title: Large-scale purification of human BACE expressed in mammalian cells and removal of the prosegment with HIV-1 protease to improve crystal diffraction.
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.
authors.affiliation: Global Research and Development, St. Louis Laboratories, Pfizer Inc., Chesterfield, MO 63017, USA.
authors.forename: T L
authors.initials: TL
authors.lastname: Emmons
authors.forename: M E
authors.initials: ME
authors.lastname: Shuck
authors.forename: M S
authors.initials: MS
authors.lastname: Babcock
authors.forename: J S
authors.initials: JS
authors.lastname: Holloway
authors.forename: J W
authors.initials: JW
authors.lastname: Leone
authors.forename: J D
authors.initials: JD
authors.lastname: Durbin
authors.forename: D J
authors.initials: DJ
authors.lastname: Paddock
authors.forename: D B
authors.initials: DB
authors.lastname: Prince
authors.forename: R L
authors.initials: RL
authors.lastname: Heinrikson
authors.forename: H D
authors.initials: HD
authors.lastname: Fischer
authors.forename: M J
authors.initials: MJ
authors.lastname: Bienkowski
authors.forename: T E
authors.initials: TE
authors.lastname: Benson
authors.forename: A G
authors.initials: AG
authors.lastname: Tomasselli
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism / Q000473:pathology; D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003460:Crystallization; D016333:HIV Protease / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011487:Protein Conformation; D011498:Protein Precursors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D020539:Sequence Analysis, Protein; D014961:X-Ray Diffraction
chemical_list: D011498:Protein Precursors; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1; C509280:BACE1 protein, human