vector pet22b  (Millipore)


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    Structured Review

    Millipore vector pet22b
    Vector Pet22b, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vector pet22b/product/Millipore
    Average 99 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    vector pet22b - by Bioz Stars, 2020-01
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Crystallization and preliminary X-ray crystallographic analysis of Aquifex aeolicus SelA, a bacterial selenocysteine synthase
    Article Snippet: .. The mutant SelA gene corresponding to SelA-ΔN (amino-acid residues 62–452) was cloned into the Nde I and Xho I restriction sites of the vector pET22b (Novagen). .. The wild-type SelA encoded in the vector does not have any artificial sequences at its N- or C-termini, while SelA-ΔN only has an artificially introduced initiating Met residue followed by residues 62–452.

    Article Title: Multi-Compartment and Multi-Host Vector Suite for Recombinant Protein Expression and Purification
    Article Snippet: .. For the generation of the pT7pelB vector, the pelB sequence was amplified from the vector pET22b (Novagen) with the primers pelBT7For and pelBRev ( Table ) and cloned into the vector pT7GFP by RF cloning to generate the construct pT7pelB. .. This construct was then used as template with the primers T7 promoter and pelBinsRev and the generated megaprimer used for the insertion of the pelB signal sequence into vectors containing the T7 promoter and the fusion protein GST (pT7GST) or MBP (pT7MBP).

    Article Title: Staphylococcus aureus FepA and FepB Proteins Drive Heme Iron Utilization in Escherichia coli
    Article Snippet: .. The PCR product was cloned at the Nde I and Xho I sites of vector pET22b+ (Novagen) containing a T7 promoter and a His-tag. .. Resulting plasmid pET22b-fepB-6His was used for transforming E. coli strain BLI5 for expression.

    Article Title: A New Group of Modular Xylanases in Glycoside Hydrolase Family 8 from Marine Bacteria
    Article Snippet: Strain DH5α was used for gene cloning, and strain BL21(DE3) was used for gene expression. .. The vector pET22b (Novagen, USA) was used for the construction of expression plasmids.

    Article Title: Crystal Structure of the Human Short Coiled Coil Protein and Insights into SCOC-FEZ1 Complex Formation
    Article Snippet: .. Purification of SCOC-FEZ1 complexes Human FEZ1(227-290) was cloned with NdeI and XhoI restriction sites into vector pET22b (Novagen) using full length FEZ1 as template for PCR [ ]. .. Both FEZ1(227-290) pET22b and SCOC(78-159) pET28a were co-transformed into BL21 (DE3) by electroporation.

    Article Title: Protein sequences conserved in prokaryotic aminoacyl-tRNA synthetases are important for the activity of the processivity factor of human mitochondrial DNA polymerase
    Article Snippet: .. This product was cloned as an Nde I– Not I fragment in the vector pET22b(+) (Novagen) to generate clone pJAC39. ..

    Article Title: Expression and specificity of a chitin deacetylase from the nematophagous fungus Pochonia chlamydosporia potentially involved in pathogenicity
    Article Snippet: .. Briefly, the vector pET22b(+) (Novagen) was used as a template to include a StrepII encoding sequence downstream of the multiple cloning site using PCR via a 50-mer phosphorylated primer pair. .. Electrocompetent E. coli DH5α cells were transformed with the pET22b(+)StrepIIC plasmid containing the Pc_2566 synthetic gene.

    Amplification:

    Article Title: Multi-Compartment and Multi-Host Vector Suite for Recombinant Protein Expression and Purification
    Article Snippet: .. For the generation of the pT7pelB vector, the pelB sequence was amplified from the vector pET22b (Novagen) with the primers pelBT7For and pelBRev ( Table ) and cloned into the vector pT7GFP by RF cloning to generate the construct pT7pelB. .. This construct was then used as template with the primers T7 promoter and pelBinsRev and the generated megaprimer used for the insertion of the pelB signal sequence into vectors containing the T7 promoter and the fusion protein GST (pT7GST) or MBP (pT7MBP).

    Article Title: Immunization of N terminus of enterovirus 71 VP4 elicits cross-protective antibody responses
    Article Snippet: To construct the plasmid expressing the fusion protein, DNA fragment encoding HBc-N149-VP4N20 was synthesized and amplified using primers P1u (5'- CCGCTCGAGCACCACGGTGGTT-3') and P1d (5'- GGAATTCCATATGGATATTGATCCGTATAAAG-3'). .. The PCR products were double-digested by XhoI and NdeI and subsequently inserted into the vector pET22b(+) (Novagen, USA).

    Article Title: Staphylococcus aureus FepA and FepB Proteins Drive Heme Iron Utilization in Escherichia coli
    Article Snippet: The fepB gene without its signal sequence was amplified by PCR with genomic DNA from S. aureus NCTC 8325 using primer pairs fepB-PS-Nde I/Xho I-fepB. .. The PCR product was cloned at the Nde I and Xho I sites of vector pET22b+ (Novagen) containing a T7 promoter and a His-tag.

    Article Title: Cystathionine ?-lyase: clinical, metabolic, genetic, and structural studies
    Article Snippet: The construction of the vector pET22b+ (Novagen) expressing CTH in E.coli was described previously by Steegborn et al [ ]. .. The CTH sequence was amplified from the template pET22b-CGL using these primers.

    Filtration:

    Article Title: Production of a mono-biotinylated EGFR nanobody in the E. coli periplasm using the pET22b vector
    Article Snippet: The EL2BH synthetic DNA sequence was ordered from GenScript and subcloned into vector pET22b (EMDMillipore) between Eco RI and Hin dIII sites. .. The eluate was concentrated by ultrafiltration (Amicon, MWCO 10 kDa), and free biotin was removed by gel filtration (Zeba spin columns, MWCO 7 kDa, ThermoFisher).

