Spiders spin a wide variety of different silk types with different biological functions that are known for their extraordinary mechanical properties. Dragline silk has predominantly captured the interest of researchers because it exhibits high tensile strength and toughness while maintaining its elasticity. This thesis has focused on the characterization of a family of small molecular weight proteins recently discovered in dragline silk. These proteins were discovered in the western black widow spider, Latrodectus hesperus, and have been termed Cysteine-Rich Proteins (CRPs) due to their high conserved cysteine content. CRP family members were used in protein-protein interaction studies to determine if there is any interaction with the major ampullate spidroins (MaSps). After affinity chromatography and co-expression studies in bacteria, there were no detectable interactions between the CRPs and MaSp1. Further studies which could be an important role in the natural silk assembly process. Further protein interaction studies in different salt and pH conditions can further determine the function of the CRPs in dragline silk formation.

Spider silk has outstanding mechanical properties, displaying high tensile strength and extensibility. The unique combination of strength and great extensibility make it one of the toughest materials in the world. Of the seven different spider silks, dragline silk, the lifeline silk of the spider, represents one of the most renowned fiber types that has extraordinary properties. As a result, many labs across the globe are racing to manufacture synthetic dragline silk fibers. With the production of synthetic dragline silk fibers, there are unlimited commercial applications. In this study, we developed several codon-optimized MaSp1 minifibroin constructs for recombinant protein expression in bacteria. These recombinant MaSp1 minifibroin constructs were engineered to contain the N-terminal domain (NTD), different copies of internal block repeats (ranging from 2 to 64 copies of 35 amino acid blocks), and the C-terminal domain (CTD). The NTD and CTDs were derived from the natural cDNA sequences of black widow spiders, while the internal block repeats were generated from synthetic DNA fragments that were codon-optimized for expression in Escherichia coli . Different numbers of internal block repeats were created using a specialized seamless cloning strategy. By applying this seamless cloning strategy, we successfully multimerized MaSp1 block repeats that approach the natural fibroin size. Moreover, through the construction of a customized NTD-CTD spidroin construct, multimerized block repeats from any fibroin can be rapidly inserted to facilitate minifibroin protein expression in bacteria. Overall, this strategy as well as the created vectors, should help advance the silk community in the production of synthetic silk fibers that have properties that more closely resemble natural fibers.