tev protease recognition sequence  (New England Biolabs)


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    Name:
    TEV Protease
    Description:
    TEV Protease also known as Tobacco Etch Virus TEV Protease is a highly specific cysteine protease that recognizes the amino acid sequence Glu Asn Leu Tyr Phe Gln Gly Ser and cleaves between the Gln and Gly Ser residues It is often used for the removal of affinity purification tags such as maltose binding protein MBP or poly histidine from fusion proteins TEV Protease has a 7xHis tag for easy removal from a reaction using nickel affinity resins and has been engineered for greater performance
    Catalog Number:
    p8112s
    Price:
    102
    Size:
    1 000 units
    Category:
    Proteases
    Buy from Supplier


    Structured Review

    New England Biolabs tev protease recognition sequence
    TEV Protease
    TEV Protease also known as Tobacco Etch Virus TEV Protease is a highly specific cysteine protease that recognizes the amino acid sequence Glu Asn Leu Tyr Phe Gln Gly Ser and cleaves between the Gln and Gly Ser residues It is often used for the removal of affinity purification tags such as maltose binding protein MBP or poly histidine from fusion proteins TEV Protease has a 7xHis tag for easy removal from a reaction using nickel affinity resins and has been engineered for greater performance
    https://www.bioz.com/result/tev protease recognition sequence/product/New England Biolabs
    Average 99 stars, based on 75 article reviews
    Price from $9.99 to $1999.99
    tev protease recognition sequence - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "A widespread family of heat-resistant obscure (Hero) proteins protect against protein instability and aggregation"

    Article Title: A widespread family of heat-resistant obscure (Hero) proteins protect against protein instability and aggregation

    Journal: bioRxiv

    doi: 10.1101/816124

    Activity of heat-soluble proteins and nucleonic acids on Ago2 molecular behavior and LDH desiccation. (A) Schematic representation of FLAG-TEV-SNAP-Ago2 immunopurified on magnetic beads. (B) FLAG-TEV-SNAP-Ago2 was immunopurified onto magnetics beads via anti-FLAG antibody and then the FLAG tag was cleaved off by TEV protease in buffer, crude lysates or their boiled supernatants from fly S2 or human HEK293T cells. PK indicates that the boiled supernatant had been mostly deproteinized by Proteinase K in advance. BZ indicates that DNA and RNA in boiled supernatant had been degraded by Benzonase. BZ/PK indicates that DNA and RNA had been degraded by Benzonase, followed by the deproteinization by Proteinase K. Eluted Ago2 (top) and Ago2 still remaining on the beads (middle) were visualized by a red fluorescent dye covalently attached to the SNAP tag. The values on the top indicate relative amounts of eluted Ago2, normalized to that with S2 crude cell lysate. Proteins remaining in the boiled supernatants have an activity to promote Ago2 elution. (C) Small RNA pull-down assay for pre-Ago2-RISC and mature Ago2-RISC assembled in the reconstitution system, containing a 32 P-radiolabeled small RNA duplex, Ago2, Dicer-2/R2D2, and the Hsp70/Hsp90 chaperone machinery. Supplementation of the boiled supernatants from S2 or HEK293T cells promoted the formation of both pre- and mature Ago2-RISC. (D) Quantification of (C). Data represent means ± SD from 3 independent experiments. (E) Heat-soluble proteins in the boiled supernatants protect the LDH activity from desiccation. LDH mixed with the indicated boiled supernatants or buffer was dried up overnight and the remaining LDH activities were measured. Data represent means ± SD from 3 independent experiments.
    Figure Legend Snippet: Activity of heat-soluble proteins and nucleonic acids on Ago2 molecular behavior and LDH desiccation. (A) Schematic representation of FLAG-TEV-SNAP-Ago2 immunopurified on magnetic beads. (B) FLAG-TEV-SNAP-Ago2 was immunopurified onto magnetics beads via anti-FLAG antibody and then the FLAG tag was cleaved off by TEV protease in buffer, crude lysates or their boiled supernatants from fly S2 or human HEK293T cells. PK indicates that the boiled supernatant had been mostly deproteinized by Proteinase K in advance. BZ indicates that DNA and RNA in boiled supernatant had been degraded by Benzonase. BZ/PK indicates that DNA and RNA had been degraded by Benzonase, followed by the deproteinization by Proteinase K. Eluted Ago2 (top) and Ago2 still remaining on the beads (middle) were visualized by a red fluorescent dye covalently attached to the SNAP tag. The values on the top indicate relative amounts of eluted Ago2, normalized to that with S2 crude cell lysate. Proteins remaining in the boiled supernatants have an activity to promote Ago2 elution. (C) Small RNA pull-down assay for pre-Ago2-RISC and mature Ago2-RISC assembled in the reconstitution system, containing a 32 P-radiolabeled small RNA duplex, Ago2, Dicer-2/R2D2, and the Hsp70/Hsp90 chaperone machinery. Supplementation of the boiled supernatants from S2 or HEK293T cells promoted the formation of both pre- and mature Ago2-RISC. (D) Quantification of (C). Data represent means ± SD from 3 independent experiments. (E) Heat-soluble proteins in the boiled supernatants protect the LDH activity from desiccation. LDH mixed with the indicated boiled supernatants or buffer was dried up overnight and the remaining LDH activities were measured. Data represent means ± SD from 3 independent experiments.

