A further reason for the lacking plant species effect was probably the rather small patch size of plant individuals given under field conditions.
We used N. benthamiana ΔXTFT as a host in a first set of experiments aimed at generating rhEPOELD with human-like N-glycosylation, i.e. lacking plant specific core α1,3-fucose and β1,2-xylose residues.
Reaction was started by the addition of 500 μl TCME of different concentrations (100 500 μg) and the change in absorbance was measured continuously at 560 nm for 20 min. A cuvette containing ferritin, ferrozine and phosphate buffer but lacking plant extract was used as the reference solution.
The transformation procedure was optimised for high level expression (up to 10 μg/ml) and successfully performed even with a transgenic glyco-engineered strain lacking plant-specific immunogenic sugar residues in N-glycans.
N. benthamiana ΔXTFT plants lacking plant-specific α1,3-fucosylation and β1,2-xylosylation were grown at 24°C with a 16-h light:8-h dark photoperiod.
Another major breakthrough towards the humanization of the plant N-glycosylation pathway was the generation of mutants lacking plant-specific β1,2-xylose and core α1,3-fucose.
To this end, we overexpressed human GnTIII, IV and V and modified versions thereof in the glycoengineered Nicotiana benthamiana, lacking plant-specific sugar residues (▵XT/FT; Strasser et al. 2008).
A secreted form of 2G12 has been constitutively expressed in the leaves of wild-type Arabidopsis plants [ 73] and in a mutant strain modified to knock out the genes encoding β1,2-xylosyltransferase (XT) and core α1,3-fucosyltransferase (FT), thus producing complex N-glycans lacking plant-specific residues [ 74].
This beneficial effect of sialic acid on protein stability likely explains why knockin strategies for plant glycoengineering in glycosylation have mainly focused on the addition of terminal β1,4-galactose and sialic acid residues to humanize N-glycan in mutant plants lacking plant-specific N-glycan residues [ 75].
WT N. benthamiana plants, the glycosylation mutants ΔXTFT (lacking plant-specific N-glycosylation, i.e. β1,2-xylose and core α1,3-fucose, [ 28]) and Gal+ (synthesizing β1,4-galactosylated structures, [ 30]), were grown at 22°C with a 16-h light: 8-h dark photoperiod.
In animals, H3K9me3 demethylation is catalyzed by members of the KDM4 subfamily [ 59- 62], which lacks plant members, suggesting that H3K9me3 demethylation in plants is catalyzed by proteins from another subfamily.
