Large-scale purification of human BACE expressed in mammalian cells and removal of the prosegment with HIV-1 protease to improve crystal diffraction.
Protein Pept Lett
; 15(2): 119-30, 2008.
Article
em En
| MEDLINE
| ID: mdl-18289104
ABSTRACT
BACE, or beta-secretase, is an attractive target in the treatment of Alzheimer's Disease because of its involvement in the generation of amyloid beta 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 Thr(1), and a derivative missing the first 24 amino acids beginning with E(25). These BACE precursors co-crystallized in the presence of inhibitors yielding structures to 3.2 A resolution. HIV-1 protease treatment of this mixture resulted in complete cleavage of the F(39)-V(40) bond, leaving the V(40)EM...ES(432) (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 A resolution.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Precursores de Proteínas
/
Ácido Aspártico Endopeptidases
/
Protease de HIV
/
Secretases da Proteína Precursora do Amiloide
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Protein Pept Lett
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2008
Tipo de documento:
Article
País de afiliação:
Estados Unidos