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Protein Expr Purif. 2014 Jun;98:32-7. doi: 10.1016/j.pep.2014.02.009. Epub 2014 Mar 3.

Expression and purification of the aortic amyloid polypeptide medin.

Author information

1
Institute of Integrative Biology, Biosciences Building, Crown Street, University of Liverpool, Liverpool L69 7ZB, United Kingdom.
2
Department of Chemistry, Lancaster University, Lancaster LA1 4YB, United Kingdom. Electronic address: d.middleton@lancaster.ac.uk.

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.

KEYWORDS:

Amyloid; Fibril; SUMO; Solid-state NMR; pOPINS

PMID:
24602872
DOI:
10.1016/j.pep.2014.02.009
[Indexed for MEDLINE]
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