Engineering, expression, purification and characterization of new mutant form of PA83 in N. benthamiana plants as antrax vaccine candida

Bacillus anthracis has long been considered a potential biological warfare agent, and therefore, developing safe, low-cost and highly efficient anthrax vaccine with long-term stability is important. Numerous studies in recent years demonstrated that plant based transient expression system is a promising technology with high expression capacity and provides safe, fast, inexpensive production of valuable recombinant proteins. Plant produced PA of B. anthracis has great potential as a target for a safe, effective, and low-cost vaccine against anthrax. Protective antigen of B. anthracis is not a glycoprotein, however, its sequence has nine potential glycosylation sites and it has been shown to be aberrantly glycosylated when expressed in Nicotiana benthamiana plants. Recently, a non-glycosylated versions of PA83 antigen was produced by in vivo deglycosylation strategy (PNGase F or Endo H based) (Mamedov et al 2012; Mamedov et al 2016; Mamedov et al 2017) or by site-directed mutagenesis-based approach, by mutation of six N-Glycosylations sites (Mamedov et al 2016). When we analyzed this mutant form of PA83, we found that Asn-39 site, which was not mutated in the previous studies has some level of glycosylation. In this study, a new mutant form by mutation of seven N-glycosylation sites (including Asn-39) of protective antigen was engineered, codon optimized, expressed and produced in N. benthamiana plant. For comparison, version of PA83 containing point mutations in six potential N-glycosylation sites was also produced in N. benthamiana. Both PA83 mutants were purified using IMAC affinity column and purified protein were subjected for stability analysis. KEYWORDS: Anthrax vaccine candidate, in vivo deglycosylation, Protective Antigen, Transient gene expression.