pmal c2 expression vector  (New England Biolabs)


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    Name:
    pMAL c5X Vector
    Description:
    pMAL c5X Vector 10 ug
    Catalog Number:
    N8108S
    Price:
    131
    Size:
    10 ug
    Category:
    E coli Expression Vectors
    Score:
    85
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    Structured Review

    New England Biolabs pmal c2 expression vector
    pMAL c5X Vector
    pMAL c5X Vector 10 ug
    https://www.bioz.com/result/pmal c2 expression vector/product/New England Biolabs
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    pmal c2 expression vector - by Bioz Stars, 2019-10
    99/100 stars

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    Clone Assay:

    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation
    Article Snippet: SCGB1A1 and SCGB1A1A coding regions were obtained by PCR amplification using the following primers (Sigma-Aldrich): SCGB1A1- F, 5′- GAA ATC TGC CAG AGC TTT GCA GAC ATC ATT CAA GGC C-3′; SCGB1A1 -R, 5′-GTC ACC TGC AGG CTA AGC ACA CAG TGG GCT CTT TGC-3′ ; SCGB1A1A -F, 5′-GGA ATC TGC CAG AGA TTG GTA GGC ATC GTT CAA GCC C-3′; SCGB1A1A -R, 5′-GTC ACC TGC AGG CTA AGC ACA CAG TGG GCT CTC TAC-3′ . .. Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON). .. This vector is designed to produce maltose-binding protein (MBP) fusion proteins without adding vector-derived residues.

    Article Title: Overexpression of the transcription factor NF-YC9 confers abscisic acid hypersensitivity in Arabidopsis
    Article Snippet: The NF-YC9-6His and ABI5-6His recombinant proteins were produced in E. coli strain BL21. .. The full-length coding sequence of the NF-YC9 or ABI5 gene was cloned into the Sal I/EcoR I sites of pMAL-c5X vector (NEB), respectively. .. The 5′ end biotin-labeled probes used for this EMSA were amplified by PCR using the primer pairs described in Supplemental Table S4, and unlabeled fragments of the same sequences were used as the competitors.

    Article Title: Early detection of Mycobacterium avium subsp. paratuberculosis infection in cattle with multiplex-bead based immunoassays
    Article Snippet: The 6 recombinant MAP proteins selected in this study were expressed as maltose binding protein (MBP) fusion proteins because previous studies demonstrated higher yields as compared to six-His tag clones [ ]. .. The full-length coding sequences for 5 of the 6 genes were amplified from MAP K-10 genomic DNA with 5’ primer containing an Xba I and 3’ primer a Hind III restriction site and cloned into the pMAL-c5 translational fusion expression vector (New England Biolabs, Beverly, MA, USA). .. The MAP1201c + 2942c was chemically synthesized, amplified and cloned in a manner similar to the other 5 genes.

    Article Title: Somatic and Reproductive Cell Development in Rice Anther Is Regulated by a Putative Glutaredoxin
    Article Snippet: These two plasmids were introduced into calli derived from seeds of plants heterozygous for the MIL1 locus using an Agrobacterium tumefaciens –mediated method as previously described ( ). .. The recombinant MIL1 protein was obtained by cloning the MIL1 ORF into the expression vector pMAL-c5X (New England Biolabs), which was transformed into Escherichia coli strain BL21 (DE3) (Ding Guo). .. The MIL1-MBP fusion protein was purified using the pMAL protein fusion and purification system following the instruction manual (New England Biolabs).

    Article Title: Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis
    Article Snippet: The amplified product was purified and cloned into the pCR4-blunt TOPO vector (Life Technologies) and confirmed by sequencing. .. An fusion construct was assembled in pMAL-c5X vector (NEB) using an Infusion strategy (Clontech) by linearizing the vector with Xmn I and Eco RI followed by amplification with the primers 4_fw and 4_rev ( ).

    Article Title: CAPN5 mutation in hereditary uveitis: the R243L mutation increases calpain catalytic activity and triggers intraocular inflammation in a mouse model
    Article Snippet: The CONSURF scores were mapped to the B-factor column of mini-CAPN1 (PDB ID 2ARY) and visualized using PyMOL ( ). .. Sequences encoding the calpain-1/5 catalytic domain and the inactive calpain-1/5-C81S catalytic domain were cloned into pMal-c5x (New England Biolabs, Ipswich, MA) with a c-terminal His tag. .. Mini-calpains were expressed as MBP-fusion proteins in BL21(DE3) E. coli and purified on an amylose resin (New England Biolabs).

    Article Title: The Arabidopsis Malectin-Like/LRR-RLK IOS1 Is Critical for BAK1-Dependent and BAK1-Independent Pattern-Triggered Immunity
    Article Snippet: Paragraph title: Cloning, Expression, and Purification of Recombinant Proteins ... MBP was expressed from pMAL-c5X (New England Biolabs).

    Article Title: Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays
    Article Snippet: An In-Fusion HD cloning kit and Stellar competent cells were purchased from Clontech (Mountain View, CA). .. The pMal-c5X vector was purchased from New England Biolabs (NEB; Ipswich, MA).

    Article Title: KBTBD13 interacts with Cullin 3 to form a functional ubiquitin ligase
    Article Snippet: The BTB domain (6–132) of KBTBD13 followed by a hexa-histidine tag was cloned into the NcoI and NdeI sites of pMAL-c5X to generate MBP-KBTBD13(6–132)-(His)6 . .. The co-expression construct was generated by inserting downstream from the multiple cloning site in pMAL-c5X (MCS-1), a linker, tac promoter, and second multiple cloning site (MCS-2). .. KBTBD13(6–132) was cloned into NcoI and NdeI sites of MCS-1 and Cul3(1–383)-(His)6 was cloned into the NotI and SbfI sites of MCS-2.

    Article Title: Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5′-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover
    Article Snippet: Paragraph title: Cloning, Expression, and Purification of Human OAT ... A commercial protein expression vector, pMAL-C5X (New England Biolabs), was digested using restriction enzymes Eco RI and Ava I, and the resulting polymerase chain reaction insert containing the OAT coding region was ligated into the expression vector using T4 DNA ligase (New England Biolabs) to yield a protein expression plasmid encoding an N-terminal maltose binding protein (MBP) linked through a TEV cleavage sequence to the full length OAT enzyme.

    Article Title: Structures of Xenopus Embryonic Epidermal Lectin Reveal a Conserved Mechanism of Microbial Glycan Recognition
    Article Snippet: We refer to this as XEELCRD to denote the carbohydrate recognition domain. .. The expression construct was synthesized as a gBlock (Integrated DNA Technologies) and then cloned into the MfeI and BamHI sites of pMAL-c5x (New England Biolabs). .. The expected protein sequence corresponds to full-length thioredoxin (underlined) followed by a linker, a histidine tag, and an enterokinase cleavage site (bold) fused to residues 22–47 of XEEL (italics): MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA GSGSGHMHHHHHHSSGT DDDDK GSCEQASISEKKEKILNLLACWTEGN .

    Article Title: Mitotic spindle scaling during Xenopus development by kif2a and importin ?
    Article Snippet: Recombinant importin α used for in vitro biochemical experiments had the first 43 amino acids deleted to prevent known autoinhibitory interactions, but did not contain phosphomimetic mutations. .. Full-length kif2a and motor domain (amino acids 204–538) were amplified by polymerase chain reaction, cloned in to pMAL-C5X (New England Biolabs, Ipswitch MA, USA), and expressed and purified as described for MBP fusion proteins ( ). .. The NLS mutant had three charge switch mutations, K427E, K428E, and K430E, introduced into the NLS sequence identified by Quikchange mutagenesis (Stratagene/Agilent Technologies, Santa Clara CA, USA).

    Article Title: Quantitative analysis of TALE-DNA interactions suggests polarity effects
    Article Snippet: Thus, the 111-42 truncation refers to a variant that retains 111 N-terminal and 42 C-terminal residues from the full-length TALE, appended to the RVD repeat region ( Supplementary Figure S1A ). .. AvrBs3254-180 and dTALEs were cloned using Bam HI/Age I and Xho I/Age I, respectively, into pMAL-TEV, a prokaryotic expression plasmid derived from pMAL-c5x (New England Biolabs) that contained a site for the Tobacco Etch Virus (TEV) protease. .. TALE reading frames were bounded by an N-terminal maltose-binding protein (MBP) tag, a TEV protease cleavage site and a His6 C-terminal His-tag ( Supplementary Figure S1B ).

    Article Title: A juvenile mouse pheromone inhibits sexual behavior through the vomeronasal system
    Article Snippet: Peptide sequences were determined using Sequest (ThermoFinnigan) . .. A gene encoding the secreted form of ESP22 (Ala23-End) was cloned into pMAL-c5x bacterial expression vector (New England Biolabs) using SacI and BamHI restriction sites. .. ESP22 was expressed and purified as a fusion protein with maltose-binding protein (MBP) in BL21(DE3) cells following manufacturer’s protocols (pMAL Protein Fusion & Purification System, New England Biolabs).

    Centrifugation:

    Article Title: The Arabidopsis Malectin-Like/LRR-RLK IOS1 Is Critical for BAK1-Dependent and BAK1-Independent Pattern-Triggered Immunity
    Article Snippet: After overnight induction at 16°C with 0.4 mM IPTG, the bacteria were pelleted by centrifugation, resuspended in 100 mL binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, and 0.04% 2-mercaptoethanol), and sonicated. .. MBP was expressed from pMAL-c5X (New England Biolabs).

