title: High yield expression of human BACE constructs in Eschericia coli for refolding, purification, and high resolution diffracting crystal forms.
abstract: BACE (beta-site APP cleaving enzyme) or beta-secretase, the enzyme responsible for processing APP to give the N-terminal portion of the Abeta peptide, is a membrane bound aspartyl protease consisting of an ectodomain catalytic unit, a C-terminal transmembrane segment and a cytoplasmic domain. Three BACE constructs, pET11a-BACE, pQE80L-BACE, and pQE70-BACE were designed to terminate at a position just before the transmembrane domain (Ser(432)) and are described schematically below. (1) pET11a-T7.Tag-G-S-M-(A-8GV......QTDES(432)), (2) pQE80L-Met-R-G-S-(His)(6)-G-S-I-E-T-D-(T(1)QH...QTDES(432)), and (3) pQE70-Met-BACE (R(36)GSFVEMG....PQTDES(432) (His) (6)) Each construct was over-expressed in Escherichia coli as inclusion bodies. The inclusion body proteins were solubilized in urea and refolded by dilution in water to yield active enzyme. Maximal activity for pET11a-BACE and pQE80L-BACE was usually reached at day 3 to 4, while construct pQE70-BACE required about 21 days. Active BACE was purified to homogeneity by anion-exchange chromatography and affinity chromatography over a column of immobilized peptide inhibitor. The process, easily scalable to 60 liters of cell culture, yielded in excess of 400 mg of active enzyme for crystallographic analysis. Highly purified pET11a-BACE and pQE70-BACE formed complexes with various inhibitors, the latter protein giving crystals diffracting up to 1.45 A resolution. In addition, a crystal form that does not require the presence of an inhibitor has been obtained for pQE70-BACE. This ligand-free crystal form has proven useful for the preparation of BACE-inhibitor complexes in soaking experiments.
authors.affiliation: Global Research and Development, St. Louis Laboratories, Pfizer Inc., Chesterfield, MO 63017, USA. alfredo.tomasselli@pfizer.com
authors.forename: A G
authors.initials: AG
authors.lastname: Tomasselli
authors.forename: D J
authors.initials: DJ
authors.lastname: Paddock
authors.forename: T L
authors.initials: TL
authors.lastname: Emmons
authors.forename: A M
authors.initials: AM
authors.lastname: Mildner
authors.forename: J W
authors.initials: JW
authors.lastname: Leone
authors.forename: J M
authors.initials: JM
authors.lastname: Lull
authors.forename: J I
authors.initials: JI
authors.lastname: Cialdella
authors.forename: D B
authors.initials: DB
authors.lastname: Prince
authors.forename: H D
authors.initials: HD
authors.lastname: Fischer
authors.forename: R L
authors.initials: RL
authors.lastname: Heinrikson
authors.forename: T E
authors.initials: TE
authors.lastname: Benson
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism / Q000473:pathology; D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016333:HIV Protease / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011487:Protein Conformation; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: D011498:Protein Precursors; D011994:Recombinant Proteins; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1; C509280:BACE1 protein, human