title: Engineered aggregation inhibitor fusion for production of highly amyloidogenic human islet amyloid polypeptide.
abstract: Human islet amyloid polypeptide (IAPP) is the major component of pancreatic amyloid deposits in type 2 diabetes. The structural conversion of IAPP from a monomeric state into amyloid assemblies is the subject of intense research. Recombinant production of IAPP is, however, difficult due to its extreme aggregation propensity. Here we describe a novel strategy for expression of IAPP in Escherichia coli, based on an engineered protein tag, which sequesters IAPP monomers and prevents IAPP aggregation. The IAPP-binding protein HI18 was selected by phage display from a β-wrapin library. Fusion of HI18 to IAPP enabled the soluble expression of the construct. IAPP was cleaved from the fusion construct and purified to homogeneity with a yield of 3mg of isotopically labeled peptide per liter of culture. In the monomeric state, IAPP was largely disordered as evidenced by far-UV CD and liquid-state NMR spectroscopy but competent to form amyloid fibrils according to atomic force microscopy. These results demonstrate the ability of the engineered β-wrapin HI18 for shielding the hydrophobic sequence of IAPP during expression and purification. Fusion of aggregation-inhibiting β-wrapins is a suitable approach for the recombinant production of aggregation-prone proteins.
authors.affiliation: Institute of Physical Biology, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany.
authors.forename: Ewa Agnieszka
authors.initials: EA
authors.lastname: Mirecka
authors.affiliation: Institute of Physical Biology, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany; Institute of Structural Biochemistry (ICS-6), Research Centre Jülich, 52425 Jülich, Germany.
authors.forename: Lothar
authors.initials: L
authors.lastname: Gremer
authors.affiliation: Institute of Physical Biology, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Schiefer
authors.affiliation: Institute of Physical Biology, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany.
authors.forename: Filipp
authors.initials: F
authors.lastname: Oesterhelt
authors.affiliation: Institute of Physical Biology, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany; Institute of Structural Biochemistry (ICS-6), Research Centre Jülich, 52425 Jülich, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Stoldt
authors.affiliation: Institute of Physical Biology, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany; Institute of Structural Biochemistry (ICS-6), Research Centre Jülich, 52425 Jülich, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Willbold
authors.affiliation: Institute of Physical Biology, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany; Institute of Structural Biochemistry (ICS-6), Research Centre Jülich, 52425 Jülich, Germany. Electronic address: wolfgang.hoyer@uni-duesseldorf.de.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hoyer
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics*; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D058228:Islet Amyloid Polypeptide / Q000096:biosynthesis* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D018625:Microscopy, Atomic Force; D066263:Protein Aggregation, Pathological / Q000235:genetics*; D017510:Protein Folding; D017433:Protein Structure, Secondary
chemical_list: D058228:Islet Amyloid Polypeptide
keywords: Islet amyloid polypeptide; Protein aggregation; Protein engineering; Recombinant expression; β-Wrapin