    Synthesized:

    Article Title: Comparing substrate specificity of two UDP-sugar Pyrophosphorylases and Efficient One-pot Enzymatic Synthesis of UDP-GlcA and UDP-GalA
    Article Snippet: Codon optimized gene of AtGlcAK, AtUSP were synthesized by Genscript (Piscataway, NJ). .. E. coli DH5α and BL21 (DE3), as well as vector pET22b were purchased from EMD Millipore (Bilerica, MA).

    Article Title: Immunization of N terminus of enterovirus 71 VP4 elicits cross-protective antibody responses
    Article Snippet: To construct the plasmid expressing the fusion protein, DNA fragment encoding HBc-N149-VP4N20 was synthesized and amplified using primers P1u (5'- CCGCTCGAGCACCACGGTGGTT-3') and P1d (5'- GGAATTCCATATGGATATTGATCCGTATAAAG-3'). .. The PCR products were double-digested by XhoI and NdeI and subsequently inserted into the vector pET22b(+) (Novagen, USA).

    Article Title: Cystathionine ?-lyase: clinical, metabolic, genetic, and structural studies
    Article Snippet: The construction of the vector pET22b+ (Novagen) expressing CTH in E.coli was described previously by Steegborn et al [ ]. .. Two complementary oligonucleotides, #621 and #622, with extensions coding for the restriction enzymes Apa I and Xho I, respectively, were synthesized ( ).

    Construct:

    Article Title: Multi-Compartment and Multi-Host Vector Suite for Recombinant Protein Expression and Purification
    Article Snippet: .. For the generation of the pT7pelB vector, the pelB sequence was amplified from the vector pET22b (Novagen) with the primers pelBT7For and pelBRev ( Table ) and cloned into the vector pT7GFP by RF cloning to generate the construct pT7pelB. .. This construct was then used as template with the primers T7 promoter and pelBinsRev and the generated megaprimer used for the insertion of the pelB signal sequence into vectors containing the T7 promoter and the fusion protein GST (pT7GST) or MBP (pT7MBP).

    Article Title: Immunization of N terminus of enterovirus 71 VP4 elicits cross-protective antibody responses
    Article Snippet: To construct the plasmid expressing the fusion protein, DNA fragment encoding HBc-N149-VP4N20 was synthesized and amplified using primers P1u (5'- CCGCTCGAGCACCACGGTGGTT-3') and P1d (5'- GGAATTCCATATGGATATTGATCCGTATAAAG-3'). .. The PCR products were double-digested by XhoI and NdeI and subsequently inserted into the vector pET22b(+) (Novagen, USA).

    Article Title: Production of a mono-biotinylated EGFR nanobody in the E. coli periplasm using the pET22b vector
    Article Snippet: The mono-biotinylated 7D12 EGFR nanobody construct will be referred to as EL2BH. .. The EL2BH synthetic DNA sequence was ordered from GenScript and subcloned into vector pET22b (EMDMillipore) between Eco RI and Hin dIII sites.

    Article Title: Staphylococcus aureus FepA and FepB Proteins Drive Heme Iron Utilization in Escherichia coli
    Article Snippet: The PCR product was cloned at the Nde I and Xho I sites of vector pET22b+ (Novagen) containing a T7 promoter and a His-tag. .. The plasmid carrying efeO was constructed by amplification of MG1655 genomic DNA using complementary oligonucleotides.

    Article Title: Cystathionine ?-lyase: clinical, metabolic, genetic, and structural studies
    Article Snippet: The construction of the vector pET22b+ (Novagen) expressing CTH in E.coli was described previously by Steegborn et al [ ]. .. The vector pGEX6-P-1(GE Healthcare), which is used for expression of glutathione- S -transferase fusion proteins, was employed to prepare the GST-CTH fusion construct.

    Article Title: The structure and dynamic properties of the complete histidine phosphotransfer domain of the chemotaxis specific histidine autokinase CheA from Thermotoga maritima
    Article Snippet: .. Protein expression and purification Full length His6-tagged P1 construct (residues 1–133) of T. maritima (or TmP1 ) was subcloned in the vector pET22b(+) (Novagen). .. Plasmids were transformed into E. coli strain BL21(DE3) (Novagen).

    Article Title: Characterization of a Soluble Ligand Binding Domain of the NMDA Receptor Regulatory Subunit NR3A
    Article Snippet: .. The resulting NR3A S1S2 construct was then inserted into a modified version of the vector pET22b(+) (Novagen, Madison, WI), with an N-terminal His8 SSGLVPRGS affinity tag and thrombin cleavage site at its N terminus. ..

    Incubation:

    Article Title: Expression and specificity of a chitin deacetylase from the nematophagous fungus Pochonia chlamydosporia potentially involved in pathogenicity
    Article Snippet: Briefly, the vector pET22b(+) (Novagen) was used as a template to include a StrepII encoding sequence downstream of the multiple cloning site using PCR via a 50-mer phosphorylated primer pair. .. Positive transformants were selected using LB medium supplemented with 0.1 mg mL−1 ampicillin and cells were grown in 10 mL of the same media and incubated overnight at 37 °C (200 rpm).

    Expressing:

    Article Title: Comparing substrate specificity of two UDP-sugar Pyrophosphorylases and Efficient One-pot Enzymatic Synthesis of UDP-GlcA and UDP-GalA
    Article Snippet: E. coli DH5α and BL21 (DE3), as well as vector pET22b were purchased from EMD Millipore (Bilerica, MA). .. BiGalK expression E. coli strain was kept by our group.

    Article Title: Immunization of N terminus of enterovirus 71 VP4 elicits cross-protective antibody responses
    Article Snippet: To construct the plasmid expressing the fusion protein, DNA fragment encoding HBc-N149-VP4N20 was synthesized and amplified using primers P1u (5'- CCGCTCGAGCACCACGGTGGTT-3') and P1d (5'- GGAATTCCATATGGATATTGATCCGTATAAAG-3'). .. The PCR products were double-digested by XhoI and NdeI and subsequently inserted into the vector pET22b(+) (Novagen, USA).