    Techniques Used: Activity Assay, Magnetic Beads, FLAG-tag, Pull Down Assay

    2) Product Images from "GoldCLIP: Gel-omitted Ligation-dependent CLIP"

    Article Title: GoldCLIP: Gel-omitted Ligation-dependent CLIP

    Journal: Genomics, Proteomics & Bioinformatics

    doi: 10.1016/j.gpb.2018.04.003

    Isolation of PTB – RNA complexes without gel purifications A. Comparison of Halo fusion proteins (input vs . unbound fractions) for the indicated samples. The Halo fusion proteins are labeled with HaloTag Alexa Fluor® 660 ligand and resolved on SDS–PAGE. Bottom panel, Western blot analysis of tubulin as a loading control. B. Western blot analysis showing that similar amounts of PTB–RNA complexes are released by TEV protease following the indicated washing conditions after Halo bead isolation from the HEK 293T Halo-PTB stable cells. Non-transfected HEK 293T cells are used as control. C. Autoradiogram (upper panel) of 32 P-labeled RNA crosslinked to PTB purified by HaloTag and released by TEV protease. RNA–protein complexes of 60–70 kD are seen only with the UV crosslinking condition. Western blot analysis (bottom panel) of TEV-released PTB proteins from equal amounts of samples prepared from the lysate of the Halo-PTB stable cell line.
    Figure Legend Snippet: Isolation of PTB – RNA complexes without gel purifications A. Comparison of Halo fusion proteins (input vs . unbound fractions) for the indicated samples. The Halo fusion proteins are labeled with HaloTag Alexa Fluor® 660 ligand and resolved on SDS–PAGE. Bottom panel, Western blot analysis of tubulin as a loading control. B. Western blot analysis showing that similar amounts of PTB–RNA complexes are released by TEV protease following the indicated washing conditions after Halo bead isolation from the HEK 293T Halo-PTB stable cells. Non-transfected HEK 293T cells are used as control. C. Autoradiogram (upper panel) of 32 P-labeled RNA crosslinked to PTB purified by HaloTag and released by TEV protease. RNA–protein complexes of 60–70 kD are seen only with the UV crosslinking condition. Western blot analysis (bottom panel) of TEV-released PTB proteins from equal amounts of samples prepared from the lysate of the Halo-PTB stable cell line.

    Techniques Used: Isolation, Labeling, SDS Page, Western Blot, Transfection, Purification, Stable Transfection

    HaloTag based GoldCLIP technology A. Schematic flow chart of GoldCLIP technology. Cells stably expressing Halo-tagged fusion RBPs are crosslinked by UV irradiation. After cell lysis, Halo-RBP complexes are then captured by magnetic beads coated with Halo ligand under native conditions and a specific 3′ linker is ligated to RNAs bound by RBPs. Following denaturing washes, purified RNAs are cloned via an iCLIP protocol for high-throughput sequencing. B. Western blot analysis showing the expression level of Halo-PTB in the HEK 293T Halo-PTB stable cells compared to endogenous PTB using a monoclonal anti-PTB antibody (BB7). Non-transfected HEK 293T cells are used as control. A diagram of Halo-PTB fusion protein is shown below. C. Localization of Halo-PTB fusion proteins in 293T cell line. HaloTag TMR ligand staining of Halo-PTB fusion protein is shown in the top panel, and immunofluorescent staining of endogenous PTB using a monoclonal PTB antibody (BB7) is shown in the bottom panel. RBP, RNA-binding protein; iCLIP, individual-nucleotide resolution CLIP; PTB, polypyrimidine tract-binding protein; TMR, tetramethylrhodamine; TEV, tobacco etch virus.
    Figure Legend Snippet: HaloTag based GoldCLIP technology A. Schematic flow chart of GoldCLIP technology. Cells stably expressing Halo-tagged fusion RBPs are crosslinked by UV irradiation. After cell lysis, Halo-RBP complexes are then captured by magnetic beads coated with Halo ligand under native conditions and a specific 3′ linker is ligated to RNAs bound by RBPs. Following denaturing washes, purified RNAs are cloned via an iCLIP protocol for high-throughput sequencing. B. Western blot analysis showing the expression level of Halo-PTB in the HEK 293T Halo-PTB stable cells compared to endogenous PTB using a monoclonal anti-PTB antibody (BB7). Non-transfected HEK 293T cells are used as control. A diagram of Halo-PTB fusion protein is shown below. C. Localization of Halo-PTB fusion proteins in 293T cell line. HaloTag TMR ligand staining of Halo-PTB fusion protein is shown in the top panel, and immunofluorescent staining of endogenous PTB using a monoclonal PTB antibody (BB7) is shown in the bottom panel. RBP, RNA-binding protein; iCLIP, individual-nucleotide resolution CLIP; PTB, polypyrimidine tract-binding protein; TMR, tetramethylrhodamine; TEV, tobacco etch virus.

