title: A facile method for expression and purification of (15)N isotope-labeled human Alzheimer's β-amyloid peptides from E. coli for NMR-based structural analysis.
abstract: Alzheimer's disease (AD) is a progressive neurodegenerative disease affecting millions of people worldwide. AD is characterized by the presence of extracellular plaques composed of aggregated/oligomerized β-amyloid peptides with Aβ42 peptide representing a major isoform in the senile plaques. Given the pathological significance of Aβ42 in the progression of AD, there is considerable interest in understanding the structural ensembles for soluble monomer and oligomeric forms of Aβ42. This report describes an efficient method to express and purify high quality (15)N isotope-labeled Aβ42 for structural studies by NMR. The protocol involves utilization of an auto induction system with (15)N isotope labeled medium, for high-level expression of Aβ42 as a fusion with IFABP. After the over-expression of the (15)N isotope-labeled IFABP-Aβ42 fusion protein in the inclusion bodies, pure (15)N isotope-labeled Aβ42 peptide is obtained following a purification method that is streamlined and improved from the method originally developed for the isolation of unlabeled Aβ42 peptide (Garai et al., 2009). We obtain a final yield of ∼ 6 mg/L culture for (15)N isotope-labeled Aβ42 peptide. Mass spectrometry and (1)H-(15)N HSQC spectra of monomeric Aβ42 peptide validate the uniform incorporation of the isotopic label. The method described here is equally applicable for the uniform isotope labeling with (15)N and (13)C in Aβ42 peptide as well as its other variants including any Aβ42 peptide mutants.
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720, USA. Electronic address: sudhir@berkeley.edu.
authors.forename: Sudhir C
authors.initials: SC
authors.lastname: Sharma
authors.affiliation: Department of Bioengineering, University of California, Berkeley, CA 94720, USA.
authors.forename: Tara
authors.initials: T
authors.lastname: Armand
authors.affiliation: Pharmaceutical Chemistry, University of California, San Francisco, CA, USA.
authors.forename: K Aurelia
authors.initials: KA
authors.lastname: Ball
authors.affiliation: Committee on Molecular Metabolism and Nutrition, University of Chicago, 1027 E. 57th Street, Chicago, IL 60637, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Chen
authors.affiliation: QB3 Institute, University of California, Berkeley, CA 94720, USA.
authors.forename: Jeffrey G
authors.initials: JG
authors.lastname: Pelton
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720, USA; QB3 Institute, University of California, Berkeley, CA 94720, USA.
authors.forename: David E
authors.initials: DE
authors.lastname: Wemmer
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720, USA; Department of Bioengineering, University of California, Berkeley, CA 94720, USA; Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, USA; QB3 Institute, University of California, Berkeley, CA 94720, USA; Chemical Sciences Division, Lawrence Berkeley National Labs, Berkeley, CA 94720, USA.
authors.forename: Teresa
authors.initials: T
authors.lastname: Head-Gordon
mesh_terms: D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D050556:Fatty Acid-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007553:Isotope Labeling; D009587:Nitrogen Isotopes / Q000032:analysis; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D016229:Amyloid beta-Peptides; D050556:Fatty Acid-Binding Proteins; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C075222:amyloid beta-protein (1-42)
keywords: Alzheimer’s disease; Amyloid β; Expression; Fatty acid binding protein; HSQC