Biosynthesis of Polypeptide Fusions: Integrating Order-Promoting and Disordered Domains For The Creation of Nanomaterials

• Caleb Murat, Chemical Engineering, University of Delaware

• April Kloxin, Chemical & Biomolecular Engineering, University of Delaware

Proteins are the fundamental building block of nature, providing unique biochemical and biophysical properties that arise from their underlying amino acid sequences. Yet, creating materials with proteins for designed properties can be challenging. Engineered building blocks for making protein-based materials with designed structure and properties remain a great need. We hypothesized that fusion of order-promoting and disordered domains would produce unique nanomaterial structures, including vesicles and fibrils that would be useful in biomedical applications for drug delivery. To test this, we selected i) bundlemers, computationally designed peptides that form coiled-coils and provide structural integrity, and ii) Resilin-Like Polypeptides (RLPs), amino acid sequences of disordered proteins responsible for jumping, flying, and stridulation in insects. By fusing RLPs of different lengths and varying the ratio of disordered to ordered domains, we aimed to probe the range of different nanostructures that could be created with this building block design. Uniquely, these fusions can be biosynthesized using only naturally-occurring amino acids, making these building blocks scalable, sustainable, and biocompatible. The goal for this summer was to establish the biosynthetic workflows and initial nanostructures that were created with the expressed and purified building block designs. The objectives for this project then were i) learn biosynthesis and characterization techniques, ii) design and execute biosynthetic workflow for production of design blocks, and iii) examine resulting nanostructures. Specifically, after bacterial transformation and protein expression, techniques such as SDS gel confirmed that our target protein was successfully produced from our DNA vector and TEM established our target structures were produced under desired conditions. At RT and 37C, we observed spherical structures, suggestive of vesicles, while at higher temperatures we saw fibrils. In addition, making use of the confocal microscope, we observed that our protein could encapsulate drugs. Future work for this project will determine drug release profiles and whether the assembled structures can be infused into a hydrogel for broader biomedical applications.