Type II diabetes is characterized by deposition of the hormone human Islet Amyloid Polypeptide (hIAPP). Formation of hIAPP amyloid fibrils and aggregates is considered to be responsible for pancreatic β-cell losses. Therefore, insight into the structure of hIAPP in the solid-state and in solution is of fundamental importance in order to better understand the action of small molecules, which can potentially dissolve protein aggregates and modulate cell toxicity. So far, no procedure has been described that allows to obtain the native human IAPP peptide at high yields. We present here a cloning, expression and purification protocol that permits the production of 2.5 and 3mg of native peptide per liter of minimal and LB medium, respectively. In the construct, hIAPP is fused to a chitin binding domain (CBD). The CBD is subsequently cleaved off making use of intein splicing reaction which yield amidation of the C-terminus. The N-terminus contains a solubilization domain which is cleaved by V8 protease, avoiding additional residues at the N-terminus. The correct formation of the disulfide bond is achieved by oxidation with H2O2.

Central European Institute of Technology Masaryk University Kamenice 5 A35 1S081 62500 Brno Czech Republic

Helmholtz Zentrum München Ingolstädter Landstr 1 85764 Neuherberg Germany

Helmholtz Zentrum München Lichtenbergstr 4 85747 Garching Germany

Munich Center for Integrated Protein Science Lichtenbergstr 4 85747 Garching Germany

Citace poskytuje Crossref.org

The redox environment triggers conformational changes and aggregation of hIAPP in Type II Diabetes