Recombinant insect cell expression and purification of human β-secretase (BACE-1) for X-ray crystallography

被引:20
作者
Bruinzeel, W [1 ]
Yon, J [1 ]
Giovannelli, S [1 ]
Masure, S [1 ]
机构
[1] Johnson & Johnson Pharmaceut Res & Dev, Dept Assay Dev & HTS, B-2340 Beerse, Belgium
关键词
D O I
10.1016/S1046-5928(02)00516-8
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Human beta-secretase (BACE-1) is a type I integral membrane aspartic protease that catalyzes the internal cleavage of the amyloid precursor protein (APP), generating the N-terminus of the A peptide, The generation and subsequent extracellular deposition of Abeta(1-42) peptide into amyloid plaques in the brain constitute one of the hallmarks of Alzheimer's disease (AD), a common debilitating neurodegenerative disorder, Inhibition of BACE-1 is considered an excellent therapeutic strategy against AD. To generate pure enzyme for protein crystallography and subsequent structure-based drug design, we have expressed a soluble, unglycosylated, 6xHis-tagged form of proBACE-1 in insect cells using baculovirus infection. To avoid production of a mixture of the pro-enzyme form and the mature form of BACE-1, the proprotein convertase furin was coexpressed with proBACE-1, leading to almost complete proteolytic activation of the recombinant enzyme. The mature enzyme was secreted in the conditioned medium of BACE-1/furin coinfected HighFive insect cells. Secreted BACE-1 protein was purified to homogeneity from the medium using subsequent Ni-chelate affinity chromatography, anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. To avoid autoproteolysis. all purification steps were performed at pH values outside the activity range of BACE-1. The purified. biologically active enzyme was homogeneous on SDS/PAGE and had the expected sequence and molecular mass determined by N-terminal amino acid sequencing and mass spectrometry, respectively. Moreover, the preparation showed a single peak of the expected size with only 17%, polydispersity using dynamic light scattering analysis. The yield of BACE-1 from fermentation cultures was approximately 0.1 mg pure enzyme per liter of cell culture medium. The purified protein was successfully used to generate BACE-1/inhibitor co-crystals and to determine the crystal structure of the complex by X-ray analysis. The availability of substantial quantities of active, homogeneous enzyme will be of great help in future structure-based drug design efforts in the search for efficient protease inhibitor drugs to treat AD. (C) 2002 Elsevier Science (USA). All rights reserved.
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页码:139 / 148
页数:10
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