(326g) Type III Secretion of Silk, Elastin and Resilin-Based Hybrid Biomaterials

Despite their integral role in the development of load-bearing materials for tissue culture and biomedical applications, recombinant expression of biopolymers remains challenging due to their inherent insolubility in cell cytoplasm.  Monomer expression and purification is difficult because this class of proteins is characterized by repetitive, glycine-rich, lengthy hydrophobic domains that are prone to aggregation and proteolytic degradation in vivo.  Engineering the type III bacterial secretion pathway enables a new strategy for customizable design, expression and export of pure biopolymer-forming proteins.  Using this approach we are able to achieve high-titer recombinant production of pure silk, elastin and resilin monomers that can be cross-linked post-secretion for the fabrication of materials with a variety of tunable mechanical and chemical properties.  We utilize a recursive cloning strategy to sequentially incorporate different functional modules into a genetic sequence encoding silk monomers, tropo-elastin and pro-resilin; we then transform the plasmid into Salmonella typhimurium and induce high-titer expression and extracellular secretion (10-100 mg/L) of the protein.  The pure monomers are then cross-linked via chemical or enzymatic methods to produce insoluble, mechanically stable hybrid biomaterials.   Due to genetic-level control over protein character, we can incorporate chemical moieties for conjugation with functionalized hydrogel-forming synthetic polymers and antibacterial peptide motifs for advanced functionality.  With this talk, we will detail the synthesis and characterization of these biopolymers, and discuss the key advantages of our microbial production system.