title: A Robust and Efficient Production and Purification Procedure of Recombinant Alzheimers Disease Methionine-Modified Amyloid-β Peptides.
abstract: 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.
authors.affiliation: Laboratoire d'Ingénierie des Systèmes Biologiques et Procédés, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France.|Laboratoire de Chimie de Coordination, CNRS, Université de Toulouse, INPT, Toulouse, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Hoarau
authors.affiliation: Laboratoire d'Ingénierie des Systèmes Biologiques et Procédés, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France.
authors.forename: Yannick
authors.initials: Y
authors.lastname: Malbert
authors.affiliation: Laboratoire d'Ingénierie des Systèmes Biologiques et Procédés, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France.
authors.forename: Romain
authors.initials: R
authors.lastname: Irague
authors.affiliation: Laboratoire de Chimie de Coordination, CNRS, Université de Toulouse, INPT, Toulouse, France.
authors.forename: Christelle
authors.initials: C
authors.lastname: Hureau
authors.affiliation: Laboratoire de Chimie de Coordination, CNRS, Université de Toulouse, INPT, Toulouse, France.
authors.forename: Peter
authors.initials: P
authors.lastname: Faller
authors.affiliation: Laboratoire de Chimie de Coordination, CNRS, Université de Toulouse, INPT, Toulouse, France.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Gras
authors.affiliation: Laboratoire d'Ingénierie des Systèmes Biologiques et Procédés, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: André
authors.affiliation: Laboratoire d'Ingénierie des Systèmes Biologiques et Procédés, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France.
authors.forename: Magali
authors.initials: M
authors.lastname: Remaud-Siméon
mesh_terms: D000544:Alzheimer Disease; D016229:Amyloid beta-Peptides / Q000096:biosynthesis* / Q000737:chemistry*; D052160:Benzothiazoles; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D008715:Methionine / Q000737:chemistry*; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013194:Staining and Labeling; D013844:Thiazoles / Q000032:analysis
chemical_list: D016229:Amyloid beta-Peptides; D052160:Benzothiazoles; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013844:Thiazoles; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42); C009462:thioflavin T; D008715:Methionine