title: Preparation of uniformly (13)C,(15)N-labeled recombinant human amylin for solid-state NMR investigation.
abstract: 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 (13)C,(15)N-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.
authors.affiliation: Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Denmark; Department of Chemistry, Aarhus University, Denmark.
authors.forename: Iga
authors.initials: I
authors.lastname: Kosicka
authors.affiliation: Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Denmark; Department of Molecular Biology and Genetics, Aarhus University, Denmark.
authors.forename: Torsten
authors.initials: T
authors.lastname: Kristensen
authors.affiliation: Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Denmark; Department of Chemistry, Aarhus University, Denmark.
authors.forename: Morten
authors.initials: M
authors.lastname: Bjerring
authors.affiliation: Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Denmark.
authors.forename: Karen
authors.initials: K
authors.lastname: Thomsen
authors.affiliation: Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Denmark; Department of Molecular Biology and Genetics, Aarhus University, Denmark.
authors.forename: Carsten
authors.initials: C
authors.lastname: Scavenius
authors.affiliation: Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Denmark; Department of Molecular Biology and Genetics, Aarhus University, Denmark.
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Enghild
authors.affiliation: Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Denmark; Department of Chemistry, Aarhus University, Denmark. Electronic address: ncn@inano.au.dk.
authors.forename: Niels Chr
authors.initials: NC
authors.lastname: Nielsen
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000648:ultrastructure; D002247:Carbon Isotopes; D002942:Circular Dichroism; D003488:Cyanogen Bromide / Q000737:chemistry; D006801:Humans; D058228:Islet Amyloid Polypeptide / Q000737:chemistry / Q000302:isolation & purification*; D007553:Isotope Labeling / Q000379:methods*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification
chemical_list: D000682:Amyloid; D002247:Carbon Isotopes; D058228:Islet Amyloid Polypeptide; D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins; D003488:Cyanogen Bromide
keywords: Amylin; Amyloid fibrils; Protein expression; Solid-state NMR