Crystallization of glycosylated human BACE protease domain expressed in Trichoplusia ni.
Human beta-amyloid precursor protein cleaving enzyme (beta-secretase, or BACE) belongs to the aspartyl protease family, and is responsible for generating the N-terminus of beta-amyloid peptide (Abeta). 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 A data set was collected on a single orthorhombic crystal with unit cell parameters a=74 A, b=130 A, c=134A. Successful molecular replacement shows two BACE molecules in the asymmetric unit.
Wang
Wenyan
W
Protein Engineering and Biochem.Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.
Reichert
Paul
P
Beyer
Brian M
BM
Liu
Jian-Jun
JJ
Lee
Julie
J
Zhang
Lili
L
Liu
Yan-Hui
YH
Taremi
S Shane
SS
Le
Hung V
HV
Strickland
Corey
C
D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003460:Crystallization; D010450:Endopeptidases; D006801:Humans; D009036:Moths / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary
D010455:Peptides; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509284:BACE2 protein, human; C509280:BACE1 protein, human