstown, NJ)/GB1 [ ], and expressed in E. coli BL21 (DE3). .. The final construct contained, sequentially, an ATG initiation codon, a purification (His)6 tag, the GB1 fusion partner for increased solubility, a 9-residue linker, and a thrombin cleavage site, LVPR’GS (‘ representing the site of thrombin-catalyzed cleavage), all sequentially inserted into the parent plasmid [ ].

    Expressing:

    Article Title: Overexpression, crystallization and preliminary X--ray crystallographic analysis of the C-terminal cytosolic domain of mouse anoctamin 1
    Article Snippet: .. Protein expression and purification mANO1-CTD was cloned into the expression vector pET-15b(+) (Novagen), adding a hexahistidine-containing 20-residue tag to the N-terminus. .. The recombinant protein was overexpressed in Escherichia coli Rosetta2(DE3)pLysS cells using Luria broth culture medium.

    Article Title: Quantitative methods for measuring DNA flexibility in vitro and in vivo
    Article Snippet: .. The S. cerevisiae Nhp6A coding region is cloned between the Nco I and Bam HI sites of bacterial expression vector pET-15b (Novagen). .. The resulting plasmid (pJ1400) encodes untagged Nhp6A protein (molecular weight 10,801) with the following amino acid sequence: MVTPREPK2 RT2 RK3 DPNAPKRALSAYMF2 ANENRDIVRSENPDITFGQVGK2 LGE-KWKALTPE2 KQPYEAKAQADK2 RYESEKELYNATLA A 6-mL overnight LB culture of E. coli strain BL21(DE3) containing carbenicillin (Cb) (50 µg/mL) is grown from a single colony, pelleted by centrifugation, washed with 1 mL fresh LB medium to remove secreted β-lactamase, and resuspended in 1 ml of medium.

    Article Title: Solution structure of the complex of VEK-30 and plasminogen kringle 2
    Article Snippet: .. Briefly, the DNA fragment containing the VEK-30 cDNA was inserted into the bacterial expression vector pET-15b (Novagen, Gibbstown, NJ)/GB1 [ ], and expressed in E. coli BL21 (DE3). .. The final construct contained, sequentially, an ATG initiation codon, a purification (His)6 tag, the GB1 fusion partner for increased solubility, a 9-residue linker, and a thrombin cleavage site, LVPR’GS (‘ representing the site of thrombin-catalyzed cleavage), all sequentially inserted into the parent plasmid [ ].

    Article Title: Deciphering the regulatory and catalytic mechanisms of an unusual SAM-dependent enzyme
    Article Snippet: .. Gene cloning, protein expression, and purification A codon-optimized gene encoding LepI, originated from A. flavus , was integrated into the expression vector pET-15b (Novagen, Madison, WI, USA) between the NdeI and XhoI sites with an N-terminal histidine tag. .. DNA sequencing and transformation into Escherichia coli BL21 (DE3) cells for expression verified the constructed plasmid pET-15b-LepI.

    Article Title: hnRNP H Is a Component of a Splicing Enhancer Complex That Activates a c-src Alternative Exon in Neuronal Cells
    Article Snippet: .. Both PCR products were digested with Xho I and Bam HI and then cloned into the expression vector pET 15b (Novagen) at the same sites. .. The recombinant N-terminal histidine-tagged proteins were expressed in Escherichia coli BL21(DE3).

    Article Title: Solution structure of the cytohesin-1 (B2-1) Sec7 domain and its interaction with the GTPase ADP ribosylation factor 1
    Article Snippet: .. The gene was cloned into the Nde I– Xho I restriction sites of the expression vector pET-15b (Novagen). .. Residues 18–181 (Δ17Arf-1) were subcloned into the Nde I– Xho I restriction sites of pET-20b. (Novagen).

    Article Title: Expression, purification and preliminary X-ray crystallographic analysis of Arf1-GDP in complex with dimeric p23 peptide
    Article Snippet: .. The PCR products were digested with Nde I and Bam HI restriction enzymes (New England Biolabs) and then inserted into expression vector pET-15b (Novagen). ..

    Article Title: DipA, a Pore-Forming Protein in the Outer Membrane of Lyme Disease Spirochetes Exhibits Specificity for the Permeation of Dicarboxylates
    Article Snippet: .. Overexpression of a recombinant fragment of DipA A fragment of DipA representing the 90 C-terminal amino acids was produced in E. coli Rosetta™ 2 (DE3) (Novagen), using expression vector pET 15b (Novagen) containing an N-terminal His6 -Tag. .. The gene fragment of 285 bp was amplified by PCR using following oligonucleotides: rbb0418_f ( 5′-CTG CATATG GAAGGAAAAACACAAATTGG-3′ ) containing NdeI restriction site and rbb0418_r ( 5′-GACTTTA GGATCC TTAAGTTATAGACATTCC-3′ ) containing BamHI restriction site.

    Article Title: Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein
    Article Snippet: .. The digested fragment was inserted into the bacterial expression vector pET-15b (Novagen, Madison, Wis.) to give NS3-ScFv-pET. .. For eucaryotic expression, the primer pair 5′-CCG GGT ACC ACA GGT GAA GCT GCA G-3′ and 5′-GCC TCT AGA CTA CCG TTT GAT TTC CAG-3′ was used to introduce Kpn I and Xba I restriction sites (underlined).

    Article Title: Human and yeast Rad52 proteins promote DNA strand exchange
    Article Snippet: .. The complete coding sequence of the HsRad52 gene was amplified by PCR from plasmid pEG2 ( ) and inserted into bacterial expression vector pET 15b (Novagen) between Nde I and Bam HI sites in frame with a 5′-end sequence coding for a series of six histidine residues, which function as a metal-binding domain in the translated fusion protein. .. The resulting plasmid was designated pEG161H1.

    Article Title: Biochemical characterization of an unclassified glutathione S-transferase of Plutella xylostella
    Article Snippet: .. After digestion of the PCR product with Nde I and Bam HI, the obtained fragment was subcloned into the Nde I– Bam HI site of expression vector pET-15b (Novagen; EMD Biosciences, Inc., Darmstadt, Germany). .. Competent Escherichia coli Rosetta (DE3) pLysS cells (Novagen; EMD Millipore, USA) were transformed with a prepared expression plasmid that harbored pxgstu1 and were grown at 37°C on Luria–Bertani medium that contained 100 µg/mL ampicillin.

