pet e coli t7 expression vectors  (Millipore)


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    Structured Review

    Millipore pet e coli t7 expression vectors
    Pet E Coli T7 Expression Vectors, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pet e coli t7 expression vectors/product/Millipore
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pet e coli t7 expression vectors - by Bioz Stars, 2020-04
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    Related Articles

    Clone Assay:

    Article Title: Serological Expression Cloning of Novel Immunoreactive Antigens of Babesia microti
    Article Snippet: .. Five clones were chosen for high-level protein expression with the pET-17b vector (Novagen) ( bmn1-2 , -9 , -15 , and -17 and mn-10 [carboxy terminus]). .. These expression constructs were engineered to include an N-terminal six-histidine tag for ease of purification with an Ni-nitrilotriacetic acid agarose column.

    Article Title: Point mutations within the streptococcal regulator of virulence (Srv) alter protein-DNA interactions and Srv function
    Article Snippet: For expression and purification of recombinant protein, srv was cloned into expression vector pET32A (renamed pSDR srv ) and transformed into E. coli NovaBlue (Novagen). .. Recombinant (His6 -tagged) Srv protein was purified from E. coli protein expression strain Origami(DE3) (Novagen).

    Article Title: BcXYG1, a Secreted Xyloglucanase from Botrytis cinerea, Triggers Both Cell Death and Plant Immune Responses 1
    Article Snippet: .. To construct the E. coli protein expression vectors, the sequence encoding mature BcXYG1 protein without the signal peptide was cloned into pET-22b (+) (Novagen) to generate the expression vector pET22b-BcXYG1-6×His. .. For transient expression of proteins in plants using the A. tumefaciens infiltration method, the sequence encoding Arabidopsis PR3 (TAIR identifier AT3G12500) signal peptide was fused upstream of the BcXYG1-HA fusion protein, and the construct was cloned into pCAMBIA3300 (Cambia) between the cauliflower mosaic virus 35S promoter and the NOS terminator.

    Article Title: Molecular Cloning and Biochemical Characterization of a Recombinant Sterol 3-O-Glucosyltransferase from Gymnema sylvestre R.Br. Catalyzing Biosynthesis of Steryl Glucosides
    Article Snippet: .. Heterologous Expression of the Gene in E. coli The full length cDNA was cloned and expressed in E. coli protein expression system, namely, pET-28a as N-terminal fusion protein with a His6 tag to facilitate its purification (Novagen). .. The complete open reading frame (ORF) was PCR amplified with full length primers ( ).

    Article Title: Application of phage display to high throughput antibody generation and characterization
    Article Snippet: Paragraph title: Antigen cloning and expression ... Our E. coli protein expression system was described previously [ ] and utilizes the T7 RNA polymerase promoter driving cytoplasmic expression in BL21-DE3 cells (Novagen, Nottingham, UK).

    Article Title: NMR Evidence for Forming Highly Populated Helical Conformations in the Partially Folded hNck2 SH3 Domain
    Article Snippet: .. To achieve high-level protein expression, the DNA fragment encoding the first hNck2 SH3 domain with Escherichia coli -preferred codons was obtained by a polymerase chain reaction-based de novo gene synthesis approach, as previously described ( , ), and subsequently cloned into pET32a vector (Novagen, Singapore). .. Similarly, the DNA fragment for the SH3 domain carrying four-residue Ala-mutations over residues Lue25 -Trp26 -Leu27 -Leu28 (designated 4AlaMut) was synthesized by the same approach, except that the two middle primers were replaced by those with the mutations.

    Article Title: Crystal Structure of Botulinum Neurotoxin A2 in Complex with the Human Protein Receptor SV2C Reveals Plasticity in Receptor Binding
    Article Snippet: The interacting part of the fourth luminal domain of synaptic vesicle glycoprotein 2C (SV2C-L4, residues 474-567 Uniprot ID Q496J9) was amplified from the pGEX4-T1 SV2C-L4 plasmid used for binding studies (primers 5′-TACTTCCAATCCATGGAGAGAGATAAATATGCAAATTTC-3′ and 5′-TATCCACCTTTACTGTCACGTCTTGTTGTGAAAAAACGAG-3′) and cloned into a pNIC28-Bsa4 (N-terminal His6-tag with TEV site, KanR) vector using LIC cloning. .. SV2C-L4 protein was expressed in E. coli BL21 (DE3) T1R (SigmaAldrich B2935, St. Louis, MO, USA), that had been transformed with pRARE2 and made competent, by induction with 0.5 mM IPTG at an OD600 of 3.0 in Terrific Broth media and further growth at 18 °C overnight.

    Centrifugation:

    Article Title: Characterization of the Raf Kinase Inhibitory Protein (RKIP) Binding Pocket: NMR-Based Screening Identifies Small-Molecule Ligands
    Article Snippet: Protein expression BL21(DE3) cells (Novagen) harboring a pMCSG7-derived expression vector for rat RKIP were grown at 37°C in the M9 media with15 N-NH3 Cl as the sole nitrogen source and supplemented with ampicillin (100 µg/ml). .. The His-tagged rRKIP expressing cells were harvested by centrifugation and stored at −80°C.

    Article Title: Crystal Structure of Botulinum Neurotoxin A2 in Complex with the Human Protein Receptor SV2C Reveals Plasticity in Receptor Binding
    Article Snippet: SV2C-L4 protein was expressed in E. coli BL21 (DE3) T1R (SigmaAldrich B2935, St. Louis, MO, USA), that had been transformed with pRARE2 and made competent, by induction with 0.5 mM IPTG at an OD600 of 3.0 in Terrific Broth media and further growth at 18 °C overnight. .. Bacteria were harvested by centrifugation and lysed in Lysis buffer (100 mM HEPES, pH 8.0, 500 mM NaCl, 10% (v /v ) glycerol, 10 mM Imidazole, 0.5 mM TCEP) after treatment with benzonase nuclease (Sigma) and protease inhibitor cocktail (Roche) using sonication followed by centrifugation.

    Article Title: A Non-Motor Microtubule Binding Site Is Essential for the High Processivity and Mitotic Function of Kinesin-8 Kif18A
    Article Snippet: Kif18A776-898 –GFP and Kif18A898 –GFP were expressed in BL21(DE3)-T1R competent Escherichia coli (Sigma B2935) transformed with pRARE (Novagen, # 70954). .. The extract was cleared by centrifugation at 10 min at 10,000 x g. The supernatant was incubated with Amylose resin (NEB) for 2 h at 4°C and the resin was washed four times with 3C-buffer (50 mM TrisHCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.01% Tween20).

    Amplification:

    Article Title: BcXYG1, a Secreted Xyloglucanase from Botrytis cinerea, Triggers Both Cell Death and Plant Immune Responses 1
    Article Snippet: The 5′ (538 bp) and 3′ (534 bp) flanks of the BcXYG1 open reading frame were amplified by PCR from genomic DNA of the wild-type strain B05.10, and the fragments were cloned into the upstream and downstream regions, respectively, of the hph cassette using the Gibson Assembly Master Mix kit (New England Biolabs). .. To construct the E. coli protein expression vectors, the sequence encoding mature BcXYG1 protein without the signal peptide was cloned into pET-22b (+) (Novagen) to generate the expression vector pET22b-BcXYG1-6×His.

    Article Title: Molecular Cloning and Biochemical Characterization of a Recombinant Sterol 3-O-Glucosyltransferase from Gymnema sylvestre R.Br. Catalyzing Biosynthesis of Steryl Glucosides
    Article Snippet: Heterologous Expression of the Gene in E. coli The full length cDNA was cloned and expressed in E. coli protein expression system, namely, pET-28a as N-terminal fusion protein with a His6 tag to facilitate its purification (Novagen). .. The complete open reading frame (ORF) was PCR amplified with full length primers ( ).

