Jasmine Hershewe1 2 Chelsea Buck3 4 Patrick Dennis3 Michael Jewett1 2

1, Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, United States
2, Center for Synthetic Biology, Northwestern University, Evanston, Illinois, United States
3, Air Force Research Laboratory, Dayton, Ohio, United States
4, Department of Materials Science, University of Dayton, Dayton, Ohio, United States

Nature has evolved exquisite systems that serve as inspiration for the design of synthetic materials with user-defined functions and properties. DNA-encoded proteins comprise many of these materials, and the molecular assembly of protein subunits into hierarchical structures is responsible for some of the most essential, sophisticated, and diverse functions in nature. With the cost of DNA synthesis quickly falling to < $0.05/base, engineering materials using synthetic biology has emerged as an interesting, scalable avenue for advancing nanobiotechnology and materials science.

Key hurdles that exist for engineering and discovering tunable protein materials are the lengthy design, build, test cycles associated with building proteins in living cells. Synthetic biology has enabled the development of several key platform technologies that could accelerate biomaterials design at the ‘build’ stage of the cycle. In particular, we have developed a high-yielding cell-free protein synthesis platform from Escherichia coli that synthesizes up to 1.8g/L of protein in batch mode in ~8 hours. With an eye toward enhancing the chemical diversity of protein biopolymers, we have recently interfaced this platform with orthogonal translation systems (OTS) to genetically encode non-canonical amino acids (ncAAs) into proteins. We have demonstrated the ability to site-specifically introduce multiple identical ncAAs into a single protein (>10) with high titers and purity.

Using this foundation, we have begun studying a promising target for engineering protein-based materials, sourced from the giant squid, called ‘suckerins.’ Suckerins are a class of structural proteins that form sucker ring teeth assemblies that display robust mechanical properties and thermoplastic behavior. We synthesized and purified milligram quantities of a 23kDa suckerin isoform, ‘suckerin-12,’ bearing the ncAA para-L-azido phenylalanine (pAzF). We systematically investigated the ion- and pH-responsiveness of suckerin-12 and exploited the biochemistry of the molecule to assemble protein core nanoparticles with tuneable diameters between tens to hundreds of nanometers. We have begun utilizing the incorporated ncAA to functionalize suckerin-12 with hydrophilic ligands to generate synthetic, conjugate nanomaterials with altered self-assembly properties. We anticipate this work will be useful for elucidating important design parameters for fabricating protein-based nanoparticles and, more broadly, for accelerated engineering of protein nanomaterials using synthetic biology.