pngase f  (New England Biolabs)


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    Name:
    PNGase F native
    Description:
    PNGase F native 75 000 units
    Catalog Number:
    P0704L
    Price:
    754
    Category:
    Glycosidases
    Size:
    75 000 units
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    New England Biolabs pngase f
    PNGase F native
    PNGase F native 75 000 units
    https://www.bioz.com/result/pngase f/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pngase f - by Bioz Stars, 2021-09
    98/100 stars

    Images

    1) Product Images from "A novel probe to assess cytosolic entry of exogenous proteins"

    Article Title: A novel probe to assess cytosolic entry of exogenous proteins

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05556-z

    Generation and characterization of ddRLuc-Fc. a . b , c ddRLuc-Fc shows proteasome inhibition- and NGLY1 deglycosylation-dependent activity. ddRLuc-Fc-transfected 293T cells were incubated with DMSO alone, 200 nM Epox in DMSO, or a combination of 200 nM Epox and 20 μm zVAD in DMSO, at 37 °C for 6 h. In b equal amounts of cell lysates were tested for luciferase activity as described in Methods; activities are normalized to that seen with Epox only, which is set to 100 (bars represent the mean +/−s.d. of one representative experiment with triplicates). In c intact cells were examined by luminescence microscopy (BF: bright field; Lumin: luminescence; scale bar = 50 μm; brightness bar inserts: 0 to 255, linear scale). d Purification of recombinant ddRLuc-Fc. Coomassie Blue staining (left panel) and western blot (right panel) of purified ddRLuc-Fc. Lane 1: purified ddRLuc-Fc in reducing sample buffer; Lane 2: purified ddRLuc-Fc in non-reducing sample buffer; Lane 3: recombinant RLuc in reducing sample buffer. Red arrow indicates glycosylated ddRLuc-Fc; blue arrow indicates unglycosylated ddRLuc-Fc; yellow arrow indicates ddRLuc-Fc dimer. e , f In vitro deglycosylation by PNGase F or Endo H followed by SDS gel electrophoresis ( e , upper panel: Coomassie Blue staining; lower panel: western blot) or luciferase assays ( f , points represent the mean +/−s.d. of one representative experiment with triplicates). g Relative stabilities of glycosylated (blue line) and deglycosylated (red line) ddRLuc-Fc. Samples were incubated at 37 °C for various times and immediately examined by luciferase assays. The horizontal dashed line refers to 50% activity compared to initial activity, and the vertical dash line indicates the t 1/2 . Points represent the mean +/− s.d. of one representative experiment with triplicates. In b – g , representative data from three independent experiments are shown
    Figure Legend Snippet: Generation and characterization of ddRLuc-Fc. a . b , c ddRLuc-Fc shows proteasome inhibition- and NGLY1 deglycosylation-dependent activity. ddRLuc-Fc-transfected 293T cells were incubated with DMSO alone, 200 nM Epox in DMSO, or a combination of 200 nM Epox and 20 μm zVAD in DMSO, at 37 °C for 6 h. In b equal amounts of cell lysates were tested for luciferase activity as described in Methods; activities are normalized to that seen with Epox only, which is set to 100 (bars represent the mean +/−s.d. of one representative experiment with triplicates). In c intact cells were examined by luminescence microscopy (BF: bright field; Lumin: luminescence; scale bar = 50 μm; brightness bar inserts: 0 to 255, linear scale). d Purification of recombinant ddRLuc-Fc. Coomassie Blue staining (left panel) and western blot (right panel) of purified ddRLuc-Fc. Lane 1: purified ddRLuc-Fc in reducing sample buffer; Lane 2: purified ddRLuc-Fc in non-reducing sample buffer; Lane 3: recombinant RLuc in reducing sample buffer. Red arrow indicates glycosylated ddRLuc-Fc; blue arrow indicates unglycosylated ddRLuc-Fc; yellow arrow indicates ddRLuc-Fc dimer. e , f In vitro deglycosylation by PNGase F or Endo H followed by SDS gel electrophoresis ( e , upper panel: Coomassie Blue staining; lower panel: western blot) or luciferase assays ( f , points represent the mean +/−s.d. of one representative experiment with triplicates). g Relative stabilities of glycosylated (blue line) and deglycosylated (red line) ddRLuc-Fc. Samples were incubated at 37 °C for various times and immediately examined by luciferase assays. The horizontal dashed line refers to 50% activity compared to initial activity, and the vertical dash line indicates the t 1/2 . Points represent the mean +/− s.d. of one representative experiment with triplicates. In b – g , representative data from three independent experiments are shown

    Techniques Used: Inhibition, Activity Assay, Transfection, Incubation, Luciferase, Microscopy, Purification, Recombinant, Staining, Western Blot, In Vitro, SDS-Gel, Electrophoresis

    2) Product Images from "Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines"

    Article Title: Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0193624

    The biochemical properties of TPO protein expressed in breast tissues (A and B) and breast-derived cell lines (C).  (A) N-linked glycan content in TPO expressed in breast tissues. Total cell extract was digested with PNGase F, then subjected to 8% SDS-PAGE, followed by Western blotting, and probing with TPO-specific mAb 47 monoclonal antibody. Controls were processed under the same conditions as the samples except that no enzyme was added. One representative immunoblot out of at least three independent experiments is shown. (B) Enzymatic activity of TPO expressed in breast tissues. Tissue lysate was incubated with TPO-specific mAb A4, then protein A agarose was added to precipitate immune complexes. TPO-antibody complexes bound to agarose were incubated with luminol in the presence of hydrogen peroxide. The intensity of luminescencent signal was measured and results were expressed as relative light units (RLU). As positive control, TPO immunoprecipitated from human thyroid tissue lysate (Graves’ disease case) was used to measure luminol oxidation. Agarose A incubated with mAb A4 alone (lysate omitted) was used as negative control. One representative of three independent experiments is shown. (C) TPO protein expression in breast epithelial normal (184A1) and cancer cell lines (MCF-7 and MDA-MB-231). Western blotting was used to detect TPO protein presence. The specificity of the reaction was verified by preabsorption of ab76935 antibody with the excess of highly purified human TPO. NTHY was used as a positive control. β-actin-specific Ab was used as a loading control. BN: peri-tumoral breast tissue; BC: breast cancer tissue; G-B: Graves’ disease thyroid tissue; NTHY: NTHY-ori 3–1 cell line; PNGase F: Peptide-N-Glycosidase F; RLU: relative light units.
    Figure Legend Snippet: The biochemical properties of TPO protein expressed in breast tissues (A and B) and breast-derived cell lines (C). (A) N-linked glycan content in TPO expressed in breast tissues. Total cell extract was digested with PNGase F, then subjected to 8% SDS-PAGE, followed by Western blotting, and probing with TPO-specific mAb 47 monoclonal antibody. Controls were processed under the same conditions as the samples except that no enzyme was added. One representative immunoblot out of at least three independent experiments is shown. (B) Enzymatic activity of TPO expressed in breast tissues. Tissue lysate was incubated with TPO-specific mAb A4, then protein A agarose was added to precipitate immune complexes. TPO-antibody complexes bound to agarose were incubated with luminol in the presence of hydrogen peroxide. The intensity of luminescencent signal was measured and results were expressed as relative light units (RLU). As positive control, TPO immunoprecipitated from human thyroid tissue lysate (Graves’ disease case) was used to measure luminol oxidation. Agarose A incubated with mAb A4 alone (lysate omitted) was used as negative control. One representative of three independent experiments is shown. (C) TPO protein expression in breast epithelial normal (184A1) and cancer cell lines (MCF-7 and MDA-MB-231). Western blotting was used to detect TPO protein presence. The specificity of the reaction was verified by preabsorption of ab76935 antibody with the excess of highly purified human TPO. NTHY was used as a positive control. β-actin-specific Ab was used as a loading control. BN: peri-tumoral breast tissue; BC: breast cancer tissue; G-B: Graves’ disease thyroid tissue; NTHY: NTHY-ori 3–1 cell line; PNGase F: Peptide-N-Glycosidase F; RLU: relative light units.

    Techniques Used: Derivative Assay, SDS Page, Western Blot, Activity Assay, Incubation, Positive Control, Immunoprecipitation, Negative Control, Expressing, Multiple Displacement Amplification, Purification

    3) Product Images from "A Disease-causing Mutation Illuminates the Protein Membrane Topology of the Kidney-expressed Prohibitin Homology (PHB) Domain Protein Podocin *"

    Article Title: A Disease-causing Mutation Illuminates the Protein Membrane Topology of the Kidney-expressed Prohibitin Homology (PHB) Domain Protein Podocin *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.521773

    The disease-causing podocin mutation P118L and its mouse equivalent P120L are N -glycosylated. A , B , and D , FLAG- or V5-tagged mouse and human podocin constructs were transiently expressed in HEK 293T cells. Cell lysates were analyzed by Western blot analysis using an antibody against the tag. A , wild-type podocin shows a protein band of the expected molecular weight, whereas podocin with the P→L mutation shows one (mouse) or two (human) additional band(s) of higher molecular weight. B , after PNGase F treatment of cell lysates, the additional bands of podocin P→L disappear. C , Schematic of mouse and human podocin depicting the hydrophobic region ( white ), the PHB domain ( dark gray ), the position of the patient mutation ( P120L/P118L ), and the potential sites of N -glycosylation ( N ). D , site-directed mutagenesis of the putative N -glycosylation site of mouse podocin Asn-201 into Gln prevents glycosylation. Mutation of one potential N -glycosylation site of human podocin (Asn-199 or Asn-355, respectively) removes the top band of the two additional bands. Mutation of both Asn residues removes both extra bands.
    Figure Legend Snippet: The disease-causing podocin mutation P118L and its mouse equivalent P120L are N -glycosylated. A , B , and D , FLAG- or V5-tagged mouse and human podocin constructs were transiently expressed in HEK 293T cells. Cell lysates were analyzed by Western blot analysis using an antibody against the tag. A , wild-type podocin shows a protein band of the expected molecular weight, whereas podocin with the P→L mutation shows one (mouse) or two (human) additional band(s) of higher molecular weight. B , after PNGase F treatment of cell lysates, the additional bands of podocin P→L disappear. C , Schematic of mouse and human podocin depicting the hydrophobic region ( white ), the PHB domain ( dark gray ), the position of the patient mutation ( P120L/P118L ), and the potential sites of N -glycosylation ( N ). D , site-directed mutagenesis of the putative N -glycosylation site of mouse podocin Asn-201 into Gln prevents glycosylation. Mutation of one potential N -glycosylation site of human podocin (Asn-199 or Asn-355, respectively) removes the top band of the two additional bands. Mutation of both Asn residues removes both extra bands.

    Techniques Used: Mutagenesis, Construct, Western Blot, Molecular Weight

    Mass spectrometric analysis of mouse podocin P120L . A , theoretical peptides and predicted masses containing the potential glycosylation site Asn-201. Deglycosylation by PNGase F treatment results in conversion of asparagine ( N ) to aspartic acid ( D ). Because of the presence of a methionine at position 1, these peptides can also be present in oxidized form. B , MALDI MS of the two Coomassie-stained bands containing podocin P120L . Following in-gel tryptic digestion, the samples were treated with PNGase F as indicated. After PNGase F treatment, the upper band contained two peptide masses corresponding to the deglycosylated form, whereas masses corresponding to non-glycosylated peptides were not detected. In the respective control sample (no PNGase F treatment), no podocin peptides were detected in this mass region. Both results suggest glycosylation of the protein at site Asn-201 in the upper band . The lower band contained in the PNGase F-treated sample as well as in the untreated sample only peptide masses that correspond to the non-glycosylated peptide (in oxidized and non-oxidized form), indicating that, in the lower band , the protein was not glycosylated at site Asn-201. C , to analyze the amino acid sequence of the peptide masses detected in MALDI MS, aliquots of the remaining samples were subjected to nano LC ESI-MS/MS analysis. Peptides identified by ESI-MS/MS confirmed that the masses correspond to podocin P120L peptides containing Asn-201 in non- and deglycosylated forms, respectively. Detected ion series in MS2 spectra are indicated in red (y ions) or blue (b ions).
    Figure Legend Snippet: Mass spectrometric analysis of mouse podocin P120L . A , theoretical peptides and predicted masses containing the potential glycosylation site Asn-201. Deglycosylation by PNGase F treatment results in conversion of asparagine ( N ) to aspartic acid ( D ). Because of the presence of a methionine at position 1, these peptides can also be present in oxidized form. B , MALDI MS of the two Coomassie-stained bands containing podocin P120L . Following in-gel tryptic digestion, the samples were treated with PNGase F as indicated. After PNGase F treatment, the upper band contained two peptide masses corresponding to the deglycosylated form, whereas masses corresponding to non-glycosylated peptides were not detected. In the respective control sample (no PNGase F treatment), no podocin peptides were detected in this mass region. Both results suggest glycosylation of the protein at site Asn-201 in the upper band . The lower band contained in the PNGase F-treated sample as well as in the untreated sample only peptide masses that correspond to the non-glycosylated peptide (in oxidized and non-oxidized form), indicating that, in the lower band , the protein was not glycosylated at site Asn-201. C , to analyze the amino acid sequence of the peptide masses detected in MALDI MS, aliquots of the remaining samples were subjected to nano LC ESI-MS/MS analysis. Peptides identified by ESI-MS/MS confirmed that the masses correspond to podocin P120L peptides containing Asn-201 in non- and deglycosylated forms, respectively. Detected ion series in MS2 spectra are indicated in red (y ions) or blue (b ions).

    Techniques Used: Mass Spectrometry, Staining, Sequencing

    4) Product Images from "Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines"

    Article Title: Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0193624

    The biochemical properties of TPO protein expressed in breast tissues (A and B) and breast-derived cell lines (C).  (A) N-linked glycan content in TPO expressed in breast tissues. Total cell extract was digested with PNGase F, then subjected to 8% SDS-PAGE, followed by Western blotting, and probing with TPO-specific mAb 47 monoclonal antibody. Controls were processed under the same conditions as the samples except that no enzyme was added. One representative immunoblot out of at least three independent experiments is shown. (B) Enzymatic activity of TPO expressed in breast tissues. Tissue lysate was incubated with TPO-specific mAb A4, then protein A agarose was added to precipitate immune complexes. TPO-antibody complexes bound to agarose were incubated with luminol in the presence of hydrogen peroxide. The intensity of luminescencent signal was measured and results were expressed as relative light units (RLU). As positive control, TPO immunoprecipitated from human thyroid tissue lysate (Graves’ disease case) was used to measure luminol oxidation. Agarose A incubated with mAb A4 alone (lysate omitted) was used as negative control. One representative of three independent experiments is shown. (C) TPO protein expression in breast epithelial normal (184A1) and cancer cell lines (MCF-7 and MDA-MB-231). Western blotting was used to detect TPO protein presence. The specificity of the reaction was verified by preabsorption of ab76935 antibody with the excess of highly purified human TPO. NTHY was used as a positive control. β-actin-specific Ab was used as a loading control. BN: peri-tumoral breast tissue; BC: breast cancer tissue; G-B: Graves’ disease thyroid tissue; NTHY: NTHY-ori 3–1 cell line; PNGase F: Peptide-N-Glycosidase F; RLU: relative light units.
    Figure Legend Snippet: The biochemical properties of TPO protein expressed in breast tissues (A and B) and breast-derived cell lines (C). (A) N-linked glycan content in TPO expressed in breast tissues. Total cell extract was digested with PNGase F, then subjected to 8% SDS-PAGE, followed by Western blotting, and probing with TPO-specific mAb 47 monoclonal antibody. Controls were processed under the same conditions as the samples except that no enzyme was added. One representative immunoblot out of at least three independent experiments is shown. (B) Enzymatic activity of TPO expressed in breast tissues. Tissue lysate was incubated with TPO-specific mAb A4, then protein A agarose was added to precipitate immune complexes. TPO-antibody complexes bound to agarose were incubated with luminol in the presence of hydrogen peroxide. The intensity of luminescencent signal was measured and results were expressed as relative light units (RLU). As positive control, TPO immunoprecipitated from human thyroid tissue lysate (Graves’ disease case) was used to measure luminol oxidation. Agarose A incubated with mAb A4 alone (lysate omitted) was used as negative control. One representative of three independent experiments is shown. (C) TPO protein expression in breast epithelial normal (184A1) and cancer cell lines (MCF-7 and MDA-MB-231). Western blotting was used to detect TPO protein presence. The specificity of the reaction was verified by preabsorption of ab76935 antibody with the excess of highly purified human TPO. NTHY was used as a positive control. β-actin-specific Ab was used as a loading control. BN: peri-tumoral breast tissue; BC: breast cancer tissue; G-B: Graves’ disease thyroid tissue; NTHY: NTHY-ori 3–1 cell line; PNGase F: Peptide-N-Glycosidase F; RLU: relative light units.

    Techniques Used: Derivative Assay, SDS Page, Western Blot, Activity Assay, Incubation, Positive Control, Immunoprecipitation, Negative Control, Expressing, Multiple Displacement Amplification, Purification

    5) Product Images from "N-glycosylation critically regulates function of oxalate transporter SLC26A6"

    Article Title: N-glycosylation critically regulates function of oxalate transporter SLC26A6

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00171.2016

    Confirmation of glycosylation of mouse and human SLC26A6. Microsomes isolated from ( A ) mouse duodenum, jejunum, and kidney and ( B ) human Caco2b cells were incubated with a cocktail of N - and O -linkage specific glycosidases (N+O), the N -linkage specific glycosidase PNGase F (N), or vehicle alone ( C ) and then subjected to SDS-PAGE and Western analysis with the rabbit anti-human SLC26A6 antibody R29. The quantity of microsomal membranes used for each digest was commensurate with the relative total expression of SLC26A6 observed in each source in preliminary Western analysis. For this series of experiments we utilized 10 μg of mouse duodenum, 20 μg of mouse jejunum, 70 μg of mouse kidney, and 13 μg of Caco2b microsomal membranes.
    Figure Legend Snippet: Confirmation of glycosylation of mouse and human SLC26A6. Microsomes isolated from ( A ) mouse duodenum, jejunum, and kidney and ( B ) human Caco2b cells were incubated with a cocktail of N - and O -linkage specific glycosidases (N+O), the N -linkage specific glycosidase PNGase F (N), or vehicle alone ( C ) and then subjected to SDS-PAGE and Western analysis with the rabbit anti-human SLC26A6 antibody R29. The quantity of microsomal membranes used for each digest was commensurate with the relative total expression of SLC26A6 observed in each source in preliminary Western analysis. For this series of experiments we utilized 10 μg of mouse duodenum, 20 μg of mouse jejunum, 70 μg of mouse kidney, and 13 μg of Caco2b microsomal membranes.

