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

Paul R. Turner, Katie Bourne, Daniel Garama, Alan Carne, Wickliffe C. Abraham, Warren P. Tate

Research output: Contribution to journalArticleResearchpeer-review

16 Citations (Scopus)


The secreted fragment of the amyloid precursor protein (sAPPα) generated following cleavage by α-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 sAPPα, or the alternative cleavage product sAPPβ. 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 NFκB transcription factor in COS-7 cells transfected with a luciferase reporter plasmid, with sAPPα 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.

Original languageEnglish
Pages (from-to)68-74
Number of pages7
JournalJournal of Neuroscience Methods
Issue number1
Publication statusPublished - 15 Aug 2007
Externally publishedYes


  • Alzheimer's disease
  • Amyloid precursor protein
  • Neuropharmacological reagent
  • One step purification
  • SAPPα
  • SAPPβ
  • Secreted fragment

Cite this