Purification and Characterization of the Human 纬-Secretase Activating Protein

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• 作者：Catherine L. Deatherage ; Arina Hadziselimovic ; Charles R. Sanders
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：June 26, 2012
• 年：2012
• 卷：51
• 期：25
• 页码：5153-5159
• 全文大小：294K
• 年卷期：v.51,no.25(June 26, 2012)
• ISSN：1520-4995

文摘

Alzheimer鈥檚 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 纬-secretase activating protein (GSAP), has been proposed to promote elevated levels of amyloid-尾 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 伪-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.