    Article Title: Application of phage display to high throughput antibody generation and characterization
    Article Snippet: All steps in antigen production, including PCR amplification, sequence confirmation, expression and purification, were electronically tracked using a custom designed laboratory information management system (unpublished). .. Our E. coli protein expression system was described previously [ ] and utilizes the T7 RNA polymerase promoter driving cytoplasmic expression in BL21-DE3 cells (Novagen, Nottingham, UK).

    Article Title: Crystal Structure of Botulinum Neurotoxin A2 in Complex with the Human Protein Receptor SV2C Reveals Plasticity in Receptor Binding
    Article Snippet: The interacting part of the fourth luminal domain of synaptic vesicle glycoprotein 2C (SV2C-L4, residues 474-567 Uniprot ID Q496J9) was amplified from the pGEX4-T1 SV2C-L4 plasmid used for binding studies (primers 5′-TACTTCCAATCCATGGAGAGAGATAAATATGCAAATTTC-3′ and 5′-TATCCACCTTTACTGTCACGTCTTGTTGTGAAAAAACGAG-3′) and cloned into a pNIC28-Bsa4 (N-terminal His6-tag with TEV site, KanR) vector using LIC cloning. .. SV2C-L4 protein was expressed in E. coli BL21 (DE3) T1R (SigmaAldrich B2935, St. Louis, MO, USA), that had been transformed with pRARE2 and made competent, by induction with 0.5 mM IPTG at an OD600 of 3.0 in Terrific Broth media and further growth at 18 °C overnight.

    Article Title: Engineering biosynthesis of the anticancer alkaloid noscapine in yeast
    Article Snippet: For DNA manipulation and amplification, we used chemically competent or electrocompetent E. coli strains TOP10 and ccdB Survival 2 T1R purchased from Life Technologies. .. BL21(DE3) and Ni-NTA agorose resin used for E. coli protein expression and hexa-histidine tagged protein purification were kindly provided by Professor Chaitan Khosla, and T7 Tag Affinity Purification Kit used for T7-tagged protein purification was purchased from EMD Millipore.

    Filtration:

    Article Title: Engineering biosynthesis of the anticancer alkaloid noscapine in yeast
    Article Snippet: BL21(DE3) and Ni-NTA agorose resin used for E. coli protein expression and hexa-histidine tagged protein purification were kindly provided by Professor Chaitan Khosla, and T7 Tag Affinity Purification Kit used for T7-tagged protein purification was purchased from EMD Millipore. .. Size-exclusion chromatography was performed on a BioSep-SEC-s4000 column (Phenomenex, kindly provided by Professor Chaitan Khosla), and the column was equilibrated with Gel Filtration Markers Kit for Protein Molecular Weights 12,000–200,000 Da purchased from Sigma.

    Synthesized:

    Article Title: NMR Evidence for Forming Highly Populated Helical Conformations in the Partially Folded hNck2 SH3 Domain
    Article Snippet: To achieve high-level protein expression, the DNA fragment encoding the first hNck2 SH3 domain with Escherichia coli -preferred codons was obtained by a polymerase chain reaction-based de novo gene synthesis approach, as previously described ( , ), and subsequently cloned into pET32a vector (Novagen, Singapore). .. Similarly, the DNA fragment for the SH3 domain carrying four-residue Ala-mutations over residues Lue25 -Trp26 -Leu27 -Leu28 (designated 4AlaMut) was synthesized by the same approach, except that the two middle primers were replaced by those with the mutations.

    Construct:

    Article Title: Serological Expression Cloning of Novel Immunoreactive Antigens of Babesia microti
    Article Snippet: Five clones were chosen for high-level protein expression with the pET-17b vector (Novagen) ( bmn1-2 , -9 , -15 , and -17 and mn-10 [carboxy terminus]). .. These expression constructs were engineered to include an N-terminal six-histidine tag for ease of purification with an Ni-nitrilotriacetic acid agarose column.

    Article Title: Characterization of the Raf Kinase Inhibitory Protein (RKIP) Binding Pocket: NMR-Based Screening Identifies Small-Molecule Ligands
    Article Snippet: Protein expression BL21(DE3) cells (Novagen) harboring a pMCSG7-derived expression vector for rat RKIP were grown at 37°C in the M9 media with15 N-NH3 Cl as the sole nitrogen source and supplemented with ampicillin (100 µg/ml). .. The expression construct consisted of a His6 tag fused in frame with a TEV protease cleavage signal and the entire open reading frame (residues 1-187) of wild-type rat RKIP (rRKIP).

    Article Title: BcXYG1, a Secreted Xyloglucanase from Botrytis cinerea, Triggers Both Cell Death and Plant Immune Responses 1
    Article Snippet: .. To construct the E. coli protein expression vectors, the sequence encoding mature BcXYG1 protein without the signal peptide was cloned into pET-22b (+) (Novagen) to generate the expression vector pET22b-BcXYG1-6×His. .. For transient expression of proteins in plants using the A. tumefaciens infiltration method, the sequence encoding Arabidopsis PR3 (TAIR identifier AT3G12500) signal peptide was fused upstream of the BcXYG1-HA fusion protein, and the construct was cloned into pCAMBIA3300 (Cambia) between the cauliflower mosaic virus 35S promoter and the NOS terminator.

    Article Title: Molecular Cloning and Biochemical Characterization of a Recombinant Sterol 3-O-Glucosyltransferase from Gymnema sylvestre R.Br. Catalyzing Biosynthesis of Steryl Glucosides
    Article Snippet: Heterologous Expression of the Gene in E. coli The full length cDNA was cloned and expressed in E. coli protein expression system, namely, pET-28a as N-terminal fusion protein with a His6 tag to facilitate its purification (Novagen). .. The 2.1 kb amplified ORF was cloned at Nde I and Bam H1 restriction sites in pET-28a expression vector and the resulting construct was used to transform E. coli BL21 (DE3) strain (Novagen).

    Article Title: Engineering biosynthesis of the anticancer alkaloid noscapine in yeast
    Article Snippet: All engineered yeast strains described in this work, as listed in , were constructed in a haploid W303α background (MATα leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 ) . .. BL21(DE3) and Ni-NTA agorose resin used for E. coli protein expression and hexa-histidine tagged protein purification were kindly provided by Professor Chaitan Khosla, and T7 Tag Affinity Purification Kit used for T7-tagged protein purification was purchased from EMD Millipore.

    Article Title: A robust and tunable mitotic oscillator in artificial cells
    Article Snippet: GFP-NLS protein was expressed in BL21 (DE3)-T-1 competent cells (Sigma Aldrich, B2935) that were induced by 0.1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside, Sigma Aldrich, I6758) overnight. .. Securin-mCherry and cyclin B1-YFP plasmids were constructed using Gibson assembly method ( ).

    Enzyme-linked Immunosorbent Assay:

    Article Title: Efficacy of recombinant measles virus expressing highly pathogenic avian influenza virus (HPAIV) antigen against HPAIV infection in monkeys
    Article Snippet: Measurement of antibody titers The titer of the antibodies directed the MV and H5 HA protein in the monkey sera were determined with ELISA. .. The H5 HA gene was ligated to the E. coli protein expression vector pET21b (Novagen), from which the recombinant protein was expressed as a fusion protein with a histidine tag.

    Incubation:

    Article Title: Efficacy of recombinant measles virus expressing highly pathogenic avian influenza virus (HPAIV) antigen against HPAIV infection in monkeys
    Article Snippet: The H5 HA gene was ligated to the E. coli protein expression vector pET21b (Novagen), from which the recombinant protein was expressed as a fusion protein with a histidine tag. .. After incubation for 2 h, the bacteria were collected, lysed with lysis buffer (50 mM Tris-HCl, 500 mM NaCl,20 mM Imidazol pH7.9), and freezed at −80 °C.

