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

• Published in: Protein expression and purification 2015-02, Vol.106, p.49-56
• Main Authors: Rodriguez Camargo, Diana C., Tripsianes, Konstantinos, Kapp, Tobias G., Mendes, Joaquim, Schubert, Jasmin, Cordes, Burghard, Reif, Bernd
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amyloid; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Cloning, Molecular - methods; Diabetes; Electrophoresis, Agar Gel; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli - metabolism; human Islet Amyloid Polypeptide (hIAPP); Humans; Islet Amyloid Polypeptide - chemistry; Islet Amyloid Polypeptide - isolation & purification; Islet Amyloid Polypeptide - metabolism; Molecular Sequence Data; Nuclear magnetic resonance (NMR); Recombinant Fusion Proteins - isolation & purification; Recombinant protein
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2014.10.012

•Expressing and purification of hIAPP is hampered due to its high amyloidogenicity.•C- and N-terminal tags (chitin binding domain, leader) increase expression levels.•Intein cleavage ensures C-terminal amidation.•hIAPP is obtained without extra residues at the N-terminus.•Oxidation yields a disulfide bridge between residues 2 and 7. 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.