    Techniques Used: Flow Cytometry, Stable Transfection, Expressing, Irradiation, Lysis, Magnetic Beads, Purification, Clone Assay, Next-Generation Sequencing, Western Blot, Transfection, Staining, RNA Binding Assay, Cross-linking Immunoprecipitation, Binding Assay

    3) Product Images from "Thermodynamic instability of viral proteins is a pathogen-associated molecular pattern targeted by human defensins"

    Article Title: Thermodynamic instability of viral proteins is a pathogen-associated molecular pattern targeted by human defensins

    Journal: Scientific Reports

    doi: 10.1038/srep32499

    Defensins’ disulfide bonds are essential for the inhibition of TEV protease activity. ( A ) Activity of TEV protease was assessed in the absence of defensins and following the incubation of TEV protease with HNP-1 (3-fold molar excess) or RC-101 (5-fold molar excess). Cleavage of MBP-PLS (5 μM; 20:1 w/w ratio to TEV protease) was monitored by SDS-PAGE as a reduction of the full-length substrate protein MBP-PLS expressed in percent and plotted versus time. ( B , C ) Under reducing conditions (10 mM TCEP), defensins do not affect the activity ( B ) or melting profile ( C ) of TEV protease.
    Figure Legend Snippet: Defensins’ disulfide bonds are essential for the inhibition of TEV protease activity. ( A ) Activity of TEV protease was assessed in the absence of defensins and following the incubation of TEV protease with HNP-1 (3-fold molar excess) or RC-101 (5-fold molar excess). Cleavage of MBP-PLS (5 μM; 20:1 w/w ratio to TEV protease) was monitored by SDS-PAGE as a reduction of the full-length substrate protein MBP-PLS expressed in percent and plotted versus time. ( B , C ) Under reducing conditions (10 mM TCEP), defensins do not affect the activity ( B ) or melting profile ( C ) of TEV protease.

    Techniques Used: Inhibition, Activity Assay, Incubation, SDS Page

    α- and θ-Defensins facilitate unfolding of viral proteins in DSF experiments. Thermal denaturation profiles of HIV-1 proteins Gag-Δp6 (10 μM), CA (20 μM), RTp51 (10 μM), and IN (5 μM), as well as PFV IN (7 μM) and TEV protease (5 μM) were shifted toward lower temperatures in the presence of 3-fold molar excess of HNP-1 or 5-fold molar excess of RC-101 over the viral proteins. HD-5 caused destabilization of HIV-1 proteins Gag-Δp6 and CA and PFV IN. β-Defensin hBD-2 had no effect on any of the tested proteins. Human structural protein plastin-3 (PLS3) and serum IgG were not significantly affected by the defensins. See also Table 1 .
    Figure Legend Snippet: α- and θ-Defensins facilitate unfolding of viral proteins in DSF experiments. Thermal denaturation profiles of HIV-1 proteins Gag-Δp6 (10 μM), CA (20 μM), RTp51 (10 μM), and IN (5 μM), as well as PFV IN (7 μM) and TEV protease (5 μM) were shifted toward lower temperatures in the presence of 3-fold molar excess of HNP-1 or 5-fold molar excess of RC-101 over the viral proteins. HD-5 caused destabilization of HIV-1 proteins Gag-Δp6 and CA and PFV IN. β-Defensin hBD-2 had no effect on any of the tested proteins. Human structural protein plastin-3 (PLS3) and serum IgG were not significantly affected by the defensins. See also Table 1 .

    Techniques Used:

    4) Product Images from "Crystal structure of a human plasma membrane phospholipid flippase"

    Article Title: Crystal structure of a human plasma membrane phospholipid flippase

    Journal: bioRxiv

    doi: 10.1101/2019.12.23.881698

    Structural determination of ATP11C-CDC50A complex (A) Construction of TP11C and CDC50A used in crystallization. See Methods for details. (B) Purification of ATP11C-CDC50A complex. Lane 1: solubilized cell lysate, lane 2: pass-through of Flag resin, lane 3: wash fraction, lane 4: elution by Flag peptide (subjected to ATPase assay), lane 5: TEV proteinase- and endoglycosidase-treated sample, lane 6: pass-through fraction of Ni-NTA and amylose resin, lane 7: concentrated peak fractions by size-exclusion chromatography (arrowhead in the left panel). Arrowheads on the right indicated as follows, a: HSP70, b: EndoHf, c: cleaved eGFP, d: TEV proteinase. The elution profile of ATP11C-CDC50A complex by size-exclusion column chromatography is shown on the left. (C) Representative X-ray diffraction obtained from a plate-like crystal shown in D. Diffraction spots better than 3.6Å were obtained along the c * -axis, whereas these are limited to around 4∼6Å in directions along with a * - and b * -axes, thus strongly anisotropic. (D,E) Three-dimensional crystals obtained from the samples purified from Expi293 cells, showing thin, but large plate-like crystals (D). In contrast, small crystals were obtained from CDC50A-KO cells (E). (F) Data acquisition strategy. We employed normal type LithoLoops for helical scan data acquisition from large single crystals. However, because crystals showed strong anisotropy, we also collected data sets by irradiating X-ray beam from the direction perpendicular to the c -axis by using 90° bent type LithoLoops. For the small but well diffracting crystals obtained from CDC50A-KO cells, data from each individual crystals was collected for 10°. All of these crystals showed identical unit cell size and symmetry regardless of crystal morphologies and expression cell types, as seen in the histograms of unit cell dimensions (G). All diffraction data from 1,588 crystals were finally merged into a single data set (H), and used for the molecular replacement.
    Figure Legend Snippet: Structural determination of ATP11C-CDC50A complex (A) Construction of TP11C and CDC50A used in crystallization. See Methods for details. (B) Purification of ATP11C-CDC50A complex. Lane 1: solubilized cell lysate, lane 2: pass-through of Flag resin, lane 3: wash fraction, lane 4: elution by Flag peptide (subjected to ATPase assay), lane 5: TEV proteinase- and endoglycosidase-treated sample, lane 6: pass-through fraction of Ni-NTA and amylose resin, lane 7: concentrated peak fractions by size-exclusion chromatography (arrowhead in the left panel). Arrowheads on the right indicated as follows, a: HSP70, b: EndoHf, c: cleaved eGFP, d: TEV proteinase. The elution profile of ATP11C-CDC50A complex by size-exclusion column chromatography is shown on the left. (C) Representative X-ray diffraction obtained from a plate-like crystal shown in D. Diffraction spots better than 3.6Å were obtained along the c * -axis, whereas these are limited to around 4∼6Å in directions along with a * - and b * -axes, thus strongly anisotropic. (D,E) Three-dimensional crystals obtained from the samples purified from Expi293 cells, showing thin, but large plate-like crystals (D). In contrast, small crystals were obtained from CDC50A-KO cells (E). (F) Data acquisition strategy. We employed normal type LithoLoops for helical scan data acquisition from large single crystals. However, because crystals showed strong anisotropy, we also collected data sets by irradiating X-ray beam from the direction perpendicular to the c -axis by using 90° bent type LithoLoops. For the small but well diffracting crystals obtained from CDC50A-KO cells, data from each individual crystals was collected for 10°. All of these crystals showed identical unit cell size and symmetry regardless of crystal morphologies and expression cell types, as seen in the histograms of unit cell dimensions (G). All diffraction data from 1,588 crystals were finally merged into a single data set (H), and used for the molecular replacement.

