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
Protein & Peptide Letters
Title: Large-Scale Purification of Human BACE Expressed in Mammalian Cells and Removal of the Prosegment with HIV-1 Protease to Improve Crystal Diffraction
Volume: 15 Issue: 2
Author(s): T. L. Emmons, A. G. Tomasselli, T. E. Benson, M. J. Bienkowski, H. D. Fischer, R. L. Heinrikson, D. B. Prince, D. J. Paddock, J. D. Durbin, J. W. Leone, J. S. Holloway, M. S. Babcock, M. E. Shuck, A. G. Tomasselli, T. E. Benson, M. J. Bienkowski, H. D. Fischer, R. L. Heinrikson, D. B. Prince, D. J. Paddock, J. D. Durbin, J. W. Leone, J. S. Holloway, M. S. Babcock, M. E. Shuck and T. L. Emmons
Affiliation:
Keywords: Alzheimer's, BACE, beta-secretase, amyloid, expression systems, HIV-1 protease
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.
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L. Emmons T., G. Tomasselli A., E. Benson T., J. Bienkowski M., D. Fischer H., L. Heinrikson R., B. Prince D., J. Paddock D., D. Durbin J., W. Leone J., S. Holloway J., S. Babcock M., E. Shuck M., Tomasselli G. A., Benson E. T., Bienkowski J. M., Fischer D. H., Heinrikson L. R., Prince B. D., Paddock J. D., Durbin D. J., Leone W. J., Holloway S. J., Babcock S. M., Shuck E. M. and Emmons L. T., Large-Scale Purification of Human BACE Expressed in Mammalian Cells and Removal of the Prosegment with HIV-1 Protease to Improve Crystal Diffraction, Protein & Peptide Letters 2008; 15 (2) . https://dx.doi.org/10.2174/092986608783489599
DOI https://dx.doi.org/10.2174/092986608783489599 |
Print ISSN 0929-8665 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5305 |
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