    Article Title: Development and Characterization of a Camelid Single Domain Antibody–Urease Conjugate That Targets Vascular Endothelial Growth Factor Receptor 2
    Article Snippet: Transformation of BL21 (DE3) competent E. coli cells (Sigma, B2935-10 × 50 µL) was according to the manufacturer’s instructions. .. Flasks were incubated in a shaker incubator at 200 rpm and 37°C.

    Article Title: A Non-Motor Microtubule Binding Site Is Essential for the High Processivity and Mitotic Function of Kinesin-8 Kif18A
    Article Snippet: Kif18A776-898 –GFP and Kif18A898 –GFP were expressed in BL21(DE3)-T1R competent Escherichia coli (Sigma B2935) transformed with pRARE (Novagen, # 70954). .. The extract was cleared by centrifugation at 10 min at 10,000 x g. The supernatant was incubated with Amylose resin (NEB) for 2 h at 4°C and the resin was washed four times with 3C-buffer (50 mM TrisHCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.01% Tween20).

    Solubility:

    Article Title: Application of phage display to high throughput antibody generation and characterization
    Article Snippet: Our E. coli protein expression system was described previously [ ] and utilizes the T7 RNA polymerase promoter driving cytoplasmic expression in BL21-DE3 cells (Novagen, Nottingham, UK). .. Most proteins were expressed with an amino-terminal decahistidine (His10) tag for affinity purification and either a thioredoxin (Trx) or MBP solubility enhancing fusion.

    Expressing:

    Article Title: Efficacy of recombinant measles virus expressing highly pathogenic avian influenza virus (HPAIV) antigen against HPAIV infection in monkeys
    Article Snippet: .. The H5 HA gene was ligated to the E. coli protein expression vector pET21b (Novagen), from which the recombinant protein was expressed as a fusion protein with a histidine tag. .. Competent BL21 cells were transformed with the plasmid to express the protein at high levels.

    Article Title: Serological Expression Cloning of Novel Immunoreactive Antigens of Babesia microti
    Article Snippet: .. Five clones were chosen for high-level protein expression with the pET-17b vector (Novagen) ( bmn1-2 , -9 , -15 , and -17 and mn-10 [carboxy terminus]). .. These expression constructs were engineered to include an N-terminal six-histidine tag for ease of purification with an Ni-nitrilotriacetic acid agarose column.

    Article Title: Characterization of the Raf Kinase Inhibitory Protein (RKIP) Binding Pocket: NMR-Based Screening Identifies Small-Molecule Ligands
    Article Snippet: .. Protein expression BL21(DE3) cells (Novagen) harboring a pMCSG7-derived expression vector for rat RKIP were grown at 37°C in the M9 media with15 N-NH3 Cl as the sole nitrogen source and supplemented with ampicillin (100 µg/ml). .. The expression construct consisted of a His6 tag fused in frame with a TEV protease cleavage signal and the entire open reading frame (residues 1-187) of wild-type rat RKIP (rRKIP).

    Article Title: Point mutations within the streptococcal regulator of virulence (Srv) alter protein-DNA interactions and Srv function
    Article Snippet: .. Recombinant (His6 -tagged) Srv protein was purified from E. coli protein expression strain Origami(DE3) (Novagen). ..

    Article Title: BcXYG1, a Secreted Xyloglucanase from Botrytis cinerea, Triggers Both Cell Death and Plant Immune Responses 1
    Article Snippet: .. To construct the E. coli protein expression vectors, the sequence encoding mature BcXYG1 protein without the signal peptide was cloned into pET-22b (+) (Novagen) to generate the expression vector pET22b-BcXYG1-6×His. .. For transient expression of proteins in plants using the A. tumefaciens infiltration method, the sequence encoding Arabidopsis PR3 (TAIR identifier AT3G12500) signal peptide was fused upstream of the BcXYG1-HA fusion protein, and the construct was cloned into pCAMBIA3300 (Cambia) between the cauliflower mosaic virus 35S promoter and the NOS terminator.

    Article Title: Molecular Cloning and Biochemical Characterization of a Recombinant Sterol 3-O-Glucosyltransferase from Gymnema sylvestre R.Br. Catalyzing Biosynthesis of Steryl Glucosides
    Article Snippet: .. Heterologous Expression of the Gene in E. coli The full length cDNA was cloned and expressed in E. coli protein expression system, namely, pET-28a as N-terminal fusion protein with a His6 tag to facilitate its purification (Novagen). .. The complete open reading frame (ORF) was PCR amplified with full length primers ( ).

    Article Title: Development and Characterization of a Camelid Single Domain Antibody–Urease Conjugate That Targets Vascular Endothelial Growth Factor Receptor 2
    Article Snippet: Paragraph title: Expression of V21H1 and V21H4 ... Transformation of BL21 (DE3) competent E. coli cells (Sigma, B2935-10 × 50 µL) was according to the manufacturer’s instructions.

    Article Title: Application of phage display to high throughput antibody generation and characterization
    Article Snippet: .. Our E. coli protein expression system was described previously [ ] and utilizes the T7 RNA polymerase promoter driving cytoplasmic expression in BL21-DE3 cells (Novagen, Nottingham, UK). ..

    Article Title: NMR Evidence for Forming Highly Populated Helical Conformations in the Partially Folded hNck2 SH3 Domain
    Article Snippet: .. To achieve high-level protein expression, the DNA fragment encoding the first hNck2 SH3 domain with Escherichia coli -preferred codons was obtained by a polymerase chain reaction-based de novo gene synthesis approach, as previously described ( , ), and subsequently cloned into pET32a vector (Novagen, Singapore). .. Similarly, the DNA fragment for the SH3 domain carrying four-residue Ala-mutations over residues Lue25 -Trp26 -Leu27 -Leu28 (designated 4AlaMut) was synthesized by the same approach, except that the two middle primers were replaced by those with the mutations.

    Article Title: Type II platelet-activating factor-acetylhydrolase is essential for epithelial morphogenesis in Caenorhabditis elegans
    Article Snippet: .. Recombinant PAF-1 and PAF-2 proteins that were expressed and purified by an E. coli pET expression system (Novagen) were used as the enzymes. .. C. elegans were lysed by sonication and were centrifuged at 20,000 × g for 20 min.

    Article Title: Crystal Structure of Botulinum Neurotoxin A2 in Complex with the Human Protein Receptor SV2C Reveals Plasticity in Receptor Binding
    Article Snippet: Cloning, expression and purification of SV2C-L4. .. SV2C-L4 protein was expressed in E. coli BL21 (DE3) T1R (SigmaAldrich B2935, St. Louis, MO, USA), that had been transformed with pRARE2 and made competent, by induction with 0.5 mM IPTG at an OD600 of 3.0 in Terrific Broth media and further growth at 18 °C overnight.

    Article Title: SetRICE391, a negative transcriptional regulator of the integrating conjugative element 391 mutagenic response
    Article Snippet: .. The ICE391 SetR protein was overproduced using the T7 protein expression system (EMD Millipore). .. Overproduced SetRICE391 was purified using protocols previously developed for the purification of LexA and DinR proteins [ , ].

    Article Title: Engineering biosynthesis of the anticancer alkaloid noscapine in yeast
    Article Snippet: .. BL21(DE3) and Ni-NTA agorose resin used for E. coli protein expression and hexa-histidine tagged protein purification were kindly provided by Professor Chaitan Khosla, and T7 Tag Affinity Purification Kit used for T7-tagged protein purification was purchased from EMD Millipore. .. NuPAGE Novex 4–12% Bis-Tris Protein Gels (1.0 mm, 10 well) used for purified protein SDS–PAGE analysis and western blot were purchased from Life Technologies and Color Prestained Protein Standard (Broad Range, 11–245 kDa) used as the protein marker was obtained from New England Biolabs.

    Article Title: A Non-Motor Microtubule Binding Site Is Essential for the High Processivity and Mitotic Function of Kinesin-8 Kif18A
    Article Snippet: Paragraph title: Protein Expression ... Kif18A776-898 –GFP and Kif18A898 –GFP were expressed in BL21(DE3)-T1R competent Escherichia coli (Sigma B2935) transformed with pRARE (Novagen, # 70954).