    Techniques Used: Crystallization Assay, Purification, ATPase Assay, Size-exclusion Chromatography, Column Chromatography, Expressing

    Related Articles

    Amplification:

    Article Title: Deciphering the Combinatorial DNA-binding Code of the CCAAT-binding Complex and the Iron-regulatory Basic Region Leucine Zipper (bZIP) Transcription Factor HapX *
    Article Snippet: .. A cDNA fragment encoding A. nidulans HapX(1–198) (covering the CBC binding domain, basic region, and coiled-coil domain) with an extended N terminus, including a cleavage site for tobacco etch virus protease, was amplified and subcloned into the pMAL-c2X (New England Biolabs) vector. .. The resulting plasmid was transformed into E. coli Rosetta 2 (DE3) cells for overnight autoinduction.

    Purification:

    Article Title: Thermodynamic instability of viral proteins is a pathogen-associated molecular pattern targeted by human defensins
    Article Snippet: .. MBP-PLS, an actin-binding domain of PLS3 in-frame with maltose binding protein (MBP) and TEV protease cleavage site, was purified using amylose resin (New England Biolabs) according to the manufacturer instructions. .. Differential scanning fluorimetry (DSF) Viral protein samples (5–20 μM) in a 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.5) supplemented with 150 mM NaCl and 1x SYPRO Orange dye (Invitrogen) were subjected to temperature denaturation in the absence or presence of defensins.

    FLAG-tag:

    Article Title: A widespread family of heat-resistant obscure (Hero) proteins protect against protein instability and aggregation
    Article Snippet: .. pCAGEN-FlagTevSnap-DEST and pCAGEN-GFP-DEST A DNA fragment containing N-terminal FLAG tag, TEV protease recognition sequence, and SNAP tag or the GFP sequence followed by the Gateway attR sites was inserted into pCAGEN by NEBuilder HiFi DNA Assembly Master Mix (NEB). .. pCAGEN-FlagTevSnap-Ago2(WT) A DNA fragment containing a codon-optimized Drosophila Ago2 CDS sequence in pENTR/D-Ago2 ( ) was inserted into pCAGEN-FlagTevSnap -DEST using Gateway LR Clonase (Invitrogen).

    Article Title: Structures of the human PGD2 receptor CRTH2 reveal novel mechanisms for ligand recognition
    Article Snippet: .. After treatment with TEV protease, 3C protease and PNGase F (NEB) at 4°C overnight, the N-terminal Flag-tag, th e C-terminal His-tag and glycosylation were removed. .. The monodispersed receptor was collected after size-exclusion chromatography using a Superdex 200 Increase column (GE healthcare).

    Incubation:

    Article Title: Crystal structure of a human plasma membrane phospholipid flippase
    Article Snippet: .. Eluted proteins were incubated with TEV protease and MBP-fusion endoglycosidase (EndoHf, New England Biolabs) at 4 °C overnight. .. Released affinity tags containing Flag-EGFP were removed from the mixture by a Ni-NTA resin (QIAGEN).

    Sequencing:

    Article Title: A widespread family of heat-resistant obscure (Hero) proteins protect against protein instability and aggregation
    Article Snippet: .. pCAGEN-FlagTevSnap-DEST and pCAGEN-GFP-DEST A DNA fragment containing N-terminal FLAG tag, TEV protease recognition sequence, and SNAP tag or the GFP sequence followed by the Gateway attR sites was inserted into pCAGEN by NEBuilder HiFi DNA Assembly Master Mix (NEB). .. pCAGEN-FlagTevSnap-Ago2(WT) A DNA fragment containing a codon-optimized Drosophila Ago2 CDS sequence in pENTR/D-Ago2 ( ) was inserted into pCAGEN-FlagTevSnap -DEST using Gateway LR Clonase (Invitrogen).

