Production, purification and functional validation of human secreted amyloid precursor proteins for use as neuropharmacological reagents.
The secreted fragment of the amyloid precursor protein (sAPPalpha) generated following cleavage by alpha-secretase is an important mediator of cell function and is both neurotrophic and neuroprotective. HEK 293T cells have been stably integrated with a fragment of the APP gene to produce and secrete either sAPPalpha, or the alternative cleavage product sAPPbeta. Heparin binding domains on the proteins have been utilised to develop a one-step fast-performance-liquid-chromatography (FPLC) purification of sAPPs from the conditioned media. Immunoblotting analyses with a sAPP specific antibody coupled with highly sensitive silver staining techniques have validated the expression and purification strategy. Functional activity of the purified fragments was demonstrated by their ability to protect COS-7 and SH-SY5Y (neuroblastoma) cells against the adverse effects of glucose deprivation in a cell viability assay. The purified sAPPs also activated the NFkappaB transcription factor in COS-7 cells transfected with a luciferase reporter plasmid, with sAPPalpha the more potent activator as expected. The simple protocol to produce these mammalian expressed proteins will facilitate their use as potential neuropharmacological reagents in the elucidation of biochemical pathways modulated by sAPPs, and in the study of Alzheimer's disease mechanisms in general.
Turner
Paul R
PR
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Bourne
Katie
K
Garama
Daniel
D
Carne
Alan
A
Abraham
Wickliffe C
WC
Tate
Warren P
WP
D000544:Alzheimer Disease / Q000188:drug therapy / Q000378:metabolism / Q000503:physiopathology; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D000818:Animals; D000918:Antibody Specificity / Q000276:immunology; D019556:COS Cells; D018929:Cell Culture Techniques; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002522:Chlorocebus aethiops; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D009448:Neurochemistry / Q000379:methods*; D018696:Neuroprotective Agents / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D017434:Protein Structure, Tertiary / Q000502:physiology; D016622:Silver Staining / Q000379:methods; D015533:Transcriptional Activation / Q000187:drug effects
D016564:Amyloid beta-Protein Precursor; D018696:Neuroprotective Agents