Figure 3

Functional analyses of human GalNAc-T2 expressed in N. benthamiana L . plants. GalNAc-T2 enzyme assay was set up in a final volume of 100 μl with UDP-GalNAc as the donor, and the hCG-β peptide as an acceptor (see "Materials and Methods"). A sample corresponding to 25 μl of the reaction mixture containing microsomal fraction of A - control plants, or B - GalNAc-T2 transgenic plants as an enzyme source, was subjected to RP-HPLC as described (see "Materials and Methods"): a - peak corresponding to the hCG-β peptide, b, c - peaks corresponding to the glycosylated forms of hCG-β peptide; C - monitoring the attachment of GalNAc to the hCG-β peptide by radioactive enzyme assay using [3H]UDP-GalNAc as a donor and 41 μg (●),, 20.5 μg (■) or 10.25 μg (▲) microsomal proteins from GalNAc-T2 plant, or 41 μg microsomal proteins from control plant (○) as an enzyme source. Aliquots of 0.5 ml from the collected fractions (2 ml fraction volume) were used to monitor the radioactivity.