    Techniques Used: Isolation, Incubation, SDS Page, Western Blot, Expressing

    Effect of enzymatic deglycosylation of plasma membrane SLC26A6 on chloride-stimulated 14 C-oxalate uptake. Seventy-two hours after transient transfection with wild-type human SLC26A6 (wt-hA6), OKP cells were exposed to PNGase F or vehicle in media for 4 h at 37°C. Cells were utilized for either evaluation of cell-surface expression of transfected SLC26A6 or chloride gradient-stimulated oxalate uptake. A : representative Western blot of surface biotinylated SLC26A6 from a single experiment confirming the efficacy of deglycosylation with PNGase F and illustrating the relative surface expression of SLC26A6 under control and PNGase F-treated conditions. B : chloride gradient-stimulated 14 C-oxalate uptake of control and PNGase F-treated OKP cells transiently transfected with human SLC26A6. n = 3 experiments of 4 replicates per experiment. * P = 0.0051, significantly different by two-tailed Student's t -test.
    Figure Legend Snippet: Effect of enzymatic deglycosylation of plasma membrane SLC26A6 on chloride-stimulated 14 C-oxalate uptake. Seventy-two hours after transient transfection with wild-type human SLC26A6 (wt-hA6), OKP cells were exposed to PNGase F or vehicle in media for 4 h at 37°C. Cells were utilized for either evaluation of cell-surface expression of transfected SLC26A6 or chloride gradient-stimulated oxalate uptake. A : representative Western blot of surface biotinylated SLC26A6 from a single experiment confirming the efficacy of deglycosylation with PNGase F and illustrating the relative surface expression of SLC26A6 under control and PNGase F-treated conditions. B : chloride gradient-stimulated 14 C-oxalate uptake of control and PNGase F-treated OKP cells transiently transfected with human SLC26A6. n = 3 experiments of 4 replicates per experiment. * P = 0.0051, significantly different by two-tailed Student's t -test.

    Techniques Used: Transfection, Expressing, Western Blot, Two Tailed Test

    Confirmation of identification of all N -linked glycosylation sites in human SLC26A6. OKP cells transfected with either wild-type (WT; wt-hA6) or double-glycosylation mutant human SLC26A6 (Δ167+172, 167+172-N/Q-hA6) were exposed to PNGase F in media for 4 h at 37°C. Cells were then surface biotinylated, processed for SDS-PAGE, and subjected to Western analysis with the rabbit anti-human SLC26A6 antibody R29.
    Figure Legend Snippet: Confirmation of identification of all N -linked glycosylation sites in human SLC26A6. OKP cells transfected with either wild-type (WT; wt-hA6) or double-glycosylation mutant human SLC26A6 (Δ167+172, 167+172-N/Q-hA6) were exposed to PNGase F in media for 4 h at 37°C. Cells were then surface biotinylated, processed for SDS-PAGE, and subjected to Western analysis with the rabbit anti-human SLC26A6 antibody R29.

    Techniques Used: Transfection, Mutagenesis, SDS Page, Western Blot

    6) Product Images from "The Expression and Characterization of Functionally Active Soluble CD83 by Pichia pastoris Using High-Density Fermentation"

    Article Title: The Expression and Characterization of Functionally Active Soluble CD83 by Pichia pastoris Using High-Density Fermentation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0089264

    The purification and characterization of sCD83. (A) The isolation of sCD83 by HisS from the fermentation supernatant. HisS: His-Select chromatography. (B) The further purification of sCD83 by S-200. S-200: size-exclusion chromatography S-200 HR, M: pre-stained protein molecular marker. (C) Approximately 20 µg of sCD83 was incubated with 2.0 IU of PNGase F according to the manufacturer’s instructions. The reaction mixture was analyzed in 15% SDS-PAGE and stained with Coomassie blue. Western blotting was performed using anti-CD83 polyclonal antibodies under reducing conditions.
    Figure Legend Snippet: The purification and characterization of sCD83. (A) The isolation of sCD83 by HisS from the fermentation supernatant. HisS: His-Select chromatography. (B) The further purification of sCD83 by S-200. S-200: size-exclusion chromatography S-200 HR, M: pre-stained protein molecular marker. (C) Approximately 20 µg of sCD83 was incubated with 2.0 IU of PNGase F according to the manufacturer’s instructions. The reaction mixture was analyzed in 15% SDS-PAGE and stained with Coomassie blue. Western blotting was performed using anti-CD83 polyclonal antibodies under reducing conditions.

    Techniques Used: Purification, Isolation, Chromatography, Size-exclusion Chromatography, Staining, Marker, Incubation, SDS Page, Western Blot

    7) Product Images from "Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion"

    Article Title: Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion

    Journal: Journal of Virology

    doi: 10.1128/JVI.01348-17

    ). The boxed region in the top diagram is enlarged below, showing the glycan moiety, glycan loop, fusion loop, and dimer interface. (B) Amino acid sequence of the rMR and the mutant (m1MR and m2MR) viruses around the VNDT motif. (C) Examination of the glycosylation status of the E protein in cells infected with the rMR, m1MR, or m2MR virus. Vero cells infected with the viruses (MOI = 1) were radiolabeled with Expre 35 S 35 S protein-labeling mix, immunoprecipitated with 4G2 antibody, digested with endo H or PNGase F, analyzed by SDS-PAGE, and detected by fluorography. Relative migration of molecular mass markers (in kilodaltons) is shown on the left. (D) Multistep growth of the rMR, m1MR, and m2MR in Vero and C6/36 cells. The graphs show mean values, with error bars representing standard deviations from the results of three independent experiments. An unpaired Student  t  test (two-tailed) was used to determine significance. ns, nonsignificant.
    Figure Legend Snippet: ). The boxed region in the top diagram is enlarged below, showing the glycan moiety, glycan loop, fusion loop, and dimer interface. (B) Amino acid sequence of the rMR and the mutant (m1MR and m2MR) viruses around the VNDT motif. (C) Examination of the glycosylation status of the E protein in cells infected with the rMR, m1MR, or m2MR virus. Vero cells infected with the viruses (MOI = 1) were radiolabeled with Expre 35 S 35 S protein-labeling mix, immunoprecipitated with 4G2 antibody, digested with endo H or PNGase F, analyzed by SDS-PAGE, and detected by fluorography. Relative migration of molecular mass markers (in kilodaltons) is shown on the left. (D) Multistep growth of the rMR, m1MR, and m2MR in Vero and C6/36 cells. The graphs show mean values, with error bars representing standard deviations from the results of three independent experiments. An unpaired Student t test (two-tailed) was used to determine significance. ns, nonsignificant.

    Techniques Used: Sequencing, Mutagenesis, Infection, Labeling, Immunoprecipitation, SDS Page, Migration, Two Tailed Test

    8) Product Images from "N-Glycosylation affects the stability and barrier function of the MUC16 mucin"

    Article Title: N-Glycosylation affects the stability and barrier function of the MUC16 mucin

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.770123

    MALDI-TOF profile of  N -glycans from transmembrane mucins isolated from human corneal epithelial cells.  The  N -linked glycans were released enzymatically by PNGase F, permethylated, and profiled by MALDI-TOF mass spectrometry. Magnified portions at  m / z  = 1500–2500, 2500–3500, and 3500–5500 are shown. Putative structures are assigned based on compositional information and known biosynthetic pathways. Most of the  N -glycans on corneal mucins have compositions consistent with bi-, tri-, and tetra-antennary complex glycans, carrying  N -acetyllactosamine residues and high mannose structures. All molecular ions detected are present in the form of [M + Na] + .
    Figure Legend Snippet: MALDI-TOF profile of N -glycans from transmembrane mucins isolated from human corneal epithelial cells. The N -linked glycans were released enzymatically by PNGase F, permethylated, and profiled by MALDI-TOF mass spectrometry. Magnified portions at m / z = 1500–2500, 2500–3500, and 3500–5500 are shown. Putative structures are assigned based on compositional information and known biosynthetic pathways. Most of the N -glycans on corneal mucins have compositions consistent with bi-, tri-, and tetra-antennary complex glycans, carrying N -acetyllactosamine residues and high mannose structures. All molecular ions detected are present in the form of [M + Na] + .

    Techniques Used: Isolation, Mass Spectrometry

    Galectin-3 interaction with MUC16 and its retention on the cell surface were N -glycan-dependent. a , 2-fold serial dilutions of rhGal-3 were applied individually to a nitrocellulose membrane in a slot-blot apparatus. Membranes were subsequently incubated with denatured cell lysates treated with or without PNGase F for 3 h at 37 °C. The enzymatic release of N -glycans significantly reduced the binding activity of MUC16 toward immobilized rhGal-3. Removal of N -glycans after PNGase F treatment was evaluated using ConA. b , treatment of stratified human corneal epithelial cells with PNGase F for 24 h significantly increased the amount of galectin-3 present in the cell culture medium. Relative amounts of N -glycans in biotinylated cell-surface proteins were evaluated using ConA. c , stratified human corneal epithelial cells were cultured in medium containing 1–10 μg/ml tunicamycin ( TM ) for the last 3 days of culture. Cells were then surface-labeled with biotin at 4 °C and pulled down using NeutrAvidin™. Galectin-3 was detected by Western blotting. d , cultures of human corneal epithelial cells were transfected with either non-targeting scramble control ( siScr ) or MGAT1 -targeting siRNA ( siMGAT1 ). As observed by cell-surface biotinylation and Western blotting, the knockdown of MGAT1 decreased the abundance of cell-surface galectin-3. Results in a and c represent at least three independent experiments. Results in b and d represent three independent experiments performed in triplicate. Data in c are represented as the mean ± S.D. The box and whisker plot show the 25 and 75 percentiles ( box ) and the median and the minimum and maximum data values ( whiskers ). Significance was determined using Student's t test ( b and d ) and one-way analysis of variance with Tukey's post hoc test ( c ). **, p
    Figure Legend Snippet: Galectin-3 interaction with MUC16 and its retention on the cell surface were N -glycan-dependent. a , 2-fold serial dilutions of rhGal-3 were applied individually to a nitrocellulose membrane in a slot-blot apparatus. Membranes were subsequently incubated with denatured cell lysates treated with or without PNGase F for 3 h at 37 °C. The enzymatic release of N -glycans significantly reduced the binding activity of MUC16 toward immobilized rhGal-3. Removal of N -glycans after PNGase F treatment was evaluated using ConA. b , treatment of stratified human corneal epithelial cells with PNGase F for 24 h significantly increased the amount of galectin-3 present in the cell culture medium. Relative amounts of N -glycans in biotinylated cell-surface proteins were evaluated using ConA. c , stratified human corneal epithelial cells were cultured in medium containing 1–10 μg/ml tunicamycin ( TM ) for the last 3 days of culture. Cells were then surface-labeled with biotin at 4 °C and pulled down using NeutrAvidin™. Galectin-3 was detected by Western blotting. d , cultures of human corneal epithelial cells were transfected with either non-targeting scramble control ( siScr ) or MGAT1 -targeting siRNA ( siMGAT1 ). As observed by cell-surface biotinylation and Western blotting, the knockdown of MGAT1 decreased the abundance of cell-surface galectin-3. Results in a and c represent at least three independent experiments. Results in b and d represent three independent experiments performed in triplicate. Data in c are represented as the mean ± S.D. The box and whisker plot show the 25 and 75 percentiles ( box ) and the median and the minimum and maximum data values ( whiskers ). Significance was determined using Student's t test ( b and d ) and one-way analysis of variance with Tukey's post hoc test ( c ). **, p

