High Yield Expression of Human BACE Constructs in Eschericia coli for Refolding, Purification, and High Resolution Diffracting Crystal Forms

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Abstract:

BACE (β-site APP cleaving enzyme) or β-secretase, the enzyme responsible for processing APP to give the Nterminal portion of the Aβ peptide, is a membrane bound aspartyl protease consisting of an ectodomain catalytic unit, a Cterminal 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 (Ser432) and are described schematically below.

(1) pET11a-T7.Tag-G-S-M-(A-8GV....QTDES432),

(2) pQE80L-Met-R-G-S-(His)6-G-S-I-E-T-D-(T1QH...QTDES432), and

(3) pQE70-Met-BACE (R36GSFVEMG....PQTDES432(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 Å 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.

Keywords: BACE; crystal; expression; inclusion bodies; refold

Document Type: Research Article

DOI: http://dx.doi.org/10.2174/092986608783489553

Publication date: February 1, 2008

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  • Protein & Peptide Letters publishes short papers in all important aspects of protein and peptide research, including structural studies, recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, drug design etc. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallisation, and preliminary structure determinations of biologically important proteins are acceptable. Purely theoretical papers are also acceptable provided they provide new insight into the principles of protein/peptide structure and function.

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