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
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
ISSN
1046-5928
Metadata
Show full item recordAbstract
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.