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Title
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High yield expression of human BACE constructs in Eschericia coli for refolding, purification, and high resolution diffracting crystal forms.
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Authors
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A.G.Tomasselli,
D.J.Paddock,
T.L.Emmons,
A.M.Mildner,
J.W.Leone,
J.M.Lull,
J.I.Cialdella,
D.B.Prince,
H.D.Fischer,
R.L.Heinrikson,
T.E.Benson.
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Ref.
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Protein Pept Lett, 2008,
15,
131-143.
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PubMed id
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Abstract
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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.
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