Properties
Description
PNGase A is a recombinant amidase derived from Oryza sativa and expressed in Pichia pastoris. It cleaves N-linked glycans with or without α(1,3)-linked core fucose residues.
Molecular Weight
63.8 kDa
Unit Definition
One unit will remove > 95% of the carbohydrate from 1 µg of denatured recombinant Avidin produced in Maize in 1 hour at 37°C in a total reaction volume of 10 µl.
Expression System
Pichia pastoris
Components
PNGase A (5,000 units/ml); 10 x GlycoBuffer (50 mM sodium acetate, pH 6, 25°C); Glycoprotein denaturing buffer (10 x); NP-40 (10%)
Reaction Conditions
1X GlycoBuffer (50 mM sodium acetate, pH 6, 25°C), Incubate at 37°C
Heat Inactivation
10 minutes at 65°C
Production Methods
Fermentation
Detection Method
One microgram of recombinant avidin undergoes denaturation in 1X glycoprotein denaturing buffer at 100°C for 10 minutes.. Subsequent to this, NP-40 and GlycoBuffer are introduced into the mixture. Thereafter, two-fold dilutions of PNGase A are added, and the reaction mixture is incubated at 37°C for one hour. The separation and visualization of the reaction products are analyzed by SDS-PAGE.
Concentration
5,000 units/ml
Buffer
20 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, pH 7.5 at 25°C
Applications
PNGase A can be used to cleave high mannose N-glycan structures from Man 3 up to Man 9.
