title: A routine method for cloning, expressing and purifying Aβ(1-42) for structural NMR studies.
abstract: Nuclear magnetic resonance (NMR) is a key technology in the biophysicist's toolbox for gaining atomic-level insight into structure and dynamics of biomolecules. Investigation of the amyloid-β peptide (Aβ) of Alzheimer's disease is one area where NMR has proven useful, and holds even more potential. A barrier to realizing this potential, however, is the expense of the isotopically enriched peptide required for most NMR work. Whereas most biomolecular NMR studies employ biosynthetic methods as a very cost-effective means to obtain isotopically enriched biomolecules, this approach has proven less than straightforward for Aβ. Furthermore, the notorious propensity of Aβ to aggregate during purification and handling reduces yields and increases the already relatively high costs of solid phase synthesis methods. Here we report our biosynthetic and purification developments that yield pure, uniformly enriched ¹⁵N and ¹³C¹⁵N Aβ(1-42), in excess of 10 mg/L of culture media. The final HPLC-purified product was stable for long periods, which we characterize by solution-state NMR, thioflavin T assays, circular dichroism, electrospray mass spectrometry, and dynamic light scattering. These developments should facilitate further investigations into Alzheimer's disease, and perhaps misfolding diseases in general.
authors.affiliation: School of Chemistry, Bio21 Institute, University of Melbourne, Melbourne, VIC, Australia.
authors.forename: Daniel K
authors.initials: DK
authors.lastname: Weber
authors.forename: Marc-Antoine
authors.initials: MA
authors.lastname: Sani
authors.forename: John D
authors.initials: JD
authors.lastname: Gehman
mesh_terms: D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002247:Carbon Isotopes; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D002942:Circular Dichroism; D003001:Cloning, Molecular; D006801:Humans; D007553:Isotope Labeling; D007700:Kinetics; D008970:Molecular Weight; D009391:Nephelometry and Turbidimetry; D009590:Nitrogen Radioisotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D066263:Protein Aggregation, Pathological / Q000378:metabolism; D011487:Protein Conformation; D059748:Proteolysis; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D016229:Amyloid beta-Peptides; D002247:Carbon Isotopes; D009590:Nitrogen Radioisotopes; D010446:Peptide Fragments; D025841:Small Ubiquitin-Related Modifier Proteins; C075222:amyloid beta-protein (1-42)