    Binding Assay:

    Article Title: Deciphering the Combinatorial DNA-binding Code of the CCAAT-binding Complex and the Iron-regulatory Basic Region Leucine Zipper (bZIP) Transcription Factor HapX *
    Article Snippet: .. A cDNA fragment encoding A. nidulans HapX(1–198) (covering the CBC binding domain, basic region, and coiled-coil domain) with an extended N terminus, including a cleavage site for tobacco etch virus protease, was amplified and subcloned into the pMAL-c2X (New England Biolabs) vector. .. The resulting plasmid was transformed into E. coli Rosetta 2 (DE3) cells for overnight autoinduction.

    Article Title: Thermodynamic instability of viral proteins is a pathogen-associated molecular pattern targeted by human defensins
    Article Snippet: .. MBP-PLS, an actin-binding domain of PLS3 in-frame with maltose binding protein (MBP) and TEV protease cleavage site, was purified using amylose resin (New England Biolabs) according to the manufacturer instructions. .. Differential scanning fluorimetry (DSF) Viral protein samples (5–20 μM) in a 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.5) supplemented with 150 mM NaCl and 1x SYPRO Orange dye (Invitrogen) were subjected to temperature denaturation in the absence or presence of defensins.

    Plasmid Preparation:

    Article Title: Deciphering the Combinatorial DNA-binding Code of the CCAAT-binding Complex and the Iron-regulatory Basic Region Leucine Zipper (bZIP) Transcription Factor HapX *
    Article Snippet: .. A cDNA fragment encoding A. nidulans HapX(1–198) (covering the CBC binding domain, basic region, and coiled-coil domain) with an extended N terminus, including a cleavage site for tobacco etch virus protease, was amplified and subcloned into the pMAL-c2X (New England Biolabs) vector. .. The resulting plasmid was transformed into E. coli Rosetta 2 (DE3) cells for overnight autoinduction.

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    New England Biolabs tev protease recognition sequence
    Activity of heat-soluble proteins and nucleonic acids on Ago2 molecular behavior and LDH desiccation. (A) Schematic representation of <t>FLAG-TEV-SNAP-Ago2</t> immunopurified on magnetic beads. (B) FLAG-TEV-SNAP-Ago2 was immunopurified onto magnetics beads via anti-FLAG antibody and then the FLAG tag was cleaved off by TEV protease in buffer, crude lysates or their boiled supernatants from fly S2 or human HEK293T cells. PK indicates that the boiled supernatant had been mostly deproteinized by Proteinase K in advance. BZ indicates that <t>DNA</t> and RNA in boiled supernatant had been degraded by Benzonase. BZ/PK indicates that DNA and RNA had been degraded by Benzonase, followed by the deproteinization by Proteinase K. Eluted Ago2 (top) and Ago2 still remaining on the beads (middle) were visualized by a red fluorescent dye covalently attached to the SNAP tag. The values on the top indicate relative amounts of eluted Ago2, normalized to that with S2 crude cell lysate. Proteins remaining in the boiled supernatants have an activity to promote Ago2 elution. (C) Small RNA pull-down assay for pre-Ago2-RISC and mature Ago2-RISC assembled in the reconstitution system, containing a 32 P-radiolabeled small RNA duplex, Ago2, Dicer-2/R2D2, and the Hsp70/Hsp90 chaperone machinery. Supplementation of the boiled supernatants from S2 or HEK293T cells promoted the formation of both pre- and mature Ago2-RISC. (D) Quantification of (C). Data represent means ± SD from 3 independent experiments. (E) Heat-soluble proteins in the boiled supernatants protect the LDH activity from desiccation. LDH mixed with the indicated boiled supernatants or buffer was dried up overnight and the remaining LDH activities were measured. Data represent means ± SD from 3 independent experiments.
    Tev Protease Recognition Sequence, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tev protease recognition sequence/product/New England Biolabs
    Average 99 stars, based on 27 article reviews
    Price from $9.99 to $1999.99
    tev protease recognition sequence - by Bioz Stars, 2020-07
    99/100 stars
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    Activity of heat-soluble proteins and nucleonic acids on Ago2 molecular behavior and LDH desiccation. (A) Schematic representation of FLAG-TEV-SNAP-Ago2 immunopurified on magnetic beads. (B) FLAG-TEV-SNAP-Ago2 was immunopurified onto magnetics beads via anti-FLAG antibody and then the FLAG tag was cleaved off by TEV protease in buffer, crude lysates or their boiled supernatants from fly S2 or human HEK293T cells. PK indicates that the boiled supernatant had been mostly deproteinized by Proteinase K in advance. BZ indicates that DNA and RNA in boiled supernatant had been degraded by Benzonase. BZ/PK indicates that DNA and RNA had been degraded by Benzonase, followed by the deproteinization by Proteinase K. Eluted Ago2 (top) and Ago2 still remaining on the beads (middle) were visualized by a red fluorescent dye covalently attached to the SNAP tag. The values on the top indicate relative amounts of eluted Ago2, normalized to that with S2 crude cell lysate. Proteins remaining in the boiled supernatants have an activity to promote Ago2 elution. (C) Small RNA pull-down assay for pre-Ago2-RISC and mature Ago2-RISC assembled in the reconstitution system, containing a 32 P-radiolabeled small RNA duplex, Ago2, Dicer-2/R2D2, and the Hsp70/Hsp90 chaperone machinery. Supplementation of the boiled supernatants from S2 or HEK293T cells promoted the formation of both pre- and mature Ago2-RISC. (D) Quantification of (C). Data represent means ± SD from 3 independent experiments. (E) Heat-soluble proteins in the boiled supernatants protect the LDH activity from desiccation. LDH mixed with the indicated boiled supernatants or buffer was dried up overnight and the remaining LDH activities were measured. Data represent means ± SD from 3 independent experiments.