    Techniques Used: Dot Blot, Incubation, Binding Assay, Activity Assay, Cell Culture, Labeling, Western Blot, Transfection, Whisker Assay

    9) Product Images from "A prokaryotic viral sequence is expressed and conserved in mammalian brain"

    Article Title: A prokaryotic viral sequence is expressed and conserved in mammalian brain

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1706110114

    Glycosylation and PK resistance of spx1. Replicate blots of mouse brain homogenates ( A ) were digested with PNGase F to remove N-linked sugars (N), or neuraminidase+O-glycosidase for O-linked sugars (O). Spx1 detection shown in lanes 1–3 and PrP in lanes 4–6. No decrease in M r from deglycosylation is seen in spx1 digests, whereas PrP, known to have N-linked sugars, is markedly reduced in size by PNGase F (lane 5), but not O-linked digestion (lane 6), as expected. ( B ) Shows resistant spx1 in GT1 control (NL) and FU-CJD infected cells. In NL and FU-CJD samples (lanes 2 and 4, respectively), 10% of spx1 was PK resistant, whereas tubulin (Tu) showed no resistance. Parallel samples show no PrP-res in NL, whereas 25% PrP-res is seen in the FU-CJD cells (compare lanes 6 and 8).
    Figure Legend Snippet: Glycosylation and PK resistance of spx1. Replicate blots of mouse brain homogenates ( A ) were digested with PNGase F to remove N-linked sugars (N), or neuraminidase+O-glycosidase for O-linked sugars (O). Spx1 detection shown in lanes 1–3 and PrP in lanes 4–6. No decrease in M r from deglycosylation is seen in spx1 digests, whereas PrP, known to have N-linked sugars, is markedly reduced in size by PNGase F (lane 5), but not O-linked digestion (lane 6), as expected. ( B ) Shows resistant spx1 in GT1 control (NL) and FU-CJD infected cells. In NL and FU-CJD samples (lanes 2 and 4, respectively), 10% of spx1 was PK resistant, whereas tubulin (Tu) showed no resistance. Parallel samples show no PrP-res in NL, whereas 25% PrP-res is seen in the FU-CJD cells (compare lanes 6 and 8).

    Techniques Used: Infection

    Related Articles

    other:

    Article Title: Sulfation of glycosaminoglycans depends on catalytic activity of a lithium-inhibited phosphatase
    Article Snippet: PNGaseF digestion10 ug of protein extract were digested with PNGaseF (NEB) according to manufacturer instructions.

    Polymerase Chain Reaction:

    Article Title: Combining functional metagenomics and glycoanalytics to identify enzymes that facilitate structural characterization of sulfated N-glycans
    Article Snippet: .. PNGaseF, proteinase K, β-agarase, FseI, β-N-Acetylhexosaminidasef , Monarch® PCR and DNA clean-up kit, PURExpress® in vitro protein synthesis kit, RNase inhibitor murine, NEBuilder® HiFi DNA assembly cloning kit, GlycoBuffer 1, Q5® Hot Start High-Fidelity 2X Master Mix, NEB 5 alpha competent cells and NEBExpress® Ni spin columns were from New England Biolabs (Ipswich, MA, USA). ..

    In Vitro:

    Article Title: Combining functional metagenomics and glycoanalytics to identify enzymes that facilitate structural characterization of sulfated N-glycans
    Article Snippet: .. PNGaseF, proteinase K, β-agarase, FseI, β-N-Acetylhexosaminidasef , Monarch® PCR and DNA clean-up kit, PURExpress® in vitro protein synthesis kit, RNase inhibitor murine, NEBuilder® HiFi DNA assembly cloning kit, GlycoBuffer 1, Q5® Hot Start High-Fidelity 2X Master Mix, NEB 5 alpha competent cells and NEBExpress® Ni spin columns were from New England Biolabs (Ipswich, MA, USA). ..

    Clone Assay:

    Article Title: Combining functional metagenomics and glycoanalytics to identify enzymes that facilitate structural characterization of sulfated N-glycans
    Article Snippet: .. PNGaseF, proteinase K, β-agarase, FseI, β-N-Acetylhexosaminidasef , Monarch® PCR and DNA clean-up kit, PURExpress® in vitro protein synthesis kit, RNase inhibitor murine, NEBuilder® HiFi DNA assembly cloning kit, GlycoBuffer 1, Q5® Hot Start High-Fidelity 2X Master Mix, NEB 5 alpha competent cells and NEBExpress® Ni spin columns were from New England Biolabs (Ipswich, MA, USA). ..

    Lysis:

    Article Title: How Do Polymer Coatings Affect the Growth and Bacterial Population of a Biofilm Formed by Total Human Salivary Bacteria?—A Study by 16S-RNA Sequencing
    Article Snippet: .. Biofilm samples were mixed 2:1 with lysis buffer comprising 5 mg/mL lysozyme (L6876 Sigma-Aldrich, Munich, Germany), 10 mg/mL proteinase K (3115836001, Roche Diagnostics, Mannheim, Germany), 9500 U/mL PNGase F (P0704, New England Biolabs, Frankfurt, Germany), 3000 U/mL RNAse Inhibitor (M0314, New England Biolabs, Frankfurt, Germany), 0.5% NP40 (ab142227, Abcam, Cambridge, UK), 5 mM Tris-HCl and 0.05 mM EDTA (TE pH 8 – 12090015, Thermo Fisher Scientific, Braunschweig, Germany). ..

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    New England Biolabs pngase f native
    Mouse ISM1 is a secreted soluble protein that is glycosylated at asparagine residues 39 and 282. (A) Western blot of ISM1 in WCL and CM samples of HEK293T cells transiently transfected with mouse ISM1 and digested with <t>PNGase</t> F or Endo H as indicated. ISM1 protein is detected as a band of ∼70 kD in both WCL and CM without digestion. (B) HEK293T cells were transiently transfected with ISM1 or its N39Q, N282Q, and N39Q/N282Q point mutated forms. WCL samples were digested with or without PNGase F and subjected to Western blotting to detect ISM1. The size of intracellular ISM1 shifted from 70 kD to ∼65 and 60 kD following single or combined N point mutations, respectively. (C) Western blot of ISM1 in WCL and CM samples of HEK293T cells transiently transfected with wild-type ISM1 or its N39Q, N282Q, and N39Q/N282Q point mutated forms, in the presence or absence of 1 ng/ml tunicamycin. The protein molecular weight markers in kilodaltons are indicated by numbers on the left side of the Western blots.
    Pngase F Native, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mouse ISM1 is a secreted soluble protein that is glycosylated at asparagine residues 39 and 282. (A) Western blot of ISM1 in WCL and CM samples of HEK293T cells transiently transfected with mouse ISM1 and digested with PNGase F or Endo H as indicated. ISM1 protein is detected as a band of ∼70 kD in both WCL and CM without digestion. (B) HEK293T cells were transiently transfected with ISM1 or its N39Q, N282Q, and N39Q/N282Q point mutated forms. WCL samples were digested with or without PNGase F and subjected to Western blotting to detect ISM1. The size of intracellular ISM1 shifted from 70 kD to ∼65 and 60 kD following single or combined N point mutations, respectively. (C) Western blot of ISM1 in WCL and CM samples of HEK293T cells transiently transfected with wild-type ISM1 or its N39Q, N282Q, and N39Q/N282Q point mutated forms, in the presence or absence of 1 ng/ml tunicamycin. The protein molecular weight markers in kilodaltons are indicated by numbers on the left side of the Western blots.

    Journal: The Journal of Cell Biology

    Article Title: ISM1 regulates NODAL signaling and asymmetric organ morphogenesis during development

    doi: 10.1083/jcb.201801081

    Figure Lengend Snippet: Mouse ISM1 is a secreted soluble protein that is glycosylated at asparagine residues 39 and 282. (A) Western blot of ISM1 in WCL and CM samples of HEK293T cells transiently transfected with mouse ISM1 and digested with PNGase F or Endo H as indicated. ISM1 protein is detected as a band of ∼70 kD in both WCL and CM without digestion. (B) HEK293T cells were transiently transfected with ISM1 or its N39Q, N282Q, and N39Q/N282Q point mutated forms. WCL samples were digested with or without PNGase F and subjected to Western blotting to detect ISM1. The size of intracellular ISM1 shifted from 70 kD to ∼65 and 60 kD following single or combined N point mutations, respectively. (C) Western blot of ISM1 in WCL and CM samples of HEK293T cells transiently transfected with wild-type ISM1 or its N39Q, N282Q, and N39Q/N282Q point mutated forms, in the presence or absence of 1 ng/ml tunicamycin. The protein molecular weight markers in kilodaltons are indicated by numbers on the left side of the Western blots.

