title: Expression and purification of the aortic amyloid polypeptide medin.
abstract: The 50-amino acid protein medin is the main fibrillar component of human aortic medial amyloid (AMA), the most common form of localised amyloid which affects 97% of Caucasians over the age of 50. Structural models for several amyloid assemblies, including the Alzheimer's amyloid-β peptides, have been defined from solid-state nuclear magnetic resonance (SSNMR) measurements on (13)C- and (15)N-labelled protein fibrils. SSNMR-derived structural information on fibrillar medin is scant, however, because studies to date have been restricted to limited measurements on site-specifically labelled protein prepared by solid-phase synthesis. Here we report a procedure for the expression of a SUMO-medin fusion protein in Escherichia coli and IMAC purification yielding pure, uniformly (13)C,(15)N-labelled medin in quantities required for SSNMR analysis. Thioflavin T fluorescence and dynamic light scattering measurements and transmission electron microscopy analysis confirm that recombinant medin assembles into amyloid-like fibrils over a 48-h period. The first (13)C and (15)N SSNMR spectra obtained for uniformly-labelled fibrils indicate that medin adopts a predominantly β-sheet conformation with some unstructured elements, and provide the basis for further, more detailed structural investigations.
authors.affiliation: Institute of Integrative Biology, Biosciences Building, Crown Street, University of Liverpool, Liverpool L69 7ZB, United Kingdom.
authors.forename: Hannah A
authors.initials: HA
authors.lastname: Davies
authors.affiliation: Institute of Integrative Biology, Biosciences Building, Crown Street, University of Liverpool, Liverpool L69 7ZB, United Kingdom.
authors.forename: Mark C
authors.initials: MC
authors.lastname: Wilkinson
authors.affiliation: Institute of Integrative Biology, Biosciences Building, Crown Street, University of Liverpool, Liverpool L69 7ZB, United Kingdom.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Gibson
authors.affiliation: Department of Chemistry, Lancaster University, Lancaster LA1 4YB, United Kingdom. Electronic address: d.middleton@lancaster.ac.uk.
authors.forename: David A
authors.initials: DA
authors.lastname: Middleton
mesh_terms: D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000954:Antigens, Surface / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D001011:Aorta / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008894:Milk Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D017433:Protein Structure, Secondary
chemical_list: D000682:Amyloid; D000954:Antigens, Surface; C098942:MFGE8 protein, human; D008894:Milk Proteins
keywords: Amyloid; Fibril; SUMO; Solid-state NMR; pOPINS