Engineered aggregation inhibitor fusion for production of highly amyloidogenic human islet amyloid polypeptide

• Published in: Journal of biotechnology Vol. 191; pp. 221 - 227
• Main Authors: Mirecka, Ewa Agnieszka, Gremer, Lothar, Schiefer, Stephanie, Oesterhelt, Filipp, Stoldt, Matthias, Willbold, Dieter, Hoyer, Wolfgang
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 10.12.2014
• Subjects: Agglomeration; Amino Acid Sequence - genetics; amyloid; atomic force microscopy; bacteriophages; Construction; Diabetes Mellitus, Type 2 - genetics; Escherichia coli; Escherichia coli - genetics; Gene Expression Regulation; Homogeneity; Human; Humans; hydrophobicity; Inhibitors; Islet amyloid polypeptide; Islet Amyloid Polypeptide - biosynthesis; Islet Amyloid Polypeptide - genetics; isotope labeling; Magnetic Resonance Spectroscopy; Microscopy, Atomic Force; noninsulin-dependent diabetes mellitus; nuclear magnetic resonance spectroscopy; Polypeptides; Protein aggregation; Protein Aggregation, Pathological - genetics; Protein engineering; Protein Folding; Protein Structure, Secondary; Proteins; Recombinant; Recombinant expression; β-Wrapin; β-Wrapin; Recombinant expression; Protein engineering; Protein aggregation; Islet amyloid polypeptide
• DOI: 10.1016/j.jbiotec.2014.06.006
• ISSN: 0168-1656; 1873-4863

•We present an expression system for an extremely aggregation-prone protein.•Fusion of an aggregation inhibitor permits soluble expression of the target protein.•The aggregation inhibitor was selected by phage display from a β-wrapin library.•The method yields pure, monomeric, aggregation-competent islet amyloid polypeptide.•The method is applicable to other aggregation-prone proteins. 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.