    Article Snippet: According to the manufacturer’s instructions, the samples were treated with PNGase F (P0704S; New England Biolabs) or Endo H (P0702S; New England Biolabs) and analyzed by Western blotting.

    Techniques: Western Blot, Transfection, Molecular Weight

    AMOP domain is required for the function of ISM1 as an antagonist of NODAL signaling. (A) Diagram of domain-deleted ISM1 constructs lacking either TSR1 or AMOP (ΔTSR1 and ΔAMOP, respectively). The N -glycosylation sites at positions N39 and N282 are indicated. N39 is located in the N terminus, 10 amino acids downstream from the signal peptide (amino acids 1–29), and N282 is located in the region between TSR1 and AMOP domains (amino acids 260–285). (B) Western blot of ISM1 in WCL and CM samples of HEK293T cells transiently transfected with wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1. Samples were digested with PNGase F or Endo H before being subjected to Western blotting. (C) Mapping of NODAL-interacting domain of ISM1. HEK293T cells were transiently transfected with FLAG-NODAL together with either wild-type ISM1 or domain-deleted ISM1 mutants. CM samples were immunoprecipitated (IP) with anti-FLAG or anti-ISM1 antibodies and subjected to Western blotting with anti-ISM1 and anti-FLAG antibodies. (D) Mapping of ACVR1B ECD -interacting domain of ISM1. HEK293T cells transiently expressing ACVR1B ECD were cotransfected with wild-type ISM1 or domain-deleted ISM1 mutants. ACVR2B ECD were also cotransfected with ACVR1B ECD to potentially improve the interaction between ISM1 and ACVR1B ECD . CM samples were immunoprecipitated with anti-ISM1 antibodies and immunoblotted with anti-HA antibodies. ISM1 interaction with ACVR1B ECD is no longer observed when AMOP domain is absent. (E) Western blot of pSMAD2 in WCL of P19C6 cells treated with 100 ng/ml NODAL in the presence of mock, HIS-CER1, FLAG-LEFTY1, wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1 CM. Western blots for HIS (CER1), FLAG (LEFTY1), and ISM1 in CM samples is shown.

    Journal: The Journal of Cell Biology

    Article Title: ISM1 regulates NODAL signaling and asymmetric organ morphogenesis during development

    doi: 10.1083/jcb.201801081

    Figure Lengend Snippet: AMOP domain is required for the function of ISM1 as an antagonist of NODAL signaling. (A) Diagram of domain-deleted ISM1 constructs lacking either TSR1 or AMOP (ΔTSR1 and ΔAMOP, respectively). The N -glycosylation sites at positions N39 and N282 are indicated. N39 is located in the N terminus, 10 amino acids downstream from the signal peptide (amino acids 1–29), and N282 is located in the region between TSR1 and AMOP domains (amino acids 260–285). (B) Western blot of ISM1 in WCL and CM samples of HEK293T cells transiently transfected with wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1. Samples were digested with PNGase F or Endo H before being subjected to Western blotting. (C) Mapping of NODAL-interacting domain of ISM1. HEK293T cells were transiently transfected with FLAG-NODAL together with either wild-type ISM1 or domain-deleted ISM1 mutants. CM samples were immunoprecipitated (IP) with anti-FLAG or anti-ISM1 antibodies and subjected to Western blotting with anti-ISM1 and anti-FLAG antibodies. (D) Mapping of ACVR1B ECD -interacting domain of ISM1. HEK293T cells transiently expressing ACVR1B ECD were cotransfected with wild-type ISM1 or domain-deleted ISM1 mutants. ACVR2B ECD were also cotransfected with ACVR1B ECD to potentially improve the interaction between ISM1 and ACVR1B ECD . CM samples were immunoprecipitated with anti-ISM1 antibodies and immunoblotted with anti-HA antibodies. ISM1 interaction with ACVR1B ECD is no longer observed when AMOP domain is absent. (E) Western blot of pSMAD2 in WCL of P19C6 cells treated with 100 ng/ml NODAL in the presence of mock, HIS-CER1, FLAG-LEFTY1, wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1 CM. Western blots for HIS (CER1), FLAG (LEFTY1), and ISM1 in CM samples is shown.

    Article Snippet: According to the manufacturer’s instructions, the samples were treated with PNGase F (P0704S; New England Biolabs) or Endo H (P0702S; New England Biolabs) and analyzed by Western blotting.

    Techniques: Construct, Western Blot, Transfection, Immunoprecipitation, Expressing

    VC induced N259-linked glycosylation on P4HA1. (A) Treatment of MEF cells for 6 hrs with VC concentration of ≥40 μM induced expression of an additional protein band reacting to P4HA1 specific antibody. (B) HFF cells and HEL cells also expressed a second protein reacting to antibody specific to P4HA1 at 6 hrs after VC (40 μM) treatment. (C) PNGaseF treatment of VC-treated and non-treated MEF cell lysate reduced the apparent molecular weight of the proteins reacting to antibody specific for P4HA1. (D) HA-tagged WT, N259Q, N113Q or N113Q/N259Q double mutants were expressed in P4ha1 -silenced MEF cells in the absence of VC. The cell lysates were immunoblotted with HA-tag antibody. The N259Q mutant showed the same apparent molecular weight as WT, whereas the apparent molecular weights of N113Q and N113Q/N259Q double mutants were equal but lower than WT (left panel). WT, N113Q, N259Q and N113Q/N259Q double mutants all showed the same motility as N113Q or N113Q/N259Q double mutants after PNGase F treatment (right panel). (E) N259Q mutated P4HA1 did not show a motility shift after VC treatment (top panel) whereas PNGaseF treatment increased motility of N259Q mutated P4HA1 with or without VC stimulation (third panel). (F) 40 μM or higher concentrations of VC induced an additional protein band reacting to HA-tagged antibody for N113Q mutated P4HA1 (top panel), which was sensitive to PNGaseF treatment (third panel).

    Journal: Cellular and molecular life sciences : CMLS

    Article Title: Ascorbate inducible N259 glycans on prolyl 4-hydroxylase subunit α1 promote hydroxylation and secretion of Type I collagen

    doi: 10.1007/s00018-019-03081-w

    Figure Lengend Snippet: VC induced N259-linked glycosylation on P4HA1. (A) Treatment of MEF cells for 6 hrs with VC concentration of ≥40 μM induced expression of an additional protein band reacting to P4HA1 specific antibody. (B) HFF cells and HEL cells also expressed a second protein reacting to antibody specific to P4HA1 at 6 hrs after VC (40 μM) treatment. (C) PNGaseF treatment of VC-treated and non-treated MEF cell lysate reduced the apparent molecular weight of the proteins reacting to antibody specific for P4HA1. (D) HA-tagged WT, N259Q, N113Q or N113Q/N259Q double mutants were expressed in P4ha1 -silenced MEF cells in the absence of VC. The cell lysates were immunoblotted with HA-tag antibody. The N259Q mutant showed the same apparent molecular weight as WT, whereas the apparent molecular weights of N113Q and N113Q/N259Q double mutants were equal but lower than WT (left panel). WT, N113Q, N259Q and N113Q/N259Q double mutants all showed the same motility as N113Q or N113Q/N259Q double mutants after PNGase F treatment (right panel). (E) N259Q mutated P4HA1 did not show a motility shift after VC treatment (top panel) whereas PNGaseF treatment increased motility of N259Q mutated P4HA1 with or without VC stimulation (third panel). (F) 40 μM or higher concentrations of VC induced an additional protein band reacting to HA-tagged antibody for N113Q mutated P4HA1 (top panel), which was sensitive to PNGaseF treatment (third panel).

    Article Snippet: PNGase F (P0704S) was purchased from New England BioLabs (Beverly, MA, USA).

    Techniques: Concentration Assay, Expressing, Molecular Weight, Mutagenesis

    SDS-PAGE analysis of purified plant produced Pfs48/45 variants. Lanes were loaded with ~1.0 μg (A) or ~2.0 μg (B) per lane for glycosylated, Endo H or PNGase F in vivo deglycosylated plant produced Pfs48/45proteins. (A) Lanes: 1- glycosylated Pfs48/45; 2- deglycosylated Pfs48/45, produced by in vivo deglycosylation of Pfs48/45, co-expressed with Endo H; 3- deglycosylated Pfs48/45, produced by in vivo deglycosylation of Pfs48/45, co-expressed with PNGase F. (B) Lanes: 1- glycosylated Pfs48/45-10C; 2- deglycosylated Pfs48/45-10C, produced by in vivo deglycosylation of Pfs48/45-10C,co-expressed with PNGase F; 3- deglycosylated Pfs48/45, produced by in vivo deglycosylation of Pfs48/45-10C, co-expressed with PNGase F. Indications of gPfs48/45, dPfs48/45, gPfs48/45-10C and dPfs48/45-10C are the same as shown in Fig 5 . M: color prestained protein standard (New England Biolabs). Arrow in B (indicating lane:2) shows aggregation of deglycosylated Pfs48/45-10C protein, produced by in vivo deglycosylation of Pfs48/45 co-expressed with PNGase F.