    Amplification:

    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation
    Article Snippet: SCGB1A1 and SCGB1A1A coding regions were obtained by PCR amplification using the following primers (Sigma-Aldrich): SCGB1A1- F, 5′- GAA ATC TGC CAG AGC TTT GCA GAC ATC ATT CAA GGC C-3′; SCGB1A1 -R, 5′-GTC ACC TGC AGG CTA AGC ACA CAG TGG GCT CTT TGC-3′ ; SCGB1A1A -F, 5′-GGA ATC TGC CAG AGA TTG GTA GGC ATC GTT CAA GCC C-3′; SCGB1A1A -R, 5′-GTC ACC TGC AGG CTA AGC ACA CAG TGG GCT CTC TAC-3′ . .. Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON).

    Article Title: CP40 from Corynebacterium pseudotuberculosis is an endo-β-N-acetylglucosaminidase
    Article Snippet: The coding sequence of cp40 was amplified by PCR using the oligonucleotide primers 5′-TGT-AGC-CAT-GG G-CGA-GTC-TGC-AAC-CTT-3′ and 5′-GAA-AGG-AAA-ACT-GGA-TCC -TCT-AGA-ACC-AGT-TGG-3′ (The restriction sites for NcoI and BamHI are italic). .. The 1140 bp PCR product was digested with the restriction enzymes NcoI and BamHI (Thermo Fisher Scientific, New York, NY), and ligated into pMAL-c5X-His vector (New England Biolabs, Berkeley, California) using DNA ligase T4 (Thermo Fisher Scientific).

    Article Title: Early detection of Mycobacterium avium subsp. paratuberculosis infection in cattle with multiplex-bead based immunoassays
    Article Snippet: The 6 recombinant MAP proteins selected in this study were expressed as maltose binding protein (MBP) fusion proteins because previous studies demonstrated higher yields as compared to six-His tag clones [ ]. .. The full-length coding sequences for 5 of the 6 genes were amplified from MAP K-10 genomic DNA with 5’ primer containing an Xba I and 3’ primer a Hind III restriction site and cloned into the pMAL-c5 translational fusion expression vector (New England Biolabs, Beverly, MA, USA). .. The MAP1201c + 2942c was chemically synthesized, amplified and cloned in a manner similar to the other 5 genes.

    Article Title: Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis
    Article Snippet: The amplified product was purified and cloned into the pCR4-blunt TOPO vector (Life Technologies) and confirmed by sequencing. .. An fusion construct was assembled in pMAL-c5X vector (NEB) using an Infusion strategy (Clontech) by linearizing the vector with Xmn I and Eco RI followed by amplification with the primers 4_fw and 4_rev ( ). .. A single colony of NEB expression Escherichia coli harboring the pMAL-c5X vector alone or the positive vector containing Zm 4 fusion was used to inoculate an overnight starter culture in 4 mL of Luria-Bertani broth incubated at 37°C with vigorous shaking.

    Article Title: Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2
    Article Snippet: Five-day-old P . patens protonemata were bombarded with the GFP-fusion constructs, and cells were observed under a confocal laser-scanning microscope. .. For preparation of MBP-PpSnRKB, the entire coding sequence of PpSnRKB cDNA ( Pp1s240_91V6.1 ) was amplified from reverse transcripts of P . patens protonemata and fused in-frame to the NdeI-digested pMAL-c5X vector (New England Biolabs). .. The MBP-fusion protein was purified using amylose resin according to the manufacturer’s instructions.

    Article Title: Mitotic spindle scaling during Xenopus development by kif2a and importin ?
    Article Snippet: Recombinant importin α used for in vitro biochemical experiments had the first 43 amino acids deleted to prevent known autoinhibitory interactions, but did not contain phosphomimetic mutations. .. Full-length kif2a and motor domain (amino acids 204–538) were amplified by polymerase chain reaction, cloned in to pMAL-C5X (New England Biolabs, Ipswitch MA, USA), and expressed and purified as described for MBP fusion proteins ( ). .. The NLS mutant had three charge switch mutations, K427E, K428E, and K430E, introduced into the NLS sequence identified by Quikchange mutagenesis (Stratagene/Agilent Technologies, Santa Clara CA, USA).

    Synthesized:

    Article Title: Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays
    Article Snippet: The pMal-c5X vector was purchased from New England Biolabs (NEB; Ipswich, MA). .. The pMal-c5X vector was purchased from New England Biolabs (NEB; Ipswich, MA).

    Article Title: Structures of Xenopus Embryonic Epidermal Lectin Reveal a Conserved Mechanism of Microbial Glycan Recognition
    Article Snippet: We refer to this as XEELCRD to denote the carbohydrate recognition domain. .. The expression construct was synthesized as a gBlock (Integrated DNA Technologies) and then cloned into the MfeI and BamHI sites of pMAL-c5x (New England Biolabs). .. The expected protein sequence corresponds to full-length thioredoxin (underlined) followed by a linker, a histidine tag, and an enterokinase cleavage site (bold) fused to residues 22–47 of XEEL (italics): MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA GSGSGHMHHHHHHSSGT DDDDK GSCEQASISEKKEKILNLLACWTEGN .

    Affinity Purification:

    Article Title: The Arabidopsis Malectin-Like/LRR-RLK IOS1 Is Critical for BAK1-Dependent and BAK1-Independent Pattern-Triggered Immunity
    Article Snippet: The soluble His-tagged proteins were affinity purified using a HisTrap FF column (GE Healthcare) according to the manufacturer's instructions. .. MBP was expressed from pMAL-c5X (New England Biolabs).

    Article Title: Quantitative analysis of TALE-DNA interactions suggests polarity effects
    Article Snippet: AvrBs3254-180 and dTALEs were cloned using Bam HI/Age I and Xho I/Age I, respectively, into pMAL-TEV, a prokaryotic expression plasmid derived from pMAL-c5x (New England Biolabs) that contained a site for the Tobacco Etch Virus (TEV) protease. .. AvrBs3254-180 and dTALEs were cloned using Bam HI/Age I and Xho I/Age I, respectively, into pMAL-TEV, a prokaryotic expression plasmid derived from pMAL-c5x (New England Biolabs) that contained a site for the Tobacco Etch Virus (TEV) protease.

    Construct:

    Article Title: Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis
    Article Snippet: The amplified product was purified and cloned into the pCR4-blunt TOPO vector (Life Technologies) and confirmed by sequencing. .. An fusion construct was assembled in pMAL-c5X vector (NEB) using an Infusion strategy (Clontech) by linearizing the vector with Xmn I and Eco RI followed by amplification with the primers 4_fw and 4_rev ( ). .. A single colony of NEB expression Escherichia coli harboring the pMAL-c5X vector alone or the positive vector containing Zm 4 fusion was used to inoculate an overnight starter culture in 4 mL of Luria-Bertani broth incubated at 37°C with vigorous shaking.

    Article Title: Catch Bond Interaction between Cell-Surface Sulfatase Sulf1 and Glycosaminoglycans
    Article Snippet: For expression in Escherichia coli ( E. coli ), a pMAL-c5X plasmid (New England Biolabs, Ipswich, MA) encoding the maltose binding protein-HD (MBP-HD) fusion protein was used. .. MBP-HD consists of an N-terminal (MBP) sequence followed by the sequence of the HD from human Sulf1 (K417 -K735 ), as schematically depicted in a .

    Article Title: The Arabidopsis Malectin-Like/LRR-RLK IOS1 Is Critical for BAK1-Dependent and BAK1-Independent Pattern-Triggered Immunity
    Article Snippet: The production of MBP-tagged FLS2 and EFR kinase domain constructs was performed as described by . .. MBP was expressed from pMAL-c5X (New England Biolabs).

    Article Title: KBTBD13 interacts with Cullin 3 to form a functional ubiquitin ligase
    Article Snippet: The BTB domain (6–132) of KBTBD13 followed by a hexa-histidine tag was cloned into the NcoI and NdeI sites of pMAL-c5X to generate MBP-KBTBD13(6–132)-(His)6 . .. The co-expression construct was generated by inserting downstream from the multiple cloning site in pMAL-c5X (MCS-1), a linker, tac promoter, and second multiple cloning site (MCS-2). .. KBTBD13(6–132) was cloned into NcoI and NdeI sites of MCS-1 and Cul3(1–383)-(His)6 was cloned into the NotI and SbfI sites of MCS-2.

    Article Title: Structures of Xenopus Embryonic Epidermal Lectin Reveal a Conserved Mechanism of Microbial Glycan Recognition
    Article Snippet: We refer to this as XEELCRD to denote the carbohydrate recognition domain. .. The expression construct was synthesized as a gBlock (Integrated DNA Technologies) and then cloned into the MfeI and BamHI sites of pMAL-c5x (New England Biolabs). .. The expected protein sequence corresponds to full-length thioredoxin (underlined) followed by a linker, a histidine tag, and an enterokinase cleavage site (bold) fused to residues 22–47 of XEEL (italics): MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA GSGSGHMHHHHHHSSGT DDDDK GSCEQASISEKKEKILNLLACWTEGN .

    Electrophoresis:

    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation
    Article Snippet: Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON). .. Each reaction was performed in a final volume of 25 µL, including 2 µL of 10 × PCR buffer, 0.2 mM dNTPs, 2 mM MgCl2 , 0.3 µM of each primer, 2 U of Platinum Taq, and 1 µL of template cDNA (100 ng).

    Incubation:

    Article Title: Overexpression of the transcription factor NF-YC9 confers abscisic acid hypersensitivity in Arabidopsis
    Article Snippet: The full-length coding sequence of the NF-YC9 or ABI5 gene was cloned into the Sal I/EcoR I sites of pMAL-c5X vector (NEB), respectively. .. The 5′ end biotin-labeled probes used for this EMSA were amplified by PCR using the primer pairs described in Supplemental Table S4, and unlabeled fragments of the same sequences were used as the competitors.

