Journal
BIOCHEMISTRY
Volume 51, Issue 44, Pages 8779-8790Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi300997e
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Funding
- Swiss National Science Foundation [31003A_134938/1]
- Swiss National Centres of Competence in Research
- Neural Plasticity and Repair
- Strauss foundation
- EPFL-Merck-Serono grant
- Swiss National Science Foundation (SNF) [31003A_134938] Funding Source: Swiss National Science Foundation (SNF)
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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.
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