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A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation
Published on: March 27, 2017
Production of recombinant amyloid-beta peptide 42 as an ubiquitin extension
Eun Kyung Lee1, Jin Ha Hwang, Dong Yeon Shin
1Department of Life Science, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea.
Abstract:
Amyloid-beta peptide 42 (Abeta42) mediates neuronal degeneration in Alzheimer's disease (AD). We sought to produce recombinant Abeta42 as an ubiquitin extension. A synthetic oligonucleotide encoding Abeta42 was constructed and cloned as an extended polypeptide of hexahistidine-tagged ubiquitin (H(6)Ub) using the pET vector. Isopropyl-beta-D-thiogalactopyranoside induction of transformed Escherichia coli resulted in the production of large amounts of insoluble H(6)Ub-Abeta42 fusion protein. H(6)Ub-Abeta42 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(6)Ub-Abeta42 was eluted, indicating that covalently attached ubiquitin prevented Abeta42 from aggregating. Abeta42 was cleaved from H(6)Ub using recombinant yeast ubiquitin hydrolase-1 (YUH-1) and purified using reverse-phase chromatography. The recombinant Abeta42 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 Abeta42.
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