title: Cloning, expression and purification of the human Islet Amyloid Polypeptide (hIAPP) from Escherichia coli.
abstract: 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.
authors.affiliation: Helmholtz Zentrum München (Deutsches Forschungszentrum für Gesundheit und Umwelt), Ingolstädter Landstr. 1, 85764 Neuherberg, Germany; Munich Center for Integrated Protein Science (CIPS-M) at Department Chemie, Technische Universität München (TUM), Lichtenbergstr. 4, 85747 Garching, Germany.
authors.forename: Diana C
authors.initials: DC
authors.lastname: Rodriguez Camargo
authors.affiliation: Central European Institute of Technology (CEITEC), Masaryk University, Kamenice 5/A35/1S081, 62500 Brno, Czech Republic.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Tripsianes
authors.affiliation: Munich Center for Integrated Protein Science (CIPS-M) at Department Chemie, Technische Universität München (TUM), Lichtenbergstr. 4, 85747 Garching, Germany.
authors.forename: Tobias G
authors.initials: TG
authors.lastname: Kapp
authors.affiliation: Helmholtz Zentrum München (Deutsches Forschungszentrum für Gesundheit und Umwelt), Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.
authors.forename: Joaquim
authors.initials: J
authors.lastname: Mendes
authors.affiliation: Helmholtz Zentrum München (Deutsches Forschungszentrum für Gesundheit und Umwelt), Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.
authors.forename: Jasmin
authors.initials: J
authors.lastname: Schubert
authors.affiliation: Munich Center for Integrated Protein Science (CIPS-M) at Department Chemie, Technische Universität München (TUM), Lichtenbergstr. 4, 85747 Garching, Germany.
authors.forename: Burghard
authors.initials: B
authors.lastname: Cordes
authors.affiliation: Helmholtz Zentrum München (Deutsches Forschungszentrum für Gesundheit und Umwelt), Ingolstädter Landstr. 1, 85764 Neuherberg, Germany; Munich Center for Integrated Protein Science (CIPS-M) at Department Chemie, Technische Universität München (TUM), Lichtenbergstr. 4, 85747 Garching, Germany. Electronic address: reif@tum.de.
authors.forename: Bernd
authors.initials: B
authors.lastname: Reif
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D003001:Cloning, Molecular / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D058228:Islet Amyloid Polypeptide / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification
chemical_list: D058228:Islet Amyloid Polypeptide; D011993:Recombinant Fusion Proteins
keywords: Amyloid; Diabetes; Escherichia coli; Nuclear magnetic resonance (NMR); Recombinant protein; human Islet Amyloid Polypeptide (hIAPP)