Fig 1: 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.
Fig 2: 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.
Fig 3: 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.
Fig 4: 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.
Fig 5: 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).
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Storage:Storage Conditions: 20 mM Tris-HCl 50 mM NaCl 5 mM EDTA 50% Glycerol pH 7.5 @ 25°C
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