Liao Yi-Hung, Chen Yun-Ru
Genomics Research Center, Academia Sinica, 128, Academia Rd., Sec. 2, Nankang Dist., Taipei 115, Taiwan.
Genomics Research Center, Academia Sinica, 128, Academia Rd., Sec. 2, Nankang Dist., Taipei 115, Taiwan.
Protein Expr Purif. 2015 Sep;113:63-71. doi: 10.1016/j.pep.2015.05.002. Epub 2015 May 9.
Amyloid-β (Aβ) is a major constituent in the senile plaques of patients with Alzheimer's disease (AD). Aβ has been intensively studied in amyloid research; however, challenges posed by data reproducibility arise from purity of synthetic Aβ and high expense for its isotope-labeling. The difficulties motivate development and optimization of recombinant Aβ (rAβ) production. Here, we report a new procedure to express and purify high quality rAβ40 from Escherichia coli. The new Aβ construct expressed insoluble Aβ fused with an N-terminal histidine-tag connected by a linker harboring TEV protease cut site. After purification and partial refolding, the fusion tag was removed by TEV protease cleavage, immobilized metal affinity chromatography (IMAC), and reversed phase-HPLC purification with a yield of 3.5 mg/L culture with and without (15)N label. The rAβ adopts classic amyloid fibrillization and is capable of binding to its clinical relevant metal ions.
淀粉样β蛋白（Aβ）是阿尔茨海默病（AD）患者脑内老年斑的主要成分。Aβ在淀粉样蛋白研究中受到广泛关注；然而，合成Aβ的纯度以及同位素标记的高成本给数据的可重复性带来了挑战。这些困难促使人们开发和优化重组Aβ（rAβ）的生产方法。在此，我们报道了一种从大肠杆菌中表达和纯化高质量rAβ40的新方法。新的Aβ构建体表达出不溶性Aβ，其与N端组氨酸标签融合，通过含有TEV蛋白酶切割位点的接头连接。经过纯化和部分复性后，通过TEV蛋白酶切割、固定化金属亲和色谱（IMAC）和反相高效液相色谱（RP-HPLC）纯化去除融合标签，无论有无（15）N标记，产量均为3.5 mg/L培养物。rAβ呈现典型的淀粉样纤维化，并且能够结合与其临床相关的金属离子。
