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Expression and purification of the aortic amyloid polypeptide medin

Research output: Contribution to Journal/MagazineJournal articlepeer-review

<mark>Journal publication date</mark>06/2014
<mark>Journal</mark>Protein Expression and Purification
Number of pages6
Pages (from-to)32-37
Publication StatusPublished
<mark>Original language</mark>English


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-p peptides, have been defined from solid-state nuclear magnetic resonance (SSNMR) measurements on C-13- and N-15-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 C-13,N-15-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 C-13 and N-15 SSNMR spectra obtained for uniformly-labelled fibrils indicate that medin adopts a predominantly p-sheet conformation with some unstructured elements, and provide the basis for further, more detailed structural investigations. (C) 2014 Published by Elsevier Inc.