Crystallization of glycosylated human BACE protease domain expressed in Trichoplusia ni

Abstract

Human β-amyloid precursor protein cleaving enzyme (β-secretase, or BACE) belongs to the aspartyl protease family, and is responsible for generating the N-terminus of β-amyloid peptide (Aβ). BACE is a type I transmembrane glycoprotein with pre-, pro- and catalytic domains, a short transmembrane helix and a cytoplasmic region. In this study, a truncated form was engineered to produce the authentic catalytic domain of BACE in Trichoplusia ni (High 5™) cells. The glycosylated BACE zymogen (proBACE) was secreted into the conditioned medium for facile purification by metal chelate and gel filtration chromatographies. The mature catalytic domain was obtained by a trans cleavage event under acidic conditions and crystallized in the absence of a bound inhibitor. A complete 3.4 Å data set was collected on a single orthorhombic crystal with unit cell parameters a=74 Å, b=130 Å, c=134Å. Successful molecular replacement shows two BACE molecules in the asymmetric unit.