Kloepper Kathryn D, Woods Wendy S, Winter Kem A, George Julia M, Rienstra Chad M
Department of Chemistry, University of Illinois, 600 S. Mathews Ave., Urbana, IL 61801, USA.
Protein Expr Purif. 2006 Jul;48(1):112-7. doi: 10.1016/j.pep.2006.02.009. Epub 2006 Mar 10.
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.
根据固态核磁共振结构研究的要求，我们报道了从同位素（13C、15N）标记的细菌生长培养基中表达和纯化与帕金森病相关的蛋白质α-突触核蛋白的过程。使用针对时间效率和产量进行优化的方案，在大肠杆菌（BL21(DE3)）中进行表达。通过化学裂解、硫酸铵沉淀粗纯化以及两步色谱法（疏水相互作用和尺寸排阻），每升生长培养基可获得30 - 35毫克的产物。通过凝胶电泳和质谱法确认纯度。此外，我们通过控制环境孵育条件证明了可重复的纤维生长。高度解析的多维固态核磁共振谱表明，在大多数α-突触核蛋白纤维结构中存在微观有序性。解析良好的残基类型（苏氨酸、丝氨酸、丙氨酸、甘氨酸、缬氨酸和异亮氨酸）的信号数量和强度与单一构象一致，通过连续制备的种子接种可重复制备该构象。通过仔细控制孵育条件，可将固态核磁共振谱中显示的纤维生长速率和结构多态性的变化降至最低。