    Article Title: Potential Alu Function: Regulation of the Activity of Double-Stranded RNA-Activated Kinase PKR
    Article Snippet: .. PKR was cloned into expression vector pET-15b (Novagen) and bacterially expressed as a hexahistidine-tagged fusion protein as described previously ( ). .. Cell culture, cell lysis and extraction, and protein purification by nickel-agarose affinity column chromatography and by gel filtration fast-performance liquid chromatography were carried out as previously described ( ) with the following modifications.

    Transformation Assay:

    Article Title: Deciphering the regulatory and catalytic mechanisms of an unusual SAM-dependent enzyme
    Article Snippet: Gene cloning, protein expression, and purification A codon-optimized gene encoding LepI, originated from A. flavus , was integrated into the expression vector pET-15b (Novagen, Madison, WI, USA) between the NdeI and XhoI sites with an N-terminal histidine tag. .. DNA sequencing and transformation into Escherichia coli BL21 (DE3) cells for expression verified the constructed plasmid pET-15b-LepI.

    Article Title: Expression, purification and preliminary X-ray crystallographic analysis of Arf1-GDP in complex with dimeric p23 peptide
    Article Snippet: The PCR products were digested with Nde I and Bam HI restriction enzymes (New England Biolabs) and then inserted into expression vector pET-15b (Novagen). .. The expression vector was transformed into Escherichia coli strain BL21 (DE3).

    Article Title: Biochemical characterization of an unclassified glutathione S-transferase of Plutella xylostella
    Article Snippet: After digestion of the PCR product with Nde I and Bam HI, the obtained fragment was subcloned into the Nde I– Bam HI site of expression vector pET-15b (Novagen; EMD Biosciences, Inc., Darmstadt, Germany). .. Competent Escherichia coli Rosetta (DE3) pLysS cells (Novagen; EMD Millipore, USA) were transformed with a prepared expression plasmid that harbored pxgstu1 and were grown at 37°C on Luria–Bertani medium that contained 100 µg/mL ampicillin.

    Over Expression:

    Article Title: DipA, a Pore-Forming Protein in the Outer Membrane of Lyme Disease Spirochetes Exhibits Specificity for the Permeation of Dicarboxylates
    Article Snippet: .. Overexpression of a recombinant fragment of DipA A fragment of DipA representing the 90 C-terminal amino acids was produced in E. coli Rosetta™ 2 (DE3) (Novagen), using expression vector pET 15b (Novagen) containing an N-terminal His6 -Tag. .. The gene fragment of 285 bp was amplified by PCR using following oligonucleotides: rbb0418_f ( 5′-CTG CATATG GAAGGAAAAACACAAATTGG-3′ ) containing NdeI restriction site and rbb0418_r ( 5′-GACTTTA GGATCC TTAAGTTATAGACATTCC-3′ ) containing BamHI restriction site.

    Article Title: Biochemical characterization of an unclassified glutathione S-transferase of Plutella xylostella
    Article Snippet: Paragraph title: 3. Overexpression and purification of the recombinant protein ... After digestion of the PCR product with Nde I and Bam HI, the obtained fragment was subcloned into the Nde I– Bam HI site of expression vector pET-15b (Novagen; EMD Biosciences, Inc., Darmstadt, Germany).

    Chromatography:

    Article Title: Potential Alu Function: Regulation of the Activity of Double-Stranded RNA-Activated Kinase PKR
    Article Snippet: PKR was cloned into expression vector pET-15b (Novagen) and bacterially expressed as a hexahistidine-tagged fusion protein as described previously ( ). .. Cell culture, cell lysis and extraction, and protein purification by nickel-agarose affinity column chromatography and by gel filtration fast-performance liquid chromatography were carried out as previously described ( ) with the following modifications.

    Introduce:

    Article Title: Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein
    Article Snippet: For Escherichia coli expression, the forward primer 5′-CCG CTC GAG CAG GTG AAG CTG CAG-3′ and reverse primer 5′-C GG ATC C TA CCG TTT GAT TTC CAG-3′ were used to amplify the NS3-ScFv to introduce 5′ Bam HI and 3′ Xho I restriction sites (underlined). .. The digested fragment was inserted into the bacterial expression vector pET-15b (Novagen, Madison, Wis.) to give NS3-ScFv-pET.

    DNA Sequencing:

    Article Title: Deciphering the regulatory and catalytic mechanisms of an unusual SAM-dependent enzyme
    Article Snippet: Gene cloning, protein expression, and purification A codon-optimized gene encoding LepI, originated from A. flavus , was integrated into the expression vector pET-15b (Novagen, Madison, WI, USA) between the NdeI and XhoI sites with an N-terminal histidine tag. .. DNA sequencing and transformation into Escherichia coli BL21 (DE3) cells for expression verified the constructed plasmid pET-15b-LepI.

    Polymerase Chain Reaction:

    Article Title: hnRNP H Is a Component of a Splicing Enhancer Complex That Activates a c-src Alternative Exon in Neuronal Cells
    Article Snippet: .. Both PCR products were digested with Xho I and Bam HI and then cloned into the expression vector pET 15b (Novagen) at the same sites. .. The recombinant N-terminal histidine-tagged proteins were expressed in Escherichia coli BL21(DE3).

    Article Title: Solution structure of the cytohesin-1 (B2-1) Sec7 domain and its interaction with the GTPase ADP ribosylation factor 1
    Article Snippet: The Arf-1 cDNA was isolated by PCR amplification from a human spleen cell cDNA library ( ) by using primers based on the Arf-1 sequence ( ). .. The gene was cloned into the Nde I– Xho I restriction sites of the expression vector pET-15b (Novagen).

    Article Title: Expression, purification and preliminary X-ray crystallographic analysis of Arf1-GDP in complex with dimeric p23 peptide
    Article Snippet: .. The PCR products were digested with Nde I and Bam HI restriction enzymes (New England Biolabs) and then inserted into expression vector pET-15b (Novagen). ..