    Journal: PLoS ONE

    Article Title: In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

    doi: 10.1371/journal.pone.0183589

    Figure Lengend Snippet: SDS-PAGE analysis of purified plant produced Pfs48/45 variants. Lanes were loaded with ~1.0 μg (A) or ~2.0 μg (B) per lane for glycosylated, Endo H or PNGase F in vivo deglycosylated plant produced Pfs48/45proteins. (A) Lanes: 1- glycosylated Pfs48/45; 2- deglycosylated Pfs48/45, produced by in vivo deglycosylation of Pfs48/45, co-expressed with Endo H; 3- deglycosylated Pfs48/45, produced by in vivo deglycosylation of Pfs48/45, co-expressed with PNGase F. (B) Lanes: 1- glycosylated Pfs48/45-10C; 2- deglycosylated Pfs48/45-10C, produced by in vivo deglycosylation of Pfs48/45-10C,co-expressed with PNGase F; 3- deglycosylated Pfs48/45, produced by in vivo deglycosylation of Pfs48/45-10C, co-expressed with PNGase F. Indications of gPfs48/45, dPfs48/45, gPfs48/45-10C and dPfs48/45-10C are the same as shown in Fig 5 . M: color prestained protein standard (New England Biolabs). Arrow in B (indicating lane:2) shows aggregation of deglycosylated Pfs48/45-10C protein, produced by in vivo deglycosylation of Pfs48/45 co-expressed with PNGase F.

    Article Snippet: Our results showed that the expression levels, solubility and purification yields of Endo H deglycosylated Pfs48/45 or Pfs48/45-10C proteins were higher than those deglycosylated by PNGase F. In addition, as can be seen in the , purified PNGase F deglycosylated Pfs48/45-10C tends to aggregate, especially at higher concentrations.

    Techniques: SDS Page, Purification, Produced, In Vivo

    SDS-PAGE analysis of plant produced, purified bacterial Endo H from  N .  benthamiana  plants and evaluation of its deglycosylating activity  in vitro . (A) SDS-PAGE analysis of purified plant produced Endo H from  N .  benthamiana  plant. Lanes: 1-About 20 μg of the crude supernatant was loaded; 2–0.75 μg purified Endo H was loaded. (B), (C) Western blot or SDS-PAGE analysis of PA83 protein incubated with either plant produced Endo H or commercial Endo H or commercial PNGase F. Lanes: 1- plant produced PA83; 2- plant produced PA83 was treated with the plant produced Endo H; 3- plant produced PA83 was treated with the commercial Endo H; 4- plant produced PA83 was treated with the commercial PNGase F. 100 ng or 2 μg PA83 protein samples were loaded in each lane in Western blot and SDS-PAGE, respectively. M1: color prestained protein standard (New England Biolabs); M2: MagicMark XP Western Protein Standard (ThermoFisher Scientific). Arrows in C indicates migration of commercial Endo H and PNGase F.

    Journal: PLoS ONE

    Article Title: In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

    doi: 10.1371/journal.pone.0183589

    Figure Lengend Snippet: SDS-PAGE analysis of plant produced, purified bacterial Endo H from N . benthamiana plants and evaluation of its deglycosylating activity in vitro . (A) SDS-PAGE analysis of purified plant produced Endo H from N . benthamiana plant. Lanes: 1-About 20 μg of the crude supernatant was loaded; 2–0.75 μg purified Endo H was loaded. (B), (C) Western blot or SDS-PAGE analysis of PA83 protein incubated with either plant produced Endo H or commercial Endo H or commercial PNGase F. Lanes: 1- plant produced PA83; 2- plant produced PA83 was treated with the plant produced Endo H; 3- plant produced PA83 was treated with the commercial Endo H; 4- plant produced PA83 was treated with the commercial PNGase F. 100 ng or 2 μg PA83 protein samples were loaded in each lane in Western blot and SDS-PAGE, respectively. M1: color prestained protein standard (New England Biolabs); M2: MagicMark XP Western Protein Standard (ThermoFisher Scientific). Arrows in C indicates migration of commercial Endo H and PNGase F.

    Article Snippet: Our results showed that the expression levels, solubility and purification yields of Endo H deglycosylated Pfs48/45 or Pfs48/45-10C proteins were higher than those deglycosylated by PNGase F. In addition, as can be seen in the , purified PNGase F deglycosylated Pfs48/45-10C tends to aggregate, especially at higher concentrations.

    Techniques: SDS Page, Produced, Purification, Activity Assay, In Vitro, Western Blot, Incubation, Migration

    Schematic representation of Endo H or PNGase F cleavages. (A) Endo H cleaves between the two GlcNAc residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one GlcNAc remaining on the asparagines (Asn). (B) Peptide - N -Glycosidase F (PNGase F), is an amidase that cleaves the bond between GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N -linked glycoproteins;

    Journal: PLoS ONE

    Article Title: In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

    doi: 10.1371/journal.pone.0183589

    Figure Lengend Snippet: Schematic representation of Endo H or PNGase F cleavages. (A) Endo H cleaves between the two GlcNAc residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one GlcNAc remaining on the asparagines (Asn). (B) Peptide - N -Glycosidase F (PNGase F), is an amidase that cleaves the bond between GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N -linked glycoproteins;

    Article Snippet: Our results showed that the expression levels, solubility and purification yields of Endo H deglycosylated Pfs48/45 or Pfs48/45-10C proteins were higher than those deglycosylated by PNGase F. In addition, as can be seen in the , purified PNGase F deglycosylated Pfs48/45-10C tends to aggregate, especially at higher concentrations.

    Techniques:

    Study of the deglycosylation efficiency of plant produced Endo-H against PNGase F in vitro . (A) SDS-PAGE analysis of purified plant produced PNGase F and Endo H. Lanes were loaded with 1.0 μg per lane. 1-plant produced PNGase F; 2- plant produced Endo H; M-color prestained protein standard (New England Biolabs). (B) Plant produced PA83 was incubated with different amounts (0, 25, 100, 200, 400, 800 ng) of plant produced Endo H or PNGase F, as indicated. After incubation, proteins were analyzed by SDS-PAGE followed by Western blot analysis. Proteins were detected using a mixture of anti-His Tag antibody to detect His tagged PA83 and anti-FLAG antibody to detect FLAG-tagged Endo H or PNGase F. M: MagicMark XP Western Protein Standard (ThermoFisher Scientific). (C) Plant produced PA83 was incubated at 37°C for 1 h with different amounts (0, 25, 50, 400 and 800 ngs) of commercial Endo H, as indicated. Lanes, C- PA83 protein was kept at 4°C for 1 h; M-color prestained protein standard (New England Biolabs). (D) Plant produced PA83 was incubated at 37°C for 1 h with different amounts (0, 25, 50, 400 and 800 ngs) of commercial PNGase F, as indicated. Lanes, C- PA83 protein was kept at 4°C for 1 h; M- color prestained protein standard (New England Biolabs).

    Journal: PLoS ONE

    Article Title: In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

    doi: 10.1371/journal.pone.0183589

    Figure Lengend Snippet: Study of the deglycosylation efficiency of plant produced Endo-H against PNGase F in vitro . (A) SDS-PAGE analysis of purified plant produced PNGase F and Endo H. Lanes were loaded with 1.0 μg per lane. 1-plant produced PNGase F; 2- plant produced Endo H; M-color prestained protein standard (New England Biolabs). (B) Plant produced PA83 was incubated with different amounts (0, 25, 100, 200, 400, 800 ng) of plant produced Endo H or PNGase F, as indicated. After incubation, proteins were analyzed by SDS-PAGE followed by Western blot analysis. Proteins were detected using a mixture of anti-His Tag antibody to detect His tagged PA83 and anti-FLAG antibody to detect FLAG-tagged Endo H or PNGase F. M: MagicMark XP Western Protein Standard (ThermoFisher Scientific). (C) Plant produced PA83 was incubated at 37°C for 1 h with different amounts (0, 25, 50, 400 and 800 ngs) of commercial Endo H, as indicated. Lanes, C- PA83 protein was kept at 4°C for 1 h; M-color prestained protein standard (New England Biolabs). (D) Plant produced PA83 was incubated at 37°C for 1 h with different amounts (0, 25, 50, 400 and 800 ngs) of commercial PNGase F, as indicated. Lanes, C- PA83 protein was kept at 4°C for 1 h; M- color prestained protein standard (New England Biolabs).

    Article Snippet: Our results showed that the expression levels, solubility and purification yields of Endo H deglycosylated Pfs48/45 or Pfs48/45-10C proteins were higher than those deglycosylated by PNGase F. In addition, as can be seen in the , purified PNGase F deglycosylated Pfs48/45-10C tends to aggregate, especially at higher concentrations.

    Techniques: Produced, In Vitro, SDS Page, Purification, Incubation, Western Blot, Next-Generation Sequencing

    Glycan detection and Western blot analysis of glycosylated and  in vivo  deglycosylated PA83 and Pfs48/45-10C variants. (A), (C) 0.25 μg of protein from each sample was run on a 10% SDS-PAGE followed by in-gel glycan detection using the Pro-Q Emerald 300 glycoprotein staining kit. Stained proteins were visualized by UV illumination. (B), (D) Western blot analysis of the same samples using anti-His Tag antibody (BioLegend). (A), (B) Lanes: 1 –plant produced glycosylated PA83; 2– deglycosylated PA83, produced by  in vivo  deglycosylation of PA83, co-expressed with Endo H; 3– deglycosylated PA83, produced by  in vivo  deglycosylation of PA83, co-expressed with PNGase F. (C), (D) Lines: 1 –plant produced glycosylated Pfs48/45-10C; 2–deglycosylated Pfs48/45-10C, produced by  in vivo  deglycosylation of Pfs48/45-10C, co-expressed with Endo H; 3– deglycosylated Pfs48/45-10C, produced by  in vivo  deglycosylation of Pfs48/45-10C, co-expressed with Endo H. M1: CandyCane glycoprotein molecular weight standards (Molecular Probes), 250 ng of each protein per lane. M2: MagicMark XP Western Protein Standard (ThermoFisher Scientific). Indications of gPA83, dPA83, gPfs48/45-10C, dPfs48/45-10C are the same as shown in   Fig 4 .

