Bitkiden üretimli deglikozile Pfs48 / 45 varyantlarının ifadesi, üretimi ve karakterizasyonu

Plasmodium falciparum is one of the deadliest parasite in human history and therefore, developing safe, low-cost and highly efficient malaria vaccine with long-term stability is urgently needed. Many recent studies have shown that the plant-based transient expression system is most promising technology that enables safe, rapid and inexpensive production of valuable, recombinant proteins with high expression capacity. Pfs48/45 is one of the leading candidates for transmission blocking (TB) vaccine development and plays a key role in parasite fertilization. Plant produced Pfs48/45 of P. falciparum could be a great potential as a TB vaccine candidate for a safe, effective, and low-cost vaccine against malaria. Pfs48/45 protein has seven potential glycosylation sites and is aberrantly glycosylated when expressed in Nicotiana benthamiana plants. Recently, to avoid the aberrant glycosylation, a strategy was developed to produce non-glycosylated proteins in plants by co-expression of target proteins of interest with bacterial enzymes PNGase F or Endo H (Mamedov et al. 2013, 2016, 2017). In this study, non-glycosylated variants of Pfs48/45 protein, was engineered, expressed and produced in N. benthamiana plant using PNGase F or Endo H in vivo deglycosylation strategy. Glycosylated and deglycosylated variants of Pfs48/45 were purified and purified protein were characterized using a conformational specific Pfs48/4 monoclonal antibody (MRA-26, IIC5-B10), a known TB antibody. We demonstrate that, in contrast to the glycosylated form, plant produced in vivo Endo H deglycosylated Pfs48/45 was recognized by conformational specific Pfs48/45 monoclonal antibody, in a manner like its PNGase F deglycosylated counterpart. This suggest that plant produced Pfs48/45 protein contain epitopes present on native Pfs48/45 and therefore, have a great potential for the development of a Pfs48/45-based TB malaria vaccine.