A Robust and Efficient Production and Purification Procedure of Recombinant Alzheimers Disease Methionine-Modified Amyloid-β Peptides

• Published in: PloS one Vol. 11; no. 8; p. e0161209
• Main Authors: Hoarau, Marie, Malbert, Yannick, Irague, Romain, Hureau, Christelle, Faller, Peter, Gras, Emmanuel, André, Isabelle, Remaud-Siméon, Magali
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 2016; Public Library of Science (PLoS)
• Subjects: Alzheimer Disease; Alzheimer's disease; Amyloid; Amyloid beta-Peptides - biosynthesis; Amyloid beta-Peptides - chemistry; Biology and Life Sciences; Chromatography; Denaturation; E coli; Electrophoresis, Polyacrylamide Gel; Engineering and Technology; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Gel electrophoresis; Humans; Inclusion bodies; Inclusion Bodies - metabolism; Life Sciences; Methionine; Methionine - chemistry; Methods; Neurodegenerative diseases; Neutralization; Peptide Fragments - biosynthesis; Peptide Fragments - chemistry; Peptides; pH effects; Physical Sciences; Protein purification; Proteins; Purification; Purity; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Research and Analysis Methods; Sodium lauryl sulfate; Spectrophotometry; Spectrophotometry, Ultraviolet; Staining and Labeling; Thiazoles - analysis; Ultrafiltration; France; facile method; n-15; a-beta-42; expression; coli; structural-analysis; aggregation; inclusion-bodies
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0161209

An improved production and purification method for Alzheimer's disease related methionine-modified amyloid-β 1-40 and 1-42 peptides is proposed, taking advantage of the formation of inclusion body in Escherichia coli. A Thioflavin-S assay was set-up to evaluate inclusion body formation during growth and optimize culture conditions for amyloid-β peptides production. A simple and fast purification protocol including first the isolation of the inclusion bodies and second, two cycles of high pH denaturation/ neutralization combined with an ultrafiltration step on 30-kDa cut-off membrane was established. Special attention was paid to purity monitoring based on a rational combination of UV spectrophotometry and SDS-PAGE analyses at the various stages of the process. It revealed that this chromatography-free protocol affords good yield of high quality peptides in term of purity. The resulting peptides were fully characterized and are appropriate models for highly reproducible in vitro aggregation studies.