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Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid beta 1-42

1
Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA
2
Purdue Institute for Drug Discovery, Purdue University, West Lafayette, IN 47907, USA
3
Purdue Institute for Integrative Neuroscience, Purdue University, West Lafayette, IN 47907, USA
4
Purdue Institute for Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, IN 47907, USA
5
Purdue Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA
*
Author to whom correspondence should be addressed.
These authors contributed equally to the paper.
Methods Protoc. 2019, 2(2), 48; https://doi.org/10.3390/mps2020048
Received: 16 April 2019 / Revised: 31 May 2019 / Accepted: 3 June 2019 / Published: 7 June 2019
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Abstract

Amyloid plaques found in the brains of Alzheimer’s disease patients primarily consists of amyloid beta 1-42 (Aβ42). Commercially, Aβ42 is synthesized using high-throughput peptide synthesizers resulting in the presence of impurities and the racemization of amino acids that affects its aggregation properties. Furthermore, the repeated purchase of even a small quantity (~1 mg) of commercial Aβ42 can be expensive for academic researchers. Here, we describe a detailed methodology for robust expression of recombinant human Aβ(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli using standard molecular biology techniques with refined and rapid one-step analytical purification techniques. The peptide is isolated and purified from transformed cells using an optimized reverse-phase high-performance liquid chromatography (HPLC) protocol with commonly available C18 columns, yielding high amounts of peptide (~15–20 mg per 1 L culture) within a short period of time. The recombinant human Aβ(M1-42) forms characteristic aggregates similar to synthetic Aβ42 aggregates as verified by western blotting and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique produces pure recombinant human Aβ(M1-42) that may be used to synthesize chemical probes and in several downstream in vitro and in vivo assays to facilitate Alzheimer’s disease research. View Full-Text
Keywords: amyloid beta; recombinant abeta; HPLC; expression; purification; neuroscience; peptide; neurodegeneration; Alzheimer’s disease amyloid beta; recombinant abeta; HPLC; expression; purification; neuroscience; peptide; neurodegeneration; Alzheimer’s disease
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Prakash, P.; Lantz, T.C.; Jethava, K.P.; Chopra, G. Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid beta 1-42. Methods Protoc. 2019, 2, 48.

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