title: Large scale refolding and purification of the catalytic domain of human BACE-2 produced in E. coli.
abstract: Evidence for a key role of beta-amyloid (Abeta) in Alzheimer's disease has led to considerable interest in potential therapeutic strategies targeting enzymes involved in processing the amyloid precursor protein (APP). Beta-site APP Cleaving Enzyme (BACE or beta-secretase) is a membrane bound aspartyl protease that has been shown to be directly involved in Abeta production and, therefore, is at the forefront of therapeutic targets in the treatment of Alzheimer's disease. BACE-2, an enzyme closely related to BACE, regulates Abeta production in a manner antagonistic to BACE, suggesting that non-selective inhibition of BACE-2 by BACE inhibitors might impair the lowering of Abeta. The design of BACE inhibitors that do not inhibit BACE-2 would be enhanced by structural and kinetic studies, efforts that typically demand considerable amounts of both enzymes. A BACE-2 construct containing 19 residues of the BACE prosegment followed by the BACE-2 catalytic domain sequence, Asp36-Trp447, was produced in E. coli inclusion bodies (IB) at 110-140 mg/L cell culture. Exploration of a variety of refolding conditions resulted in an efficient method for refolding the resulting pro-BACE-2 construct, and this protein undergoes facile autocatalytic cleavage, optimal at pH 4, at the Leu40- downward arrow-Ala41 bond. Refolded BACE-2 was purified by anion exchange, molecular sieve, and affinity chromatographies, yielding 105 mg of homogeneous enzyme (kcat/ Km = 1.2 x 10(4) x M(-1) x sec(-1)) from 8 liters of E. coli cell culture.
authors.affiliation: Global Research and Development, St. Louis Laboratories, Pfizer Inc., 700 Chesterfield Parkway West, Chesterfield, MO, USA.
authors.forename: T L
authors.initials: TL
authors.lastname: Emmons
authors.forename: A M
authors.initials: AM
authors.lastname: Mildner
authors.forename: J M
authors.initials: JM
authors.lastname: Lull
authors.forename: J W
authors.initials: JW
authors.lastname: Leone
authors.forename: H D
authors.initials: HD
authors.lastname: Fischer
authors.forename: R L
authors.initials: RL
authors.lastname: Heinrikson
authors.forename: A G
authors.initials: AG
authors.lastname: Tomasselli
mesh_terms: D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D008969:Molecular Sequence Data; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509284:BACE2 protein, human