Overexpression and Purification of Human APP and BACE

• Main Authors: Ju-Chieh Wung, 翁如潔
• Other Authors: Fan-Lu Kung
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/85242406835546939531

碩士 === 國立臺灣大學 === 藥學研究所 === 92 === Alzheimer’s disease is a neurodegenerative disorder of which one of the major pathological features is the presence of amyloid plaque and neurofibrillary tangle in brain cortex and hippocampus. Amyloid plaque is mostly composed of Abeta, which is generated from proteolytic cleavage of amyloid precursor protein (APP) by beta-secretase and gamma-secretase in sequential. A novel protein designated as beta-site APP cleaving enzyme (BACE) contains all the known characteristics of beta-secretase. To investigate how BACE catalyzes the proteolytic reaction of APP, it is essential to obtain large amounts of APP and BACE. To obtain the substrate of BACE, a construct encoding glutathion S-transferase and APP695 was used to overexpress the GST-APP695 fusion protein in Escherichia coli, which was later purified using affinity chromatography. The plasmid containing the full length BACE gene was transformed into E. coli and used to overexpress GST-BACE as GST-APP695. It was found that the fusion protein formed inclusion bodies when being overexpressed. It was attempted to develop a denaturation/refolding scheme to obtain active BACE from inclusion bodies. To assess the effects of N-glycosylated on BACE activity, the full length BACE gene was transfected into Spodoptera frugiperda cells to obtain N-glycosylated active BACE. Hopefully an overexpression/purification scheme can be established to obtain large quantities of APP and BACE efficiently to investigate the substrate recognition of BACE using in vitro kinetics studies.