title: Production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expressing bacterial PNGase F.
abstract: Application of tools of molecular biology and genomics is increasingly leading towards the development of recombinant protein-based biologics. As such, it is leading to an increased diversity of targets that have important health applications and require more flexible approaches for expression because of complex post-translational modifications. For example, Plasmodium parasites may have complex post-translationally modified proteins such as Pfs48/45 that do not carry N-linked glycans (Exp. Parasitol. 1998; 90, 165.) but contain potential N-linked glycosylation sites that can be aberrantly glycosylated during expression in mammalian and plant systems. Therefore, it is important to develop strategies for producing non-glycosylated forms of these targets to preserve biological activity and native conformation. In this study, we are describing in vivo deglycosylation of recombinant N-glycosylated proteins as a result of their transient co-expression with bacterial PNGase F (Peptide: N-glycosidase F). In addition, we show that the recognition of an in vivo deglycosylated plant-produced malaria vaccine candidate, Pfs48F1, by monoclonal antibodies I, III and V raised against various epitopes (I, III and V) of native Pfs48/45 of Plasmodium falciparum, was significantly stronger compared to that of the glycosylated form of plant-produced Pfs48F1. To our knowledge, neither in vivo enzymatic protein deglycosylation has been previously achieved in any eukaryotic system, including plants, nor has bacterial PNGase F been expressed in the plant system. Thus, here, we report for the first time the expression in plants of an active bacterial enzyme PNGase F and the production of recombinant proteins of interest in a non-glycosylated form.
authors.affiliation: Fraunhofer USA Center for Molecular Biotechnology, Newark, DE, USA. tmammedov@fraunhofer-cmb.org
authors.forename: Tarlan
authors.initials: T
authors.lastname: Mamedov
authors.forename: Ananya
authors.initials: A
authors.lastname: Ghosh
authors.forename: R Mark
authors.initials: RM
authors.lastname: Jones
authors.forename: Vadim
authors.initials: V
authors.lastname: Mett
authors.forename: Christine E
authors.initials: CE
authors.lastname: Farrance
authors.forename: Konstantin
authors.initials: K
authors.lastname: Musiychuk
authors.forename: April
authors.initials: A
authors.lastname: Horsey
authors.forename: Vidadi
authors.initials: V
authors.lastname: Yusibov
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology; D001419:Bacteria / Q000201:enzymology*; D001427:Bacterial Toxins / Q000276:immunology; D001709:Biotechnology / Q000379:methods*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D013058:Mass Spectrometry; D010449:Peptide Mapping; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000302:isolation & purification / Q000378:metabolism*; D030821:Plants, Genetically Modified; D010963:Plasmodium falciparum / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012995:Solubility; D014026:Nicotiana / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011134:Polysaccharides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C030325:anthrax toxin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase