Cloning, expression, purification and characterization of factor IX variants in N. benthamiana plant

In this study, human FIX was codon optimized, cloned, and expressed in N. benthamiana plant alone or with modified enzymes, VKD-CG and Furin. In addition, to study the specific effect of plant glycosylation on stability and activity of plant produced FIX, FIX was also co-expressed with deglycosylating enzymes, PNGase F or Endo H. Plant produced FIX variants were then partially purified using IMAC column and their comperative enzymatic activities were performed. Activities of plant produced partially purified FIX variants were determined by chromogenic assay. The results showed that plant produced FIX possess about 10% relative activity to human FIX. However, we observed very little or no difference between FIX activities, produced alone or produced with co-expression with furin or VKD-GC. About 12% relative activity to human FIX was observed for plant produced FIX, produced with co-expression with furin or furin plus VKD-GC genes. In addition, we also performed a stability and activity analysis of different variants of plant produced FIX. The activities of Endo H and PNGase F deglycosylated FIX variants were 13,6 and 12,5% relative to human FIX, respectively. The stability of plant produced glycosylated and in vivo deglycosylated forms of FIX were examined after incubation at 37˚C for 24 hours. In vitro half-life (time to 50% remaining) of glycosylated, PNGase F in vivo deglycosylated, and Endo H in vivo deglycosylated forms of FIX were calculated by analyzing protein bands on Western blot, which were 25.80, 37.99 and 80.57 hours respectively. These results demonstrate that the plant produced Endo H in vivo deglycosylated form of FIX appeared to be more stable compared to glycosylated FIX or PNGase F in vivo deglycosylated counterparts at elevated temperatures. KEYWORDS: Carboxylation, Hemophilia B disease, in vivo deglycosylation, Proteolytic cleavage, Transient gene expression.