    Journal: bioRxiv

    Article Title: A widespread family of heat-resistant obscure (Hero) proteins protect against protein instability and aggregation

    doi: 10.1101/816124

    Figure Lengend Snippet: Activity of heat-soluble proteins and nucleonic acids on Ago2 molecular behavior and LDH desiccation. (A) Schematic representation of FLAG-TEV-SNAP-Ago2 immunopurified on magnetic beads. (B) FLAG-TEV-SNAP-Ago2 was immunopurified onto magnetics beads via anti-FLAG antibody and then the FLAG tag was cleaved off by TEV protease in buffer, crude lysates or their boiled supernatants from fly S2 or human HEK293T cells. PK indicates that the boiled supernatant had been mostly deproteinized by Proteinase K in advance. BZ indicates that DNA and RNA in boiled supernatant had been degraded by Benzonase. BZ/PK indicates that DNA and RNA had been degraded by Benzonase, followed by the deproteinization by Proteinase K. Eluted Ago2 (top) and Ago2 still remaining on the beads (middle) were visualized by a red fluorescent dye covalently attached to the SNAP tag. The values on the top indicate relative amounts of eluted Ago2, normalized to that with S2 crude cell lysate. Proteins remaining in the boiled supernatants have an activity to promote Ago2 elution. (C) Small RNA pull-down assay for pre-Ago2-RISC and mature Ago2-RISC assembled in the reconstitution system, containing a 32 P-radiolabeled small RNA duplex, Ago2, Dicer-2/R2D2, and the Hsp70/Hsp90 chaperone machinery. Supplementation of the boiled supernatants from S2 or HEK293T cells promoted the formation of both pre- and mature Ago2-RISC. (D) Quantification of (C). Data represent means ± SD from 3 independent experiments. (E) Heat-soluble proteins in the boiled supernatants protect the LDH activity from desiccation. LDH mixed with the indicated boiled supernatants or buffer was dried up overnight and the remaining LDH activities were measured. Data represent means ± SD from 3 independent experiments.

    Article Snippet: pCAGEN-FlagTevSnap-DEST and pCAGEN-GFP-DEST A DNA fragment containing N-terminal FLAG tag, TEV protease recognition sequence, and SNAP tag or the GFP sequence followed by the Gateway attR sites was inserted into pCAGEN by NEBuilder HiFi DNA Assembly Master Mix (NEB).

    Techniques: Activity Assay, Magnetic Beads, FLAG-tag, Pull Down Assay

    Isolation of PTB – RNA complexes without gel purifications A. Comparison of Halo fusion proteins (input vs . unbound fractions) for the indicated samples. The Halo fusion proteins are labeled with HaloTag Alexa Fluor® 660 ligand and resolved on SDS–PAGE. Bottom panel, Western blot analysis of tubulin as a loading control. B. Western blot analysis showing that similar amounts of PTB–RNA complexes are released by TEV protease following the indicated washing conditions after Halo bead isolation from the HEK 293T Halo-PTB stable cells. Non-transfected HEK 293T cells are used as control. C. Autoradiogram (upper panel) of 32 P-labeled RNA crosslinked to PTB purified by HaloTag and released by TEV protease. RNA–protein complexes of 60–70 kD are seen only with the UV crosslinking condition. Western blot analysis (bottom panel) of TEV-released PTB proteins from equal amounts of samples prepared from the lysate of the Halo-PTB stable cell line.

    Journal: Genomics, Proteomics & Bioinformatics

    Article Title: GoldCLIP: Gel-omitted Ligation-dependent CLIP

    doi: 10.1016/j.gpb.2018.04.003

    Figure Lengend Snippet: Isolation of PTB – RNA complexes without gel purifications A. Comparison of Halo fusion proteins (input vs . unbound fractions) for the indicated samples. The Halo fusion proteins are labeled with HaloTag Alexa Fluor® 660 ligand and resolved on SDS–PAGE. Bottom panel, Western blot analysis of tubulin as a loading control. B. Western blot analysis showing that similar amounts of PTB–RNA complexes are released by TEV protease following the indicated washing conditions after Halo bead isolation from the HEK 293T Halo-PTB stable cells. Non-transfected HEK 293T cells are used as control. C. Autoradiogram (upper panel) of 32 P-labeled RNA crosslinked to PTB purified by HaloTag and released by TEV protease. RNA–protein complexes of 60–70 kD are seen only with the UV crosslinking condition. Western blot analysis (bottom panel) of TEV-released PTB proteins from equal amounts of samples prepared from the lysate of the Halo-PTB stable cell line.

    Article Snippet: Finally, PTB–RNA complexes were cleaved off the beads by TEV protease and digested with protease K (catalog No. P8102S; New England Biolabs) at 37 °C for 30 min.

