Large-Scale Purification of Human BACE Expressed in Mammalian Cells and Removal of the Prosegment with HIV-1 Protease to Improve Crystal Diffraction

Author(s): A. G. Tomasselli, T. L. Emmons, M. E. Shuck, M. S. Babcock, J. S. Holloway, J. W. Leone, J. D. Durbin, D. J. Paddock, D. B. Prince, R. L. Heinrikson, H. D. Fischer, M. J. Bienkowski, T. E. Benson, T. L. Emmons, M. E. Shuck, M. S. Babcock, J. S. Holloway, J. W. Leone, J. D. Durbin, D. J. Paddock, D. B. Prince, R. L. Heinrikson, H. D. Fischer, M. J. Bienkowski, T. E. Benson, A. G. Tomasselli

Journal Name: Protein & Peptide Letters

Volume 15 , Issue 2 , 2008


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Abstract:

BACE, or β-secretase, is an attractive target in the treatment of Alzheimers Disease because of its involvement in the generation of amyloid β peptides. BACE is a type I transmembrane aspartyl protease composed of pre-, pro-, catalytic, transmembrane and cytoplasmic domains. For the present study, the coding sequence was truncated just before the transmembrane domain and the resulting construct was extended with the C-terminal addition of a (His)6 and expressed in several mammalian host cells. The enzyme expressed in CHO cells had the best crystallographic behavior and was purified in large quantities in a three step procedure. The purified BACE was comprised of two forms, namely the full length proBACE construct beginning with Thr1, and a derivative missing the first 24 amino acids beginning with E25. These BACE precursors co-crystallized in the presence of inhibitors yielding structures to 3.2 Å resolution. HIV-1 protease treatment of this mixture resulted in complete cleavage of the F39-V40 bond, leaving the V40EM … ES432 (His)6 derivative that was purified yielding an enzyme that was no more active than untreated BACE but co-crystallized with inhibitors producing well shaped, bipyramidal co-crystals diffracting to 2.6 Å resolution.

Keywords: Alzheimer's, BACE, beta-secretase, amyloid, expression systems, HIV-1 protease

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Article Details

VOLUME: 15
ISSUE: 2
Year: 2008
Published on: 01 March, 2012
Page: [119 - 130]
Pages: 12
DOI: 10.2174/092986608783489599
Price: $65

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