    Article Title: Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5′-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover
    Article Snippet: A commercial protein expression vector, pMAL-C5X (New England Biolabs), was digested using restriction enzymes Eco RI and Ava I, and the resulting polymerase chain reaction insert containing the OAT coding region was ligated into the expression vector using T4 DNA ligase (New England Biolabs) to yield a protein expression plasmid encoding an N-terminal maltose binding protein (MBP) linked through a TEV cleavage sequence to the full length OAT enzyme. .. For protein production and purification, pMAL-t-OAT was used to transform E. coli BL21(DE3) cells.

    Activity Assay:

    Article Title: CAPN5 mutation in hereditary uveitis: the R243L mutation increases calpain catalytic activity and triggers intraocular inflammation in a mouse model
    Article Snippet: Paragraph title: CAPN5 cloning, purification and activity assay ... Sequences encoding the calpain-1/5 catalytic domain and the inactive calpain-1/5-C81S catalytic domain were cloned into pMal-c5x (New England Biolabs, Ipswich, MA) with a c-terminal His tag.

    Expressing:

    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation
    Article Snippet: SCGB1A1 and SCGB1A1A coding regions were obtained by PCR amplification using the following primers (Sigma-Aldrich): SCGB1A1- F, 5′- GAA ATC TGC CAG AGC TTT GCA GAC ATC ATT CAA GGC C-3′; SCGB1A1 -R, 5′-GTC ACC TGC AGG CTA AGC ACA CAG TGG GCT CTT TGC-3′ ; SCGB1A1A -F, 5′-GGA ATC TGC CAG AGA TTG GTA GGC ATC GTT CAA GCC C-3′; SCGB1A1A -R, 5′-GTC ACC TGC AGG CTA AGC ACA CAG TGG GCT CTC TAC-3′ . .. Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON). .. This vector is designed to produce maltose-binding protein (MBP) fusion proteins without adding vector-derived residues.

    Article Title: CP40 from Corynebacterium pseudotuberculosis is an endo-β-N-acetylglucosaminidase
    Article Snippet: Paragraph title: Recombinant expression of CP40 and sequencing of the cp40 gene ... The 1140 bp PCR product was digested with the restriction enzymes NcoI and BamHI (Thermo Fisher Scientific, New York, NY), and ligated into pMAL-c5X-His vector (New England Biolabs, Berkeley, California) using DNA ligase T4 (Thermo Fisher Scientific).

    Article Title: Intrinsic disorder within AKAP79 fine-tunes anchored phosphatase activity toward substrates and drug sensitivity
    Article Snippet: Paragraph title: Protein expression and purification ... AKAP79 was expressed as an MBP fusion in a modified pMAL c5x backbone (NEB).

    Article Title: Early detection of Mycobacterium avium subsp. paratuberculosis infection in cattle with multiplex-bead based immunoassays
    Article Snippet: The 6 recombinant MAP proteins selected in this study were expressed as maltose binding protein (MBP) fusion proteins because previous studies demonstrated higher yields as compared to six-His tag clones [ ]. .. The full-length coding sequences for 5 of the 6 genes were amplified from MAP K-10 genomic DNA with 5’ primer containing an Xba I and 3’ primer a Hind III restriction site and cloned into the pMAL-c5 translational fusion expression vector (New England Biolabs, Beverly, MA, USA). .. The MAP1201c + 2942c was chemically synthesized, amplified and cloned in a manner similar to the other 5 genes.

    Article Title: Somatic and Reproductive Cell Development in Rice Anther Is Regulated by a Putative Glutaredoxin
    Article Snippet: These two plasmids were introduced into calli derived from seeds of plants heterozygous for the MIL1 locus using an Agrobacterium tumefaciens –mediated method as previously described ( ). .. The recombinant MIL1 protein was obtained by cloning the MIL1 ORF into the expression vector pMAL-c5X (New England Biolabs), which was transformed into Escherichia coli strain BL21 (DE3) (Ding Guo). .. The MIL1-MBP fusion protein was purified using the pMAL protein fusion and purification system following the instruction manual (New England Biolabs).

    Article Title: Catch Bond Interaction between Cell-Surface Sulfatase Sulf1 and Glycosaminoglycans
    Article Snippet: In this study, several hundred unbinding events are plotted in a histogram and the most probable dissociation forces F max were estimated by approximation of a (bimodal) Gaussian distribution. .. For expression in Escherichia coli ( E. coli ), a pMAL-c5X plasmid (New England Biolabs, Ipswich, MA) encoding the maltose binding protein-HD (MBP-HD) fusion protein was used. .. MBP-HD consists of an N-terminal (MBP) sequence followed by the sequence of the HD from human Sulf1 (K417 -K735 ), as schematically depicted in a .

    Article Title: The Arabidopsis Malectin-Like/LRR-RLK IOS1 Is Critical for BAK1-Dependent and BAK1-Independent Pattern-Triggered Immunity
    Article Snippet: Paragraph title: Cloning, Expression, and Purification of Recombinant Proteins ... MBP was expressed from pMAL-c5X (New England Biolabs).

    Article Title: Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5′-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover
    Article Snippet: A sequence encoding the cleavage site for tobacco etch virus (TEV) protease (ENLYFQG) was inserted at the 5′ end of the OAT coding region using forward primer 5′GCGCT-CGGGGAAAACCTGTATTTTCAGGGCGCCTCTGCTAC-ATCTGTTGCAAC3′ and reverse primer 5′GCGGAATTCT-CAGAAAGACAAGATGGTC3′; restriction sites Ava I and Eco RI were added to the 5′ and 3′ ends, respectively. .. A commercial protein expression vector, pMAL-C5X (New England Biolabs), was digested using restriction enzymes Eco RI and Ava I, and the resulting polymerase chain reaction insert containing the OAT coding region was ligated into the expression vector using T4 DNA ligase (New England Biolabs) to yield a protein expression plasmid encoding an N-terminal maltose binding protein (MBP) linked through a TEV cleavage sequence to the full length OAT enzyme. .. The resulting vector (pMAL-t-OAT) was used to transform E. coli DH5 α cells for plasmid storage and amplification.

    Article Title: Structures of Xenopus Embryonic Epidermal Lectin Reveal a Conserved Mechanism of Microbial Glycan Recognition
    Article Snippet: We refer to this as XEELCRD to denote the carbohydrate recognition domain. .. The expression construct was synthesized as a gBlock (Integrated DNA Technologies) and then cloned into the MfeI and BamHI sites of pMAL-c5x (New England Biolabs). .. The expected protein sequence corresponds to full-length thioredoxin (underlined) followed by a linker, a histidine tag, and an enterokinase cleavage site (bold) fused to residues 22–47 of XEEL (italics): MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA GSGSGHMHHHHHHSSGT DDDDK GSCEQASISEKKEKILNLLACWTEGN .

    Article Title: Quantitative analysis of TALE-DNA interactions suggests polarity effects
    Article Snippet: Thus, the 111-42 truncation refers to a variant that retains 111 N-terminal and 42 C-terminal residues from the full-length TALE, appended to the RVD repeat region ( Supplementary Figure S1A ). .. AvrBs3254-180 and dTALEs were cloned using Bam HI/Age I and Xho I/Age I, respectively, into pMAL-TEV, a prokaryotic expression plasmid derived from pMAL-c5x (New England Biolabs) that contained a site for the Tobacco Etch Virus (TEV) protease. .. TALE reading frames were bounded by an N-terminal maltose-binding protein (MBP) tag, a TEV protease cleavage site and a His6 C-terminal His-tag ( Supplementary Figure S1B ).

    Article Title: A juvenile mouse pheromone inhibits sexual behavior through the vomeronasal system
    Article Snippet: Peptide sequences were determined using Sequest (ThermoFinnigan) . .. A gene encoding the secreted form of ESP22 (Ala23-End) was cloned into pMAL-c5x bacterial expression vector (New England Biolabs) using SacI and BamHI restriction sites. .. ESP22 was expressed and purified as a fusion protein with maltose-binding protein (MBP) in BL21(DE3) cells following manufacturer’s protocols (pMAL Protein Fusion & Purification System, New England Biolabs).

    Modification:

    Article Title: Intrinsic disorder within AKAP79 fine-tunes anchored phosphatase activity toward substrates and drug sensitivity
    Article Snippet: All proteins were transformed and expressed in BL21 (DE3) pLysS cells (Life Technologies). .. AKAP79 was expressed as an MBP fusion in a modified pMAL c5x backbone (NEB). .. In addition, a 10x His-tag was placed at the C-terminus of the AKAP79 sequence.

    Transformation Assay:

    Article Title: CP40 from Corynebacterium pseudotuberculosis is an endo-β-N-acetylglucosaminidase
    Article Snippet: The 1140 bp PCR product was digested with the restriction enzymes NcoI and BamHI (Thermo Fisher Scientific, New York, NY), and ligated into pMAL-c5X-His vector (New England Biolabs, Berkeley, California) using DNA ligase T4 (Thermo Fisher Scientific). .. The pMAL-c5X-His vector encodes both Maltose-binding protein (MBP) and a 6xhistidine (His) tags Generated pMAL-c5X-His-cp40 plasmid was transformed to E. coli Top10 chemically competent cells.

    Article Title: CP40 from Corynebacterium pseudotuberculosis is an endo-β-N-acetylglucosaminidase
    Article Snippet: The 1140 bp PCR product was digested with the restriction enzymes NcoI and BamHI (Thermo Fisher Scientific, New York, NY), and ligated into pMAL-c5X-His vector (New England Biolabs, Berkeley, California) using DNA ligase T4 (Thermo Fisher Scientific). .. The pMAL-c5X-His vector encodes both Maltose-binding protein (MBP) and a 6xhistidine (His) tags Generated pMAL-c5X-His-cp40 plasmid was transformed to E. coli Top10 chemically competent cells. .. Plasmids with correct insert were transformed into the E. coli expression strain BL21 (DE3) pLysE.

    Article Title: Intrinsic disorder within AKAP79 fine-tunes anchored phosphatase activity toward substrates and drug sensitivity
    Article Snippet: All proteins were transformed and expressed in BL21 (DE3) pLysS cells (Life Technologies). .. AKAP79 was expressed as an MBP fusion in a modified pMAL c5x backbone (NEB).

    Article Title: Early detection of Mycobacterium avium subsp. paratuberculosis infection in cattle with multiplex-bead based immunoassays
    Article Snippet: The full-length coding sequences for 5 of the 6 genes were amplified from MAP K-10 genomic DNA with 5’ primer containing an Xba I and 3’ primer a Hind III restriction site and cloned into the pMAL-c5 translational fusion expression vector (New England Biolabs, Beverly, MA, USA). .. The vector and amplification products were each digested with Xba I and Hind III, followed by overnight ligation at 4°C.

    Article Title: Somatic and Reproductive Cell Development in Rice Anther Is Regulated by a Putative Glutaredoxin
    Article Snippet: These two plasmids were introduced into calli derived from seeds of plants heterozygous for the MIL1 locus using an Agrobacterium tumefaciens –mediated method as previously described ( ). .. The recombinant MIL1 protein was obtained by cloning the MIL1 ORF into the expression vector pMAL-c5X (New England Biolabs), which was transformed into Escherichia coli strain BL21 (DE3) (Ding Guo). .. The MIL1-MBP fusion protein was purified using the pMAL protein fusion and purification system following the instruction manual (New England Biolabs).

    Article Title: Structures of Xenopus Embryonic Epidermal Lectin Reveal a Conserved Mechanism of Microbial Glycan Recognition
    Article Snippet: The expression construct was synthesized as a gBlock (Integrated DNA Technologies) and then cloned into the MfeI and BamHI sites of pMAL-c5x (New England Biolabs). .. The expected protein sequence corresponds to full-length thioredoxin (underlined) followed by a linker, a histidine tag, and an enterokinase cleavage site (bold) fused to residues 22–47 of XEEL (italics): MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA GSGSGHMHHHHHHSSGT DDDDK GSCEQASISEKKEKILNLLACWTEGN .

    Article Title: Quantitative analysis of TALE-DNA interactions suggests polarity effects
    Article Snippet: AvrBs3254-180 and dTALEs were cloned using Bam HI/Age I and Xho I/Age I, respectively, into pMAL-TEV, a prokaryotic expression plasmid derived from pMAL-c5x (New England Biolabs) that contained a site for the Tobacco Etch Virus (TEV) protease. .. Tandem affinity purification allowed isolation of homogeneous full-length MBP-TALE–His6 fusion proteins ( Supplementary Figure S2B ).

    Derivative Assay:

    Article Title: Quantitative analysis of TALE-DNA interactions suggests polarity effects
    Article Snippet: Thus, the 111-42 truncation refers to a variant that retains 111 N-terminal and 42 C-terminal residues from the full-length TALE, appended to the RVD repeat region ( Supplementary Figure S1A ). .. AvrBs3254-180 and dTALEs were cloned using Bam HI/Age I and Xho I/Age I, respectively, into pMAL-TEV, a prokaryotic expression plasmid derived from pMAL-c5x (New England Biolabs) that contained a site for the Tobacco Etch Virus (TEV) protease. .. TALE reading frames were bounded by an N-terminal maltose-binding protein (MBP) tag, a TEV protease cleavage site and a His6 C-terminal His-tag ( Supplementary Figure S1B ).

    High Performance Liquid Chromatography:

    Article Title: Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays
    Article Snippet: The pMal-c5X vector was purchased from New England Biolabs (NEB; Ipswich, MA). .. Luciferase and d -luciferin were purchased from Promega (Madison, WI).

    Chromatography:

    Article Title: Catch Bond Interaction between Cell-Surface Sulfatase Sulf1 and Glycosaminoglycans
    Article Snippet: For expression in Escherichia coli ( E. coli ), a pMAL-c5X plasmid (New England Biolabs, Ipswich, MA) encoding the maltose binding protein-HD (MBP-HD) fusion protein was used. .. For expression in Escherichia coli ( E. coli ), a pMAL-c5X plasmid (New England Biolabs, Ipswich, MA) encoding the maltose binding protein-HD (MBP-HD) fusion protein was used.

    Ligation:

    Article Title: Early detection of Mycobacterium avium subsp. paratuberculosis infection in cattle with multiplex-bead based immunoassays
    Article Snippet: The full-length coding sequences for 5 of the 6 genes were amplified from MAP K-10 genomic DNA with 5’ primer containing an Xba I and 3’ primer a Hind III restriction site and cloned into the pMAL-c5 translational fusion expression vector (New England Biolabs, Beverly, MA, USA). .. The MAP1201c + 2942c was chemically synthesized, amplified and cloned in a manner similar to the other 5 genes.

    Protease Inhibitor:

    Article Title: Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays
    Article Snippet: LB medium, bovine serum albumin (BSA), NaCl, Triton X-100, protease inhibitor cocktail, ATP sulfurylase, Tris-HCl (pH 7.5), and HisPur Ni-nitrilotriacetic acid (NTA) agarose resin were purchased from Thermo Fisher Scientific (Waltham, MA). .. The pMal-c5X vector was purchased from New England Biolabs (NEB; Ipswich, MA).

    Generated:

    Article Title: CP40 from Corynebacterium pseudotuberculosis is an endo-β-N-acetylglucosaminidase
    Article Snippet: The 1140 bp PCR product was digested with the restriction enzymes NcoI and BamHI (Thermo Fisher Scientific, New York, NY), and ligated into pMAL-c5X-His vector (New England Biolabs, Berkeley, California) using DNA ligase T4 (Thermo Fisher Scientific). .. The pMAL-c5X-His vector encodes both Maltose-binding protein (MBP) and a 6xhistidine (His) tags Generated pMAL-c5X-His-cp40 plasmid was transformed to E. coli Top10 chemically competent cells. .. Plasmids with correct insert were transformed into the E. coli expression strain BL21 (DE3) pLysE.

    Article Title: Accurate high-throughput structure mapping and prediction with transition metal ion FRET
    Article Snippet: Maltose Binding Protein (MBP) was subcloned from the pMAL-c5x vector (New England Biolabs). .. Minigenes with cysteine and di-histidine mutations were synthesized (Biobasic Inc.) and introduced into the wtMBP plasmid.

    Article Title: KBTBD13 interacts with Cullin 3 to form a functional ubiquitin ligase
    Article Snippet: The BTB domain (6–132) of KBTBD13 followed by a hexa-histidine tag was cloned into the NcoI and NdeI sites of pMAL-c5X to generate MBP-KBTBD13(6–132)-(His)6 . .. The co-expression construct was generated by inserting downstream from the multiple cloning site in pMAL-c5X (MCS-1), a linker, tac promoter, and second multiple cloning site (MCS-2). .. KBTBD13(6–132) was cloned into NcoI and NdeI sites of MCS-1 and Cul3(1–383)-(His)6 was cloned into the NotI and SbfI sites of MCS-2.

    DNA Sequencing:

    Article Title: Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5′-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover
    Article Snippet: A commercial protein expression vector, pMAL-C5X (New England Biolabs), was digested using restriction enzymes Eco RI and Ava I, and the resulting polymerase chain reaction insert containing the OAT coding region was ligated into the expression vector using T4 DNA ligase (New England Biolabs) to yield a protein expression plasmid encoding an N-terminal maltose binding protein (MBP) linked through a TEV cleavage sequence to the full length OAT enzyme. .. The resulting vector (pMAL-t-OAT) was used to transform E. coli DH5 α cells for plasmid storage and amplification.

    Sequencing:

    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation
    Article Snippet: Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON). .. Each reaction was performed in a final volume of 25 µL, including 2 µL of 10 × PCR buffer, 0.2 mM dNTPs, 2 mM MgCl2 , 0.3 µM of each primer, 2 U of Platinum Taq, and 1 µL of template cDNA (100 ng).

    Article Title: CP40 from Corynebacterium pseudotuberculosis is an endo-β-N-acetylglucosaminidase
    Article Snippet: Paragraph title: Recombinant expression of CP40 and sequencing of the cp40 gene ... The 1140 bp PCR product was digested with the restriction enzymes NcoI and BamHI (Thermo Fisher Scientific, New York, NY), and ligated into pMAL-c5X-His vector (New England Biolabs, Berkeley, California) using DNA ligase T4 (Thermo Fisher Scientific).

    Article Title: Overexpression of the transcription factor NF-YC9 confers abscisic acid hypersensitivity in Arabidopsis
    Article Snippet: The NF-YC9-6His and ABI5-6His recombinant proteins were produced in E. coli strain BL21. .. The full-length coding sequence of the NF-YC9 or ABI5 gene was cloned into the Sal I/EcoR I sites of pMAL-c5X vector (NEB), respectively. .. The 5′ end biotin-labeled probes used for this EMSA were amplified by PCR using the primer pairs described in Supplemental Table S4, and unlabeled fragments of the same sequences were used as the competitors.

    Article Title: Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis
    Article Snippet: The amplified product was purified and cloned into the pCR4-blunt TOPO vector (Life Technologies) and confirmed by sequencing. .. An fusion construct was assembled in pMAL-c5X vector (NEB) using an Infusion strategy (Clontech) by linearizing the vector with Xmn I and Eco RI followed by amplification with the primers 4_fw and 4_rev ( ).

