During cleavage reactions, it is recommended that samples be removed at various time points and analyzed by SDS-PAGE to estimate the yield, purity, and extent of digestion. The amount of PreScission Protease, temperature and length of incubation required for complete digestion of a given GST fusion partner may vary depending on the fusion partner. Optimal conditions for each fusion should be determined in pilot experiments.Digestion may be improved by adding TritonTM X-100, TweenTM 20, NonidetTM, or NP40 to a concentration of 0.01%. Concentrations of these detergents up to 1% do not inhibit PreScission Protease.
Category
Enzymes
Background
PreScission Protease is a fusion protein of glutathione S-transferase (GST) and human rhinovirus (HRV) type 14 3C protease . The protease specifically recognizes a subset of sequences which include the core amino acid sequence Leu-Phe-Gln/Gly-Pro cleaving between the Gln and Gly residues . Substrate recognition and cleavage are likely to be dependent not only upon primary structural signals, but also upon the secondary and tertiary structures of the fusion protein as well.