Active Peptide-N4-N-Acetyl-Beta-D-Glucosaminyl Asparagine Amidase F (PNGaseF)

PNGase F (Peptide-N-glycosidase F) is a kind of enzymes for the deglycosylation of glycoproteins. The enzyme releases asparagine-linked oligosaccharides from glycoproteins and glycopeptides by hydrolyzing the amide of the asparagine (Asn) side chain. Thus, the activity of recombinant PNGase F measured by deglycosylating Interferon Gamma (IFNg) under denatured conditions. Prepare 10×denaturing buffer(5% SDS,400mM DTT), dilute IFNg to 1 μg/μl by 1×denaturing buffer,then heat the solution to 100℃for 10 minutes to denature the glycoprotein. Cool to room temperature and microcentrifuge briefly. Dobule dilution of recombinant PNGase F by assay buffer(50mmol/L Tris(pH7.4),1% NP-40), add 10 μl denatured IFNg to 10 μl different concentration of recombinant PNGase F, incubate reaction mixture at 37℃ for 1 hour. Stop the reaction by heating to 100℃ for 5 minutes, assess deglycosylation by SDS-PAGE. In the 10 μl reaction system, the amount of PNGase F needed to remove more than 95% carbohydrates from 10 μg denatured IFNg in 1 hour at 37℃ was defined as a unit. In this procedure, one unit is equal to 2.5ng recombinant PNGase F . The results are shown in Figure 1.