Herein, we describe a simple and efficient approach to produce recombinant human α-synuclein (hAS) with high purity from Escherichia coli (E. coli). The cDNA for hAS was inserted into plasmid pET32a and expressed in E. coli BL21 (DE3) with an N-terminal tag containing E. coli thioredoxin (trx), followed by a histidine hexapeptide, and a tobacco etch virus (TEV) protease cleavage site (trx-6His-TEV). The fusion protein, trx-hAS, was initially released by osmotic shock treatment from the host cells and subsequently purified using a nickel affinity chromatography. A TEV protease cleavage step was performed to liberate the target protein, hAS, from the fusion partner, trx. Finally, an additional nickel affinity chromatography was performed to further purify the digested product. The yield of this method is ∼25 mg of tag-less protein (with ∼99% purity) per liter of culture volume. Reverse phase HPLC (RP-HPLC) and electrospray ionization (ESI) mass spectrometry confirmed the purity and authenticity of the purified protein. Thioflavin T (ThT) fluorescence assay, transmission electron microscopy (TEM), and circular dichroism (CD) spectroscopy demonstrated that the purified proteins form fibrils. Our protocol not only provides a convenient procedure for preparing highly pure hAS, but also requires very little specialized laboratory techniques.

在本文中，我们描述了一种简单有效的方法，可从大肠杆菌（E. coli）生产高纯度的重组人α-突触核蛋白（hAS ）。用于hAS的cDNA插入质粒pET32a中，并在大肠杆菌BL21（DE3）中表达，带有包含大肠杆菌的N端标签。硫氧还蛋白（trx），然后是组氨酸六肽，以及烟草蚀刻病毒（TEV）蛋白酶切割位点（trx-6His-TEV）。融合蛋白trx-hAS首先通过渗透休克处理从宿主细胞中释放出来，然后使用镍亲和色谱法纯化。进行TEV蛋白酶切割步骤以从融合伴侣trx释放靶蛋白hAS。最后，进行另一次镍亲和层析以进一步纯化消化的产物。该方法的产量为每升培养液约25 mg的无标签蛋白（纯度约99％）。反相HPLC（RP-HPLC）和电喷雾电离（ESI）质谱证实了纯化蛋白的纯度和真实性。硫黄素T（ThT）荧光测定，透射电子显微镜（TEM），圆二色性（CD）光谱证明纯化的蛋白质形成原纤维。我们的方案不仅提供了制备高纯度hAS的简便程序，而且所需的专业化实验室技术也非常少。