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Expression and purification of the aortic amyloid polypeptide medin.

Authors :
Davies HA
Wilkinson MC
Gibson RP
Middleton DA
Source :
Protein expression and purification [Protein Expr Purif] 2014 Jun; Vol. 98, pp. 32-7. Date of Electronic Publication: 2014 Mar 03.
Publication Year :
2014

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.<br /> (Copyright © 2014. Published by Elsevier Inc.)

Details

Language :
English
ISSN :
1096-0279
Volume :
98
Database :
MEDLINE
Journal :
Protein expression and purification
Publication Type :
Academic Journal
Accession number :
24602872
Full Text :
https://doi.org/10.1016/j.pep.2014.02.009