Rapid purification of active γ-secretase, an intramembrane protease implicated in Alzheimer's disease

Abstract

γ-Secretase is an unconventional aspartyl protease that processes many type 1 membrane proteins within the lipid bilayer. Because its cleavage of amyloid-β precursor protein generates the amyloid-β protein (Aβ) of Alzheimer's disease, partially inhibiting γ-secretase is an attractive therapeutic strategy, but the structure of the protease remains poorly understood. We recently used electron microscopy and single particle image analysis on the purified enzyme to generate the first 3D reconstruction of γ-secretase, but at low resolution (15 Å). The limited amount of purified γ-secretase that can be produced using currently available cell lines and procedures has prevented the achievement of a high resolution crystal structure by X-ray crystallography or 2D crystallization. We report here the generation and characterization of a new mammalian cell line (S-20) that overexpresses strikingly high levels of all four γ-secretase components (presenilin, nicastrin, Aph-1 and Pen-2). We then used these cells to develop a rapid protocol for the high-grade purification of proteolytically active γ-secretase. The cells and purification methods detailed here provide a key step towards crystallographic studies of this ubiquitous enzyme. © 2007 The Authors.