Open AccessA Robust and Efficient Production and Purification Procedure of Recombinant Alzheimers Disease Methionine-Modified Amyloid-β Peptides
Author(s) -
Marie Hoarau,
Yannick Malbert,
Romain Irague,
Christelle Hureau,
Peter Faller,
Emmanuel Gras,
Isabelle André,
Magali RemaudSiméon
Publication year - 2016
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0161209
Subject(s) - thioflavin , biochemistry , methionine , amyloid (mycology) , inclusion bodies , escherichia coli , recombinant dna , chemistry , peptide , alzheimer's disease , chromatography , amino acid , medicine , disease , inorganic chemistry , pathology , gene
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.