Generation of Monoclonal Antibody Fragments Binding the Native γ-Secretase Complex for Use in Structural Studies

A detailed understanding of gamma-secretase structure is crucially needed to elucidate its unique properties of intramembrane protein cleavage and to design therapeutic compounds for the safe treatment of Alzheimer's disease. gamma-Secretase is an enzyme complex composed of four membrane proteins, and the scarcity of its supply associated with the challenges of crystallizing membrane proteins is a major hurdle for the determination of its high-resolution structure. This study addresses some of these issues, first by adapting CHO cells overexpressing gamma-secretase to growth in suspension, thus yielding multiliter cultures and milligram quantities of highly purified, active gamma-secretase. Next, the amounts of gamma-secretase were sufficient for immunization of mice and allowed generation of Nicastrin- and Aph-1-specific monoclonal antibodies, from which Fab fragments were proteolytically prepared and subsequently purified. The amounts of gamma-secretase produced are compatible with robot assisted crystallogenesis using nanoliter technologies. In addition, our Fab fragments bind exposed regions of native gamma-secretase in a dose-dependent manner without interfering with its catalytic properties and can therefore be used as specific tools to facilitate crystal formation.