Expression, purification and activity measurement of soluble BACE protein in E.coli.
To express BACE (beta-site APP cleaving enzyme), an aspartic protease related to Alzheimer's disease in E.coli to obtain the active protein after refolding and purification, the sequence for soluble form of BACE was inserted into prokaryotic expression vector pET11a. After expression in E.coli BL21(DE3), the protein was obtained as inclusion bodies, after refolding and purification. The proteolytic activity of the protein was measured by HPLC and MS. After refolding and purification, the protein exhibited beta-secretase activity by cleaving the synthetic peptide, which was designed according to the beta-secretase site at Swedish mutant of APP, and according Hanes method the kinetics constants for the synthetic peptide of BACE protein were measured. The study will facilitate BACE protein structure determination and inhibitor development efforts, which may lead to the evolution of promising and effective treatments for Alzheimer's disease.
Wei
Xiao-Chao
XC
Laboratory of Biological Macromolecule, College of Life Sciences, Peking University, Beijing 100871, China. haofy@bio.pku.edu.cn
Li
Heng
H
Ji
Jian-Guo
JG
Li
Ru
R
Ren
Yan-Fei
YF
Feng
Jin-Hui
JH
Hao
Fu-Ying
FY
D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D013058:Mass Spectrometry / Q000379:methods; D009842:Oligopeptides / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
D009842:Oligopeptides; D011994:Recombinant Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases