کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8360573 | 1542343 | 2014 | 6 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Expression and purification of the aortic amyloid polypeptide medin
ترجمه فارسی عنوان
بیان و پاکسازی پلی پپتید آمینوئید مدین آئورت
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موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
چکیده انگلیسی
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 13C- and 15N-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 13C,15N-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 13C and 15N 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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 98, June 2014, Pages 32-37
Journal: Protein Expression and Purification - Volume 98, June 2014, Pages 32-37
نویسندگان
Hannah A. Davies, Mark C. Wilkinson, Robert P. Gibson, David A. Middleton,