کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2020464 1542342 2014 12 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Preparation of uniformly 13C,15N-labeled recombinant human amylin for solid-state NMR investigation
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Preparation of uniformly 13C,15N-labeled recombinant human amylin for solid-state NMR investigation
چکیده انگلیسی


- We developed a method of high yield production of recombinant human amylin.
- Fibril growth and cytotoxicity of recombinant amylin was demonstrated.
- Recombinant amylin is a well suited material for structural investigations by NMR.

A number of diseases are caused by the formation of amyloid fibrils. Detailed understanding of structural features of amyloid fibers is of great importance for our understanding of disease progression and design of agents for diagnostics or potential prevention of protein aggregation. In lack of 3D crystal ordering, solid-state NMR forms the most suited method to determine the structures of the fibrils with atomic resolution. To exploit this potential, large amounts of isotopic-labeled protein need to be obtained through recombinant protein expression. However, expression and purification of amyloidogenic proteins in large amounts remains challenging due to their aggregation potential, toxicity for cells and difficult purification. In this work, we report a method for the production of large amounts of uniformly labeled 13C,15N-human amylin, being one of the most amyloidogenic peptides known. This method utilizes inclusion bodies-directed expression and cheap chemical cleavage with cyanogen bromide in order to minimize the cost of the procedure compared to the use of less efficient proteolytic enzymes. We demonstrate the formation of amylin fibrils in vitro characterized using biophysical methods and electron microscopy, show toxicity towards human cells, and demonstrate that produced material may form the basis for structure determination using solid-state NMR.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 99, July 2014, Pages 119-130
نویسندگان
, , , , , , ,