Production of recombinant amyloid-β peptide 42 as an ubiquitin extension

Abstract

Amyloid-β peptide 42 (Aβ42) mediates neuronal degeneration in Alzheimer’s disease (AD). We sought to produce recombinant Aβ42 as an ubiquitin extension. A synthetic oligonucleotide encoding Aβ42 was constructed and cloned as an extended polypeptide of hexahistidine-tagged ubiquitin (H 6Ub) using the pET vector. Isopropyl-β- d-thiogalactopyranoside induction of transformed Escherichia coli resulted in the production of large amounts of insoluble H 6Ub–Aβ42 fusion protein. H 6Ub–Aβ42 was solubilized in 8 M urea and applied to a nickel–nitrilotriacetic acid affinity column for purification. Column washing removed the urea and soluble H 6Ub–Aβ42 was eluted, indicating that covalently attached ubiquitin prevented Aβ42 from aggregating. Aβ42 was cleaved from H 6Ub using recombinant yeast ubiquitin hydrolase-1 (YUH-1) and purified using reverse-phase chromatography. The recombinant Aβ42 prepared in this study has the same toxic effect on human neuroblastoma SH-SY5Y cells comparing with chemically synthesized, commercial one. The peptide yield was more than 4 mg/L culture, indicating this ubiquitin fusion technique is an attractive method for production of aggregation-prone peptides such as Aβ42.