Preparation of alpha-synuclein fibrils for solid-state NMR: expression, purification, and incubation of wild-type and mutant forms.
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.
Kloepper
Kathryn D
KD
Department of Chemistry, University of Illinois, 600 S. Mathews Ave., Urbana, IL 61801, USA.
Woods
Wendy S
WS
Winter
Kem A
KA
George
Julia M
JM
Rienstra
Chad M
CM
D002247:Carbon Isotopes / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009154:Mutation; D009587:Nitrogen Isotopes / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D051844:alpha-Synuclein