    Planar Chromatography:

    Article Title: Engineering biosynthesis of the anticancer alkaloid noscapine in yeast
    Article Snippet: BL21(DE3) and Ni-NTA agorose resin used for E. coli protein expression and hexa-histidine tagged protein purification were kindly provided by Professor Chaitan Khosla, and T7 Tag Affinity Purification Kit used for T7-tagged protein purification was purchased from EMD Millipore. .. The Anti-6X His tag antibody (horseradish peroxidase (HRP), ab1269) and Anti-T7 tag antibody (HRP, ab21477) used for western blot of PsMT2/PsMT3 and PsMT2/Ps6OMT heterodimers were purchased from Abcam PLC.

    Western Blot:

    Article Title: Type II platelet-activating factor-acetylhydrolase is essential for epithelial morphogenesis in Caenorhabditis elegans
    Article Snippet: For Western blotting and PAF-AH assays, mixed-stage animals were heat-shocked at 33°C for 2 h and were collected from plates 24 h after heat treatment. .. Recombinant PAF-1 and PAF-2 proteins that were expressed and purified by an E. coli pET expression system (Novagen) were used as the enzymes.

    Article Title: Engineering biosynthesis of the anticancer alkaloid noscapine in yeast
    Article Snippet: BL21(DE3) and Ni-NTA agorose resin used for E. coli protein expression and hexa-histidine tagged protein purification were kindly provided by Professor Chaitan Khosla, and T7 Tag Affinity Purification Kit used for T7-tagged protein purification was purchased from EMD Millipore. .. NuPAGE Novex 4–12% Bis-Tris Protein Gels (1.0 mm, 10 well) used for purified protein SDS–PAGE analysis and western blot were purchased from Life Technologies and Color Prestained Protein Standard (Broad Range, 11–245 kDa) used as the protein marker was obtained from New England Biolabs.

    Transformation Assay:

    Article Title: Efficacy of recombinant measles virus expressing highly pathogenic avian influenza virus (HPAIV) antigen against HPAIV infection in monkeys
    Article Snippet: The H5 HA gene was ligated to the E. coli protein expression vector pET21b (Novagen), from which the recombinant protein was expressed as a fusion protein with a histidine tag. .. Competent BL21 cells were transformed with the plasmid to express the protein at high levels.

    Article Title: Point mutations within the streptococcal regulator of virulence (Srv) alter protein-DNA interactions and Srv function
    Article Snippet: For expression and purification of recombinant protein, srv was cloned into expression vector pET32A (renamed pSDR srv ) and transformed into E. coli NovaBlue (Novagen). .. Recombinant (His6 -tagged) Srv protein was purified from E. coli protein expression strain Origami(DE3) (Novagen).

    Article Title: Development and Characterization of a Camelid Single Domain Antibody–Urease Conjugate That Targets Vascular Endothelial Growth Factor Receptor 2
    Article Snippet: .. Transformation of BL21 (DE3) competent E. coli cells (Sigma, B2935-10 × 50 µL) was according to the manufacturer’s instructions. .. One colony from a transformation plate was aseptically inoculated to 200 mL of LB broth (LB media EZ mix, Sigma cat #L76581, 20 g/L) supplemented with 50 mg/L kanamycin.

    Article Title: Crystal Structure of Botulinum Neurotoxin A2 in Complex with the Human Protein Receptor SV2C Reveals Plasticity in Receptor Binding
    Article Snippet: .. SV2C-L4 protein was expressed in E. coli BL21 (DE3) T1R (SigmaAldrich B2935, St. Louis, MO, USA), that had been transformed with pRARE2 and made competent, by induction with 0.5 mM IPTG at an OD600 of 3.0 in Terrific Broth media and further growth at 18 °C overnight. .. Bacteria were harvested by centrifugation and lysed in Lysis buffer (100 mM HEPES, pH 8.0, 500 mM NaCl, 10% (v /v ) glycerol, 10 mM Imidazole, 0.5 mM TCEP) after treatment with benzonase nuclease (Sigma) and protease inhibitor cocktail (Roche) using sonication followed by centrifugation.

    Article Title: A Non-Motor Microtubule Binding Site Is Essential for the High Processivity and Mitotic Function of Kinesin-8 Kif18A
    Article Snippet: .. Kif18A776-898 –GFP and Kif18A898 –GFP were expressed in BL21(DE3)-T1R competent Escherichia coli (Sigma B2935) transformed with pRARE (Novagen, # 70954). .. Expression was induced at OD 0.6 with 0.5 mM IPTG over night at 18°C.

    Over Expression:

    Article Title: BcXYG1, a Secreted Xyloglucanase from Botrytis cinerea, Triggers Both Cell Death and Plant Immune Responses 1
    Article Snippet: To construct the BcXYG1 overexpression vector, the full-length BcXYG1 open reading frame fused with an HA tag at the C terminus was cloned into the pH2G vector between the B. cinerea histone H2B promoter ( identifier ) and the endo-β-1,4-glucanase precursor terminator ( identifier ). .. To construct the E. coli protein expression vectors, the sequence encoding mature BcXYG1 protein without the signal peptide was cloned into pET-22b (+) (Novagen) to generate the expression vector pET22b-BcXYG1-6×His.

    Protease Inhibitor:

    Article Title: Crystal Structure of Botulinum Neurotoxin A2 in Complex with the Human Protein Receptor SV2C Reveals Plasticity in Receptor Binding
    Article Snippet: SV2C-L4 protein was expressed in E. coli BL21 (DE3) T1R (SigmaAldrich B2935, St. Louis, MO, USA), that had been transformed with pRARE2 and made competent, by induction with 0.5 mM IPTG at an OD600 of 3.0 in Terrific Broth media and further growth at 18 °C overnight. .. Bacteria were harvested by centrifugation and lysed in Lysis buffer (100 mM HEPES, pH 8.0, 500 mM NaCl, 10% (v /v ) glycerol, 10 mM Imidazole, 0.5 mM TCEP) after treatment with benzonase nuclease (Sigma) and protease inhibitor cocktail (Roche) using sonication followed by centrifugation.

    Cell Culture:

    Article Title: NMR Evidence for Forming Highly Populated Helical Conformations in the Partially Folded hNck2 SH3 Domain
    Article Snippet: To achieve high-level protein expression, the DNA fragment encoding the first hNck2 SH3 domain with Escherichia coli -preferred codons was obtained by a polymerase chain reaction-based de novo gene synthesis approach, as previously described ( , ), and subsequently cloned into pET32a vector (Novagen, Singapore). .. Briefly, the cells were cultured at 37°C to reach an optical density at 600 nm of 0.4, and then isopropyl β -D-1-thiogalactopyranoside was added to a final concentration of 0.4 mM to induce the expression of the recombinant protein overnight at 20°C.

    Article Title: Engineering biosynthesis of the anticancer alkaloid noscapine in yeast
    Article Snippet: Yeast strains were cultured at 25 or 30 °C in complex yeast extract peptone dextrose (YPD, all components from BD Diagnostics) medium or SDM containing yeast nitrogen base (YNB) without Amino Acids (BD Diagnostics), ammonium sulfate (Fisher Scientific), 2% dextrose and the appropriate dropout solution for plasmid maintenance. .. BL21(DE3) and Ni-NTA agorose resin used for E. coli protein expression and hexa-histidine tagged protein purification were kindly provided by Professor Chaitan Khosla, and T7 Tag Affinity Purification Kit used for T7-tagged protein purification was purchased from EMD Millipore.

    Article Title: A Non-Motor Microtubule Binding Site Is Essential for the High Processivity and Mitotic Function of Kinesin-8 Kif18A
    Article Snippet: Cells were cultured in serum free SF900II Medium (Invitrogen). .. Kif18A776-898 –GFP and Kif18A898 –GFP were expressed in BL21(DE3)-T1R competent Escherichia coli (Sigma B2935) transformed with pRARE (Novagen, # 70954).

