Preparation of α-synuclein fibrils for solid-state NMR: Expression, purification, and incubation of wild-type and mutant forms

Kathryn D. Kloepper, Wendy S. Woods, Kem A. Winter, Julia M. George, Chad M. Rienstra

Research output: Contribution to journalArticlepeer-review

Abstract

We report the expression and purification of α-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.

Original languageEnglish (US)
Pages (from-to)112-117
Number of pages6
JournalProtein Expression and Purification
Volume48
Issue number1
DOIs
StatePublished - Jul 2006

Keywords

  • Fibrillation
  • Isotopic labeling
  • Magic-angle spinning
  • Neurodegenerative disease
  • Polymorphism

ASJC Scopus subject areas

  • Biotechnology

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