Comparative stability studies of endo H in vivo deglycosylated PA83 with other PA83 forms in Nicotiana benthamiana plants

Bacillus anthracis, the causative organism of anthrax, usually occurs in countries where herbivorous animals cannot be widely vaccinated. These bacteria can be transmitted to humans by cutaneous, gastrointestinal or inhalation form, which is caused by inhalation of spores. The spore form of the bacterium has led scientists to develop recombinant vaccines with an improved safety profile that quickly and effectively protects immunity, with its high mortality rates and acceptance as a potential biological warfar reagent for a long while. Plants among the recombinant protein expression systems represent a promising platform with low costs and appropriate safety profiles compared to other expression systems. However, B. anthracis PA contains potential N-linked glycosylation sites that can be abnormally glycosylated during expression in eukaryotic systems. Previous studies have produced a non-glycosylated form of plant produced PA83 by using an in vivo deglycosylation approach based on its co-expression with the peptide-N-glycosidase F (PNGase F) enzyme in Nicotiana benthamiana (Mamedov et al. 2016). Recently, for production of non-deglycosylated proteins another strategy, Endo H (Endo-β-N-acetylglucosaminidase) in vivo deglycosylation strategy was developed (Mamedov et al., 2017), which causes no amino acid change in the resulted proteins. In this thesis, the deep stability analysis of PA83 protein produced by Endo H in vivo degycosylation strategy was performed. Thus, deglycosylated PA83 proteins, produced by PNGase F or Endo H strategy were subjected to long stability tests at different time periods and temperatures after purification experiments. Endo H deglycosylated PA83 has been shown to show better stability than other forms of PA83, even over long periods of time. These results show that plant produce dPA83 can be a good candidate for safe, effective and low cost recombinant anthrax subunit vaccine development.