    Article Title: Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2
    Article Snippet: Five-day-old P . patens protonemata were bombarded with the GFP-fusion constructs, and cells were observed under a confocal laser-scanning microscope. .. For preparation of MBP-PpSnRKB, the entire coding sequence of PpSnRKB cDNA ( Pp1s240_91V6.1 ) was amplified from reverse transcripts of P . patens protonemata and fused in-frame to the NdeI-digested pMAL-c5X vector (New England Biolabs). .. The MBP-fusion protein was purified using amylose resin according to the manufacturer’s instructions.

    Article Title: The Arabidopsis Malectin-Like/LRR-RLK IOS1 Is Critical for BAK1-Dependent and BAK1-Independent Pattern-Triggered Immunity
    Article Snippet: All constructs were confirmed by Sanger sequencing and purified Trx-6xHis-IOS1KD and Trx-6xHis-IOS1KDm were analyzed by . .. MBP was expressed from pMAL-c5X (New England Biolabs).

    Article Title: Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5′-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover
    Article Snippet: A sequence encoding the cleavage site for tobacco etch virus (TEV) protease (ENLYFQG) was inserted at the 5′ end of the OAT coding region using forward primer 5′GCGCT-CGGGGAAAACCTGTATTTTCAGGGCGCCTCTGCTAC-ATCTGTTGCAAC3′ and reverse primer 5′GCGGAATTCT-CAGAAAGACAAGATGGTC3′; restriction sites Ava I and Eco RI were added to the 5′ and 3′ ends, respectively. .. A commercial protein expression vector, pMAL-C5X (New England Biolabs), was digested using restriction enzymes Eco RI and Ava I, and the resulting polymerase chain reaction insert containing the OAT coding region was ligated into the expression vector using T4 DNA ligase (New England Biolabs) to yield a protein expression plasmid encoding an N-terminal maltose binding protein (MBP) linked through a TEV cleavage sequence to the full length OAT enzyme. .. The resulting vector (pMAL-t-OAT) was used to transform E. coli DH5 α cells for plasmid storage and amplification.

    Article Title: A juvenile mouse pheromone inhibits sexual behavior through the vomeronasal system
    Article Snippet: A gene encoding the secreted form of ESP22 (Ala23-End) was cloned into pMAL-c5x bacterial expression vector (New England Biolabs) using SacI and BamHI restriction sites. .. A gene encoding the secreted form of ESP22 (Ala23-End) was cloned into pMAL-c5x bacterial expression vector (New England Biolabs) using SacI and BamHI restriction sites.

    Sonication:

    Article Title: The Arabidopsis Malectin-Like/LRR-RLK IOS1 Is Critical for BAK1-Dependent and BAK1-Independent Pattern-Triggered Immunity
    Article Snippet: After overnight induction at 16°C with 0.4 mM IPTG, the bacteria were pelleted by centrifugation, resuspended in 100 mL binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, and 0.04% 2-mercaptoethanol), and sonicated. .. MBP was expressed from pMAL-c5X (New England Biolabs).

    Injection:

    Article Title: Somatic and Reproductive Cell Development in Rice Anther Is Regulated by a Putative Glutaredoxin
    Article Snippet: The recombinant MIL1 protein was obtained by cloning the MIL1 ORF into the expression vector pMAL-c5X (New England Biolabs), which was transformed into Escherichia coli strain BL21 (DE3) (Ding Guo). .. The recombinant MIL1 protein was obtained by cloning the MIL1 ORF into the expression vector pMAL-c5X (New England Biolabs), which was transformed into Escherichia coli strain BL21 (DE3) (Ding Guo).

    Binding Assay:

    Article Title: Overexpression of the transcription factor NF-YC9 confers abscisic acid hypersensitivity in Arabidopsis
    Article Snippet: The full-length coding sequence of the NF-YC9 or ABI5 gene was cloned into the Sal I/EcoR I sites of pMAL-c5X vector (NEB), respectively. .. The 5′ end biotin-labeled probes used for this EMSA were amplified by PCR using the primer pairs described in Supplemental Table S4, and unlabeled fragments of the same sequences were used as the competitors.

    Article Title: Early detection of Mycobacterium avium subsp. paratuberculosis infection in cattle with multiplex-bead based immunoassays
    Article Snippet: The 6 recombinant MAP proteins selected in this study were expressed as maltose binding protein (MBP) fusion proteins because previous studies demonstrated higher yields as compared to six-His tag clones [ ]. .. The full-length coding sequences for 5 of the 6 genes were amplified from MAP K-10 genomic DNA with 5’ primer containing an Xba I and 3’ primer a Hind III restriction site and cloned into the pMAL-c5 translational fusion expression vector (New England Biolabs, Beverly, MA, USA).

    Article Title: Accurate high-throughput structure mapping and prediction with transition metal ion FRET
    Article Snippet: Combined with other structural methods including X-ray crystallography, small angle X-ray scattering, NMR, electron microscopy, and computational modeling, the full range of a protein’s conformational space should be revealed ( ; ). .. Maltose Binding Protein (MBP) was subcloned from the pMAL-c5x vector (New England Biolabs). .. Minigenes with cysteine and di-histidine mutations were synthesized (Biobasic Inc.) and introduced into the wtMBP plasmid.

    Article Title: Catch Bond Interaction between Cell-Surface Sulfatase Sulf1 and Glycosaminoglycans
    Article Snippet: In this study, several hundred unbinding events are plotted in a histogram and the most probable dissociation forces F max were estimated by approximation of a (bimodal) Gaussian distribution. .. For expression in Escherichia coli ( E. coli ), a pMAL-c5X plasmid (New England Biolabs, Ipswich, MA) encoding the maltose binding protein-HD (MBP-HD) fusion protein was used. .. MBP-HD consists of an N-terminal (MBP) sequence followed by the sequence of the HD from human Sulf1 (K417 -K735 ), as schematically depicted in a .

    Article Title: The Arabidopsis Malectin-Like/LRR-RLK IOS1 Is Critical for BAK1-Dependent and BAK1-Independent Pattern-Triggered Immunity
    Article Snippet: After overnight induction at 16°C with 0.4 mM IPTG, the bacteria were pelleted by centrifugation, resuspended in 100 mL binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, and 0.04% 2-mercaptoethanol), and sonicated. .. MBP was expressed from pMAL-c5X (New England Biolabs).

    Article Title: Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5′-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover
    Article Snippet: A sequence encoding the cleavage site for tobacco etch virus (TEV) protease (ENLYFQG) was inserted at the 5′ end of the OAT coding region using forward primer 5′GCGCT-CGGGGAAAACCTGTATTTTCAGGGCGCCTCTGCTAC-ATCTGTTGCAAC3′ and reverse primer 5′GCGGAATTCT-CAGAAAGACAAGATGGTC3′; restriction sites Ava I and Eco RI were added to the 5′ and 3′ ends, respectively. .. A commercial protein expression vector, pMAL-C5X (New England Biolabs), was digested using restriction enzymes Eco RI and Ava I, and the resulting polymerase chain reaction insert containing the OAT coding region was ligated into the expression vector using T4 DNA ligase (New England Biolabs) to yield a protein expression plasmid encoding an N-terminal maltose binding protein (MBP) linked through a TEV cleavage sequence to the full length OAT enzyme. .. The resulting vector (pMAL-t-OAT) was used to transform E. coli DH5 α cells for plasmid storage and amplification.

    Cellular Antioxidant Activity Assay:

    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation
    Article Snippet: SCGB1A1 and SCGB1A1A coding regions were obtained by PCR amplification using the following primers (Sigma-Aldrich): SCGB1A1- F, 5′- GAA ATC TGC CAG AGC TTT GCA GAC ATC ATT CAA GGC C-3′; SCGB1A1 -R, 5′-GTC ACC TGC AGG CTA AGC ACA CAG TGG GCT CTT TGC-3′ ; SCGB1A1A -F, 5′-GGA ATC TGC CAG AGA TTG GTA GGC ATC GTT CAA GCC C-3′; SCGB1A1A -R, 5′-GTC ACC TGC AGG CTA AGC ACA CAG TGG GCT CTC TAC-3′ . .. Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON).

    Mutagenesis:

    Article Title: Mitotic spindle scaling during Xenopus development by kif2a and importin ?
    Article Snippet: Recombinant importin α utilized for all extract and embryo experiments was a multiple phosphomimetic mutant proficient for nuclear import in egg extracts ( ). .. Full-length kif2a and motor domain (amino acids 204–538) were amplified by polymerase chain reaction, cloned in to pMAL-C5X (New England Biolabs, Ipswitch MA, USA), and expressed and purified as described for MBP fusion proteins ( ).

    Isolation:

    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation
    Article Snippet: Total RNA was isolated from equine frozen lung tissues (RNeasy, Qiagen, Mississauga, ON) and reverse transcribed into complementary DNA (cDNA) _ENREF_2 . .. Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON).

    Article Title: CP40 from Corynebacterium pseudotuberculosis is an endo-β-N-acetylglucosaminidase
    Article Snippet: The 1140 bp PCR product was digested with the restriction enzymes NcoI and BamHI (Thermo Fisher Scientific, New York, NY), and ligated into pMAL-c5X-His vector (New England Biolabs, Berkeley, California) using DNA ligase T4 (Thermo Fisher Scientific). .. Expression of MBP-cp40-His was induced by 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) (VWR International, Radnor, PA) for 2 h at 37 °C.

    Article Title: Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis
    Article Snippet: Paragraph title: Isolation of the Maize Embryo-Specific Zm 4 ... An fusion construct was assembled in pMAL-c5X vector (NEB) using an Infusion strategy (Clontech) by linearizing the vector with Xmn I and Eco RI followed by amplification with the primers 4_fw and 4_rev ( ).

