Production, purification and functional validation of human secreted amyloid precursor proteins for use as neuropharmacological reagents

The secreted fragment of the amyloid precursor protein (sAPPu) 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 zalene to produce and secrete either sAPP alpha, or the alternative cleavage product sAPPP. 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 NF kappa B transcription factor in COS-7 cells transfected with a luciferase reporter plasmid, with sAPP alpha 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. (C) 2007 Elsevier B.V. All rights reserved.