    Generated:

    Article Title: BcXYG1, a Secreted Xyloglucanase from Botrytis cinerea, Triggers Both Cell Death and Plant Immune Responses 1
    Article Snippet: The BcXYG1 replacement construct was generated as described ( ). .. To construct the E. coli protein expression vectors, the sequence encoding mature BcXYG1 protein without the signal peptide was cloned into pET-22b (+) (Novagen) to generate the expression vector pET22b-BcXYG1-6×His.

    Polymerase Chain Reaction:

    Article Title: BcXYG1, a Secreted Xyloglucanase from Botrytis cinerea, Triggers Both Cell Death and Plant Immune Responses 1
    Article Snippet: The 5′ (538 bp) and 3′ (534 bp) flanks of the BcXYG1 open reading frame were amplified by PCR from genomic DNA of the wild-type strain B05.10, and the fragments were cloned into the upstream and downstream regions, respectively, of the hph cassette using the Gibson Assembly Master Mix kit (New England Biolabs). .. To construct the E. coli protein expression vectors, the sequence encoding mature BcXYG1 protein without the signal peptide was cloned into pET-22b (+) (Novagen) to generate the expression vector pET22b-BcXYG1-6×His.

    Article Title: Molecular Cloning and Biochemical Characterization of a Recombinant Sterol 3-O-Glucosyltransferase from Gymnema sylvestre R.Br. Catalyzing Biosynthesis of Steryl Glucosides
    Article Snippet: Heterologous Expression of the Gene in E. coli The full length cDNA was cloned and expressed in E. coli protein expression system, namely, pET-28a as N-terminal fusion protein with a His6 tag to facilitate its purification (Novagen). .. The complete open reading frame (ORF) was PCR amplified with full length primers ( ).

    Article Title: Application of phage display to high throughput antibody generation and characterization
    Article Snippet: All steps in antigen production, including PCR amplification, sequence confirmation, expression and purification, were electronically tracked using a custom designed laboratory information management system (unpublished). .. Our E. coli protein expression system was described previously [ ] and utilizes the T7 RNA polymerase promoter driving cytoplasmic expression in BL21-DE3 cells (Novagen, Nottingham, UK).

    Sonication:

    Article Title: Type II platelet-activating factor-acetylhydrolase is essential for epithelial morphogenesis in Caenorhabditis elegans
    Article Snippet: Recombinant PAF-1 and PAF-2 proteins that were expressed and purified by an E. coli pET expression system (Novagen) were used as the enzymes. .. C. elegans were lysed by sonication and were centrifuged at 20,000 × g for 20 min.

    Article Title: Crystal Structure of Botulinum Neurotoxin A2 in Complex with the Human Protein Receptor SV2C Reveals Plasticity in Receptor Binding
    Article Snippet: SV2C-L4 protein was expressed in E. coli BL21 (DE3) T1R (SigmaAldrich B2935, St. Louis, MO, USA), that had been transformed with pRARE2 and made competent, by induction with 0.5 mM IPTG at an OD600 of 3.0 in Terrific Broth media and further growth at 18 °C overnight. .. Bacteria were harvested by centrifugation and lysed in Lysis buffer (100 mM HEPES, pH 8.0, 500 mM NaCl, 10% (v /v ) glycerol, 10 mM Imidazole, 0.5 mM TCEP) after treatment with benzonase nuclease (Sigma) and protease inhibitor cocktail (Roche) using sonication followed by centrifugation.

    Affinity Purification:

    Article Title: Application of phage display to high throughput antibody generation and characterization
    Article Snippet: Our E. coli protein expression system was described previously [ ] and utilizes the T7 RNA polymerase promoter driving cytoplasmic expression in BL21-DE3 cells (Novagen, Nottingham, UK). .. Most proteins were expressed with an amino-terminal decahistidine (His10) tag for affinity purification and either a thioredoxin (Trx) or MBP solubility enhancing fusion.

    Article Title: Engineering biosynthesis of the anticancer alkaloid noscapine in yeast
    Article Snippet: .. BL21(DE3) and Ni-NTA agorose resin used for E. coli protein expression and hexa-histidine tagged protein purification were kindly provided by Professor Chaitan Khosla, and T7 Tag Affinity Purification Kit used for T7-tagged protein purification was purchased from EMD Millipore. .. NuPAGE Novex 4–12% Bis-Tris Protein Gels (1.0 mm, 10 well) used for purified protein SDS–PAGE analysis and western blot were purchased from Life Technologies and Color Prestained Protein Standard (Broad Range, 11–245 kDa) used as the protein marker was obtained from New England Biolabs.

    Recombinant:

    Article Title: Efficacy of recombinant measles virus expressing highly pathogenic avian influenza virus (HPAIV) antigen against HPAIV infection in monkeys
    Article Snippet: .. The H5 HA gene was ligated to the E. coli protein expression vector pET21b (Novagen), from which the recombinant protein was expressed as a fusion protein with a histidine tag. .. Competent BL21 cells were transformed with the plasmid to express the protein at high levels.

    Article Title: Serological Expression Cloning of Novel Immunoreactive Antigens of Babesia microti
    Article Snippet: Paragraph title: Recombinant protein expression and purification. ... Five clones were chosen for high-level protein expression with the pET-17b vector (Novagen) ( bmn1-2 , -9 , -15 , and -17 and mn-10 [carboxy terminus]).

    Article Title: Point mutations within the streptococcal regulator of virulence (Srv) alter protein-DNA interactions and Srv function
    Article Snippet: .. Recombinant (His6 -tagged) Srv protein was purified from E. coli protein expression strain Origami(DE3) (Novagen). ..

    Article Title: NMR Evidence for Forming Highly Populated Helical Conformations in the Partially Folded hNck2 SH3 Domain
    Article Snippet: To achieve high-level protein expression, the DNA fragment encoding the first hNck2 SH3 domain with Escherichia coli -preferred codons was obtained by a polymerase chain reaction-based de novo gene synthesis approach, as previously described ( , ), and subsequently cloned into pET32a vector (Novagen, Singapore). .. The recombinant SH3 domains were overexpressed in E. coli strain BL21 cells.

    Article Title: Type II platelet-activating factor-acetylhydrolase is essential for epithelial morphogenesis in Caenorhabditis elegans
    Article Snippet: .. Recombinant PAF-1 and PAF-2 proteins that were expressed and purified by an E. coli pET expression system (Novagen) were used as the enzymes. .. C. elegans were lysed by sonication and were centrifuged at 20,000 × g for 20 min.

    Fluorescence:

    Article Title: A robust and tunable mitotic oscillator in artificial cells
    Article Snippet: Paragraph title: Fluorescence-labeled reporters ... GFP-NLS protein was expressed in BL21 (DE3)-T-1 competent cells (Sigma Aldrich, B2935) that were induced by 0.1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside, Sigma Aldrich, I6758) overnight.

    Size-exclusion Chromatography:

    Article Title: Engineering biosynthesis of the anticancer alkaloid noscapine in yeast
    Article Snippet: BL21(DE3) and Ni-NTA agorose resin used for E. coli protein expression and hexa-histidine tagged protein purification were kindly provided by Professor Chaitan Khosla, and T7 Tag Affinity Purification Kit used for T7-tagged protein purification was purchased from EMD Millipore. .. Size-exclusion chromatography was performed on a BioSep-SEC-s4000 column (Phenomenex, kindly provided by Professor Chaitan Khosla), and the column was equilibrated with Gel Filtration Markers Kit for Protein Molecular Weights 12,000–200,000 Da purchased from Sigma.

    Purification:

    Article Title: Serological Expression Cloning of Novel Immunoreactive Antigens of Babesia microti
    Article Snippet: Paragraph title: Recombinant protein expression and purification. ... Five clones were chosen for high-level protein expression with the pET-17b vector (Novagen) ( bmn1-2 , -9 , -15 , and -17 and mn-10 [carboxy terminus]).

