Production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expressing bacterial PNGase F

Mamedov T., Ghosh A., Jones R. M., Mett V., Farrance C. E., Musiychuk K., ...More

PLANT BIOTECHNOLOGY JOURNAL, vol.10, no.7, pp.773-782, 2012 (SCI-Expanded) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 10 Issue: 7
  • Publication Date: 2012
  • Doi Number: 10.1111/j.1467-7652.2012.00694.x
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Page Numbers: pp.773-782
  • Keywords: N-linked glycosylation, PNGase F, deglycosylation, plant transient expression, recombinant proteins, malaria vaccine candidate Pfs48, 45, PLASMODIUM-FALCIPARUM, ANTIGEN PFS48/45, N-GLYCOSYLATION, MONOCLONAL-ANTIBODY, TUNICAMYCIN, CLONING, CHALLENGE, RECEPTORS, SEQUENCE, VECTOR
  • Akdeniz University Affiliated: No


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.