    Article Title: Staphylococcus aureus FepA and FepB Proteins Drive Heme Iron Utilization in Escherichia coli
    Article Snippet: The PCR product was cloned at the Nde I and Xho I sites of vector pET22b+ (Novagen) containing a T7 promoter and a His-tag. .. Resulting plasmid pET22b-fepB-6His was used for transforming E. coli strain BLI5 for expression.

    Article Title: A New Group of Modular Xylanases in Glycoside Hydrolase Family 8 from Marine Bacteria
    Article Snippet: .. The vector pET22b (Novagen, USA) was used for the construction of expression plasmids. .. Insoluble beechwood xylan was purchased from Sigma (USA), and soluble wheat arabinoxylan (low viscosity) was obtained from Megazyme (Ireland).

    Article Title: Cystathionine ?-lyase: clinical, metabolic, genetic, and structural studies
    Article Snippet: .. The construction of the vector pET22b+ (Novagen) expressing CTH in E.coli was described previously by Steegborn et al [ ]. .. The vector pGEX6-P-1(GE Healthcare), which is used for expression of glutathione- S -transferase fusion proteins, was employed to prepare the GST-CTH fusion construct.

    Article Title: Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma gondii
    Article Snippet: The expression plasmid pROP18KD3 (beginning at Ser181 numbered from second ATG of ) was used to purify wild-type ROP18 for kinase assays and as backbone for making point mutants. .. The ROP18 gene was inserted into vector pET22b (Novagen) Nde I site (5′ end) and Hin dIII (3′end) resulting in the addition of seven extra amino acids (KLAAALE) preceding the C-terminal His6 tag.

    Article Title: Protein sequences conserved in prokaryotic aminoacyl-tRNA synthetases are important for the activity of the processivity factor of human mitochondrial DNA polymerase
    Article Snippet: Paragraph title: Cloning and expression of human pol γB ... This product was cloned as an Nde I– Not I fragment in the vector pET22b(+) (Novagen) to generate clone pJAC39.

    Article Title: The structure and dynamic properties of the complete histidine phosphotransfer domain of the chemotaxis specific histidine autokinase CheA from Thermotoga maritima
    Article Snippet: .. Protein expression and purification Full length His6-tagged P1 construct (residues 1–133) of T. maritima (or TmP1 ) was subcloned in the vector pET22b(+) (Novagen). .. Plasmids were transformed into E. coli strain BL21(DE3) (Novagen).

    Article Title: Expression and specificity of a chitin deacetylase from the nematophagous fungus Pochonia chlamydosporia potentially involved in pathogenicity
    Article Snippet: Paragraph title: Construction of a Pc CDA expression plasmid ... Briefly, the vector pET22b(+) (Novagen) was used as a template to include a StrepII encoding sequence downstream of the multiple cloning site using PCR via a 50-mer phosphorylated primer pair.

    Article Title: Characterization of a Soluble Ligand Binding Domain of the NMDA Receptor Regulatory Subunit NR3A
    Article Snippet: Paragraph title: Protein expression and purification. ... The resulting NR3A S1S2 construct was then inserted into a modified version of the vector pET22b(+) (Novagen, Madison, WI), with an N-terminal His8 SSGLVPRGS affinity tag and thrombin cleavage site at its N terminus.

    Modification:

    Article Title: Characterization of a Soluble Ligand Binding Domain of the NMDA Receptor Regulatory Subunit NR3A
    Article Snippet: .. The resulting NR3A S1S2 construct was then inserted into a modified version of the vector pET22b(+) (Novagen, Madison, WI), with an N-terminal His8 SSGLVPRGS affinity tag and thrombin cleavage site at its N terminus. ..

    Transformation Assay:

    Article Title: Production of a mono-biotinylated EGFR nanobody in the E. coli periplasm using the pET22b vector
    Article Snippet: The EL2BH synthetic DNA sequence was ordered from GenScript and subcloned into vector pET22b (EMDMillipore) between Eco RI and Hin dIII sites. .. The EL2BH nanobody was isolated from the periplasmic space of E. coli BL21-DE3 (EMDMillipore) that we previously transformed with plasmid pBirACm (Avidity) encoding an IPTG inducible biotin ligase.

    Article Title: Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma gondii
    Article Snippet: The ROP18 gene was inserted into vector pET22b (Novagen) Nde I site (5′ end) and Hin dIII (3′end) resulting in the addition of seven extra amino acids (KLAAALE) preceding the C-terminal His6 tag. .. Plasmids were transformed into BL21-CodonPlus(DE3)-RP cells (Stratagene) and expression was induced by culture in 1 mM IGTP for 3–4 h. Bacterial cell pellets were lysed in CelLytic B cell lysis reagent (Sigma-Aldrich, St Louis, MO).

    Article Title: The structure and dynamic properties of the complete histidine phosphotransfer domain of the chemotaxis specific histidine autokinase CheA from Thermotoga maritima
    Article Snippet: Protein expression and purification Full length His6-tagged P1 construct (residues 1–133) of T. maritima (or TmP1 ) was subcloned in the vector pET22b(+) (Novagen). .. Plasmids were transformed into E. coli strain BL21(DE3) (Novagen).

    Article Title: Expression and specificity of a chitin deacetylase from the nematophagous fungus Pochonia chlamydosporia potentially involved in pathogenicity
    Article Snippet: Briefly, the vector pET22b(+) (Novagen) was used as a template to include a StrepII encoding sequence downstream of the multiple cloning site using PCR via a 50-mer phosphorylated primer pair. .. Electrocompetent E. coli DH5α cells were transformed with the pET22b(+)StrepIIC plasmid containing the Pc_2566 synthetic gene.

    Over Expression:

    Article Title: Crystallization and preliminary X-ray crystallographic analysis of Aquifex aeolicus SelA, a bacterial selenocysteine synthase
    Article Snippet: Paragraph title: 2.1. Overexpression and purification of SelA ... The mutant SelA gene corresponding to SelA-ΔN (amino-acid residues 62–452) was cloned into the Nde I and Xho I restriction sites of the vector pET22b (Novagen).

