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

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BACE, or β-secretase, is an attractive target in the treatment of Alzheimer's 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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Keywords: Alzheimer's; BACE; HIV-1 protease; amyloid; beta-secretase; expression systems

Document Type: Research Article

Publication date: 2008-02-01

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  • Protein & Peptide Letters publishes short papers in all important aspects of protein and peptide research, including structural studies, recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, drug design etc. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallisation, and preliminary structure determinations of biologically important proteins are acceptable. Purely theoretical papers are also acceptable provided they provide new insight into the principles of protein/peptide structure and function.
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