    Journal: PLoS ONE

    Article Title: In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

    doi: 10.1371/journal.pone.0183589

    Figure Lengend Snippet: Glycan detection and Western blot analysis of glycosylated and in vivo deglycosylated PA83 and Pfs48/45-10C variants. (A), (C) 0.25 μg of protein from each sample was run on a 10% SDS-PAGE followed by in-gel glycan detection using the Pro-Q Emerald 300 glycoprotein staining kit. Stained proteins were visualized by UV illumination. (B), (D) Western blot analysis of the same samples using anti-His Tag antibody (BioLegend). (A), (B) Lanes: 1 –plant produced glycosylated PA83; 2– deglycosylated PA83, produced by in vivo deglycosylation of PA83, co-expressed with Endo H; 3– deglycosylated PA83, produced by in vivo deglycosylation of PA83, co-expressed with PNGase F. (C), (D) Lines: 1 –plant produced glycosylated Pfs48/45-10C; 2–deglycosylated Pfs48/45-10C, produced by in vivo deglycosylation of Pfs48/45-10C, co-expressed with Endo H; 3– deglycosylated Pfs48/45-10C, produced by in vivo deglycosylation of Pfs48/45-10C, co-expressed with Endo H. M1: CandyCane glycoprotein molecular weight standards (Molecular Probes), 250 ng of each protein per lane. M2: MagicMark XP Western Protein Standard (ThermoFisher Scientific). Indications of gPA83, dPA83, gPfs48/45-10C, dPfs48/45-10C are the same as shown in Fig 4 .

    Article Snippet: Our results showed that the expression levels, solubility and purification yields of Endo H deglycosylated Pfs48/45 or Pfs48/45-10C proteins were higher than those deglycosylated by PNGase F. In addition, as can be seen in the , purified PNGase F deglycosylated Pfs48/45-10C tends to aggregate, especially at higher concentrations.

    Techniques: Western Blot, In Vivo, SDS Page, Staining, Produced, Molecular Weight

    Study of stability of glycosylated and deglycosylated PA83 variants using SDS-PAGE analysis. Plant produced, glycosylated PA83, and in vivo Endo H or PNGase F deglycosylated forms of PA83 were purified as described in Materials and Methods. (A) The purified plant produced PA83 variants were stored for 1 hour at 37°C or for 72 hours at 4°C and analyzed by SDS-PAGE. Lanes were loaded with ~8.0 μg per lane for glycosylated, Endo H or PNGase F in vivo deglycosylated plant produced PA83 proteins. (B) Purified, plant produced, glycosylated PA83, and in vivo deglycosylated (co-expressed with Endo H or PNGase F) and in vitro deglycosylated (by commercial Endo H) proteins were incubated at 37°C for 1, 4, 8, 16 and 24 hours, and analyzed in SDS-PAGE. Lanes were loaded with ~5.0 μg per lane for each sample. M- color prestained protein standard (New England Biolabs).

    Journal: PLoS ONE

    Article Title: In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

    doi: 10.1371/journal.pone.0183589

    Figure Lengend Snippet: Study of stability of glycosylated and deglycosylated PA83 variants using SDS-PAGE analysis. Plant produced, glycosylated PA83, and in vivo Endo H or PNGase F deglycosylated forms of PA83 were purified as described in Materials and Methods. (A) The purified plant produced PA83 variants were stored for 1 hour at 37°C or for 72 hours at 4°C and analyzed by SDS-PAGE. Lanes were loaded with ~8.0 μg per lane for glycosylated, Endo H or PNGase F in vivo deglycosylated plant produced PA83 proteins. (B) Purified, plant produced, glycosylated PA83, and in vivo deglycosylated (co-expressed with Endo H or PNGase F) and in vitro deglycosylated (by commercial Endo H) proteins were incubated at 37°C for 1, 4, 8, 16 and 24 hours, and analyzed in SDS-PAGE. Lanes were loaded with ~5.0 μg per lane for each sample. M- color prestained protein standard (New England Biolabs).

    Article Snippet: Our results showed that the expression levels, solubility and purification yields of Endo H deglycosylated Pfs48/45 or Pfs48/45-10C proteins were higher than those deglycosylated by PNGase F. In addition, as can be seen in the , purified PNGase F deglycosylated Pfs48/45-10C tends to aggregate, especially at higher concentrations.

    Techniques: SDS Page, Produced, In Vivo, Purification, In Vitro, Incubation

    Western blot analysis of Pfs45/48-10C variants using MRA-26 antibody, a conformational specific Pfs48/45 mAb. Western blot analysis of Pfs45/48-10C variants using the MRA-26 antibody compared with the anti-His tag antibody. (A) Native PAGE followed by Western blot analysis of Pfs45/48-10C variants using the MRA-26 antibody. (B) Samples that were analyzed by Native PAGE, were also analyzed on SDS-PAGE, and proteins were probed with anti-His tag antibody. Lanes: 1- glycosylated Pfs48/45-10C; 2- deglycosylated Pfs48/45-10C, produced by  in vivo  deglycosylation of Pfs48/45, co-expressed with Endo H; 3- deglycosylated Pfs48/45-10C, produced by  in vivo  deglycosylation of Pfs48/45, co-expressed with PNGase F. Reduced (R) and non-reduced (N) samples were prepared as described in the Materials and Methods. M1: color prestained protein standard (New England Biolabs); M2: MagicMark XP Western Protein Standard (ThermoFisher Scientific). Western blot using a conformation-specific anti-Pfs48/45 antibody showed that reduction of the plant produced Pfs48/45 recombinant protein prevents recognition by antibody when compared with Western analysis using a His Tag antibody.

    Journal: PLoS ONE

    Article Title: In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

    doi: 10.1371/journal.pone.0183589

    Figure Lengend Snippet: Western blot analysis of Pfs45/48-10C variants using MRA-26 antibody, a conformational specific Pfs48/45 mAb. Western blot analysis of Pfs45/48-10C variants using the MRA-26 antibody compared with the anti-His tag antibody. (A) Native PAGE followed by Western blot analysis of Pfs45/48-10C variants using the MRA-26 antibody. (B) Samples that were analyzed by Native PAGE, were also analyzed on SDS-PAGE, and proteins were probed with anti-His tag antibody. Lanes: 1- glycosylated Pfs48/45-10C; 2- deglycosylated Pfs48/45-10C, produced by in vivo deglycosylation of Pfs48/45, co-expressed with Endo H; 3- deglycosylated Pfs48/45-10C, produced by in vivo deglycosylation of Pfs48/45, co-expressed with PNGase F. Reduced (R) and non-reduced (N) samples were prepared as described in the Materials and Methods. M1: color prestained protein standard (New England Biolabs); M2: MagicMark XP Western Protein Standard (ThermoFisher Scientific). Western blot using a conformation-specific anti-Pfs48/45 antibody showed that reduction of the plant produced Pfs48/45 recombinant protein prevents recognition by antibody when compared with Western analysis using a His Tag antibody.

    Article Snippet: Our results showed that the expression levels, solubility and purification yields of Endo H deglycosylated Pfs48/45 or Pfs48/45-10C proteins were higher than those deglycosylated by PNGase F. In addition, as can be seen in the , purified PNGase F deglycosylated Pfs48/45-10C tends to aggregate, especially at higher concentrations.

    Techniques: Western Blot, Clear Native PAGE, SDS Page, Produced, In Vivo, Recombinant

    Western blot analysis of bacterial Endo H or PNGase F produced in Nicotiana benthamiana plants. N . benthamiana plants were infiltrated with pBI-Endo H or pBI-PNGase F constructs to produce Endo H or PNGase F. Lanes: 1-crude extract prepared from control plant; 2- crude extract prepared from plant infiltrated with bacterial Endo H (pBI-Endo H) and 10, 20 and 40 fold diluted samples were loaded into gel; 3- crude extract prepared from plant infiltrated with bacterial PNGase F (pBI-PNGase F),and 2, 5 or 10 fold diluted samples were loaded into gel; 4- purified plant produced Endo H used as a standard protein; 10 or 25 ng were loaded into gel. M: MagicMark XP Western Protein Standard (ThermoFisher Scientific).

    Journal: PLoS ONE

    Article Title: In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

    doi: 10.1371/journal.pone.0183589

    Figure Lengend Snippet: Western blot analysis of bacterial Endo H or PNGase F produced in Nicotiana benthamiana plants. N . benthamiana plants were infiltrated with pBI-Endo H or pBI-PNGase F constructs to produce Endo H or PNGase F. Lanes: 1-crude extract prepared from control plant; 2- crude extract prepared from plant infiltrated with bacterial Endo H (pBI-Endo H) and 10, 20 and 40 fold diluted samples were loaded into gel; 3- crude extract prepared from plant infiltrated with bacterial PNGase F (pBI-PNGase F),and 2, 5 or 10 fold diluted samples were loaded into gel; 4- purified plant produced Endo H used as a standard protein; 10 or 25 ng were loaded into gel. M: MagicMark XP Western Protein Standard (ThermoFisher Scientific).

    Article Snippet: Our results showed that the expression levels, solubility and purification yields of Endo H deglycosylated Pfs48/45 or Pfs48/45-10C proteins were higher than those deglycosylated by PNGase F. In addition, as can be seen in the , purified PNGase F deglycosylated Pfs48/45-10C tends to aggregate, especially at higher concentrations.