    Derivative Assay:

    Article Title: Multi-Compartment and Multi-Host Vector Suite for Recombinant Protein Expression and Purification
    Article Snippet: For the generation of the vector pT7GST, the GST gene derived from Schistosoma japonicum was amplified from the vector pGEX-4T-1 (GE Healthcare) with the primers T7GSTFor and GSTRev ( Table ) and cloned into pT7-Trx-GFP substituting the Trx moiety with GST gene. .. For the generation of the pT7pelB vector, the pelB sequence was amplified from the vector pET22b (Novagen) with the primers pelBT7For and pelBRev ( Table ) and cloned into the vector pT7GFP by RF cloning to generate the construct pT7pelB.

    Electroporation:

    Article Title: Crystal Structure of the Human Short Coiled Coil Protein and Insights into SCOC-FEZ1 Complex Formation
    Article Snippet: Purification of SCOC-FEZ1 complexes Human FEZ1(227-290) was cloned with NdeI and XhoI restriction sites into vector pET22b (Novagen) using full length FEZ1 as template for PCR [ ]. .. Both FEZ1(227-290) pET22b and SCOC(78-159) pET28a were co-transformed into BL21 (DE3) by electroporation.

    Ligand Binding Assay:

    Article Title: Characterization of a Soluble Ligand Binding Domain of the NMDA Receptor Regulatory Subunit NR3A
    Article Snippet: Based on amino acid sequence alignments with the NR1 and NR2A ligand binding domains, the crystal structures of which have been solved ( ; ), two peptides, N511-R660 and E776-K915, were isolated by PCR from the cDNA for full-length NR3A and joined by a GT dipeptide linker. .. The resulting NR3A S1S2 construct was then inserted into a modified version of the vector pET22b(+) (Novagen, Madison, WI), with an N-terminal His8 SSGLVPRGS affinity tag and thrombin cleavage site at its N terminus.

    Generated:

    Article Title: Multi-Compartment and Multi-Host Vector Suite for Recombinant Protein Expression and Purification
    Article Snippet: For the generation of the pT7pelB vector, the pelB sequence was amplified from the vector pET22b (Novagen) with the primers pelBT7For and pelBRev ( Table ) and cloned into the vector pT7GFP by RF cloning to generate the construct pT7pelB. .. This construct was then used as template with the primers T7 promoter and pelBinsRev and the generated megaprimer used for the insertion of the pelB signal sequence into vectors containing the T7 promoter and the fusion protein GST (pT7GST) or MBP (pT7MBP).

    DNA Sequencing:

    Article Title: Expression and specificity of a chitin deacetylase from the nematophagous fungus Pochonia chlamydosporia potentially involved in pathogenicity
    Article Snippet: Briefly, the vector pET22b(+) (Novagen) was used as a template to include a StrepII encoding sequence downstream of the multiple cloning site using PCR via a 50-mer phosphorylated primer pair. .. The plasmid was then extracted using QIA Prep Spin MiniPrep Kit (Qiagen), and Pc_2566 gene sequence was verified by DNA sequencing (Fig. ).

    Polymerase Chain Reaction:

    Article Title: Multi-Compartment and Multi-Host Vector Suite for Recombinant Protein Expression and Purification
    Article Snippet: PCR reaction was carried out as follows, 95°C for 3 min, 25 cycles of 95°C for 30 s, 67°C for 30 s, and 72°C for 2 min followed by a final extension step at 72°C for 5 min. .. For the generation of the pT7pelB vector, the pelB sequence was amplified from the vector pET22b (Novagen) with the primers pelBT7For and pelBRev ( Table ) and cloned into the vector pT7GFP by RF cloning to generate the construct pT7pelB.

    Article Title: Immunization of N terminus of enterovirus 71 VP4 elicits cross-protective antibody responses
    Article Snippet: .. The PCR products were double-digested by XhoI and NdeI and subsequently inserted into the vector pET22b(+) (Novagen, USA). .. DNA fragment encoding HBc-N149 was amplified by using the primers P1u, P2d (5′-TGGGCAGCAATCTGGAAGATCCGGCGAGCCGCGAACTG-3′), P2u (5′- ACCAGTTCGCGGCTCGCCGGATCTTCCAGATTGCTGCCCA-3′) and P1d by using HBc-N149-VP4N20-encoding gene as a template and further inserted into the vector pET22b (+).

    Article Title: Staphylococcus aureus FepA and FepB Proteins Drive Heme Iron Utilization in Escherichia coli
    Article Snippet: .. The PCR product was cloned at the Nde I and Xho I sites of vector pET22b+ (Novagen) containing a T7 promoter and a His-tag. .. Resulting plasmid pET22b-fepB-6His was used for transforming E. coli strain BLI5 for expression.

    Article Title: Crystal Structure of the Human Short Coiled Coil Protein and Insights into SCOC-FEZ1 Complex Formation
    Article Snippet: .. Purification of SCOC-FEZ1 complexes Human FEZ1(227-290) was cloned with NdeI and XhoI restriction sites into vector pET22b (Novagen) using full length FEZ1 as template for PCR [ ]. .. Both FEZ1(227-290) pET22b and SCOC(78-159) pET28a were co-transformed into BL21 (DE3) by electroporation.

    Article Title: Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma gondii
    Article Snippet: The ROP18 gene was inserted into vector pET22b (Novagen) Nde I site (5′ end) and Hin dIII (3′end) resulting in the addition of seven extra amino acids (KLAAALE) preceding the C-terminal His6 tag. .. Changes were verified by PCR-based cycle sequencing using BigDye (SeqWright DNA Technology Services, Houston, TX).

    Article Title: Protein sequences conserved in prokaryotic aminoacyl-tRNA synthetases are important for the activity of the processivity factor of human mitochondrial DNA polymerase
    Article Snippet: Primers HGBNDE (5′-ACTTCGGAGACATATGCGCTCT-CGTGTAGCCG-3′) and HGBNOT (5′-CAAATATAGCGG-CCGCTACATTCTTAGCTGATGAT-3′) were used to amplify a 1.5 kb PCR product from a HeLa Marathon-ready cDNA (Clontech), using Pfu Turbo DNA polymerase (Stratagene). .. This product was cloned as an Nde I– Not I fragment in the vector pET22b(+) (Novagen) to generate clone pJAC39.

