Preparation of uniformly 13C,15N-labeled recombinant human amylin for solid-state NMR investigation

被引:6
|
作者
Kosicka, Iga [1 ,2 ]
Kristensen, Torsten [1 ,3 ]
Bjerring, Morten [1 ,2 ]
Thomsen, Karen [1 ]
Scavenius, Carsten [1 ,3 ]
Enghild, Jan J. [1 ,3 ]
Nielsen, Niels Chr. [1 ,2 ]
机构
[1] Aarhus Univ, Interdisciplinary Nanosci Ctr iNANO, Ctr Insoluble Prot Struct inSPIN, DK-8000 Aarhus C, Denmark
[2] Aarhus Univ, Dept Chem, DK-8000 Aarhus C, Denmark
[3] Aarhus Univ, Dept Mol Biol & Genet, DK-8000 Aarhus C, Denmark
基金
新加坡国家研究基金会;
关键词
Amylin; Amyloid fibrils; Protein expression; Solid-state NMR; ISLET AMYLOID POLYPEPTIDE; PANCREATIC BETA-CELLS; ESCHERICHIA-COLI; DIABETES-MELLITUS; PEPTIDE; PROTEIN; FIBRILS; FIBRILLATION; EXPRESSION; FUSION;
D O I
10.1016/j.pep.2014.04.002
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 C-13,N-15-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. (C) 2014 Elsevier Inc. All rights reserved.
引用
收藏
页码:119 / 130
页数:12
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