    Techniques: Isolation, Labeling, SDS Page, Western Blot, Transfection, Purification, Stable Transfection

    HaloTag based GoldCLIP technology A. Schematic flow chart of GoldCLIP technology. Cells stably expressing Halo-tagged fusion RBPs are crosslinked by UV irradiation. After cell lysis, Halo-RBP complexes are then captured by magnetic beads coated with Halo ligand under native conditions and a specific 3′ linker is ligated to RNAs bound by RBPs. Following denaturing washes, purified RNAs are cloned via an iCLIP protocol for high-throughput sequencing. B. Western blot analysis showing the expression level of Halo-PTB in the HEK 293T Halo-PTB stable cells compared to endogenous PTB using a monoclonal anti-PTB antibody (BB7). Non-transfected HEK 293T cells are used as control. A diagram of Halo-PTB fusion protein is shown below. C. Localization of Halo-PTB fusion proteins in 293T cell line. HaloTag TMR ligand staining of Halo-PTB fusion protein is shown in the top panel, and immunofluorescent staining of endogenous PTB using a monoclonal PTB antibody (BB7) is shown in the bottom panel. RBP, RNA-binding protein; iCLIP, individual-nucleotide resolution CLIP; PTB, polypyrimidine tract-binding protein; TMR, tetramethylrhodamine; TEV, tobacco etch virus.

    Journal: Genomics, Proteomics & Bioinformatics

    Article Title: GoldCLIP: Gel-omitted Ligation-dependent CLIP

    doi: 10.1016/j.gpb.2018.04.003

    Figure Lengend Snippet: HaloTag based GoldCLIP technology A. Schematic flow chart of GoldCLIP technology. Cells stably expressing Halo-tagged fusion RBPs are crosslinked by UV irradiation. After cell lysis, Halo-RBP complexes are then captured by magnetic beads coated with Halo ligand under native conditions and a specific 3′ linker is ligated to RNAs bound by RBPs. Following denaturing washes, purified RNAs are cloned via an iCLIP protocol for high-throughput sequencing. B. Western blot analysis showing the expression level of Halo-PTB in the HEK 293T Halo-PTB stable cells compared to endogenous PTB using a monoclonal anti-PTB antibody (BB7). Non-transfected HEK 293T cells are used as control. A diagram of Halo-PTB fusion protein is shown below. C. Localization of Halo-PTB fusion proteins in 293T cell line. HaloTag TMR ligand staining of Halo-PTB fusion protein is shown in the top panel, and immunofluorescent staining of endogenous PTB using a monoclonal PTB antibody (BB7) is shown in the bottom panel. RBP, RNA-binding protein; iCLIP, individual-nucleotide resolution CLIP; PTB, polypyrimidine tract-binding protein; TMR, tetramethylrhodamine; TEV, tobacco etch virus.

    Article Snippet: Finally, PTB–RNA complexes were cleaved off the beads by TEV protease and digested with protease K (catalog No. P8102S; New England Biolabs) at 37 °C for 30 min.

    Techniques: Flow Cytometry, Stable Transfection, Expressing, Irradiation, Lysis, Magnetic Beads, Purification, Clone Assay, Next-Generation Sequencing, Western Blot, Transfection, Staining, RNA Binding Assay, Cross-linking Immunoprecipitation, Binding Assay

    Defensins’ disulfide bonds are essential for the inhibition of TEV protease activity. ( A ) Activity of TEV protease was assessed in the absence of defensins and following the incubation of TEV protease with HNP-1 (3-fold molar excess) or RC-101 (5-fold molar excess). Cleavage of MBP-PLS (5 μM; 20:1 w/w ratio to TEV protease) was monitored by SDS-PAGE as a reduction of the full-length substrate protein MBP-PLS expressed in percent and plotted versus time. ( B , C ) Under reducing conditions (10 mM TCEP), defensins do not affect the activity ( B ) or melting profile ( C ) of TEV protease.

    Journal: Scientific Reports

    Article Title: Thermodynamic instability of viral proteins is a pathogen-associated molecular pattern targeted by human defensins

    doi: 10.1038/srep32499

    Figure Lengend Snippet: Defensins’ disulfide bonds are essential for the inhibition of TEV protease activity. ( A ) Activity of TEV protease was assessed in the absence of defensins and following the incubation of TEV protease with HNP-1 (3-fold molar excess) or RC-101 (5-fold molar excess). Cleavage of MBP-PLS (5 μM; 20:1 w/w ratio to TEV protease) was monitored by SDS-PAGE as a reduction of the full-length substrate protein MBP-PLS expressed in percent and plotted versus time. ( B , C ) Under reducing conditions (10 mM TCEP), defensins do not affect the activity ( B ) or melting profile ( C ) of TEV protease.

    Article Snippet: MBP-PLS, an actin-binding domain of PLS3 in-frame with maltose binding protein (MBP) and TEV protease cleavage site, was purified using amylose resin (New England Biolabs) according to the manufacturer instructions.

    Techniques: Inhibition, Activity Assay, Incubation, SDS Page

    α- and θ-Defensins facilitate unfolding of viral proteins in DSF experiments. Thermal denaturation profiles of HIV-1 proteins Gag-Δp6 (10 μM), CA (20 μM), RTp51 (10 μM), and IN (5 μM), as well as PFV IN (7 μM) and TEV protease (5 μM) were shifted toward lower temperatures in the presence of 3-fold molar excess of HNP-1 or 5-fold molar excess of RC-101 over the viral proteins. HD-5 caused destabilization of HIV-1 proteins Gag-Δp6 and CA and PFV IN. β-Defensin hBD-2 had no effect on any of the tested proteins. Human structural protein plastin-3 (PLS3) and serum IgG were not significantly affected by the defensins. See also Table 1 .