    Article Title: DipA, a Pore-Forming Protein in the Outer Membrane of Lyme Disease Spirochetes Exhibits Specificity for the Permeation of Dicarboxylates
    Article Snippet: Overexpression of a recombinant fragment of DipA A fragment of DipA representing the 90 C-terminal amino acids was produced in E. coli Rosetta™ 2 (DE3) (Novagen), using expression vector pET 15b (Novagen) containing an N-terminal His6 -Tag. .. The gene fragment of 285 bp was amplified by PCR using following oligonucleotides: rbb0418_f ( 5′-CTG CATATG GAAGGAAAAACACAAATTGG-3′ ) containing NdeI restriction site and rbb0418_r ( 5′-GACTTTA GGATCC TTAAGTTATAGACATTCC-3′ ) containing BamHI restriction site.

    Article Title: Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein
    Article Snippet: The assembled NS3-ScFv cDNA fragment was directly ligated to the TA cloning vector pCR 2.1 (Invitrogen, San Diego, Calif.). .. The digested fragment was inserted into the bacterial expression vector pET-15b (Novagen, Madison, Wis.) to give NS3-ScFv-pET.

    Article Title: Human and yeast Rad52 proteins promote DNA strand exchange
    Article Snippet: .. The complete coding sequence of the HsRad52 gene was amplified by PCR from plasmid pEG2 ( ) and inserted into bacterial expression vector pET 15b (Novagen) between Nde I and Bam HI sites in frame with a 5′-end sequence coding for a series of six histidine residues, which function as a metal-binding domain in the translated fusion protein. .. The resulting plasmid was designated pEG161H1.

    Article Title: Biochemical characterization of an unclassified glutathione S-transferase of Plutella xylostella
    Article Snippet: .. After digestion of the PCR product with Nde I and Bam HI, the obtained fragment was subcloned into the Nde I– Bam HI site of expression vector pET-15b (Novagen; EMD Biosciences, Inc., Darmstadt, Germany). .. Competent Escherichia coli Rosetta (DE3) pLysS cells (Novagen; EMD Millipore, USA) were transformed with a prepared expression plasmid that harbored pxgstu1 and were grown at 37°C on Luria–Bertani medium that contained 100 µg/mL ampicillin.

    Sonication:

    Article Title: Overexpression, crystallization and preliminary X--ray crystallographic analysis of the C-terminal cytosolic domain of mouse anoctamin 1
    Article Snippet: Protein expression and purification mANO1-CTD was cloned into the expression vector pET-15b(+) (Novagen), adding a hexahistidine-containing 20-residue tag to the N-terminus. .. Protein expression was induced by 0.5 mM isopropyl β-d -1-thiogalactopyranoside and the cells were incubated for 16 h at 293 K following growth to mid-log phase at 310 K. The cells were lysed by sonication in lysis buffer (20 mM Tris–HCl pH 7.5, 500 mM NaCl, 35 mM imidazole and 1 mM phenylmethanesulfonylfluoride).

    Article Title: Biochemical characterization of an unclassified glutathione S-transferase of Plutella xylostella
    Article Snippet: After digestion of the PCR product with Nde I and Bam HI, the obtained fragment was subcloned into the Nde I– Bam HI site of expression vector pET-15b (Novagen; EMD Biosciences, Inc., Darmstadt, Germany). .. After further incubation overnight at 30°C, cells were harvested by centrifugation; homogenized in 20 mM Tris-HCl buffer (pH 8.0) that contained 0.5 M NaCl, 4 mg/mL of lysozyme, and 2×103 U Cryonase Cold-active Nuclease (Takara, Tokyo, Japan); and disrupted by sonication.

    Recombinant:

    Article Title: Overexpression, crystallization and preliminary X--ray crystallographic analysis of the C-terminal cytosolic domain of mouse anoctamin 1
    Article Snippet: Protein expression and purification mANO1-CTD was cloned into the expression vector pET-15b(+) (Novagen), adding a hexahistidine-containing 20-residue tag to the N-terminus. .. The recombinant protein was overexpressed in Escherichia coli Rosetta2(DE3)pLysS cells using Luria broth culture medium.

    Article Title: Solution structure of the complex of VEK-30 and plasminogen kringle 2
    Article Snippet: For 15 N labeling of the peptide, the recombinant yeast cultures were grown in medium containing (15 NH4 )2 SO4 (99%, Cambridge Isotope Laboratories, Andover, MA). .. Briefly, the DNA fragment containing the VEK-30 cDNA was inserted into the bacterial expression vector pET-15b (Novagen, Gibbstown, NJ)/GB1 [ ], and expressed in E. coli BL21 (DE3).

    Article Title: hnRNP H Is a Component of a Splicing Enhancer Complex That Activates a c-src Alternative Exon in Neuronal Cells
    Article Snippet: Paragraph title: Cloning and expression of recombinant hnRNPs H and F. ... Both PCR products were digested with Xho I and Bam HI and then cloned into the expression vector pET 15b (Novagen) at the same sites.

    Article Title: DipA, a Pore-Forming Protein in the Outer Membrane of Lyme Disease Spirochetes Exhibits Specificity for the Permeation of Dicarboxylates
    Article Snippet: .. Overexpression of a recombinant fragment of DipA A fragment of DipA representing the 90 C-terminal amino acids was produced in E. coli Rosetta™ 2 (DE3) (Novagen), using expression vector pET 15b (Novagen) containing an N-terminal His6 -Tag. .. The gene fragment of 285 bp was amplified by PCR using following oligonucleotides: rbb0418_f ( 5′-CTG CATATG GAAGGAAAAACACAAATTGG-3′ ) containing NdeI restriction site and rbb0418_r ( 5′-GACTTTA GGATCC TTAAGTTATAGACATTCC-3′ ) containing BamHI restriction site.

    Article Title: Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein
    Article Snippet: The VH and VL of NS3 MAb ZX10 were amplified from cDNA by PCR with a recombinant phase antibody system (Pharmacia Biotech, Uppsala, Sweden). .. The digested fragment was inserted into the bacterial expression vector pET-15b (Novagen, Madison, Wis.) to give NS3-ScFv-pET.

    Article Title: Biochemical characterization of an unclassified glutathione S-transferase of Plutella xylostella
    Article Snippet: Paragraph title: 3. Overexpression and purification of the recombinant protein ... After digestion of the PCR product with Nde I and Bam HI, the obtained fragment was subcloned into the Nde I– Bam HI site of expression vector pET-15b (Novagen; EMD Biosciences, Inc., Darmstadt, Germany).

