SDS-PAGE analysis of plant produced, purified bacterial Endo H from <i>N</i>. <i>benthamiana</i> plants and evaluation of its deglycosylating activity <i>in vitro</i>.

<p>(A) SDS-PAGE analysis of purified plant produced Endo H from <i>N</i>. <i>benthamiana</i> 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.</p

Glycan detection and Western blot analysis of glycosylated and <i>in vivo</i> deglycosylated PA83 and Pfs48/45-10C variants.

<p>(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 <i>in vivo</i> deglycosylation of PA83, co-expressed with Endo H; 3– deglycosylated PA83, produced by <i>in vivo</i> 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 <i>in vivo</i> deglycosylation of Pfs48/45-10C, co-expressed with Endo H; 3– deglycosylated Pfs48/45-10C, produced by <i>in vivo</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183589#pone.0183589.g004" target="_blank">Fig 4</a>.</p

Schematic representation of Endo H or PNGase F cleavages.

<p>(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 -<i>N</i>-Glycosidase F (PNGase F), is an amidase that cleaves the bond between GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from <i>N</i>-linked glycoproteins;</p

Evaluation of the deglycosylation efficiency of Pfs48/45-10C by Endo H or PNGase F <i>in vivo</i>.

<p>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.</p

SDS-PAGE analysis of purified plant produced Pfs48/45 variants.

<p>Lanes were loaded with ~1.0 μg (A) or ~2.0 μg (B) per lane for glycosylated, Endo H or PNGase F <i>in vivo</i> deglycosylated plant produced Pfs48/45proteins. (A) Lanes: 1- glycosylated Pfs48/45; 2- deglycosylated Pfs48/45, produced by <i>in vivo</i> deglycosylation of Pfs48/45, co-expressed with Endo H; 3- deglycosylated Pfs48/45, produced by <i>in vivo</i> deglycosylation of Pfs48/45, co-expressed with PNGase F. (B) Lanes: 1- glycosylated Pfs48/45-10C; 2- deglycosylated Pfs48/45-10C, produced by <i>in vivo</i> deglycosylation of Pfs48/45-10C,co-expressed with PNGase F; 3- deglycosylated Pfs48/45, produced by <i>in vivo</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183589#pone.0183589.g005" target="_blank">Fig 5</a>. M: color prestained protein standard (New England Biolabs). Arrow in B (indicating lane:2) shows aggregation of deglycosylated Pfs48/45-10C protein, produced by <i>in vivo</i> deglycosylation of Pfs48/45 co-expressed with PNGase F.</p

Study of stability of glycosylated and deglycosylated PA83 variants using SDS-PAGE analysis.

<p>Plant produced, glycosylated PA83, and <i>in vivo</i> 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 <i>in vivo</i> deglycosylated plant produced PA83 proteins. (B) Purified, plant produced, glycosylated PA83, and <i>in vivo</i> deglycosylated (co-expressed with Endo H or PNGase F) and <i>in vitro</i> 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).</p

Western blot analysis of Pfs45/48 variants using the MRA-26 antibody, a conformational specific Pfs48/45 mAb.

<p>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 <i>in vivo</i> deglycosylation of Pfs48/45, co-expressed with Endo H; 3- deglycosylated Pfs48/45, produced by <i>in vivo</i> 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.</p

Western blot analysis of bacterial Endo H or PNGase F produced in <i>Nicotiana benthamiana</i> plants.

<p><i>N</i>. <i>benthamiana</i> 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).</p

Western blot analysis of co-expression <i>Bacillus anthracis</i> PA83 (A), Pfs48/45 (B) and Pfs48/45-10C with bacterial Endo H or PNGase F in <i>N</i>. <i>benthamiana</i> plants.

<p>(A) Western blot analysis of co-expression of PA83. Lanes: 1- <i>N</i>. <i>benthamiana</i> plant was infiltrated with pBI-PA83 construct, for the production of glycosylated PA83, 2,3- <i>N</i>. <i>benthamiana</i> 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-<i>N</i>. <i>benthamiana</i> plant was infiltrated with pEAQ-Pfs48/45 construct for the production of glycosylated Pfs48/45;2,3- <i>N</i>. <i>benthamiana</i> 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- <i>N</i>. <i>benthamiana</i> plant was infiltrated with pEAQ-Pfs48/45-10C construct for the production of glycosylated Pfs48/45-10C; 2,3- <i>N</i>. <i>benthamiana</i> 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).</p