Generation of monoclonal antibody fragments binding the native γ-secretase complex for use in structural studies.
A detailed understanding of γ-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. γ-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 γ-secretase to growth in suspension, thus yielding multiliter cultures and milligram quantities of highly purified, active γ-secretase. Next, the amounts of γ-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 γ-secretase produced are compatible with robot-assisted crystallogenesis using nanoliter technologies. In addition, our Fab fragments bind exposed regions of native γ-secretase in a dose-dependent manner without interfering with its catalytic properties and can therefore be used as specific tools to facilitate crystal formation.
Brain Mind Institute, School of Life Sciences, Swiss Federal Institute of Technology, Lausanne, Switzerland.
Jean-René
JR
Alattia
Claude
C
Schweizer
Matthias
M
Cacquevel
Mitko
M
Dimitrov
Lorène
L
Aeschbach
Mustapha
M
Oulad-Abdelghani
Patrick C
PC
Fraering
D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D002793:Cholic Acids; D006224:Cricetinae; D003412:Cricetulus; D003460:Crystallization; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D053764:Presenilin-1
D000911:Antibodies, Monoclonal; D002793:Cholic Acids; D007140:Immunoglobulin Fab Fragments; D008565:Membrane Proteins; D053764:Presenilin-1; C048531:chapso; D053829:Amyloid Precursor Protein Secretases