    Article Title: Potential Alu Function: Regulation of the Activity of Double-Stranded RNA-Activated Kinase PKR
    Article Snippet: Paragraph title: Preparation of recombinant PKR. ... PKR was cloned into expression vector pET-15b (Novagen) and bacterially expressed as a hexahistidine-tagged fusion protein as described previously ( ).

    Molecular Weight:

    Article Title: Quantitative methods for measuring DNA flexibility in vitro and in vivo
    Article Snippet: The S. cerevisiae Nhp6A coding region is cloned between the Nco I and Bam HI sites of bacterial expression vector pET-15b (Novagen). .. The resulting plasmid (pJ1400) encodes untagged Nhp6A protein (molecular weight 10,801) with the following amino acid sequence: MVTPREPK2 RT2 RK3 DPNAPKRALSAYMF2 ANENRDIVRSENPDITFGQVGK2 LGE-KWKALTPE2 KQPYEAKAQADK2 RYESEKELYNATLA A 6-mL overnight LB culture of E. coli strain BL21(DE3) containing carbenicillin (Cb) (50 µg/mL) is grown from a single colony, pelleted by centrifugation, washed with 1 mL fresh LB medium to remove secreted β-lactamase, and resuspended in 1 ml of medium.

    Mutagenesis:

    Article Title: Solution structure of the cytohesin-1 (B2-1) Sec7 domain and its interaction with the GTPase ADP ribosylation factor 1
    Article Snippet: Paragraph title: Cloning, Mutagenesis, and Expression. ... The gene was cloned into the Nde I– Xho I restriction sites of the expression vector pET-15b (Novagen).

    Isolation:

    Article Title: Solution structure of the cytohesin-1 (B2-1) Sec7 domain and its interaction with the GTPase ADP ribosylation factor 1
    Article Snippet: The Arf-1 cDNA was isolated by PCR amplification from a human spleen cell cDNA library ( ) by using primers based on the Arf-1 sequence ( ). .. The gene was cloned into the Nde I– Xho I restriction sites of the expression vector pET-15b (Novagen).

    Labeling:

    Article Title: Solution structure of the complex of VEK-30 and plasminogen kringle 2
    Article Snippet: For 15 N and 13 C labeling, the medium for 15 N feeding was used, and glucose and methanol were replaced by [13 C]-glucose (99%; Isotec, Champaign, IL) and [13 C]-methanol (99%, Isotec). .. Briefly, the DNA fragment containing the VEK-30 cDNA was inserted into the bacterial expression vector pET-15b (Novagen, Gibbstown, NJ)/GB1 [ ], and expressed in E. coli BL21 (DE3).

    Purification:

    Article Title: Overexpression, crystallization and preliminary X--ray crystallographic analysis of the C-terminal cytosolic domain of mouse anoctamin 1
    Article Snippet: .. Protein expression and purification mANO1-CTD was cloned into the expression vector pET-15b(+) (Novagen), adding a hexahistidine-containing 20-residue tag to the N-terminus. .. The recombinant protein was overexpressed in Escherichia coli Rosetta2(DE3)pLysS cells using Luria broth culture medium.

    Article Title: Quantitative methods for measuring DNA flexibility in vitro and in vivo
    Article Snippet: Paragraph title: Purification of HMGB proteins ... The S. cerevisiae Nhp6A coding region is cloned between the Nco I and Bam HI sites of bacterial expression vector pET-15b (Novagen).

    Article Title: Solution structure of the complex of VEK-30 and plasminogen kringle 2
    Article Snippet: Briefly, the DNA fragment containing the VEK-30 cDNA was inserted into the bacterial expression vector pET-15b (Novagen, Gibbstown, NJ)/GB1 [ ], and expressed in E. coli BL21 (DE3). .. The final construct contained, sequentially, an ATG initiation codon, a purification (His)6 tag, the GB1 fusion partner for increased solubility, a 9-residue linker, and a thrombin cleavage site, LVPR’GS (‘ representing the site of thrombin-catalyzed cleavage), all sequentially inserted into the parent plasmid [ ].

    Article Title: Deciphering the regulatory and catalytic mechanisms of an unusual SAM-dependent enzyme
    Article Snippet: .. Gene cloning, protein expression, and purification A codon-optimized gene encoding LepI, originated from A. flavus , was integrated into the expression vector pET-15b (Novagen, Madison, WI, USA) between the NdeI and XhoI sites with an N-terminal histidine tag. .. DNA sequencing and transformation into Escherichia coli BL21 (DE3) cells for expression verified the constructed plasmid pET-15b-LepI.

    Article Title: hnRNP H Is a Component of a Splicing Enhancer Complex That Activates a c-src Alternative Exon in Neuronal Cells
    Article Snippet: Both PCR products were digested with Xho I and Bam HI and then cloned into the expression vector pET 15b (Novagen) at the same sites. .. The bacterial cells were grown in LB medium containing ampicillin (100 μg/ml) at 37°C to an optical density of 0.7 at 600 nm before induction with 1 mM isopropyl-β- d -thiogalactopyranoside (IPTG) for 3 h. For the purification of hnRNPs H and F, 1 liter each of the IPTG-induced cells was pelleted by centrifugation and washed with 50 mM Tris-HCl (pH 8.0) containing 0.1 M NaCl.

    Article Title: Expression, purification and preliminary X-ray crystallographic analysis of Arf1-GDP in complex with dimeric p23 peptide
    Article Snippet: Paragraph title: 2.1. Cloning, expression and purification  ... The PCR products were digested with Nde I and Bam HI restriction enzymes (New England Biolabs) and then inserted into expression vector pET-15b (Novagen).

    Article Title: Human and yeast Rad52 proteins promote DNA strand exchange
    Article Snippet: Purification of HsRad52 Protein and Its Truncated Derivatives. .. The complete coding sequence of the HsRad52 gene was amplified by PCR from plasmid pEG2 ( ) and inserted into bacterial expression vector pET 15b (Novagen) between Nde I and Bam HI sites in frame with a 5′-end sequence coding for a series of six histidine residues, which function as a metal-binding domain in the translated fusion protein.

    Article Title: Biochemical characterization of an unclassified glutathione S-transferase of Plutella xylostella
    Article Snippet: Paragraph title: 3. Overexpression and purification of the recombinant protein ... After digestion of the PCR product with Nde I and Bam HI, the obtained fragment was subcloned into the Nde I– Bam HI site of expression vector pET-15b (Novagen; EMD Biosciences, Inc., Darmstadt, Germany).

