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Charles Sanders
Faculty Member
Last active: 3/3/2020

Purification and characterization of the human γ-secretase activating protein.

Deatherage CL, Hadziselimovic A, Sanders CR
Biochemistry. 2012 51 (25): 5153-9

PMID: 22681044 · PMCID: PMC3386298 · DOI:10.1021/bi300605u

Alzheimer's disease is a fatal neurological disorder that is a leading cause of death, with its prevalence increasing as the average life expectancy increases worldwide. There is an urgent need to develop new therapeutics for this disease. A newly described protein, the γ-secretase activating protein (GSAP), has been proposed to promote elevated levels of amyloid-β production, an activity that seems to be inhibited using the well-establish cancer drug, imatinib (Gleevec). Despite much interest in this protein, there has been little biochemical characterization of GSAP. Here we report protocols for the recombinant bacterial expression and purification of this potentially important protein. GSAP is expressed in inclusion bodies, which can be solubilized using harsh detergents or urea; however, traditional methods of refolding were not successful in generating soluble forms of the protein that contained well-ordered and homogeneous tertiary structure. However, GSAP could be solubilized in detergent micelle solutions, where it was seen to be largely α-helical but to adopt only heterogeneous tertiary structure. Under these same conditions, GSAP did not associate with either imatinib or the 99-residue transmembrane C-terminal domain of the amyloid precursor protein. These results highlight the challenges that will be faced in attempts to manipulate and characterize this protein.

MeSH Terms (11)

Escherichia coli Genetic Vectors Humans Inclusion Bodies Plasmids Protein Folding Proteins Protein Structure, Secondary Protein Structure, Tertiary Recombinant Fusion Proteins Thioredoxins

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