    Article Title: Expression and specificity of a chitin deacetylase from the nematophagous fungus Pochonia chlamydosporia potentially involved in pathogenicity
    Article Snippet: .. Briefly, the vector pET22b(+) (Novagen) was used as a template to include a StrepII encoding sequence downstream of the multiple cloning site using PCR via a 50-mer phosphorylated primer pair. .. Electrocompetent E. coli DH5α cells were transformed with the pET22b(+)StrepIIC plasmid containing the Pc_2566 synthetic gene.

    Article Title: Characterization of a Soluble Ligand Binding Domain of the NMDA Receptor Regulatory Subunit NR3A
    Article Snippet: Based on amino acid sequence alignments with the NR1 and NR2A ligand binding domains, the crystal structures of which have been solved ( ; ), two peptides, N511-R660 and E776-K915, were isolated by PCR from the cDNA for full-length NR3A and joined by a GT dipeptide linker. .. The resulting NR3A S1S2 construct was then inserted into a modified version of the vector pET22b(+) (Novagen, Madison, WI), with an N-terminal His8 SSGLVPRGS affinity tag and thrombin cleavage site at its N terminus.

    Affinity Purification:

    Article Title: Production of a mono-biotinylated EGFR nanobody in the E. coli periplasm using the pET22b vector
    Article Snippet: The EL2BH synthetic DNA sequence was ordered from GenScript and subcloned into vector pET22b (EMDMillipore) between Eco RI and Hin dIII sites. .. The periplasmic extract was prepared essentially according to the pET system manual (TB055 rev C 11th edition, Novagen/EMDMillipore) and the sterile filtered extract in 5 mM MgSO4 was affinity purified using streptavidin-mutein gel (Roche) according to manufacturer’s instructions.

    Binding Assay:

    Article Title: Staphylococcus aureus FepA and FepB Proteins Drive Heme Iron Utilization in Escherichia coli
    Article Snippet: The FepA potential iron binding sequence HKIE was mutagenized into AKIA on the plasmid pMD1-fepAB by a two-step PCR leading to plasmid pMD1-fepAAKIA -fepB. .. The PCR product was cloned at the Nde I and Xho I sites of vector pET22b+ (Novagen) containing a T7 promoter and a His-tag.

    DNA Extraction:

    Article Title: A New Group of Modular Xylanases in Glycoside Hydrolase Family 8 from Marine Bacteria
    Article Snippet: The vector pET22b (Novagen, USA) was used for the construction of expression plasmids. .. A bacterial genomic DNA extraction kit (Biotech, China) and a high-purity plasmid preparation kit (Biotech, China) were used for gene and plasmid preparation, respectively.

    Mutagenesis:

    Article Title: Crystallization and preliminary X-ray crystallographic analysis of Aquifex aeolicus SelA, a bacterial selenocysteine synthase
    Article Snippet: .. The mutant SelA gene corresponding to SelA-ΔN (amino-acid residues 62–452) was cloned into the Nde I and Xho I restriction sites of the vector pET22b (Novagen). .. The wild-type SelA encoded in the vector does not have any artificial sequences at its N- or C-termini, while SelA-ΔN only has an artificially introduced initiating Met residue followed by residues 62–452.

    Article Title: Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma gondii
    Article Snippet: Paragraph title: Mutagenesis ... The ROP18 gene was inserted into vector pET22b (Novagen) Nde I site (5′ end) and Hin dIII (3′end) resulting in the addition of seven extra amino acids (KLAAALE) preceding the C-terminal His6 tag.

    Isolation:

    Article Title: Production of a mono-biotinylated EGFR nanobody in the E. coli periplasm using the pET22b vector
    Article Snippet: The EL2BH synthetic DNA sequence was ordered from GenScript and subcloned into vector pET22b (EMDMillipore) between Eco RI and Hin dIII sites. .. The EL2BH nanobody was isolated from the periplasmic space of E. coli BL21-DE3 (EMDMillipore) that we previously transformed with plasmid pBirACm (Avidity) encoding an IPTG inducible biotin ligase.

    Article Title: The Linker between the Dimerization and Catalytic Domains of the CheA Histidine Kinase Propagates Changes in Structure and Dynamics That Are Important for Enzymatic Activity
    Article Snippet: Protein Preparation Genes encoding P4 (residues 356–540), P4l (residues 346–540), and P3P4 (residues 290–540) of CheA from Thermotoga maritima were subcloned into vector pET22b (Novagen), and the expressed proteins with C-terminal His6 tags were purified using Ni-NTA affinity chromatography (Qiagen). .. CheA, CheW, CheY, and Tsr-containing membranes were isolated and purified following published protocols.

    Article Title: Characterization of a Soluble Ligand Binding Domain of the NMDA Receptor Regulatory Subunit NR3A
    Article Snippet: Based on amino acid sequence alignments with the NR1 and NR2A ligand binding domains, the crystal structures of which have been solved ( ; ), two peptides, N511-R660 and E776-K915, were isolated by PCR from the cDNA for full-length NR3A and joined by a GT dipeptide linker. .. The resulting NR3A S1S2 construct was then inserted into a modified version of the vector pET22b(+) (Novagen, Madison, WI), with an N-terminal His8 SSGLVPRGS affinity tag and thrombin cleavage site at its N terminus.

    Labeling:

    Article Title: The structure and dynamic properties of the complete histidine phosphotransfer domain of the chemotaxis specific histidine autokinase CheA from Thermotoga maritima
    Article Snippet: Protein expression and purification Full length His6-tagged P1 construct (residues 1–133) of T. maritima (or TmP1 ) was subcloned in the vector pET22b(+) (Novagen). .. Cells containing isotopically labeled proteins were grown in minimal media which consisted of 1 mM magnesium sulfate, 0.1 mM calcium chloride, 0.5 μg/ml of thiamine, and 100 μg/ml of ampicillin with the addition of 1 g/l 15 NH4 Cl and 2 g/l 13 C glucose (Cambridge Isotope Laboratories) for double labeled (15 N/13 C) samples or 1 g/l 15 NH4 Cl for single labeled (15 N) samples.

