An artificial gene encoding the amino acid repeat sequence (AEAEAKAKAEAEAKAK)9 was constructed and expressed in E. coli. affording a 17 236 Da His-tagged fusion protein, poly-EAK9. Circular dichroism and FTIR results suggest that poly-EAK9 adopts an extended β-strand conformation in an antiparallel β-sheet supramolecular aggregate. The structure appears highly stable and resistant to denaturation up to 6 M urea and over a range of pH and temperature conditions. Congo Red dye-binding assays support the presence of amyloid-like fibrils, and scanning electron microscopy studies confirm that these supramolecular structures are in fact smooth, well-defined fibrils, approximately 10−20 nm in diameter. Triggered gelation occurs under physiological conditions, suggesting the potential use of poly-EAK9 gels as biocompatible and biodegradable materials. Rheological and SEM experiments are consistent with the formation of an entangled polymer network, in which the polymer mesh size appears to be inversely related to protein concentration. Rheological measurements indicate that the material is significantly more elastic than a lower molecular weight synthetic peptide of the same structure. These results demonstrate that a high molecular weight, biosynthetic protein polymer of minimal complexity can be designed to self-assemble in solution to form β-sheet fibrils and transparent, self-supporting gels.
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