In vitro glycosylation of peptide (RKDVY) and RNase A by PNGase F

The in vitro glycosylation of pentapeptide (Arg-Lys-Asp-Val-Tyr; RKDVY) and RNase A was carried out using PNGase F (peptide-N-glycosidase F), and the results were analyzed using MALDI-TOF-MS. Aminated N,N-diacetyl chitobiose was used as the sugar in the glycosylation reaction, and the amination yield of N,N′-diacetyl chitobiose was about 60%. To reduce the water activity and shift the reaction equilibrium to a reverse reaction, 1,4-dioxane or ethylene glycol was used as the organic solvent in the enzymatic glycosylation. A certain extent of nonenzymatic glycosylation, known as the Maillard reaction, was also observed, which occurs on an arginine or lysine residue when the length of the sugar residue is one or two. However, the extent of glycosylation was much higher in the enzymatic reaction, indicating that PNGase F can be effectively used to produce glycopeptides and glycoproteins in vitro.