    Protein Purification:

    Article Title: Potential Alu Function: Regulation of the Activity of Double-Stranded RNA-Activated Kinase PKR
    Article Snippet: PKR was cloned into expression vector pET-15b (Novagen) and bacterially expressed as a hexahistidine-tagged fusion protein as described previously ( ). .. Cell culture, cell lysis and extraction, and protein purification by nickel-agarose affinity column chromatography and by gel filtration fast-performance liquid chromatography were carried out as previously described ( ) with the following modifications.

    Sequencing:

    Article Title: Quantitative methods for measuring DNA flexibility in vitro and in vivo
    Article Snippet: The S. cerevisiae Nhp6A coding region is cloned between the Nco I and Bam HI sites of bacterial expression vector pET-15b (Novagen). .. The resulting plasmid (pJ1400) encodes untagged Nhp6A protein (molecular weight 10,801) with the following amino acid sequence: MVTPREPK2 RT2 RK3 DPNAPKRALSAYMF2 ANENRDIVRSENPDITFGQVGK2 LGE-KWKALTPE2 KQPYEAKAQADK2 RYESEKELYNATLA A 6-mL overnight LB culture of E. coli strain BL21(DE3) containing carbenicillin (Cb) (50 µg/mL) is grown from a single colony, pelleted by centrifugation, washed with 1 mL fresh LB medium to remove secreted β-lactamase, and resuspended in 1 ml of medium.

    Article Title: Solution structure of the cytohesin-1 (B2-1) Sec7 domain and its interaction with the GTPase ADP ribosylation factor 1
    Article Snippet: The Arf-1 cDNA was isolated by PCR amplification from a human spleen cell cDNA library ( ) by using primers based on the Arf-1 sequence ( ). .. The gene was cloned into the Nde I– Xho I restriction sites of the expression vector pET-15b (Novagen).

    Article Title: Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein
    Article Snippet: The NS3-ScFv DNA was reamplified to introduce the cloning sites and to omit the leader sequence. .. The digested fragment was inserted into the bacterial expression vector pET-15b (Novagen, Madison, Wis.) to give NS3-ScFv-pET.

    Article Title: Human and yeast Rad52 proteins promote DNA strand exchange
    Article Snippet: .. The complete coding sequence of the HsRad52 gene was amplified by PCR from plasmid pEG2 ( ) and inserted into bacterial expression vector pET 15b (Novagen) between Nde I and Bam HI sites in frame with a 5′-end sequence coding for a series of six histidine residues, which function as a metal-binding domain in the translated fusion protein. .. The resulting plasmid was designated pEG161H1.

    Cell Culture:

    Article Title: Potential Alu Function: Regulation of the Activity of Double-Stranded RNA-Activated Kinase PKR
    Article Snippet: PKR was cloned into expression vector pET-15b (Novagen) and bacterially expressed as a hexahistidine-tagged fusion protein as described previously ( ). .. Cell culture, cell lysis and extraction, and protein purification by nickel-agarose affinity column chromatography and by gel filtration fast-performance liquid chromatography were carried out as previously described ( ) with the following modifications.

    Affinity Column:

    Article Title: Solution structure of the complex of VEK-30 and plasminogen kringle 2
    Article Snippet: Briefly, the DNA fragment containing the VEK-30 cDNA was inserted into the bacterial expression vector pET-15b (Novagen, Gibbstown, NJ)/GB1 [ ], and expressed in E. coli BL21 (DE3). .. After induction with 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 3 hr at 37 °C, the fusion protein was purified using a Ni2+ -Sepharose affinity chromatography column (HisTrap HP; GE Healthcare, Piscataway, NJ).

    Article Title: Potential Alu Function: Regulation of the Activity of Double-Stranded RNA-Activated Kinase PKR
    Article Snippet: PKR was cloned into expression vector pET-15b (Novagen) and bacterially expressed as a hexahistidine-tagged fusion protein as described previously ( ). .. Cell culture, cell lysis and extraction, and protein purification by nickel-agarose affinity column chromatography and by gel filtration fast-performance liquid chromatography were carried out as previously described ( ) with the following modifications.

    cDNA Library Assay:

    Article Title: Solution structure of the cytohesin-1 (B2-1) Sec7 domain and its interaction with the GTPase ADP ribosylation factor 1
    Article Snippet: The Arf-1 cDNA was isolated by PCR amplification from a human spleen cell cDNA library ( ) by using primers based on the Arf-1 sequence ( ). .. The gene was cloned into the Nde I– Xho I restriction sites of the expression vector pET-15b (Novagen).

    Article Title: Expression, purification and preliminary X-ray crystallographic analysis of Arf1-GDP in complex with dimeric p23 peptide
    Article Snippet: The gene encoding human Arf1 (residues 18–181; UniProt accession No. ) was amplified from a human brain cDNA library (Stratagene) using upstream primer 5′-GGAATTCCATATGATGCGCATCCTCATGGTGGGCCTGG-3′ and downstream primer 5′-CGCGGATCCTCACTTCTGGTTCCGGAGCTGATTGGACAG-3′ which contained Nde I and Bam HI restriction sites. .. The PCR products were digested with Nde I and Bam HI restriction enzymes (New England Biolabs) and then inserted into expression vector pET-15b (Novagen).

    Automated Dye Terminator DNA Sequencing:

    Article Title: Solution structure of the cytohesin-1 (B2-1) Sec7 domain and its interaction with the GTPase ADP ribosylation factor 1
    Article Snippet: The gene was cloned into the Nde I– Xho I restriction sites of the expression vector pET-15b (Novagen). .. All vector sequences were confirmed by dye-terminator DNA sequencing by using an Applied Biosystems Prism 300 Genetic Analyzer (Perkin–Elmer).

    Liquid Chromatography:

    Article Title: Potential Alu Function: Regulation of the Activity of Double-Stranded RNA-Activated Kinase PKR
    Article Snippet: PKR was cloned into expression vector pET-15b (Novagen) and bacterially expressed as a hexahistidine-tagged fusion protein as described previously ( ). .. Cell culture, cell lysis and extraction, and protein purification by nickel-agarose affinity column chromatography and by gel filtration fast-performance liquid chromatography were carried out as previously described ( ) with the following modifications.

