Expression and purification of the aortic amyloid polypeptide medin

Davies, Hannah A. and Wilkinson, Mark C. and Gibson, Robert P. and Middleton, David A. (2014) Expression and purification of the aortic amyloid polypeptide medin. Protein Expression and Purification, 98. pp. 32-37. ISSN 1046-5928

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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-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.

Item Type:
Journal Article
Journal or Publication Title:
Protein Expression and Purification
Uncontrolled Keywords:
/dk/atira/pure/subjectarea/asjc/1300/1305
Subjects:
ID Code:
72096
Deposited By:
Deposited On:
10 Dec 2014 08:57
Refereed?:
Yes
Published?:
Published
Last Modified:
07 Oct 2020 03:22