    Article Title: Quantitative analysis of TALE-DNA interactions suggests polarity effects
    Article Snippet: AvrBs3254-180 and dTALEs were cloned using Bam HI/Age I and Xho I/Age I, respectively, into pMAL-TEV, a prokaryotic expression plasmid derived from pMAL-c5x (New England Biolabs) that contained a site for the Tobacco Etch Virus (TEV) protease. .. AvrBs3254-180 and dTALEs were cloned using Bam HI/Age I and Xho I/Age I, respectively, into pMAL-TEV, a prokaryotic expression plasmid derived from pMAL-c5x (New England Biolabs) that contained a site for the Tobacco Etch Virus (TEV) protease.

    Size-exclusion Chromatography:

    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation
    Article Snippet: Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON). .. Each reaction was performed in a final volume of 25 µL, including 2 µL of 10 × PCR buffer, 0.2 mM dNTPs, 2 mM MgCl2 , 0.3 µM of each primer, 2 U of Platinum Taq, and 1 µL of template cDNA (100 ng).

    Electrophoretic Mobility Shift Assay:

    Article Title: Overexpression of the transcription factor NF-YC9 confers abscisic acid hypersensitivity in Arabidopsis
    Article Snippet: Paragraph title: Electrophoretic mobility shift assay (EMSA) ... The full-length coding sequence of the NF-YC9 or ABI5 gene was cloned into the Sal I/EcoR I sites of pMAL-c5X vector (NEB), respectively.

    Purification:

    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation
    Article Snippet: Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON). .. Each reaction was performed in a final volume of 25 µL, including 2 µL of 10 × PCR buffer, 0.2 mM dNTPs, 2 mM MgCl2 , 0.3 µM of each primer, 2 U of Platinum Taq, and 1 µL of template cDNA (100 ng).

    Article Title: CP40 from Corynebacterium pseudotuberculosis is an endo-β-N-acetylglucosaminidase
    Article Snippet: The 1140 bp PCR product was digested with the restriction enzymes NcoI and BamHI (Thermo Fisher Scientific, New York, NY), and ligated into pMAL-c5X-His vector (New England Biolabs, Berkeley, California) using DNA ligase T4 (Thermo Fisher Scientific). .. The 1140 bp PCR product was digested with the restriction enzymes NcoI and BamHI (Thermo Fisher Scientific, New York, NY), and ligated into pMAL-c5X-His vector (New England Biolabs, Berkeley, California) using DNA ligase T4 (Thermo Fisher Scientific).

    Article Title: Intrinsic disorder within AKAP79 fine-tunes anchored phosphatase activity toward substrates and drug sensitivity
    Article Snippet: Paragraph title: Protein expression and purification ... AKAP79 was expressed as an MBP fusion in a modified pMAL c5x backbone (NEB).

    Article Title: Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis
    Article Snippet: The amplified product was purified and cloned into the pCR4-blunt TOPO vector (Life Technologies) and confirmed by sequencing. .. An fusion construct was assembled in pMAL-c5X vector (NEB) using an Infusion strategy (Clontech) by linearizing the vector with Xmn I and Eco RI followed by amplification with the primers 4_fw and 4_rev ( ).

    Article Title: Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2
    Article Snippet: For preparation of MBP-PpSnRKB, the entire coding sequence of PpSnRKB cDNA ( Pp1s240_91V6.1 ) was amplified from reverse transcripts of P . patens protonemata and fused in-frame to the NdeI-digested pMAL-c5X vector (New England Biolabs). .. For preparation of MBP-PpSnRKB, the entire coding sequence of PpSnRKB cDNA ( Pp1s240_91V6.1 ) was amplified from reverse transcripts of P . patens protonemata and fused in-frame to the NdeI-digested pMAL-c5X vector (New England Biolabs).

    Article Title: Catch Bond Interaction between Cell-Surface Sulfatase Sulf1 and Glycosaminoglycans
    Article Snippet: Paragraph title: Protein expression and purification ... For expression in Escherichia coli ( E. coli ), a pMAL-c5X plasmid (New England Biolabs, Ipswich, MA) encoding the maltose binding protein-HD (MBP-HD) fusion protein was used.

    Article Title: CAPN5 mutation in hereditary uveitis: the R243L mutation increases calpain catalytic activity and triggers intraocular inflammation in a mouse model
    Article Snippet: Paragraph title: CAPN5 cloning, purification and activity assay ... Sequences encoding the calpain-1/5 catalytic domain and the inactive calpain-1/5-C81S catalytic domain were cloned into pMal-c5x (New England Biolabs, Ipswich, MA) with a c-terminal His tag.

    Article Title: The Arabidopsis Malectin-Like/LRR-RLK IOS1 Is Critical for BAK1-Dependent and BAK1-Independent Pattern-Triggered Immunity
    Article Snippet: Paragraph title: Cloning, Expression, and Purification of Recombinant Proteins ... MBP was expressed from pMAL-c5X (New England Biolabs).

    Article Title: Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays
    Article Snippet: The pMal-c5X vector was purchased from New England Biolabs (NEB; Ipswich, MA). .. Luciferase and d -luciferin were purchased from Promega (Madison, WI).

    Article Title: Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5′-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover
    Article Snippet: Paragraph title: Cloning, Expression, and Purification of Human OAT ... A commercial protein expression vector, pMAL-C5X (New England Biolabs), was digested using restriction enzymes Eco RI and Ava I, and the resulting polymerase chain reaction insert containing the OAT coding region was ligated into the expression vector using T4 DNA ligase (New England Biolabs) to yield a protein expression plasmid encoding an N-terminal maltose binding protein (MBP) linked through a TEV cleavage sequence to the full length OAT enzyme.

    Article Title: Structures of Xenopus Embryonic Epidermal Lectin Reveal a Conserved Mechanism of Microbial Glycan Recognition
    Article Snippet: Paragraph title: Expression and Purification of Thioredoxin-fused XEEL (Residues 22–47) ... The expression construct was synthesized as a gBlock (Integrated DNA Technologies) and then cloned into the MfeI and BamHI sites of pMAL-c5x (New England Biolabs).

    Article Title: Mitotic spindle scaling during Xenopus development by kif2a and importin ?
    Article Snippet: Recombinant importin α used for in vitro biochemical experiments had the first 43 amino acids deleted to prevent known autoinhibitory interactions, but did not contain phosphomimetic mutations. .. Full-length kif2a and motor domain (amino acids 204–538) were amplified by polymerase chain reaction, cloned in to pMAL-C5X (New England Biolabs, Ipswitch MA, USA), and expressed and purified as described for MBP fusion proteins ( ). .. The NLS mutant had three charge switch mutations, K427E, K428E, and K430E, introduced into the NLS sequence identified by Quikchange mutagenesis (Stratagene/Agilent Technologies, Santa Clara CA, USA).

    Article Title: A juvenile mouse pheromone inhibits sexual behavior through the vomeronasal system
    Article Snippet: A gene encoding the secreted form of ESP22 (Ala23-End) was cloned into pMAL-c5x bacterial expression vector (New England Biolabs) using SacI and BamHI restriction sites. .. A gene encoding the secreted form of ESP22 (Ala23-End) was cloned into pMAL-c5x bacterial expression vector (New England Biolabs) using SacI and BamHI restriction sites.

    Protein Purification:

    Article Title: Mitotic spindle scaling during Xenopus development by kif2a and importin ?
    Article Snippet: Paragraph title: Protein purification ... Full-length kif2a and motor domain (amino acids 204–538) were amplified by polymerase chain reaction, cloned in to pMAL-C5X (New England Biolabs, Ipswitch MA, USA), and expressed and purified as described for MBP fusion proteins ( ).

    Polymerase Chain Reaction:

    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation
    Article Snippet: SCGB1A1 and SCGB1A1A coding regions were obtained by PCR amplification using the following primers (Sigma-Aldrich): SCGB1A1- F, 5′- GAA ATC TGC CAG AGC TTT GCA GAC ATC ATT CAA GGC C-3′; SCGB1A1 -R, 5′-GTC ACC TGC AGG CTA AGC ACA CAG TGG GCT CTT TGC-3′ ; SCGB1A1A -F, 5′-GGA ATC TGC CAG AGA TTG GTA GGC ATC GTT CAA GCC C-3′; SCGB1A1A -R, 5′-GTC ACC TGC AGG CTA AGC ACA CAG TGG GCT CTC TAC-3′ . .. Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON).

    Article Title: CP40 from Corynebacterium pseudotuberculosis is an endo-β-N-acetylglucosaminidase
    Article Snippet: The coding sequence of cp40 was amplified by PCR using the oligonucleotide primers 5′-TGT-AGC-CAT-GG G-CGA-GTC-TGC-AAC-CTT-3′ and 5′-GAA-AGG-AAA-ACT-GGA-TCC -TCT-AGA-ACC-AGT-TGG-3′ (The restriction sites for NcoI and BamHI are italic). .. The 1140 bp PCR product was digested with the restriction enzymes NcoI and BamHI (Thermo Fisher Scientific, New York, NY), and ligated into pMAL-c5X-His vector (New England Biolabs, Berkeley, California) using DNA ligase T4 (Thermo Fisher Scientific). .. The pMAL-c5X-His vector encodes both Maltose-binding protein (MBP) and a 6xhistidine (His) tags Generated pMAL-c5X-His-cp40 plasmid was transformed to E. coli Top10 chemically competent cells.

    Article Title: Early detection of Mycobacterium avium subsp. paratuberculosis infection in cattle with multiplex-bead based immunoassays
    Article Snippet: The full-length coding sequences for 5 of the 6 genes were amplified from MAP K-10 genomic DNA with 5’ primer containing an Xba I and 3’ primer a Hind III restriction site and cloned into the pMAL-c5 translational fusion expression vector (New England Biolabs, Beverly, MA, USA). .. The products were transformed into E . coli DH5α and selected on LB agar plates containing 0.10 mg/ml ampicillin.

