A novel method for expression and purification of authentic amyloid-β with and without (15)N labels

Protein Expr Purif. 2015 Sep:113:63-71. doi: 10.1016/j.pep.2015.05.002. Epub 2015 May 9.

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.

Keywords: Alzheimer’s disease; Amyloid-β; His-tag; Metal ions; NMR; Partial refolding; TEV protease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amyloid beta-Peptides / chemistry
  • Amyloid beta-Peptides / genetics
  • Amyloid beta-Peptides / isolation & purification*
  • Amyloid beta-Peptides / metabolism
  • Cloning, Molecular
  • Endopeptidases / genetics
  • Histidine / chemistry
  • Histidine / genetics
  • Histidine / metabolism*
  • Nitrogen Isotopes / chemistry
  • Nitrogen Isotopes / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification*
  • Recombinant Fusion Proteins / metabolism

Substances

  • Amyloid beta-Peptides
  • Nitrogen Isotopes
  • Recombinant Fusion Proteins
  • Histidine
  • Endopeptidases
  • TEV protease