Highly efficient soluble expression, purification and characterization of recombinant Aβ42 from Escherichia coli. Issue 33 (21st May 2018)

• Record Type: Journal Article
• Authors: Jia, Longgang; Wang, Wenjuan; Shang, Jinzhao; Zhao, Wenping; Wei, Wei; Wang, Ying; Li, Li; Lu, Fuping; Liu, Fufeng
• Is Part Of: RSC advances. Volume 8:Issue 33(2018)
• Page Start: 18434
• Page End: 18441
• Publication Date: 2018-05-21
• Subjects: Chemistry -- Periodicals; 540.5
• DOI: 10.1039/c8ra00042e
• Languages: English
• ISSNs: 2046-2069

Abstract

Abstract : A novel high-yield expression and purification method for Aβ42 based on a fusion with maltose binding protein followed by the soluble polypeptide linker (NANP)3 and a modified tobacco etch virus cleavage site before the Aβ42 was developed. Abstract : Aggregation of amyloid-β protein (Aβ) is hypothesized to be a seminal neuropathological event in Alzheimer's disease (AD). Recombinant expression and purification of Aβ represents a common basis for investigating the molecular mechanisms of amyloid formation and toxicity. Herein, we report a novel high-yield expression and purification method for Aβ42 based on fusion with maltose binding protein (MBP) followed by the soluble polypeptide linker (NANP)3 and a modified tobacco etch virus (TEV) cleavage site before the Aβ42. We obtained a final yield of ∼18 mg L −1 of recombinant Aβ42 that was confirmed by SDS-PAGE, protein immunoblotting and MALDI-TOF. Finally, thioflavin T fluorescence and atomic force microscopy revealed that the recombinant Aβ42 aggregated into long, branched fibrils. Furthermore, the aggregates of the recombinant peptide had a strong cytotoxic effect on PC12 cells. The method described here can therefore be used to efficiently express the soluble fusion protein MBP-Aβ42 and obtain high-purity Aβ42 peptide, which can be used to understand the molecular mechanism of Aβ42 fibrillization and screen new candidate drugs for AD.