title: A novel method for expression and purification of authentic amyloid-β with and without (15)N labels.
abstract: 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.
authors.affiliation: Genomics Research Center, Academia Sinica, 128, Academia Rd., Sec. 2, Nankang Dist., Taipei 115, Taiwan.
authors.forename: Yi-Hung
authors.initials: YH
authors.lastname: Liao
authors.affiliation: Genomics Research Center, Academia Sinica, 128, Academia Rd., Sec. 2, Nankang Dist., Taipei 115, Taiwan. Electronic address: yrchen@gate.sinica.edu.tw.
authors.forename: Yun-Ru
authors.initials: YR
authors.lastname: Chen
mesh_terms: D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009587:Nitrogen Isotopes / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D016229:Amyloid beta-Peptides; D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins; D006639:Histidine; D010450:Endopeptidases; C099488:TEV protease
keywords: Alzheimer’s disease; Amyloid-β; His-tag; Metal ions; NMR; Partial refolding; TEV protease