Publication details

Cloning, expression and purification of the human Islet Amyloid Polypeptide (hIAPP) from Escherichia coli


CAMARGO Diana C. Rodrigues TRIPSIANES Konstantinos KAPP Tobias G. MENDES Joaquim SCHUBERT Jasmin CORDES Burghard REIF Bernd

Year of publication 2015
Type Article in Periodical
Magazine / Source Protein Expression and Purification
MU Faculty or unit

Central European Institute of Technology

Field Genetics and molecular biology
Keywords Amyloid; Diabetes; Recombinant protein; human Islet Amyloid Polypeptide (hIAPP); Escherichia coli; Nuclear magnetic resonance (NMR)
Description 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 beta-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 3 mg 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. (C) 2014 Elsevier Inc. All rights reserved.

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