    Plasmid Preparation:

    Article Title: Overexpression, crystallization and preliminary X--ray crystallographic analysis of the C-terminal cytosolic domain of mouse anoctamin 1
    Article Snippet: .. Protein expression and purification mANO1-CTD was cloned into the expression vector pET-15b(+) (Novagen), adding a hexahistidine-containing 20-residue tag to the N-terminus. .. The recombinant protein was overexpressed in Escherichia coli Rosetta2(DE3)pLysS cells using Luria broth culture medium.

    Article Title: Quantitative methods for measuring DNA flexibility in vitro and in vivo
    Article Snippet: .. The S. cerevisiae Nhp6A coding region is cloned between the Nco I and Bam HI sites of bacterial expression vector pET-15b (Novagen). .. The resulting plasmid (pJ1400) encodes untagged Nhp6A protein (molecular weight 10,801) with the following amino acid sequence: MVTPREPK2 RT2 RK3 DPNAPKRALSAYMF2 ANENRDIVRSENPDITFGQVGK2 LGE-KWKALTPE2 KQPYEAKAQADK2 RYESEKELYNATLA A 6-mL overnight LB culture of E. coli strain BL21(DE3) containing carbenicillin (Cb) (50 µg/mL) is grown from a single colony, pelleted by centrifugation, washed with 1 mL fresh LB medium to remove secreted β-lactamase, and resuspended in 1 ml of medium.

    Article Title: Solution structure of the complex of VEK-30 and plasminogen kringle 2
    Article Snippet: .. Briefly, the DNA fragment containing the VEK-30 cDNA was inserted into the bacterial expression vector pET-15b (Novagen, Gibbstown, NJ)/GB1 [ ], and expressed in E. coli BL21 (DE3). .. The final construct contained, sequentially, an ATG initiation codon, a purification (His)6 tag, the GB1 fusion partner for increased solubility, a 9-residue linker, and a thrombin cleavage site, LVPR’GS (‘ representing the site of thrombin-catalyzed cleavage), all sequentially inserted into the parent plasmid [ ].

    Article Title: Deciphering the regulatory and catalytic mechanisms of an unusual SAM-dependent enzyme
    Article Snippet: .. Gene cloning, protein expression, and purification A codon-optimized gene encoding LepI, originated from A. flavus , was integrated into the expression vector pET-15b (Novagen, Madison, WI, USA) between the NdeI and XhoI sites with an N-terminal histidine tag. .. DNA sequencing and transformation into Escherichia coli BL21 (DE3) cells for expression verified the constructed plasmid pET-15b-LepI.

    Article Title: hnRNP H Is a Component of a Splicing Enhancer Complex That Activates a c-src Alternative Exon in Neuronal Cells
    Article Snippet: .. Both PCR products were digested with Xho I and Bam HI and then cloned into the expression vector pET 15b (Novagen) at the same sites. .. The recombinant N-terminal histidine-tagged proteins were expressed in Escherichia coli BL21(DE3).

    Article Title: Solution structure of the cytohesin-1 (B2-1) Sec7 domain and its interaction with the GTPase ADP ribosylation factor 1
    Article Snippet: .. The gene was cloned into the Nde I– Xho I restriction sites of the expression vector pET-15b (Novagen). .. Residues 18–181 (Δ17Arf-1) were subcloned into the Nde I– Xho I restriction sites of pET-20b. (Novagen).

    Article Title: Expression, purification and preliminary X-ray crystallographic analysis of Arf1-GDP in complex with dimeric p23 peptide
    Article Snippet: .. The PCR products were digested with Nde I and Bam HI restriction enzymes (New England Biolabs) and then inserted into expression vector pET-15b (Novagen). ..

    Article Title: DipA, a Pore-Forming Protein in the Outer Membrane of Lyme Disease Spirochetes Exhibits Specificity for the Permeation of Dicarboxylates
    Article Snippet: .. Overexpression of a recombinant fragment of DipA A fragment of DipA representing the 90 C-terminal amino acids was produced in E. coli Rosetta™ 2 (DE3) (Novagen), using expression vector pET 15b (Novagen) containing an N-terminal His6 -Tag. .. The gene fragment of 285 bp was amplified by PCR using following oligonucleotides: rbb0418_f ( 5′-CTG CATATG GAAGGAAAAACACAAATTGG-3′ ) containing NdeI restriction site and rbb0418_r ( 5′-GACTTTA GGATCC TTAAGTTATAGACATTCC-3′ ) containing BamHI restriction site.

    Article Title: Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein
    Article Snippet: .. The digested fragment was inserted into the bacterial expression vector pET-15b (Novagen, Madison, Wis.) to give NS3-ScFv-pET. .. For eucaryotic expression, the primer pair 5′-CCG GGT ACC ACA GGT GAA GCT GCA G-3′ and 5′-GCC TCT AGA CTA CCG TTT GAT TTC CAG-3′ was used to introduce Kpn I and Xba I restriction sites (underlined).

    Article Title: Human and yeast Rad52 proteins promote DNA strand exchange
    Article Snippet: .. The complete coding sequence of the HsRad52 gene was amplified by PCR from plasmid pEG2 ( ) and inserted into bacterial expression vector pET 15b (Novagen) between Nde I and Bam HI sites in frame with a 5′-end sequence coding for a series of six histidine residues, which function as a metal-binding domain in the translated fusion protein. .. The resulting plasmid was designated pEG161H1.

    Article Title: Biochemical characterization of an unclassified glutathione S-transferase of Plutella xylostella
    Article Snippet: .. After digestion of the PCR product with Nde I and Bam HI, the obtained fragment was subcloned into the Nde I– Bam HI site of expression vector pET-15b (Novagen; EMD Biosciences, Inc., Darmstadt, Germany). .. Competent Escherichia coli Rosetta (DE3) pLysS cells (Novagen; EMD Millipore, USA) were transformed with a prepared expression plasmid that harbored pxgstu1 and were grown at 37°C on Luria–Bertani medium that contained 100 µg/mL ampicillin.

