A faster migrating variant masquerades as NICD when performing in vitro γ-secretase assays with bacterially expressed notch substrates

Intramembrane proteolysis is a new and rapidly growing field. In vitro assays utilizing recombinant substrates for γ-secretase, an intramembrane-cleaving enzyme, are critically important in order to characterize the biochemical properties of this unusual enzyme. Several recombinant Notch proteins of varying length are commonly used as in vitro substrates for CHAPSO-solubilized γ-secretase. Here we report that several recombinant Notch constructs undergo limited or no proteolysis in vitro. Instead, upon incubation with or without γ-secretase, variants of the intact protein migrate during SDS-PAGE at the location expected for the γ-secretase specific cleavage products. In addition, we show that addition of aspartyl- and γ-secretase specific protease inhibitors are able to retard the formation of these variants independent of γ-secretase, which could lead to the erroneous conclusion that Notch cleavage by solubilized γ-secretase was achieved in vitro even when no proteolysis occurred. In contrast, substrates produced in mammalian or insect cells are cleaved efficiently in vitro. These observations suggest that in vitro studies reliant on recombinant, bacterially produced Notch TMD should be performed with the inclusion of additional controls able to differentiate between actual cleavage and this potential artifact.