TY - JOUR

T1 - Expression and purification of the aortic amyloid polypeptide medin

AU - Davies, Hannah A.

AU - Wilkinson, Mark C.

AU - Gibson, Robert P.

AU - Middleton, David A.

PY - 2014/6

Y1 - 2014/6

N2 - 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.

AB - 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.

KW - Amyloid

KW - Fibril

KW - SUMO

KW - pOPINS

KW - Solid-state NMR

KW - SOLID-STATE NMR

KW - MALTOSE-BINDING PROTEIN

KW - ALZHEIMERS-DISEASE

KW - MOLECULAR-LEVEL

KW - BETA

KW - FIBRILS

KW - OLIGOMERS

KW - PEPTIDE

KW - SPECTROSCOPY

KW - AGGREGATION

U2 - 10.1016/j.pep.2014.02.009

DO - 10.1016/j.pep.2014.02.009

M3 - Journal article

VL - 98

SP - 32

EP - 37

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

ER -