    Article Title: Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis
    Article Snippet: The of Zm 4 embryo-specific (protein accession no. ; ) was obtained by -PCR. .. An fusion construct was assembled in pMAL-c5X vector (NEB) using an Infusion strategy (Clontech) by linearizing the vector with Xmn I and Eco RI followed by amplification with the primers 4_fw and 4_rev ( ).

    Article Title: Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5′-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover
    Article Snippet: A sequence encoding the cleavage site for tobacco etch virus (TEV) protease (ENLYFQG) was inserted at the 5′ end of the OAT coding region using forward primer 5′GCGCT-CGGGGAAAACCTGTATTTTCAGGGCGCCTCTGCTAC-ATCTGTTGCAAC3′ and reverse primer 5′GCGGAATTCT-CAGAAAGACAAGATGGTC3′; restriction sites Ava I and Eco RI were added to the 5′ and 3′ ends, respectively. .. A commercial protein expression vector, pMAL-C5X (New England Biolabs), was digested using restriction enzymes Eco RI and Ava I, and the resulting polymerase chain reaction insert containing the OAT coding region was ligated into the expression vector using T4 DNA ligase (New England Biolabs) to yield a protein expression plasmid encoding an N-terminal maltose binding protein (MBP) linked through a TEV cleavage sequence to the full length OAT enzyme. .. The resulting vector (pMAL-t-OAT) was used to transform E. coli DH5 α cells for plasmid storage and amplification.

    Article Title: Mitotic spindle scaling during Xenopus development by kif2a and importin ?
    Article Snippet: Recombinant importin α used for in vitro biochemical experiments had the first 43 amino acids deleted to prevent known autoinhibitory interactions, but did not contain phosphomimetic mutations. .. Full-length kif2a and motor domain (amino acids 204–538) were amplified by polymerase chain reaction, cloned in to pMAL-C5X (New England Biolabs, Ipswitch MA, USA), and expressed and purified as described for MBP fusion proteins ( ). .. The NLS mutant had three charge switch mutations, K427E, K428E, and K430E, introduced into the NLS sequence identified by Quikchange mutagenesis (Stratagene/Agilent Technologies, Santa Clara CA, USA).

    Labeling:

    Article Title: Overexpression of the transcription factor NF-YC9 confers abscisic acid hypersensitivity in Arabidopsis
    Article Snippet: The full-length coding sequence of the NF-YC9 or ABI5 gene was cloned into the Sal I/EcoR I sites of pMAL-c5X vector (NEB), respectively. .. The 5′ end biotin-labeled probes used for this EMSA were amplified by PCR using the primer pairs described in Supplemental Table S4, and unlabeled fragments of the same sequences were used as the competitors.

    Article Title: Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays
    Article Snippet: The pMal-c5X vector was purchased from New England Biolabs (NEB; Ipswich, MA). .. Luciferase and d -luciferin were purchased from Promega (Madison, WI).

    IA:

    Article Title: Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays
    Article Snippet: The pMal-c5X vector was purchased from New England Biolabs (NEB; Ipswich, MA). .. Luciferase and d -luciferin were purchased from Promega (Madison, WI).

    Antiviral Assay:

    Article Title: Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5′-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover
    Article Snippet: A sequence encoding the cleavage site for tobacco etch virus (TEV) protease (ENLYFQG) was inserted at the 5′ end of the OAT coding region using forward primer 5′GCGCT-CGGGGAAAACCTGTATTTTCAGGGCGCCTCTGCTAC-ATCTGTTGCAAC3′ and reverse primer 5′GCGGAATTCT-CAGAAAGACAAGATGGTC3′; restriction sites Ava I and Eco RI were added to the 5′ and 3′ ends, respectively. .. A commercial protein expression vector, pMAL-C5X (New England Biolabs), was digested using restriction enzymes Eco RI and Ava I, and the resulting polymerase chain reaction insert containing the OAT coding region was ligated into the expression vector using T4 DNA ligase (New England Biolabs) to yield a protein expression plasmid encoding an N-terminal maltose binding protein (MBP) linked through a TEV cleavage sequence to the full length OAT enzyme. .. The resulting vector (pMAL-t-OAT) was used to transform E. coli DH5 α cells for plasmid storage and amplification.

    Plasmid Preparation:

    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation
    Article Snippet: SCGB1A1 and SCGB1A1A coding regions were obtained by PCR amplification using the following primers (Sigma-Aldrich): SCGB1A1- F, 5′- GAA ATC TGC CAG AGC TTT GCA GAC ATC ATT CAA GGC C-3′; SCGB1A1 -R, 5′-GTC ACC TGC AGG CTA AGC ACA CAG TGG GCT CTT TGC-3′ ; SCGB1A1A -F, 5′-GGA ATC TGC CAG AGA TTG GTA GGC ATC GTT CAA GCC C-3′; SCGB1A1A -R, 5′-GTC ACC TGC AGG CTA AGC ACA CAG TGG GCT CTC TAC-3′ . .. Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON). .. This vector is designed to produce maltose-binding protein (MBP) fusion proteins without adding vector-derived residues.

    Article Title: CP40 from Corynebacterium pseudotuberculosis is an endo-β-N-acetylglucosaminidase
    Article Snippet: The coding sequence of cp40 was amplified by PCR using the oligonucleotide primers 5′-TGT-AGC-CAT-GG G-CGA-GTC-TGC-AAC-CTT-3′ and 5′-GAA-AGG-AAA-ACT-GGA-TCC -TCT-AGA-ACC-AGT-TGG-3′ (The restriction sites for NcoI and BamHI are italic). .. The 1140 bp PCR product was digested with the restriction enzymes NcoI and BamHI (Thermo Fisher Scientific, New York, NY), and ligated into pMAL-c5X-His vector (New England Biolabs, Berkeley, California) using DNA ligase T4 (Thermo Fisher Scientific). .. The pMAL-c5X-His vector encodes both Maltose-binding protein (MBP) and a 6xhistidine (His) tags Generated pMAL-c5X-His-cp40 plasmid was transformed to E. coli Top10 chemically competent cells.

    Article Title: CP40 from Corynebacterium pseudotuberculosis is an endo-β-N-acetylglucosaminidase
    Article Snippet: The 1140 bp PCR product was digested with the restriction enzymes NcoI and BamHI (Thermo Fisher Scientific, New York, NY), and ligated into pMAL-c5X-His vector (New England Biolabs, Berkeley, California) using DNA ligase T4 (Thermo Fisher Scientific). .. The pMAL-c5X-His vector encodes both Maltose-binding protein (MBP) and a 6xhistidine (His) tags Generated pMAL-c5X-His-cp40 plasmid was transformed to E. coli Top10 chemically competent cells. .. Plasmids with correct insert were transformed into the E. coli expression strain BL21 (DE3) pLysE.

    Article Title: Overexpression of the transcription factor NF-YC9 confers abscisic acid hypersensitivity in Arabidopsis
    Article Snippet: The NF-YC9-6His and ABI5-6His recombinant proteins were produced in E. coli strain BL21. .. The full-length coding sequence of the NF-YC9 or ABI5 gene was cloned into the Sal I/EcoR I sites of pMAL-c5X vector (NEB), respectively. .. The 5′ end biotin-labeled probes used for this EMSA were amplified by PCR using the primer pairs described in Supplemental Table S4, and unlabeled fragments of the same sequences were used as the competitors.

    Article Title: Accurate high-throughput structure mapping and prediction with transition metal ion FRET
    Article Snippet: Combined with other structural methods including X-ray crystallography, small angle X-ray scattering, NMR, electron microscopy, and computational modeling, the full range of a protein’s conformational space should be revealed ( ; ). .. Maltose Binding Protein (MBP) was subcloned from the pMAL-c5x vector (New England Biolabs). .. Minigenes with cysteine and di-histidine mutations were synthesized (Biobasic Inc.) and introduced into the wtMBP plasmid.

    Article Title: Somatic and Reproductive Cell Development in Rice Anther Is Regulated by a Putative Glutaredoxin
    Article Snippet: These two plasmids were introduced into calli derived from seeds of plants heterozygous for the MIL1 locus using an Agrobacterium tumefaciens –mediated method as previously described ( ). .. The recombinant MIL1 protein was obtained by cloning the MIL1 ORF into the expression vector pMAL-c5X (New England Biolabs), which was transformed into Escherichia coli strain BL21 (DE3) (Ding Guo). .. The MIL1-MBP fusion protein was purified using the pMAL protein fusion and purification system following the instruction manual (New England Biolabs).

    Article Title: Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis
    Article Snippet: The amplified product was purified and cloned into the pCR4-blunt TOPO vector (Life Technologies) and confirmed by sequencing. .. An fusion construct was assembled in pMAL-c5X vector (NEB) using an Infusion strategy (Clontech) by linearizing the vector with Xmn I and Eco RI followed by amplification with the primers 4_fw and 4_rev ( ). .. A single colony of NEB expression Escherichia coli harboring the pMAL-c5X vector alone or the positive vector containing Zm 4 fusion was used to inoculate an overnight starter culture in 4 mL of Luria-Bertani broth incubated at 37°C with vigorous shaking.

    Article Title: Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2
    Article Snippet: Five-day-old P . patens protonemata were bombarded with the GFP-fusion constructs, and cells were observed under a confocal laser-scanning microscope. .. For preparation of MBP-PpSnRKB, the entire coding sequence of PpSnRKB cDNA ( Pp1s240_91V6.1 ) was amplified from reverse transcripts of P . patens protonemata and fused in-frame to the NdeI-digested pMAL-c5X vector (New England Biolabs). .. The MBP-fusion protein was purified using amylose resin according to the manufacturer’s instructions.