    Journal: Scientific Reports

    Article Title: Thermodynamic instability of viral proteins is a pathogen-associated molecular pattern targeted by human defensins

    doi: 10.1038/srep32499

    Figure Lengend Snippet: α- and θ-Defensins facilitate unfolding of viral proteins in DSF experiments. Thermal denaturation profiles of HIV-1 proteins Gag-Δp6 (10 μM), CA (20 μM), RTp51 (10 μM), and IN (5 μM), as well as PFV IN (7 μM) and TEV protease (5 μM) were shifted toward lower temperatures in the presence of 3-fold molar excess of HNP-1 or 5-fold molar excess of RC-101 over the viral proteins. HD-5 caused destabilization of HIV-1 proteins Gag-Δp6 and CA and PFV IN. β-Defensin hBD-2 had no effect on any of the tested proteins. Human structural protein plastin-3 (PLS3) and serum IgG were not significantly affected by the defensins. See also Table 1 .

    Article Snippet: MBP-PLS, an actin-binding domain of PLS3 in-frame with maltose binding protein (MBP) and TEV protease cleavage site, was purified using amylose resin (New England Biolabs) according to the manufacturer instructions.

    Techniques:

    Structural determination of ATP11C-CDC50A complex (A) Construction of TP11C and CDC50A used in crystallization. See Methods for details. (B) Purification of ATP11C-CDC50A complex. Lane 1: solubilized cell lysate, lane 2: pass-through of Flag resin, lane 3: wash fraction, lane 4: elution by Flag peptide (subjected to ATPase assay), lane 5: TEV proteinase- and endoglycosidase-treated sample, lane 6: pass-through fraction of Ni-NTA and amylose resin, lane 7: concentrated peak fractions by size-exclusion chromatography (arrowhead in the left panel). Arrowheads on the right indicated as follows, a: HSP70, b: EndoHf, c: cleaved eGFP, d: TEV proteinase. The elution profile of ATP11C-CDC50A complex by size-exclusion column chromatography is shown on the left. (C) Representative X-ray diffraction obtained from a plate-like crystal shown in D. Diffraction spots better than 3.6Å were obtained along the c * -axis, whereas these are limited to around 4∼6Å in directions along with a * - and b * -axes, thus strongly anisotropic. (D,E) Three-dimensional crystals obtained from the samples purified from Expi293 cells, showing thin, but large plate-like crystals (D). In contrast, small crystals were obtained from CDC50A-KO cells (E). (F) Data acquisition strategy. We employed normal type LithoLoops for helical scan data acquisition from large single crystals. However, because crystals showed strong anisotropy, we also collected data sets by irradiating X-ray beam from the direction perpendicular to the c -axis by using 90° bent type LithoLoops. For the small but well diffracting crystals obtained from CDC50A-KO cells, data from each individual crystals was collected for 10°. All of these crystals showed identical unit cell size and symmetry regardless of crystal morphologies and expression cell types, as seen in the histograms of unit cell dimensions (G). All diffraction data from 1,588 crystals were finally merged into a single data set (H), and used for the molecular replacement.

    Journal: bioRxiv

    Article Title: Crystal structure of a human plasma membrane phospholipid flippase

    doi: 10.1101/2019.12.23.881698

    Figure Lengend Snippet: Structural determination of ATP11C-CDC50A complex (A) Construction of TP11C and CDC50A used in crystallization. See Methods for details. (B) Purification of ATP11C-CDC50A complex. Lane 1: solubilized cell lysate, lane 2: pass-through of Flag resin, lane 3: wash fraction, lane 4: elution by Flag peptide (subjected to ATPase assay), lane 5: TEV proteinase- and endoglycosidase-treated sample, lane 6: pass-through fraction of Ni-NTA and amylose resin, lane 7: concentrated peak fractions by size-exclusion chromatography (arrowhead in the left panel). Arrowheads on the right indicated as follows, a: HSP70, b: EndoHf, c: cleaved eGFP, d: TEV proteinase. The elution profile of ATP11C-CDC50A complex by size-exclusion column chromatography is shown on the left. (C) Representative X-ray diffraction obtained from a plate-like crystal shown in D. Diffraction spots better than 3.6Å were obtained along the c * -axis, whereas these are limited to around 4∼6Å in directions along with a * - and b * -axes, thus strongly anisotropic. (D,E) Three-dimensional crystals obtained from the samples purified from Expi293 cells, showing thin, but large plate-like crystals (D). In contrast, small crystals were obtained from CDC50A-KO cells (E). (F) Data acquisition strategy. We employed normal type LithoLoops for helical scan data acquisition from large single crystals. However, because crystals showed strong anisotropy, we also collected data sets by irradiating X-ray beam from the direction perpendicular to the c -axis by using 90° bent type LithoLoops. For the small but well diffracting crystals obtained from CDC50A-KO cells, data from each individual crystals was collected for 10°. All of these crystals showed identical unit cell size and symmetry regardless of crystal morphologies and expression cell types, as seen in the histograms of unit cell dimensions (G). All diffraction data from 1,588 crystals were finally merged into a single data set (H), and used for the molecular replacement.

    Article Snippet: Eluted proteins were incubated with TEV protease and MBP-fusion endoglycosidase (EndoHf, New England Biolabs) at 4 °C overnight.

    Techniques: Crystallization Assay, Purification, ATPase Assay, Size-exclusion Chromatography, Column Chromatography, Expressing