    Article Title: Point mutations within the streptococcal regulator of virulence (Srv) alter protein-DNA interactions and Srv function
    Article Snippet: .. Recombinant (His6 -tagged) Srv protein was purified from E. coli protein expression strain Origami(DE3) (Novagen). ..

    Article Title: Molecular Cloning and Biochemical Characterization of a Recombinant Sterol 3-O-Glucosyltransferase from Gymnema sylvestre R.Br. Catalyzing Biosynthesis of Steryl Glucosides
    Article Snippet: .. Heterologous Expression of the Gene in E. coli The full length cDNA was cloned and expressed in E. coli protein expression system, namely, pET-28a as N-terminal fusion protein with a His6 tag to facilitate its purification (Novagen). .. The complete open reading frame (ORF) was PCR amplified with full length primers ( ).

    Article Title: Application of phage display to high throughput antibody generation and characterization
    Article Snippet: All steps in antigen production, including PCR amplification, sequence confirmation, expression and purification, were electronically tracked using a custom designed laboratory information management system (unpublished). .. Our E. coli protein expression system was described previously [ ] and utilizes the T7 RNA polymerase promoter driving cytoplasmic expression in BL21-DE3 cells (Novagen, Nottingham, UK).

    Article Title: NMR Evidence for Forming Highly Populated Helical Conformations in the Partially Folded hNck2 SH3 Domain
    Article Snippet: Paragraph title: Cloning, expression, and purification ... To achieve high-level protein expression, the DNA fragment encoding the first hNck2 SH3 domain with Escherichia coli -preferred codons was obtained by a polymerase chain reaction-based de novo gene synthesis approach, as previously described ( , ), and subsequently cloned into pET32a vector (Novagen, Singapore).

    Article Title: Type II platelet-activating factor-acetylhydrolase is essential for epithelial morphogenesis in Caenorhabditis elegans
    Article Snippet: .. Recombinant PAF-1 and PAF-2 proteins that were expressed and purified by an E. coli pET expression system (Novagen) were used as the enzymes. .. C. elegans were lysed by sonication and were centrifuged at 20,000 × g for 20 min.

    Article Title: Crystal Structure of Botulinum Neurotoxin A2 in Complex with the Human Protein Receptor SV2C Reveals Plasticity in Receptor Binding
    Article Snippet: Cloning, expression and purification of SV2C-L4. .. SV2C-L4 protein was expressed in E. coli BL21 (DE3) T1R (SigmaAldrich B2935, St. Louis, MO, USA), that had been transformed with pRARE2 and made competent, by induction with 0.5 mM IPTG at an OD600 of 3.0 in Terrific Broth media and further growth at 18 °C overnight.

    Article Title: SetRICE391, a negative transcriptional regulator of the integrating conjugative element 391 mutagenic response
    Article Snippet: Paragraph title: 2.5. Expression and purification of native SetRICE391 ... The ICE391 SetR protein was overproduced using the T7 protein expression system (EMD Millipore).

    Article Title: Engineering biosynthesis of the anticancer alkaloid noscapine in yeast
    Article Snippet: BL21(DE3) and Ni-NTA agorose resin used for E. coli protein expression and hexa-histidine tagged protein purification were kindly provided by Professor Chaitan Khosla, and T7 Tag Affinity Purification Kit used for T7-tagged protein purification was purchased from EMD Millipore. .. NuPAGE Novex 4–12% Bis-Tris Protein Gels (1.0 mm, 10 well) used for purified protein SDS–PAGE analysis and western blot were purchased from Life Technologies and Color Prestained Protein Standard (Broad Range, 11–245 kDa) used as the protein marker was obtained from New England Biolabs.

    Protein Purification:

    Article Title: Point mutations within the streptococcal regulator of virulence (Srv) alter protein-DNA interactions and Srv function
    Article Snippet: Paragraph title: Protein purification ... Recombinant (His6 -tagged) Srv protein was purified from E. coli protein expression strain Origami(DE3) (Novagen).

    Article Title: Engineering biosynthesis of the anticancer alkaloid noscapine in yeast
    Article Snippet: .. BL21(DE3) and Ni-NTA agorose resin used for E. coli protein expression and hexa-histidine tagged protein purification were kindly provided by Professor Chaitan Khosla, and T7 Tag Affinity Purification Kit used for T7-tagged protein purification was purchased from EMD Millipore. .. NuPAGE Novex 4–12% Bis-Tris Protein Gels (1.0 mm, 10 well) used for purified protein SDS–PAGE analysis and western blot were purchased from Life Technologies and Color Prestained Protein Standard (Broad Range, 11–245 kDa) used as the protein marker was obtained from New England Biolabs.

    Sequencing:

    Article Title: BcXYG1, a Secreted Xyloglucanase from Botrytis cinerea, Triggers Both Cell Death and Plant Immune Responses 1
    Article Snippet: .. To construct the E. coli protein expression vectors, the sequence encoding mature BcXYG1 protein without the signal peptide was cloned into pET-22b (+) (Novagen) to generate the expression vector pET22b-BcXYG1-6×His. .. For transient expression of proteins in plants using the A. tumefaciens infiltration method, the sequence encoding Arabidopsis PR3 (TAIR identifier AT3G12500) signal peptide was fused upstream of the BcXYG1-HA fusion protein, and the construct was cloned into pCAMBIA3300 (Cambia) between the cauliflower mosaic virus 35S promoter and the NOS terminator.

    Article Title: Application of phage display to high throughput antibody generation and characterization
    Article Snippet: Three design strategies were employed for the inner primers: EC+SIGP-euk, coordinates 1 to minus 5 amino acids upstream of the TM, minus a stop codon for HEK293E expression with carboxy-terminal tags; EC-SIGP-pro, coordinates plus 1 amino acid downstream of the SIGP to minus 5 amino acids upstream of the TM plus a stop codon for E. coli expression with amino-terminal tags; and IC-pro, coordinates plus 5 amino acids downstream of the TM to the end of the coding sequence plus a stop codon for E. coli expression with amino-terminal tags. .. Our E. coli protein expression system was described previously [ ] and utilizes the T7 RNA polymerase promoter driving cytoplasmic expression in BL21-DE3 cells (Novagen, Nottingham, UK).

    Protein Extraction:

    Article Title: Point mutations within the streptococcal regulator of virulence (Srv) alter protein-DNA interactions and Srv function
    Article Snippet: Recombinant (His6 -tagged) Srv protein was purified from E. coli protein expression strain Origami(DE3) (Novagen). .. Cells were lysed in BugBuster Protein Extraction Reagent (Novagen).

    Selection:

    Article Title: Molecular Cloning and Biochemical Characterization of a Recombinant Sterol 3-O-Glucosyltransferase from Gymnema sylvestre R.Br. Catalyzing Biosynthesis of Steryl Glucosides
    Article Snippet: Heterologous Expression of the Gene in E. coli The full length cDNA was cloned and expressed in E. coli protein expression system, namely, pET-28a as N-terminal fusion protein with a His6 tag to facilitate its purification (Novagen). .. Transformants were grown at 37°C in LB liquid medium containing 50 mg/mL−1 chloramphenicol and 50 mg/mL−1 kanamycin as selection antibiotics.

    Article Title: Development and Characterization of a Camelid Single Domain Antibody–Urease Conjugate That Targets Vascular Endothelial Growth Factor Receptor 2
    Article Snippet: Expression of V21H1 and V21H4 Both antibodies were expressed in the E. coli BL21 (DE3) pT7 system with kanamycin as the selection antibiotic. .. Transformation of BL21 (DE3) competent E. coli cells (Sigma, B2935-10 × 50 µL) was according to the manufacturer’s instructions.