    Article Title: Potential Alu Function: Regulation of the Activity of Double-Stranded RNA-Activated Kinase PKR
    Article Snippet: .. PKR was cloned into expression vector pET-15b (Novagen) and bacterially expressed as a hexahistidine-tagged fusion protein as described previously ( ). .. Cell culture, cell lysis and extraction, and protein purification by nickel-agarose affinity column chromatography and by gel filtration fast-performance liquid chromatography were carried out as previously described ( ) with the following modifications.

    Functional Assay:

    Article Title: Solution structure of the complex of VEK-30 and plasminogen kringle 2
    Article Snippet: To prepare the functional internal peptide, VEK-30, a (His)6 -B1 immunoglobulin-binding domain of streptococcal protein G (GB1)-tagged fusion expression system was employed. .. Briefly, the DNA fragment containing the VEK-30 cDNA was inserted into the bacterial expression vector pET-15b (Novagen, Gibbstown, NJ)/GB1 [ ], and expressed in E. coli BL21 (DE3).

    Positron Emission Tomography:

    Article Title: Overexpression, crystallization and preliminary X--ray crystallographic analysis of the C-terminal cytosolic domain of mouse anoctamin 1
    Article Snippet: .. Protein expression and purification mANO1-CTD was cloned into the expression vector pET-15b(+) (Novagen), adding a hexahistidine-containing 20-residue tag to the N-terminus. .. The recombinant protein was overexpressed in Escherichia coli Rosetta2(DE3)pLysS cells using Luria broth culture medium.

    Article Title: Quantitative methods for measuring DNA flexibility in vitro and in vivo
    Article Snippet: .. The S. cerevisiae Nhp6A coding region is cloned between the Nco I and Bam HI sites of bacterial expression vector pET-15b (Novagen). .. The resulting plasmid (pJ1400) encodes untagged Nhp6A protein (molecular weight 10,801) with the following amino acid sequence: MVTPREPK2 RT2 RK3 DPNAPKRALSAYMF2 ANENRDIVRSENPDITFGQVGK2 LGE-KWKALTPE2 KQPYEAKAQADK2 RYESEKELYNATLA A 6-mL overnight LB culture of E. coli strain BL21(DE3) containing carbenicillin (Cb) (50 µg/mL) is grown from a single colony, pelleted by centrifugation, washed with 1 mL fresh LB medium to remove secreted β-lactamase, and resuspended in 1 ml of medium.

    Article Title: Solution structure of the complex of VEK-30 and plasminogen kringle 2
    Article Snippet: .. Briefly, the DNA fragment containing the VEK-30 cDNA was inserted into the bacterial expression vector pET-15b (Novagen, Gibbstown, NJ)/GB1 [ ], and expressed in E. coli BL21 (DE3). .. The final construct contained, sequentially, an ATG initiation codon, a purification (His)6 tag, the GB1 fusion partner for increased solubility, a 9-residue linker, and a thrombin cleavage site, LVPR’GS (‘ representing the site of thrombin-catalyzed cleavage), all sequentially inserted into the parent plasmid [ ].

    Article Title: Deciphering the regulatory and catalytic mechanisms of an unusual SAM-dependent enzyme
    Article Snippet: .. Gene cloning, protein expression, and purification A codon-optimized gene encoding LepI, originated from A. flavus , was integrated into the expression vector pET-15b (Novagen, Madison, WI, USA) between the NdeI and XhoI sites with an N-terminal histidine tag. .. DNA sequencing and transformation into Escherichia coli BL21 (DE3) cells for expression verified the constructed plasmid pET-15b-LepI.

    Article Title: hnRNP H Is a Component of a Splicing Enhancer Complex That Activates a c-src Alternative Exon in Neuronal Cells
    Article Snippet: .. Both PCR products were digested with Xho I and Bam HI and then cloned into the expression vector pET 15b (Novagen) at the same sites. .. The recombinant N-terminal histidine-tagged proteins were expressed in Escherichia coli BL21(DE3).

    Article Title: Solution structure of the cytohesin-1 (B2-1) Sec7 domain and its interaction with the GTPase ADP ribosylation factor 1
    Article Snippet: .. The gene was cloned into the Nde I– Xho I restriction sites of the expression vector pET-15b (Novagen). .. Residues 18–181 (Δ17Arf-1) were subcloned into the Nde I– Xho I restriction sites of pET-20b. (Novagen).

    Article Title: Expression, purification and preliminary X-ray crystallographic analysis of Arf1-GDP in complex with dimeric p23 peptide
    Article Snippet: .. The PCR products were digested with Nde I and Bam HI restriction enzymes (New England Biolabs) and then inserted into expression vector pET-15b (Novagen). ..

    Article Title: DipA, a Pore-Forming Protein in the Outer Membrane of Lyme Disease Spirochetes Exhibits Specificity for the Permeation of Dicarboxylates
    Article Snippet: .. Overexpression of a recombinant fragment of DipA A fragment of DipA representing the 90 C-terminal amino acids was produced in E. coli Rosetta™ 2 (DE3) (Novagen), using expression vector pET 15b (Novagen) containing an N-terminal His6 -Tag. .. The gene fragment of 285 bp was amplified by PCR using following oligonucleotides: rbb0418_f ( 5′-CTG CATATG GAAGGAAAAACACAAATTGG-3′ ) containing NdeI restriction site and rbb0418_r ( 5′-GACTTTA GGATCC TTAAGTTATAGACATTCC-3′ ) containing BamHI restriction site.

    Article Title: Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein
    Article Snippet: .. The digested fragment was inserted into the bacterial expression vector pET-15b (Novagen, Madison, Wis.) to give NS3-ScFv-pET. .. For eucaryotic expression, the primer pair 5′-CCG GGT ACC ACA GGT GAA GCT GCA G-3′ and 5′-GCC TCT AGA CTA CCG TTT GAT TTC CAG-3′ was used to introduce Kpn I and Xba I restriction sites (underlined).

    Article Title: Human and yeast Rad52 proteins promote DNA strand exchange
    Article Snippet: .. The complete coding sequence of the HsRad52 gene was amplified by PCR from plasmid pEG2 ( ) and inserted into bacterial expression vector pET 15b (Novagen) between Nde I and Bam HI sites in frame with a 5′-end sequence coding for a series of six histidine residues, which function as a metal-binding domain in the translated fusion protein. .. The resulting plasmid was designated pEG161H1.

