Large-scale purification of human BACE expressed in mammalian cells and removal of the prosegment with HIV-1 protease to improve crystal diffraction.
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.
Emmons
T L
TL
Global Research and Development, St. Louis Laboratories, Pfizer Inc., Chesterfield, MO 63017, USA.
Shuck
M E
ME
Babcock
M S
MS
Holloway
J S
JS
Leone
J W
JW
Durbin
J D
JD
Paddock
D J
DJ
Prince
D B
DB
Heinrikson
R L
RL
Fischer
H D
HD
Bienkowski
M J
MJ
Benson
T E
TE
Tomasselli
A G
AG
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
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