Document Detail

Production, purification and functional validation of human secreted amyloid precursor proteins for use as neuropharmacological reagents.
MedLine Citation:
PMID:  17537517     Owner:  NLM     Status:  MEDLINE    
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.
Paul R Turner; Katie Bourne; Daniel Garama; Alan Carne; Wickliffe C Abraham; Warren P Tate
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Validation Studies     Date:  2007-04-06
Journal Detail:
Title:  Journal of neuroscience methods     Volume:  164     ISSN:  0165-0270     ISO Abbreviation:  J. Neurosci. Methods     Publication Date:  2007 Aug 
Date Detail:
Created Date:  2007-06-25     Completed Date:  2007-09-27     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  7905558     Medline TA:  J Neurosci Methods     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  68-74     Citation Subset:  IM    
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
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MeSH Terms
Alzheimer Disease / drug therapy,  metabolism,  physiopathology
Amyloid beta-Protein Precursor / chemistry*,  isolation & purification*,  pharmacology
Antibody Specificity / immunology
COS Cells
Cell Culture Techniques
Cell Line
Cell Line, Tumor
Cell Survival / drug effects,  physiology
Cercopithecus aethiops
Chromatography, Liquid / methods*
Immunoblotting / methods
Neurochemistry / methods*
Neuroprotective Agents / chemistry*,  isolation & purification*,  pharmacology
Protein Structure, Tertiary / physiology
Silver Staining / methods
Transcriptional Activation / drug effects
Reg. No./Substance:
0/Amyloid beta-Protein Precursor; 0/Neuroprotective Agents

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