    Article Title: Catch Bond Interaction between Cell-Surface Sulfatase Sulf1 and Glycosaminoglycans
    Article Snippet: In this study, several hundred unbinding events are plotted in a histogram and the most probable dissociation forces F max were estimated by approximation of a (bimodal) Gaussian distribution. .. For expression in Escherichia coli ( E. coli ), a pMAL-c5X plasmid (New England Biolabs, Ipswich, MA) encoding the maltose binding protein-HD (MBP-HD) fusion protein was used. .. MBP-HD consists of an N-terminal (MBP) sequence followed by the sequence of the HD from human Sulf1 (K417 -K735 ), as schematically depicted in a .

    Article Title: Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays
    Article Snippet: An In-Fusion HD cloning kit and Stellar competent cells were purchased from Clontech (Mountain View, CA). .. The pMal-c5X vector was purchased from New England Biolabs (NEB; Ipswich, MA). .. Luciferase and d -luciferin were purchased from Promega (Madison, WI).

    Article Title: Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5′-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover
    Article Snippet: A sequence encoding the cleavage site for tobacco etch virus (TEV) protease (ENLYFQG) was inserted at the 5′ end of the OAT coding region using forward primer 5′GCGCT-CGGGGAAAACCTGTATTTTCAGGGCGCCTCTGCTAC-ATCTGTTGCAAC3′ and reverse primer 5′GCGGAATTCT-CAGAAAGACAAGATGGTC3′; restriction sites Ava I and Eco RI were added to the 5′ and 3′ ends, respectively. .. A commercial protein expression vector, pMAL-C5X (New England Biolabs), was digested using restriction enzymes Eco RI and Ava I, and the resulting polymerase chain reaction insert containing the OAT coding region was ligated into the expression vector using T4 DNA ligase (New England Biolabs) to yield a protein expression plasmid encoding an N-terminal maltose binding protein (MBP) linked through a TEV cleavage sequence to the full length OAT enzyme. .. The resulting vector (pMAL-t-OAT) was used to transform E. coli DH5 α cells for plasmid storage and amplification.

    Article Title: Structures of Xenopus Embryonic Epidermal Lectin Reveal a Conserved Mechanism of Microbial Glycan Recognition
    Article Snippet: The expression construct was synthesized as a gBlock (Integrated DNA Technologies) and then cloned into the MfeI and BamHI sites of pMAL-c5x (New England Biolabs). .. The expected protein sequence corresponds to full-length thioredoxin (underlined) followed by a linker, a histidine tag, and an enterokinase cleavage site (bold) fused to residues 22–47 of XEEL (italics): MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA GSGSGHMHHHHHHSSGT DDDDK GSCEQASISEKKEKILNLLACWTEGN .

    Article Title: Quantitative analysis of TALE-DNA interactions suggests polarity effects
    Article Snippet: Thus, the 111-42 truncation refers to a variant that retains 111 N-terminal and 42 C-terminal residues from the full-length TALE, appended to the RVD repeat region ( Supplementary Figure S1A ). .. AvrBs3254-180 and dTALEs were cloned using Bam HI/Age I and Xho I/Age I, respectively, into pMAL-TEV, a prokaryotic expression plasmid derived from pMAL-c5x (New England Biolabs) that contained a site for the Tobacco Etch Virus (TEV) protease. .. TALE reading frames were bounded by an N-terminal maltose-binding protein (MBP) tag, a TEV protease cleavage site and a His6 C-terminal His-tag ( Supplementary Figure S1B ).

    Article Title: A juvenile mouse pheromone inhibits sexual behavior through the vomeronasal system
    Article Snippet: Peptide sequences were determined using Sequest (ThermoFinnigan) . .. A gene encoding the secreted form of ESP22 (Ala23-End) was cloned into pMAL-c5x bacterial expression vector (New England Biolabs) using SacI and BamHI restriction sites. .. ESP22 was expressed and purified as a fusion protein with maltose-binding protein (MBP) in BL21(DE3) cells following manufacturer’s protocols (pMAL Protein Fusion & Purification System, New England Biolabs).

    Positron Emission Tomography:

    Article Title: A juvenile mouse pheromone inhibits sexual behavior through the vomeronasal system
    Article Snippet: A gene encoding the secreted form of ESP22 (Ala23-End) was cloned into pMAL-c5x bacterial expression vector (New England Biolabs) using SacI and BamHI restriction sites. .. A gene encoding the secreted form of ESP22 (Ala23-End) was cloned into pMAL-c5x bacterial expression vector (New England Biolabs) using SacI and BamHI restriction sites.

    In Vitro:

    Article Title: Mitotic spindle scaling during Xenopus development by kif2a and importin ?
    Article Snippet: Recombinant importin α used for in vitro biochemical experiments had the first 43 amino acids deleted to prevent known autoinhibitory interactions, but did not contain phosphomimetic mutations. .. Full-length kif2a and motor domain (amino acids 204–538) were amplified by polymerase chain reaction, cloned in to pMAL-C5X (New England Biolabs, Ipswitch MA, USA), and expressed and purified as described for MBP fusion proteins ( ).

    Produced:

    Article Title: Overexpression of the transcription factor NF-YC9 confers abscisic acid hypersensitivity in Arabidopsis
    Article Snippet: The NF-YC9-6His and ABI5-6His recombinant proteins were produced in E. coli strain BL21. .. The full-length coding sequence of the NF-YC9 or ABI5 gene was cloned into the Sal I/EcoR I sites of pMAL-c5X vector (NEB), respectively.

    Concentration Assay:

    Article Title: Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2
    Article Snippet: For preparation of MBP-PpSnRKB, the entire coding sequence of PpSnRKB cDNA ( Pp1s240_91V6.1 ) was amplified from reverse transcripts of P . patens protonemata and fused in-frame to the NdeI-digested pMAL-c5X vector (New England Biolabs). .. The GST-fusion protein was purified using glutathione-Sepharose resin.

    Recombinant:

    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation
    Article Snippet: Paragraph title: Cloning and production of equine recombinant SCGB 1A1 and 1A1A ... Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON).

    Article Title: CP40 from Corynebacterium pseudotuberculosis is an endo-β-N-acetylglucosaminidase
    Article Snippet: Paragraph title: Recombinant expression of CP40 and sequencing of the cp40 gene ... The 1140 bp PCR product was digested with the restriction enzymes NcoI and BamHI (Thermo Fisher Scientific, New York, NY), and ligated into pMAL-c5X-His vector (New England Biolabs, Berkeley, California) using DNA ligase T4 (Thermo Fisher Scientific).

    Article Title: Overexpression of the transcription factor NF-YC9 confers abscisic acid hypersensitivity in Arabidopsis
    Article Snippet: The NF-YC9-6His and ABI5-6His recombinant proteins were produced in E. coli strain BL21. .. The full-length coding sequence of the NF-YC9 or ABI5 gene was cloned into the Sal I/EcoR I sites of pMAL-c5X vector (NEB), respectively.

    Article Title: Early detection of Mycobacterium avium subsp. paratuberculosis infection in cattle with multiplex-bead based immunoassays
    Article Snippet: Paragraph title: Preparation of recombinant proteins ... The full-length coding sequences for 5 of the 6 genes were amplified from MAP K-10 genomic DNA with 5’ primer containing an Xba I and 3’ primer a Hind III restriction site and cloned into the pMAL-c5 translational fusion expression vector (New England Biolabs, Beverly, MA, USA).

    Article Title: Somatic and Reproductive Cell Development in Rice Anther Is Regulated by a Putative Glutaredoxin
    Article Snippet: These two plasmids were introduced into calli derived from seeds of plants heterozygous for the MIL1 locus using an Agrobacterium tumefaciens –mediated method as previously described ( ). .. The recombinant MIL1 protein was obtained by cloning the MIL1 ORF into the expression vector pMAL-c5X (New England Biolabs), which was transformed into Escherichia coli strain BL21 (DE3) (Ding Guo). .. The MIL1-MBP fusion protein was purified using the pMAL protein fusion and purification system following the instruction manual (New England Biolabs).

    Article Title: Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2
    Article Snippet: Paragraph title: Preparation of Recombinant Proteins. ... For preparation of MBP-PpSnRKB, the entire coding sequence of PpSnRKB cDNA ( Pp1s240_91V6.1 ) was amplified from reverse transcripts of P . patens protonemata and fused in-frame to the NdeI-digested pMAL-c5X vector (New England Biolabs).

    Article Title: The Arabidopsis Malectin-Like/LRR-RLK IOS1 Is Critical for BAK1-Dependent and BAK1-Independent Pattern-Triggered Immunity
    Article Snippet: Paragraph title: Cloning, Expression, and Purification of Recombinant Proteins ... MBP was expressed from pMAL-c5X (New England Biolabs).

    Article Title: Mitotic spindle scaling during Xenopus development by kif2a and importin ?
    Article Snippet: Recombinant importin α used for in vitro biochemical experiments had the first 43 amino acids deleted to prevent known autoinhibitory interactions, but did not contain phosphomimetic mutations. .. Full-length kif2a and motor domain (amino acids 204–538) were amplified by polymerase chain reaction, cloned in to pMAL-C5X (New England Biolabs, Ipswitch MA, USA), and expressed and purified as described for MBP fusion proteins ( ).

    Article Title: A juvenile mouse pheromone inhibits sexual behavior through the vomeronasal system
    Article Snippet: Paragraph title: Recombinant proteins ... A gene encoding the secreted form of ESP22 (Ala23-End) was cloned into pMAL-c5x bacterial expression vector (New England Biolabs) using SacI and BamHI restriction sites.

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    New England Biolabs pmal c2 expression vector
    Pmal C2 Expression Vector, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs mbp fusion protein expression vector pmal c2
    Mbp Fusion Protein Expression Vector Pmal C2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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