Purification and characterization of the human γ-secretase activating protein.
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.
Department of Biochemistry, Center for Structural Biology, and Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-8725, USA.
Catherine L
CL
Deatherage
Arina
A
Hadziselimovic
Charles R
CR
Sanders
D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D005822:Genetic Vectors / Q000138:chemical synthesis; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000138:chemical synthesis; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
C552289:GSAP protein, human; D011506:Proteins; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins