title: Expression and purification of 15N- and 13C-isotope labeled 40-residue human Alzheimer's β-amyloid peptide for NMR-based structural analysis.
abstract: Amyloid fibrils of Alzheimer's β-amyloid peptide (Aβ) are a primary component of amyloid plaques, a hallmark of Alzheimer's disease (AD). Enormous attention has been given to the structural features and functions of Aβ in amyloid fibrils and other type of aggregates in associated with development of AD. This report describes an efficient protocol to express and purify high-quality 40-residue Aβ(1-40), the most abundant Aβ in brains, for structural studies by NMR spectroscopy. Over-expression of Aβ(1-40) with glutathione S-transferase (GST) tag connected by a Factor Xa recognition site (IEGR(▾)) in Escherichia coli resulted in the formation of insoluble inclusion bodies even with the soluble GST tag. This problem was resolved by efficient recovery of the GST-Aβ fusion protein from the inclusion bodies using 0.5% (w/v) sodium lauroyl sarcosinate as solubilizing agent and subsequent purification by affinity chromatography using a glutathione agarose column. The removal of the GST tag by Factor Xa enzymatic cleavage and purification by HPLC yielded as much as ∼7 mg and ∼1.5mg of unlabeled Aβ(1-40) and uniformly (15)N- and/or (13)C-protein Aβ(1-40) from 1L of the cell culture, respectively. Mass spectroscopy of unlabeled and labeled Aβ and (1)H/(15)N HSQC solution NMR spectrum of the obtained (15)N-labeled Aβ in the monomeric form confirmed the expression of native Aβ(1-40). It was also confirmed by electron micrography and solid-state NMR analysis that the purified Aβ(1-40) self-assembles into β-sheet rich amyloid fibrils. To the best of our knowledge, our protocol offers the highest yields among published protocols for production of recombinant Aβ(1-40) samples that are amendable for an NMR-based structural analysis. The protocol may be applied to efficient preparation of other amyloid-forming proteins and peptides that are (13)C- and (15)N-labeled for NMR experiments.
authors.affiliation: Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, United States.
authors.forename: Fei
authors.initials: F
authors.lastname: Long
authors.forename: Wonhwa
authors.initials: W
authors.lastname: Cho
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Ishii
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism*; D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000648:ultrastructure; D002247:Carbon Isotopes / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D012521:Sarcosine / Q000031:analogs & derivatives / Q000737:chemistry; D012995:Solubility
chemical_list: D016229:Amyloid beta-Peptides; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C078592:amyloid beta-protein (1-40); C025231:sarkosyl; D005982:Glutathione Transferase; D012521:Sarcosine