Expression,
Purification and Activity Measurement of Soluble BACE Protein in E.coli
WEI Xiao-Chao, LI Heng, JI Jian-Guo, LI
Ru, REN Yan-Fei, FENG Jin-Hui, HAO Fu-Ying*
( Laboratory of Biological Macromolecule, College of Life Sciences, Peking
University, Beijing 100871, China )
Abstract 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.
Key words BACE; AD; ¦Â secretase; refolding
*Corresponding author: Tel,
86-10-62752010; e-mail, [email protected]