    Techniques: Western Blot, Produced, Construct, Purification

    Western blot analysis of co-expression Bacillus anthracis PA83 (A), Pfs48/45 (B) and Pfs48/45-10C with bacterial Endo H or PNGase F in N . benthamiana plants. (A) Western blot analysis of co-expression of PA83. Lanes: 1- N . benthamiana plant was infiltrated with pBI-PA83 construct, for the production of glycosylated PA83, 2,3- N . benthamiana plants were infiltrated with combinations of the pBI-Endo H/pBI-PA83 or pBI-PNGase F/pBI-PA83 constructs, for the production of Endo H (2) or PNGase F (3) deglycosylated PA83 proteins. (B) Western blot analysis of co-expression of Pfs48/45. Lanes: 1- N . benthamiana plant was infiltrated with pEAQ-Pfs48/45 construct for the production of glycosylated Pfs48/45;2,3- N . benthamiana plants were infiltrated with combinations of the pBI-Endo H/pEAQ-Pfs48/45 or pBI-PNGase F/pEAQ-Pfs48/45constructs for the production of Endo H (2) and PNGase F (3) deglycosylated Pfs48/45 proteins. (C) Western blot analysis of co-expression of Pfs48/45-10C. Lanes: 1- N . benthamiana plant was infiltrated with pEAQ-Pfs48/45-10C construct for the production of glycosylated Pfs48/45-10C; 2,3- N . benthamiana plants were infiltrated with combinations of the pBI-Endo H/pEAQ-Pfs48/45 or pBI-PNGase F/pEAQ-Pfs48/45constructs for the production of Endo H (2) and PNGase F (3) deglycosylated Pfs48/45-10C proteins. gPA83- glycosylated PA83; dPA83- deglycosylated PA83; gPfs48/45: glycosylated Pfs48/45; dPfs48/45: deglycosylated Pfs48/45; gPfs48/45-10C: glycosylated Pfs48/45-10C; dPfs48/45-10C: deglycosylated Pfs48/45-10C.M: MagicMark XP Western Protein Standard (ThermoFisher Scientific). PA83 proteins were detected using the anti-Bacillus anthracis protective antigen antibody BAP0101 (Cat. No. ab1988, Abcam); Ps48/45, Endo H or PNGase F proteins were detected using the anti-FLAG antibody (BioLegend). Pfs48/45-10C protein was detected using the purified anti-His Tag antibody (Cat. No. 652502, BioLegend).

    Journal: PLoS ONE

    Article Title: In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

    doi: 10.1371/journal.pone.0183589

    Figure Lengend Snippet: Western blot analysis of co-expression Bacillus anthracis PA83 (A), Pfs48/45 (B) and Pfs48/45-10C with bacterial Endo H or PNGase F in N . benthamiana plants. (A) Western blot analysis of co-expression of PA83. Lanes: 1- N . benthamiana plant was infiltrated with pBI-PA83 construct, for the production of glycosylated PA83, 2,3- N . benthamiana plants were infiltrated with combinations of the pBI-Endo H/pBI-PA83 or pBI-PNGase F/pBI-PA83 constructs, for the production of Endo H (2) or PNGase F (3) deglycosylated PA83 proteins. (B) Western blot analysis of co-expression of Pfs48/45. Lanes: 1- N . benthamiana plant was infiltrated with pEAQ-Pfs48/45 construct for the production of glycosylated Pfs48/45;2,3- N . benthamiana plants were infiltrated with combinations of the pBI-Endo H/pEAQ-Pfs48/45 or pBI-PNGase F/pEAQ-Pfs48/45constructs for the production of Endo H (2) and PNGase F (3) deglycosylated Pfs48/45 proteins. (C) Western blot analysis of co-expression of Pfs48/45-10C. Lanes: 1- N . benthamiana plant was infiltrated with pEAQ-Pfs48/45-10C construct for the production of glycosylated Pfs48/45-10C; 2,3- N . benthamiana plants were infiltrated with combinations of the pBI-Endo H/pEAQ-Pfs48/45 or pBI-PNGase F/pEAQ-Pfs48/45constructs for the production of Endo H (2) and PNGase F (3) deglycosylated Pfs48/45-10C proteins. gPA83- glycosylated PA83; dPA83- deglycosylated PA83; gPfs48/45: glycosylated Pfs48/45; dPfs48/45: deglycosylated Pfs48/45; gPfs48/45-10C: glycosylated Pfs48/45-10C; dPfs48/45-10C: deglycosylated Pfs48/45-10C.M: MagicMark XP Western Protein Standard (ThermoFisher Scientific). PA83 proteins were detected using the anti-Bacillus anthracis protective antigen antibody BAP0101 (Cat. No. ab1988, Abcam); Ps48/45, Endo H or PNGase F proteins were detected using the anti-FLAG antibody (BioLegend). Pfs48/45-10C protein was detected using the purified anti-His Tag antibody (Cat. No. 652502, BioLegend).

    Article Snippet: Our results showed that the expression levels, solubility and purification yields of Endo H deglycosylated Pfs48/45 or Pfs48/45-10C proteins were higher than those deglycosylated by PNGase F. In addition, as can be seen in the , purified PNGase F deglycosylated Pfs48/45-10C tends to aggregate, especially at higher concentrations.

    Techniques: Western Blot, Expressing, Construct, Purification

    Western blot analysis of Pfs45/48 variants using the MRA-26 antibody, a conformational specific Pfs48/45 mAb. Western blot analysis of Pfs45/48 variants using the MRA-26 antibody compared with the anti-FLAG antibody (A) Native PAGE followed by Western blot analysis of Pfs45/48 variants using the MRA-26 antibody. (B) Samples that were analyzed by Native PAGE, were also analyzed on SDS-PAGE, and proteins were probed with anti-FLAG antibody. Lanes: 1- glycosylated Pfs48/45; 2- deglycosylated Pfs48/45, produced by  in vivo  deglycosylation of Pfs48/45, co-expressed with Endo H; 3- deglycosylated Pfs48/45, produced by  in vivo  deglycosylation of Pfs48/45, co-expressed with PNGase F. Reduced (R) and non-reduced (N) samples were prepared as described in the Materials and Methods. M1: color prestained protein standard (New England Biolabs); M2: MagicMark XP Western Protein Standard (ThermoFisher Scientific). Western blot using a conformation-specific anti-Pfs48/45 antibody showed that reduction of the plant produced Pfs48/45 recombinant protein prevents recognition by antibody when compared with Western analysis using a FLAG antibody.

    Journal: PLoS ONE

    Article Title: In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

    doi: 10.1371/journal.pone.0183589

    Figure Lengend Snippet: Western blot analysis of Pfs45/48 variants using the MRA-26 antibody, a conformational specific Pfs48/45 mAb. Western blot analysis of Pfs45/48 variants using the MRA-26 antibody compared with the anti-FLAG antibody (A) Native PAGE followed by Western blot analysis of Pfs45/48 variants using the MRA-26 antibody. (B) Samples that were analyzed by Native PAGE, were also analyzed on SDS-PAGE, and proteins were probed with anti-FLAG antibody. Lanes: 1- glycosylated Pfs48/45; 2- deglycosylated Pfs48/45, produced by in vivo deglycosylation of Pfs48/45, co-expressed with Endo H; 3- deglycosylated Pfs48/45, produced by in vivo deglycosylation of Pfs48/45, co-expressed with PNGase F. Reduced (R) and non-reduced (N) samples were prepared as described in the Materials and Methods. M1: color prestained protein standard (New England Biolabs); M2: MagicMark XP Western Protein Standard (ThermoFisher Scientific). Western blot using a conformation-specific anti-Pfs48/45 antibody showed that reduction of the plant produced Pfs48/45 recombinant protein prevents recognition by antibody when compared with Western analysis using a FLAG antibody.

    Article Snippet: Our results showed that the expression levels, solubility and purification yields of Endo H deglycosylated Pfs48/45 or Pfs48/45-10C proteins were higher than those deglycosylated by PNGase F. In addition, as can be seen in the , purified PNGase F deglycosylated Pfs48/45-10C tends to aggregate, especially at higher concentrations.

    Techniques: Western Blot, Clear Native PAGE, SDS Page, Produced, In Vivo, Recombinant

    Evaluation of the deglycosylation efficiency of Pfs48/45-10C by Endo H or PNGase F in vivo . Pfs48/45-10C (A), Pfs48/45 (B) and PA83 (C) were co-expressed with Endo H or PNGase F at different ratios of OD600 of Agrobacteria carrying Endo H, PNGase F and target genes, as indicated. The efficiency of deglycosylation of target proteins by Endo H or PNGase F was evaluated by Western blot analysis. Size reduction of Pfs48/45-10C, Pfs48/45 and PA83 show as an indicator of glycan removal. Proteins were probed with the anti-4xHis Tag mAb (BioLegend) (A,C) or anti-FLAG antibody (B). Indications of proteins in figures are the same as shown above.

    Journal: PLoS ONE

    Article Title: In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

    doi: 10.1371/journal.pone.0183589

    Figure Lengend Snippet: Evaluation of the deglycosylation efficiency of Pfs48/45-10C by Endo H or PNGase F in vivo . Pfs48/45-10C (A), Pfs48/45 (B) and PA83 (C) were co-expressed with Endo H or PNGase F at different ratios of OD600 of Agrobacteria carrying Endo H, PNGase F and target genes, as indicated. The efficiency of deglycosylation of target proteins by Endo H or PNGase F was evaluated by Western blot analysis. Size reduction of Pfs48/45-10C, Pfs48/45 and PA83 show as an indicator of glycan removal. Proteins were probed with the anti-4xHis Tag mAb (BioLegend) (A,C) or anti-FLAG antibody (B). Indications of proteins in figures are the same as shown above.

    Article Snippet: Our results showed that the expression levels, solubility and purification yields of Endo H deglycosylated Pfs48/45 or Pfs48/45-10C proteins were higher than those deglycosylated by PNGase F. In addition, as can be seen in the , purified PNGase F deglycosylated Pfs48/45-10C tends to aggregate, especially at higher concentrations.

    Techniques: In Vivo, Western Blot