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    Preparation of alpha-synuclein fibrils for solid-state NMR: expression, purification, and incubation of wild-type and mutant forms. 
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    • Preparation of alpha-synuclein fibrils for solid-state NMR: expression, purification, and incubation of wild-type and mutant forms.
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    • School of Biological and Chemical Sciences
    • School of Biological and Chemical Sciences
    • Preparation of alpha-synuclein fibrils for solid-state NMR: expression, purification, and incubation of wild-type and mutant forms.
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    Preparation of alpha-synuclein fibrils for solid-state NMR: expression, purification, and incubation of wild-type and mutant forms.

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    Published version
    Embargoed until: 2100-01-01
    Reason: VoR
    Volume
    48
    Pagination
    112 - 117
    DOI
    10.1016/j.pep.2006.02.009
    Journal
    Protein Expr Purif
    Issue
    1
    ISSN
    1046-5928
    Metadata
    Show full item record
    Abstract
    We report the expression and purification of alpha-synuclein, a protein implicated in Parkinson's disease, from isotopically (13C, 15N) labeled bacterial growth media, as required for solid-state NMR structural studies. Expression from Escherichia coli (BL21(DE3)) was performed with a protocol optimized for time efficiency and yield. Chemical lysis, crude purification by ammonium sulfate precipitation, and two chromatography steps (hydrophobic interaction and size exclusion) yield 30-35 mg/L of growth medium. Purity is confirmed by gel electrophoresis and mass spectrometry. Furthermore, we demonstrate reproducible fibril growth by control of environmental incubation conditions. Highly resolved multidimensional solid-state NMR spectra indicate microscopic order throughout the majority of the AS fibril structure. The number of signals and intensities of well-resolved residue types (Thr, Ser, Ala, Gly, Val, and Ile) are consistent with a single conformation, which is reproducibly prepared by seeding consecutive preparations. Variations in the fibril growth rates and structural polymorphisms exhibited in the solid-state NMR spectra are minimized by careful control of incubation conditions.
    Authors
    Kloepper, KD; Woods, WS; Winter, KA; George, JM; Rienstra, CM
    URI
    http://qmro.qmul.ac.uk/xmlui/handle/123456789/3134
    Collections
    • School of Biological and Chemical Sciences [1659]
    Language
    eng
    Licence information
    https://doi.org/10.1016/j.pep.2006.02.009
    Copyright statements
    Copyright © 2006 Elsevier Inc.
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