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Expression and purification of the aortic amyloid polypeptide medin

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Expression and purification of the aortic amyloid polypeptide medin. / Davies, Hannah A.; Wilkinson, Mark C.; Gibson, Robert P. et al.

In: Protein Expression and Purification, Vol. 98, 06.2014, p. 32-37.

Research output: Contribution to Journal/MagazineJournal articlepeer-review

Harvard

Davies, HA, Wilkinson, MC, Gibson, RP & Middleton, DA 2014, 'Expression and purification of the aortic amyloid polypeptide medin', Protein Expression and Purification, vol. 98, pp. 32-37. https://doi.org/10.1016/j.pep.2014.02.009

APA

Davies, H. A., Wilkinson, M. C., Gibson, R. P., & Middleton, D. A. (2014). Expression and purification of the aortic amyloid polypeptide medin. Protein Expression and Purification, 98, 32-37. https://doi.org/10.1016/j.pep.2014.02.009

Vancouver

Davies HA, Wilkinson MC, Gibson RP, Middleton DA. Expression and purification of the aortic amyloid polypeptide medin. Protein Expression and Purification. 2014 Jun;98:32-37. doi: 10.1016/j.pep.2014.02.009

Author

Davies, Hannah A. ; Wilkinson, Mark C. ; Gibson, Robert P. et al. / Expression and purification of the aortic amyloid polypeptide medin. In: Protein Expression and Purification. 2014 ; Vol. 98. pp. 32-37.

Bibtex

@article{76b17902e2044aecae1484797707e925,
title = "Expression and purification of the aortic amyloid polypeptide medin",
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.",
keywords = "Amyloid, Fibril, SUMO, pOPINS, Solid-state NMR, SOLID-STATE NMR, MALTOSE-BINDING PROTEIN, ALZHEIMERS-DISEASE, MOLECULAR-LEVEL, BETA, FIBRILS, OLIGOMERS, PEPTIDE, SPECTROSCOPY, AGGREGATION",
author = "Davies, {Hannah A.} and Wilkinson, {Mark C.} and Gibson, {Robert P.} and Middleton, {David A.}",
year = "2014",
month = jun,
doi = "10.1016/j.pep.2014.02.009",
language = "English",
volume = "98",
pages = "32--37",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press Inc.",

}

RIS

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 -