alexa High yield expression of human BACE constructs in Eschericia coli for refolding, purification, and high resolution diffracting crystal forms.
Psychiatry

Psychiatry

Journal of Addiction Research & Therapy

Author(s): Tomasselli AG, Paddock DJ, Emmons TL, Mildner AM, Leone JW,

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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.
This article was published in Protein Pept Lett and referenced in Journal of Addiction Research & Therapy

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