    Article Title: Engineering biosynthesis of the anticancer alkaloid noscapine in yeast
    Article Snippet: 200 mg l−1 G418 sulfate (Calbiochem) or 200 mg l−1 Hygromycin B (Life Technologies) were used in YPD medium for selection. .. BL21(DE3) and Ni-NTA agorose resin used for E. coli protein expression and hexa-histidine tagged protein purification were kindly provided by Professor Chaitan Khosla, and T7 Tag Affinity Purification Kit used for T7-tagged protein purification was purchased from EMD Millipore.

    Lysis:

    Article Title: Efficacy of recombinant measles virus expressing highly pathogenic avian influenza virus (HPAIV) antigen against HPAIV infection in monkeys
    Article Snippet: The H5 HA gene was ligated to the E. coli protein expression vector pET21b (Novagen), from which the recombinant protein was expressed as a fusion protein with a histidine tag. .. After incubation for 2 h, the bacteria were collected, lysed with lysis buffer (50 mM Tris-HCl, 500 mM NaCl,20 mM Imidazol pH7.9), and freezed at −80 °C.

    Article Title: Crystal Structure of Botulinum Neurotoxin A2 in Complex with the Human Protein Receptor SV2C Reveals Plasticity in Receptor Binding
    Article Snippet: SV2C-L4 protein was expressed in E. coli BL21 (DE3) T1R (SigmaAldrich B2935, St. Louis, MO, USA), that had been transformed with pRARE2 and made competent, by induction with 0.5 mM IPTG at an OD600 of 3.0 in Terrific Broth media and further growth at 18 °C overnight. .. Bacteria were harvested by centrifugation and lysed in Lysis buffer (100 mM HEPES, pH 8.0, 500 mM NaCl, 10% (v /v ) glycerol, 10 mM Imidazole, 0.5 mM TCEP) after treatment with benzonase nuclease (Sigma) and protease inhibitor cocktail (Roche) using sonication followed by centrifugation.

    SDS Page:

    Article Title: Crystal Structure of Botulinum Neurotoxin A2 in Complex with the Human Protein Receptor SV2C Reveals Plasticity in Receptor Binding
    Article Snippet: Fractions containing HcA2 were pooled and purity was analyzed on SDS-PAGE. .. SV2C-L4 protein was expressed in E. coli BL21 (DE3) T1R (SigmaAldrich B2935, St. Louis, MO, USA), that had been transformed with pRARE2 and made competent, by induction with 0.5 mM IPTG at an OD600 of 3.0 in Terrific Broth media and further growth at 18 °C overnight.

    Article Title: Engineering biosynthesis of the anticancer alkaloid noscapine in yeast
    Article Snippet: BL21(DE3) and Ni-NTA agorose resin used for E. coli protein expression and hexa-histidine tagged protein purification were kindly provided by Professor Chaitan Khosla, and T7 Tag Affinity Purification Kit used for T7-tagged protein purification was purchased from EMD Millipore. .. NuPAGE Novex 4–12% Bis-Tris Protein Gels (1.0 mm, 10 well) used for purified protein SDS–PAGE analysis and western blot were purchased from Life Technologies and Color Prestained Protein Standard (Broad Range, 11–245 kDa) used as the protein marker was obtained from New England Biolabs.

    Plasmid Preparation:

    Article Title: Efficacy of recombinant measles virus expressing highly pathogenic avian influenza virus (HPAIV) antigen against HPAIV infection in monkeys
    Article Snippet: .. The H5 HA gene was ligated to the E. coli protein expression vector pET21b (Novagen), from which the recombinant protein was expressed as a fusion protein with a histidine tag. .. Competent BL21 cells were transformed with the plasmid to express the protein at high levels.

    Article Title: Serological Expression Cloning of Novel Immunoreactive Antigens of Babesia microti
    Article Snippet: .. Five clones were chosen for high-level protein expression with the pET-17b vector (Novagen) ( bmn1-2 , -9 , -15 , and -17 and mn-10 [carboxy terminus]). .. These expression constructs were engineered to include an N-terminal six-histidine tag for ease of purification with an Ni-nitrilotriacetic acid agarose column.

    Article Title: Characterization of the Raf Kinase Inhibitory Protein (RKIP) Binding Pocket: NMR-Based Screening Identifies Small-Molecule Ligands
    Article Snippet: .. Protein expression BL21(DE3) cells (Novagen) harboring a pMCSG7-derived expression vector for rat RKIP were grown at 37°C in the M9 media with15 N-NH3 Cl as the sole nitrogen source and supplemented with ampicillin (100 µg/ml). .. The expression construct consisted of a His6 tag fused in frame with a TEV protease cleavage signal and the entire open reading frame (residues 1-187) of wild-type rat RKIP (rRKIP).

    Article Title: Point mutations within the streptococcal regulator of virulence (Srv) alter protein-DNA interactions and Srv function
    Article Snippet: For expression and purification of recombinant protein, srv was cloned into expression vector pET32A (renamed pSDR srv ) and transformed into E. coli NovaBlue (Novagen). .. Recombinant (His6 -tagged) Srv protein was purified from E. coli protein expression strain Origami(DE3) (Novagen).

    Article Title: BcXYG1, a Secreted Xyloglucanase from Botrytis cinerea, Triggers Both Cell Death and Plant Immune Responses 1
    Article Snippet: .. To construct the E. coli protein expression vectors, the sequence encoding mature BcXYG1 protein without the signal peptide was cloned into pET-22b (+) (Novagen) to generate the expression vector pET22b-BcXYG1-6×His. .. For transient expression of proteins in plants using the A. tumefaciens infiltration method, the sequence encoding Arabidopsis PR3 (TAIR identifier AT3G12500) signal peptide was fused upstream of the BcXYG1-HA fusion protein, and the construct was cloned into pCAMBIA3300 (Cambia) between the cauliflower mosaic virus 35S promoter and the NOS terminator.

    Article Title: Molecular Cloning and Biochemical Characterization of a Recombinant Sterol 3-O-Glucosyltransferase from Gymnema sylvestre R.Br. Catalyzing Biosynthesis of Steryl Glucosides
    Article Snippet: Heterologous Expression of the Gene in E. coli The full length cDNA was cloned and expressed in E. coli protein expression system, namely, pET-28a as N-terminal fusion protein with a His6 tag to facilitate its purification (Novagen). .. The 2.1 kb amplified ORF was cloned at Nde I and Bam H1 restriction sites in pET-28a expression vector and the resulting construct was used to transform E. coli BL21 (DE3) strain (Novagen).

    Article Title: NMR Evidence for Forming Highly Populated Helical Conformations in the Partially Folded hNck2 SH3 Domain
    Article Snippet: .. To achieve high-level protein expression, the DNA fragment encoding the first hNck2 SH3 domain with Escherichia coli -preferred codons was obtained by a polymerase chain reaction-based de novo gene synthesis approach, as previously described ( , ), and subsequently cloned into pET32a vector (Novagen, Singapore). .. Similarly, the DNA fragment for the SH3 domain carrying four-residue Ala-mutations over residues Lue25 -Trp26 -Leu27 -Leu28 (designated 4AlaMut) was synthesized by the same approach, except that the two middle primers were replaced by those with the mutations.

    Article Title: Crystal Structure of Botulinum Neurotoxin A2 in Complex with the Human Protein Receptor SV2C Reveals Plasticity in Receptor Binding
    Article Snippet: The interacting part of the fourth luminal domain of synaptic vesicle glycoprotein 2C (SV2C-L4, residues 474-567 Uniprot ID Q496J9) was amplified from the pGEX4-T1 SV2C-L4 plasmid used for binding studies (primers 5′-TACTTCCAATCCATGGAGAGAGATAAATATGCAAATTTC-3′ and 5′-TATCCACCTTTACTGTCACGTCTTGTTGTGAAAAAACGAG-3′) and cloned into a pNIC28-Bsa4 (N-terminal His6-tag with TEV site, KanR) vector using LIC cloning. .. SV2C-L4 protein was expressed in E. coli BL21 (DE3) T1R (SigmaAldrich B2935, St. Louis, MO, USA), that had been transformed with pRARE2 and made competent, by induction with 0.5 mM IPTG at an OD600 of 3.0 in Terrific Broth media and further growth at 18 °C overnight.