    Purification:

    Article Title: Crystallization and preliminary X-ray crystallographic analysis of Aquifex aeolicus SelA, a bacterial selenocysteine synthase
    Article Snippet: Paragraph title: 2.1. Overexpression and purification of SelA ... The mutant SelA gene corresponding to SelA-ΔN (amino-acid residues 62–452) was cloned into the Nde I and Xho I restriction sites of the vector pET22b (Novagen).

    Article Title: Crystal Structure of the Human Short Coiled Coil Protein and Insights into SCOC-FEZ1 Complex Formation
    Article Snippet: .. Purification of SCOC-FEZ1 complexes Human FEZ1(227-290) was cloned with NdeI and XhoI restriction sites into vector pET22b (Novagen) using full length FEZ1 as template for PCR [ ]. .. Both FEZ1(227-290) pET22b and SCOC(78-159) pET28a were co-transformed into BL21 (DE3) by electroporation.

    Article Title: The Linker between the Dimerization and Catalytic Domains of the CheA Histidine Kinase Propagates Changes in Structure and Dynamics That Are Important for Enzymatic Activity
    Article Snippet: .. Protein Preparation Genes encoding P4 (residues 356–540), P4l (residues 346–540), and P3P4 (residues 290–540) of CheA from Thermotoga maritima were subcloned into vector pET22b (Novagen), and the expressed proteins with C-terminal His6 tags were purified using Ni-NTA affinity chromatography (Qiagen). .. Purified proteins were dialyzed against the buffer containing 50 mM Na2 HPO4 (pH7.4) and 20 mM NaCl and concentrated to a final concentration of 0.5–1.5 mM.

    Article Title: Characterization of a Soluble Ligand Binding Domain of the NMDA Receptor Regulatory Subunit NR3A
    Article Snippet: Paragraph title: Protein expression and purification. ... The resulting NR3A S1S2 construct was then inserted into a modified version of the vector pET22b(+) (Novagen, Madison, WI), with an N-terminal His8 SSGLVPRGS affinity tag and thrombin cleavage site at its N terminus.

    Sequencing:

    Article Title: Multi-Compartment and Multi-Host Vector Suite for Recombinant Protein Expression and Purification
    Article Snippet: .. For the generation of the pT7pelB vector, the pelB sequence was amplified from the vector pET22b (Novagen) with the primers pelBT7For and pelBRev ( Table ) and cloned into the vector pT7GFP by RF cloning to generate the construct pT7pelB. .. This construct was then used as template with the primers T7 promoter and pelBinsRev and the generated megaprimer used for the insertion of the pelB signal sequence into vectors containing the T7 promoter and the fusion protein GST (pT7GST) or MBP (pT7MBP).

    Article Title: Production of a mono-biotinylated EGFR nanobody in the E. coli periplasm using the pET22b vector
    Article Snippet: .. The EL2BH synthetic DNA sequence was ordered from GenScript and subcloned into vector pET22b (EMDMillipore) between Eco RI and Hin dIII sites. .. The EL2BH nanobody was isolated from the periplasmic space of E. coli BL21-DE3 (EMDMillipore) that we previously transformed with plasmid pBirACm (Avidity) encoding an IPTG inducible biotin ligase.

    Article Title: Staphylococcus aureus FepA and FepB Proteins Drive Heme Iron Utilization in Escherichia coli
    Article Snippet: The fepB gene without its signal sequence was amplified by PCR with genomic DNA from S. aureus NCTC 8325 using primer pairs fepB-PS-Nde I/Xho I-fepB. .. The PCR product was cloned at the Nde I and Xho I sites of vector pET22b+ (Novagen) containing a T7 promoter and a His-tag.

    Article Title: Cystathionine ?-lyase: clinical, metabolic, genetic, and structural studies
    Article Snippet: The construction of the vector pET22b+ (Novagen) expressing CTH in E.coli was described previously by Steegborn et al [ ]. .. The CTH sequence was amplified from the template pET22b-CGL using these primers.

    Article Title: Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma gondii
    Article Snippet: The ROP18 gene was inserted into vector pET22b (Novagen) Nde I site (5′ end) and Hin dIII (3′end) resulting in the addition of seven extra amino acids (KLAAALE) preceding the C-terminal His6 tag. .. Changes were verified by PCR-based cycle sequencing using BigDye (SeqWright DNA Technology Services, Houston, TX).

    Article Title: Protein sequences conserved in prokaryotic aminoacyl-tRNA synthetases are important for the activity of the processivity factor of human mitochondrial DNA polymerase
    Article Snippet: This product was cloned as an Nde I– Not I fragment in the vector pET22b(+) (Novagen) to generate clone pJAC39. .. Sequencing of this clone revealed the presence of several differences with the previously published sequence, including two frame shifts.

    Article Title: Expression and specificity of a chitin deacetylase from the nematophagous fungus Pochonia chlamydosporia potentially involved in pathogenicity
    Article Snippet: .. Briefly, the vector pET22b(+) (Novagen) was used as a template to include a StrepII encoding sequence downstream of the multiple cloning site using PCR via a 50-mer phosphorylated primer pair. .. Electrocompetent E. coli DH5α cells were transformed with the pET22b(+)StrepIIC plasmid containing the Pc_2566 synthetic gene.

    Article Title: Characterization of a Soluble Ligand Binding Domain of the NMDA Receptor Regulatory Subunit NR3A
    Article Snippet: Based on amino acid sequence alignments with the NR1 and NR2A ligand binding domains, the crystal structures of which have been solved ( ; ), two peptides, N511-R660 and E776-K915, were isolated by PCR from the cDNA for full-length NR3A and joined by a GT dipeptide linker. .. The resulting NR3A S1S2 construct was then inserted into a modified version of the vector pET22b(+) (Novagen, Madison, WI), with an N-terminal His8 SSGLVPRGS affinity tag and thrombin cleavage site at its N terminus.

