کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2022211 | 1069286 | 2006 | 6 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Preparation of α-synuclein fibrils for solid-state NMR: Expression, purification, and incubation of wild-type and mutant forms Preparation of α-synuclein fibrils for solid-state NMR: Expression, purification, and incubation of wild-type and mutant forms](/preview/png/2022211.png)
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.
Journal: Protein Expression and Purification - Volume 48, Issue 1, July 2006, Pages 112–117