Efficient Expression of Functionally Active Aflibercept with Designed N-glycans.

Aflibercept is a therapeutic recombinant fusion protein comprising extracellular domains of human vascular endothelial growth factor receptors (VEGFRs) and IgG1-Fc. It is a highly glycosylated protein with five N-glycosylation sites that might impact it structurally and/or functionally. Aflibercept is produced in mammalian cells and exhibits large glycan heterogeneity, which hampers glycan-associated investigations. Here, we report the expression of aflibercept in a plant-based system with targeted N-glycosylation profiles. Nicotiana benthamiana-based glycoengineering resulted in the production of aflibercept variants carrying designed carbohydrates, namely, N-glycans with terminal GlcNAc and sialic acid residues, herein referred to as AFLIGnGn and AFLISia, respectively. Both variants were transiently expressed in unusually high amounts (2 g/kg fresh leaf material) in leaves and properly assembled to dimers. Mass spectrometric site-specific glycosylation analyses of purified aflibercept showed the presence of two to four glycoforms in a consistent manner. We also demonstrate incomplete occupancy of some glycosites. Both AFLIGnGn and AFLISia displayed similar binding potency to VEGF165, with a tendency of lower binding to variants with increased sialylation. Collectively, we show the expression of functionally active aflibercept in significant amounts with controlled glycosylation. The results provide the basis for further studies in order to generate optimized products in the best-case scenario.

Callus induction and somatic embryogenesis in Stevia rebaudiana Bertoni as a medicinal plant.

Stevia rebaudiana (Bert.) from Asteraceae family is a useful medicinal plant that prevents and cures diabetes, blood pressure, weight gain and tooth decay. Due to self-incompatibility in stevia, somatic embryo investigation for artificial seed production is valuable in this plant. In order to evaluate the callus induction characteristics in stevia, a factorial experiment was laid out based on a completely randomized design with three replications. The factors included ten hormone combinations and control, two kinds of media (MS and B5) and two types of explants (leaf and internode). Callus induction characters including the percentage of callus formation, days to callus induction, fresh and dry callus weight were recorded. Analysis of variance showed significant differences (p<0.01) among hormone combinations, media and explant types as well as their interactions. The best treatment for callus induction with minimum time to callus formation was 1 mg/l NAA+1 mg/l BAP. The highest fresh and dry callus weight were obtained on B5 medium supplemented by 1 mg/l 2,4-D+1 mg/l BAP (in leaf explant) and 0.25 mg/l 2,4-D+ 0.1 mg/l BAP (in internode explant). These results can be used in suspension culture. To induce somatic embryogenesis in suspension culture, six hormone treatments were investigated. The highest somatic embryogenesis percentage was obtained in MS medium supplemented by 2 mg/l 2,4-D+ 0.5 mg/l NAA+0.5 mg/l BAP.