    Positron Emission Tomography:

    Article Title: Production of a mono-biotinylated EGFR nanobody in the E. coli periplasm using the pET22b vector
    Article Snippet: The EL2BH synthetic DNA sequence was ordered from GenScript and subcloned into vector pET22b (EMDMillipore) between Eco RI and Hin dIII sites. .. The periplasmic extract was prepared essentially according to the pET system manual (TB055 rev C 11th edition, Novagen/EMDMillipore) and the sterile filtered extract in 5 mM MgSO4 was affinity purified using streptavidin-mutein gel (Roche) according to manufacturer’s instructions.

    Lysis:

    Article Title: Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma gondii
    Article Snippet: The ROP18 gene was inserted into vector pET22b (Novagen) Nde I site (5′ end) and Hin dIII (3′end) resulting in the addition of seven extra amino acids (KLAAALE) preceding the C-terminal His6 tag. .. Plasmids were transformed into BL21-CodonPlus(DE3)-RP cells (Stratagene) and expression was induced by culture in 1 mM IGTP for 3–4 h. Bacterial cell pellets were lysed in CelLytic B cell lysis reagent (Sigma-Aldrich, St Louis, MO).

    Plasmid Preparation:

    Article Title: Crystallization and preliminary X-ray crystallographic analysis of Aquifex aeolicus SelA, a bacterial selenocysteine synthase
    Article Snippet: .. The mutant SelA gene corresponding to SelA-ΔN (amino-acid residues 62–452) was cloned into the Nde I and Xho I restriction sites of the vector pET22b (Novagen). .. The wild-type SelA encoded in the vector does not have any artificial sequences at its N- or C-termini, while SelA-ΔN only has an artificially introduced initiating Met residue followed by residues 62–452.

    Article Title: Comparing substrate specificity of two UDP-sugar Pyrophosphorylases and Efficient One-pot Enzymatic Synthesis of UDP-GlcA and UDP-GalA
    Article Snippet: .. E. coli DH5α and BL21 (DE3), as well as vector pET22b were purchased from EMD Millipore (Bilerica, MA). .. Vector pQE80Lwas purchased from Qiagen (Germantown, MD).

    Article Title: Multi-Compartment and Multi-Host Vector Suite for Recombinant Protein Expression and Purification
    Article Snippet: .. For the generation of the pT7pelB vector, the pelB sequence was amplified from the vector pET22b (Novagen) with the primers pelBT7For and pelBRev ( Table ) and cloned into the vector pT7GFP by RF cloning to generate the construct pT7pelB. .. This construct was then used as template with the primers T7 promoter and pelBinsRev and the generated megaprimer used for the insertion of the pelB signal sequence into vectors containing the T7 promoter and the fusion protein GST (pT7GST) or MBP (pT7MBP).

    Article Title: Immunization of N terminus of enterovirus 71 VP4 elicits cross-protective antibody responses
    Article Snippet: .. The PCR products were double-digested by XhoI and NdeI and subsequently inserted into the vector pET22b(+) (Novagen, USA). .. DNA fragment encoding HBc-N149 was amplified by using the primers P1u, P2d (5′-TGGGCAGCAATCTGGAAGATCCGGCGAGCCGCGAACTG-3′), P2u (5′- ACCAGTTCGCGGCTCGCCGGATCTTCCAGATTGCTGCCCA-3′) and P1d by using HBc-N149-VP4N20-encoding gene as a template and further inserted into the vector pET22b (+).

    Article Title: Production of a mono-biotinylated EGFR nanobody in the E. coli periplasm using the pET22b vector
    Article Snippet: .. The EL2BH synthetic DNA sequence was ordered from GenScript and subcloned into vector pET22b (EMDMillipore) between Eco RI and Hin dIII sites. .. The EL2BH nanobody was isolated from the periplasmic space of E. coli BL21-DE3 (EMDMillipore) that we previously transformed with plasmid pBirACm (Avidity) encoding an IPTG inducible biotin ligase.

    Article Title: Staphylococcus aureus FepA and FepB Proteins Drive Heme Iron Utilization in Escherichia coli
    Article Snippet: .. The PCR product was cloned at the Nde I and Xho I sites of vector pET22b+ (Novagen) containing a T7 promoter and a His-tag. .. Resulting plasmid pET22b-fepB-6His was used for transforming E. coli strain BLI5 for expression.

    Article Title: A New Group of Modular Xylanases in Glycoside Hydrolase Family 8 from Marine Bacteria
    Article Snippet: .. The vector pET22b (Novagen, USA) was used for the construction of expression plasmids. .. Insoluble beechwood xylan was purchased from Sigma (USA), and soluble wheat arabinoxylan (low viscosity) was obtained from Megazyme (Ireland).

    Article Title: Crystal Structure of the Human Short Coiled Coil Protein and Insights into SCOC-FEZ1 Complex Formation
    Article Snippet: .. Purification of SCOC-FEZ1 complexes Human FEZ1(227-290) was cloned with NdeI and XhoI restriction sites into vector pET22b (Novagen) using full length FEZ1 as template for PCR [ ]. .. Both FEZ1(227-290) pET22b and SCOC(78-159) pET28a were co-transformed into BL21 (DE3) by electroporation.

    Article Title: Cystathionine ?-lyase: clinical, metabolic, genetic, and structural studies
    Article Snippet: .. The construction of the vector pET22b+ (Novagen) expressing CTH in E.coli was described previously by Steegborn et al [ ]. .. The vector pGEX6-P-1(GE Healthcare), which is used for expression of glutathione- S -transferase fusion proteins, was employed to prepare the GST-CTH fusion construct.