    Article Title: Engineering biosynthesis of the anticancer alkaloid noscapine in yeast
    Article Snippet: Yeast strains were cultured at 25 or 30 °C in complex yeast extract peptone dextrose (YPD, all components from BD Diagnostics) medium or SDM containing yeast nitrogen base (YNB) without Amino Acids (BD Diagnostics), ammonium sulfate (Fisher Scientific), 2% dextrose and the appropriate dropout solution for plasmid maintenance. .. BL21(DE3) and Ni-NTA agorose resin used for E. coli protein expression and hexa-histidine tagged protein purification were kindly provided by Professor Chaitan Khosla, and T7 Tag Affinity Purification Kit used for T7-tagged protein purification was purchased from EMD Millipore.

    Binding Assay:

    Article Title: Crystal Structure of Botulinum Neurotoxin A2 in Complex with the Human Protein Receptor SV2C Reveals Plasticity in Receptor Binding
    Article Snippet: The interacting part of the fourth luminal domain of synaptic vesicle glycoprotein 2C (SV2C-L4, residues 474-567 Uniprot ID Q496J9) was amplified from the pGEX4-T1 SV2C-L4 plasmid used for binding studies (primers 5′-TACTTCCAATCCATGGAGAGAGATAAATATGCAAATTTC-3′ and 5′-TATCCACCTTTACTGTCACGTCTTGTTGTGAAAAAACGAG-3′) and cloned into a pNIC28-Bsa4 (N-terminal His6-tag with TEV site, KanR) vector using LIC cloning. .. SV2C-L4 protein was expressed in E. coli BL21 (DE3) T1R (SigmaAldrich B2935, St. Louis, MO, USA), that had been transformed with pRARE2 and made competent, by induction with 0.5 mM IPTG at an OD600 of 3.0 in Terrific Broth media and further growth at 18 °C overnight.

    Produced:

    Article Title: Efficacy of recombinant measles virus expressing highly pathogenic avian influenza virus (HPAIV) antigen against HPAIV infection in monkeys
    Article Snippet: To measure the anti-H5 HA antibodies, recombinant H5 HA protein was produced. .. The H5 HA gene was ligated to the E. coli protein expression vector pET21b (Novagen), from which the recombinant protein was expressed as a fusion protein with a histidine tag.

    Concentration Assay:

    Article Title: NMR Evidence for Forming Highly Populated Helical Conformations in the Partially Folded hNck2 SH3 Domain
    Article Snippet: To achieve high-level protein expression, the DNA fragment encoding the first hNck2 SH3 domain with Escherichia coli -preferred codons was obtained by a polymerase chain reaction-based de novo gene synthesis approach, as previously described ( , ), and subsequently cloned into pET32a vector (Novagen, Singapore). .. Briefly, the cells were cultured at 37°C to reach an optical density at 600 nm of 0.4, and then isopropyl β -D-1-thiogalactopyranoside was added to a final concentration of 0.4 mM to induce the expression of the recombinant protein overnight at 20°C.

    Article Title: Crystal Structure of Botulinum Neurotoxin A2 in Complex with the Human Protein Receptor SV2C Reveals Plasticity in Receptor Binding
    Article Snippet: Concentration was determined using a calculated extinction coefficient of 88,950 M−1 s−1 . .. SV2C-L4 protein was expressed in E. coli BL21 (DE3) T1R (SigmaAldrich B2935, St. Louis, MO, USA), that had been transformed with pRARE2 and made competent, by induction with 0.5 mM IPTG at an OD600 of 3.0 in Terrific Broth media and further growth at 18 °C overnight.

    BAC Assay:

    Article Title: A Non-Motor Microtubule Binding Site Is Essential for the High Processivity and Mitotic Function of Kinesin-8 Kif18A
    Article Snippet: Protein Expression The BAC-TO-BAC expression system (Invitrogen, Paisley, UK) was used to express 6xHis-Kif18A (aa 1-898)-GFP-Avi and 6xHis-Kif18A (aa 1-777)-GFP-Avi in insect cells SF+ (obtained from protein science). .. Kif18A776-898 –GFP and Kif18A898 –GFP were expressed in BL21(DE3)-T1R competent Escherichia coli (Sigma B2935) transformed with pRARE (Novagen, # 70954).

    Marker:

    Article Title: Engineering biosynthesis of the anticancer alkaloid noscapine in yeast
    Article Snippet: BL21(DE3) and Ni-NTA agorose resin used for E. coli protein expression and hexa-histidine tagged protein purification were kindly provided by Professor Chaitan Khosla, and T7 Tag Affinity Purification Kit used for T7-tagged protein purification was purchased from EMD Millipore. .. NuPAGE Novex 4–12% Bis-Tris Protein Gels (1.0 mm, 10 well) used for purified protein SDS–PAGE analysis and western blot were purchased from Life Technologies and Color Prestained Protein Standard (Broad Range, 11–245 kDa) used as the protein marker was obtained from New England Biolabs.

    Staining:

    Article Title: A robust and tunable mitotic oscillator in artificial cells
    Article Snippet: GFP-NLS protein was expressed in BL21 (DE3)-T-1 competent cells (Sigma Aldrich, B2935) that were induced by 0.1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside, Sigma Aldrich, I6758) overnight. .. 200 mg/ml Hoechst 33342 (Sigma Aldrich, B2261) was added to stain chromosomes.

    Positron Emission Tomography:

    Article Title: Serological Expression Cloning of Novel Immunoreactive Antigens of Babesia microti
    Article Snippet: .. Five clones were chosen for high-level protein expression with the pET-17b vector (Novagen) ( bmn1-2 , -9 , -15 , and -17 and mn-10 [carboxy terminus]). .. These expression constructs were engineered to include an N-terminal six-histidine tag for ease of purification with an Ni-nitrilotriacetic acid agarose column.

    Article Title: BcXYG1, a Secreted Xyloglucanase from Botrytis cinerea, Triggers Both Cell Death and Plant Immune Responses 1
    Article Snippet: .. To construct the E. coli protein expression vectors, the sequence encoding mature BcXYG1 protein without the signal peptide was cloned into pET-22b (+) (Novagen) to generate the expression vector pET22b-BcXYG1-6×His. .. For transient expression of proteins in plants using the A. tumefaciens infiltration method, the sequence encoding Arabidopsis PR3 (TAIR identifier AT3G12500) signal peptide was fused upstream of the BcXYG1-HA fusion protein, and the construct was cloned into pCAMBIA3300 (Cambia) between the cauliflower mosaic virus 35S promoter and the NOS terminator.

    Article Title: Molecular Cloning and Biochemical Characterization of a Recombinant Sterol 3-O-Glucosyltransferase from Gymnema sylvestre R.Br. Catalyzing Biosynthesis of Steryl Glucosides
    Article Snippet: .. Heterologous Expression of the Gene in E. coli The full length cDNA was cloned and expressed in E. coli protein expression system, namely, pET-28a as N-terminal fusion protein with a His6 tag to facilitate its purification (Novagen). .. The complete open reading frame (ORF) was PCR amplified with full length primers ( ).

    Article Title: Type II platelet-activating factor-acetylhydrolase is essential for epithelial morphogenesis in Caenorhabditis elegans
    Article Snippet: .. Recombinant PAF-1 and PAF-2 proteins that were expressed and purified by an E. coli pET expression system (Novagen) were used as the enzymes. .. C. elegans were lysed by sonication and were centrifuged at 20,000 × g for 20 min.

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    Millipore pet 9c vector
    Pet 9c Vector, supplied by Millipore, used in various techniques. Bioz Stars score: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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