    Article Title: Biochemical characterization of an unclassified glutathione S-transferase of Plutella xylostella
    Article Snippet: .. After digestion of the PCR product with Nde I and Bam HI, the obtained fragment was subcloned into the Nde I– Bam HI site of expression vector pET-15b (Novagen; EMD Biosciences, Inc., Darmstadt, Germany). .. Competent Escherichia coli Rosetta (DE3) pLysS cells (Novagen; EMD Millipore, USA) were transformed with a prepared expression plasmid that harbored pxgstu1 and were grown at 37°C on Luria–Bertani medium that contained 100 µg/mL ampicillin.

    Article Title: Potential Alu Function: Regulation of the Activity of Double-Stranded RNA-Activated Kinase PKR
    Article Snippet: .. PKR was cloned into expression vector pET-15b (Novagen) and bacterially expressed as a hexahistidine-tagged fusion protein as described previously ( ). .. Cell culture, cell lysis and extraction, and protein purification by nickel-agarose affinity column chromatography and by gel filtration fast-performance liquid chromatography were carried out as previously described ( ) with the following modifications.

    Produced:

    Article Title: DipA, a Pore-Forming Protein in the Outer Membrane of Lyme Disease Spirochetes Exhibits Specificity for the Permeation of Dicarboxylates
    Article Snippet: .. Overexpression of a recombinant fragment of DipA A fragment of DipA representing the 90 C-terminal amino acids was produced in E. coli Rosetta™ 2 (DE3) (Novagen), using expression vector pET 15b (Novagen) containing an N-terminal His6 -Tag. .. The gene fragment of 285 bp was amplified by PCR using following oligonucleotides: rbb0418_f ( 5′-CTG CATATG GAAGGAAAAACACAAATTGG-3′ ) containing NdeI restriction site and rbb0418_r ( 5′-GACTTTA GGATCC TTAAGTTATAGACATTCC-3′ ) containing BamHI restriction site.

    Concentration Assay:

    Article Title: DipA, a Pore-Forming Protein in the Outer Membrane of Lyme Disease Spirochetes Exhibits Specificity for the Permeation of Dicarboxylates
    Article Snippet: Overexpression of a recombinant fragment of DipA A fragment of DipA representing the 90 C-terminal amino acids was produced in E. coli Rosetta™ 2 (DE3) (Novagen), using expression vector pET 15b (Novagen) containing an N-terminal His6 -Tag. .. The E. coli cells carrying expression plasmids were grown at 37°C to OD600 = 0.6 in LB medium containing 50 µg of carbecillin per ml and protein expression was induced by addition of isopropyl-β-d-thiogalactopyranoside (IPTG) to a final concentration of 1 mM.

    Article Title: Biochemical characterization of an unclassified glutathione S-transferase of Plutella xylostella
    Article Snippet: After digestion of the PCR product with Nde I and Bam HI, the obtained fragment was subcloned into the Nde I– Bam HI site of expression vector pET-15b (Novagen; EMD Biosciences, Inc., Darmstadt, Germany). .. After the cell density reached 0.7 OD600 , isopropyl 1-thio-β-D-galactoside was added for a final concentration of 1 mM to induce the production of the recombinant protein.

    CTG Assay:

    Article Title: Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein
    Article Snippet: For Escherichia coli expression, the forward primer 5′-CCG CTC GAG CAG GTG AAG CTG CAG-3′ and reverse primer 5′-C GG ATC C TA CCG TTT GAT TTC CAG-3′ were used to amplify the NS3-ScFv to introduce 5′ Bam HI and 3′ Xho I restriction sites (underlined). .. The digested fragment was inserted into the bacterial expression vector pET-15b (Novagen, Madison, Wis.) to give NS3-ScFv-pET.

    Lysis:

    Article Title: Overexpression, crystallization and preliminary X--ray crystallographic analysis of the C-terminal cytosolic domain of mouse anoctamin 1
    Article Snippet: Protein expression and purification mANO1-CTD was cloned into the expression vector pET-15b(+) (Novagen), adding a hexahistidine-containing 20-residue tag to the N-terminus. .. Protein expression was induced by 0.5 mM isopropyl β-d -1-thiogalactopyranoside and the cells were incubated for 16 h at 293 K following growth to mid-log phase at 310 K. The cells were lysed by sonication in lysis buffer (20 mM Tris–HCl pH 7.5, 500 mM NaCl, 35 mM imidazole and 1 mM phenylmethanesulfonylfluoride).

    Article Title: Deciphering the regulatory and catalytic mechanisms of an unusual SAM-dependent enzyme
    Article Snippet: Gene cloning, protein expression, and purification A codon-optimized gene encoding LepI, originated from A. flavus , was integrated into the expression vector pET-15b (Novagen, Madison, WI, USA) between the NdeI and XhoI sites with an N-terminal histidine tag. .. When the cell density reached an OD 600 nm of 0.6, the culture temperature was decreased from 37 °C to 15 °C, and the culture was supplemented with 0.3 mmol/L isopropyl-d-1-thiogalactopyranoside to induce the expression of LepI for 18 h. Cell pellets harvested by 4000 rpm centrifugation for 15 min were resuspended in 300 mL lysis buffer (25 mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride) and disrupted using a low-temperature high-pressure cell disruptor.

    Article Title: Potential Alu Function: Regulation of the Activity of Double-Stranded RNA-Activated Kinase PKR
    Article Snippet: PKR was cloned into expression vector pET-15b (Novagen) and bacterially expressed as a hexahistidine-tagged fusion protein as described previously ( ). .. Cell culture, cell lysis and extraction, and protein purification by nickel-agarose affinity column chromatography and by gel filtration fast-performance liquid chromatography were carried out as previously described ( ) with the following modifications.

    Variant Assay:

    Article Title: Solution structure of the complex of VEK-30 and plasminogen kringle 2
    Article Snippet: Human K2Pg (C4G/E56D/L72Y), a triple variant of K2Pg that displays enhanced affinity for lysine analogs and VEK-30 compared to wild-type-K2Pg , was expressed in Pichia pastoris GS115 cells as described in detail previously [ ]. .. Briefly, the DNA fragment containing the VEK-30 cDNA was inserted into the bacterial expression vector pET-15b (Novagen, Gibbstown, NJ)/GB1 [ ], and expressed in E. coli BL21 (DE3).

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    Millipore expression vector pet 15b
    Expression Vector Pet 15b, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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