    Article Title: The Linker between the Dimerization and Catalytic Domains of the CheA Histidine Kinase Propagates Changes in Structure and Dynamics That Are Important for Enzymatic Activity
    Article Snippet: .. Protein Preparation Genes encoding P4 (residues 356–540), P4l (residues 346–540), and P3P4 (residues 290–540) of CheA from Thermotoga maritima were subcloned into vector pET22b (Novagen), and the expressed proteins with C-terminal His6 tags were purified using Ni-NTA affinity chromatography (Qiagen). .. Purified proteins were dialyzed against the buffer containing 50 mM Na2 HPO4 (pH7.4) and 20 mM NaCl and concentrated to a final concentration of 0.5–1.5 mM.

    Article Title: Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma gondii
    Article Snippet: .. The ROP18 gene was inserted into vector pET22b (Novagen) Nde I site (5′ end) and Hin dIII (3′end) resulting in the addition of seven extra amino acids (KLAAALE) preceding the C-terminal His6 tag. .. Mutants were introduced into the pROP18KD3 plasmid by QuickChange site-directed mutagenesis (Stratagene) using oligonucleotides bearing nucleotide mismatches.

    Article Title: Protein sequences conserved in prokaryotic aminoacyl-tRNA synthetases are important for the activity of the processivity factor of human mitochondrial DNA polymerase
    Article Snippet: .. This product was cloned as an Nde I– Not I fragment in the vector pET22b(+) (Novagen) to generate clone pJAC39. ..

    Article Title: Expression and specificity of a chitin deacetylase from the nematophagous fungus Pochonia chlamydosporia potentially involved in pathogenicity
    Article Snippet: .. Briefly, the vector pET22b(+) (Novagen) was used as a template to include a StrepII encoding sequence downstream of the multiple cloning site using PCR via a 50-mer phosphorylated primer pair. .. Electrocompetent E. coli DH5α cells were transformed with the pET22b(+)StrepIIC plasmid containing the Pc_2566 synthetic gene.

    Article Title: Characterization of a Soluble Ligand Binding Domain of the NMDA Receptor Regulatory Subunit NR3A
    Article Snippet: .. The resulting NR3A S1S2 construct was then inserted into a modified version of the vector pET22b(+) (Novagen, Madison, WI), with an N-terminal His8 SSGLVPRGS affinity tag and thrombin cleavage site at its N terminus. ..

    Software:

    Article Title: Expression and specificity of a chitin deacetylase from the nematophagous fungus Pochonia chlamydosporia potentially involved in pathogenicity
    Article Snippet: Construction of a Pc CDA expression plasmid A synthetic gene encoding for Pochonia chlamydosporia chitin deacetylase was designed using the predicted protein sequence and codon-optimised for E. coli expression (GeneOptimizer™ software, GeneArt® Gene Synthesis service, ThermoFisher) . .. Briefly, the vector pET22b(+) (Novagen) was used as a template to include a StrepII encoding sequence downstream of the multiple cloning site using PCR via a 50-mer phosphorylated primer pair.

    Selection:

    Article Title: Multi-Compartment and Multi-Host Vector Suite for Recombinant Protein Expression and Purification
    Article Snippet: Selection of positive clones was performed by colony PCR by using Taq polymerase (InvitrogenTM ) with the same primers used for megaprimer generation. .. For the generation of the pT7pelB vector, the pelB sequence was amplified from the vector pET22b (Novagen) with the primers pelBT7For and pelBRev ( Table ) and cloned into the vector pT7GFP by RF cloning to generate the construct pT7pelB.

    Histone Deacetylase Assay:

    Article Title: Expression and specificity of a chitin deacetylase from the nematophagous fungus Pochonia chlamydosporia potentially involved in pathogenicity
    Article Snippet: Construction of a Pc CDA expression plasmid A synthetic gene encoding for Pochonia chlamydosporia chitin deacetylase was designed using the predicted protein sequence and codon-optimised for E. coli expression (GeneOptimizer™ software, GeneArt® Gene Synthesis service, ThermoFisher) . .. Briefly, the vector pET22b(+) (Novagen) was used as a template to include a StrepII encoding sequence downstream of the multiple cloning site using PCR via a 50-mer phosphorylated primer pair.

    Affinity Chromatography:

    Article Title: The Linker between the Dimerization and Catalytic Domains of the CheA Histidine Kinase Propagates Changes in Structure and Dynamics That Are Important for Enzymatic Activity
    Article Snippet: .. Protein Preparation Genes encoding P4 (residues 356–540), P4l (residues 346–540), and P3P4 (residues 290–540) of CheA from Thermotoga maritima were subcloned into vector pET22b (Novagen), and the expressed proteins with C-terminal His6 tags were purified using Ni-NTA affinity chromatography (Qiagen). .. Purified proteins were dialyzed against the buffer containing 50 mM Na2 HPO4 (pH7.4) and 20 mM NaCl and concentrated to a final concentration of 0.5–1.5 mM.

    Concentration Assay:

    Article Title: The Linker between the Dimerization and Catalytic Domains of the CheA Histidine Kinase Propagates Changes in Structure and Dynamics That Are Important for Enzymatic Activity
    Article Snippet: Protein Preparation Genes encoding P4 (residues 356–540), P4l (residues 346–540), and P3P4 (residues 290–540) of CheA from Thermotoga maritima were subcloned into vector pET22b (Novagen), and the expressed proteins with C-terminal His6 tags were purified using Ni-NTA affinity chromatography (Qiagen). .. Purified proteins were dialyzed against the buffer containing 50 mM Na2 HPO4 (pH7.4) and 20 mM NaCl and concentrated to a final concentration of 0.5–1.5 mM.

    Recombinant:

    Article Title: Characterization of a Soluble Ligand Binding Domain of the NMDA Receptor Regulatory Subunit NR3A
    Article Snippet: The resulting NR3A S1S2 construct was then inserted into a modified version of the vector pET22b(+) (Novagen, Madison, WI), with an N-terminal His8 SSGLVPRGS affinity tag and thrombin cleavage site at its N terminus. .. NR3A S1S2 was expressed as a recombinant protein in OrigamiB (DE3) Escherichia coli (Novagen).

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    Millipore pet22b vector
    Pet22b Vector, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore nde i hind iii digested expression vector pet22b
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    Millipore pet22b
    Pet22b, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 257 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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