Ghimire, Anupama2025-12-102025-12-102025-12-09https://hdl.handle.net/10222/85553Spider silks are protein-based biomaterials renowned for their exceptional strength and extensibility, with potential applications in the biomedical, textile, and automotive industries. Orb-weaver spiders produce up to seven silk types for functions such as locomotion, web construction, egg protection, and prey wrapping. The cannibalistic and territorial behavior of spiders limits large-scale harvesting of natural silk, highlighting the importance of recombinant silk production. This thesis focuses on the design, production, and characterization of recombinant chimeric silks with tailored properties. A pyriform-aciniform silk fusion protein, Py2W2, was engineered by fusing two pyriform (Py) and two aciniform (W) silk repeat units. When wet-spun into fibers and stretched in air, water, or ethanol, Py2W2 exhibited tunable mechanics. Air-stretched fibers reached ~157% extensibility, comparable to natural flagelliform silk, while ethanol-stretched fibers achieved superior strength. These properties exhibited a correlation with secondary structure, where increased β-sheet content enhanced strength. Py2W2 fibers also showed water-compatibility absent in Py2 fibers and outperformed Py2 and W2 fibers in mechanical tests. As an alternative strategy, composite fibers (Py2+W2) were produced by mixing Py2 and W2 proteins prior to spinning. Like Py2W2, composite fibers displayed tunable mechanics influenced by post-spin stretching, though variability was observed in some spinning conditions. Further optimization of spinning process offers a practical route for generating tunable composite fibers without complex fusion designs. To introduce biological functionality, a chimeric protein W2Cma2ap-55 was designed by combining aciniform repeats (W2), a major ampullate silk C-terminal domain (Cma2), and the G-protein-coupled receptor (GPCR) ligand apelin-55 (ap-55). Films and fibers derived from W2Cma2ap-55 retained intact ap-55, as confirmed by antibody recognition. Compared with W2Cma2 fibers, W2Cma2ap-55 fibers exhibited higher strength and toughness with similar extensibility. Both film types were noncytotoxic to HEK 293A cells, and W2Cma2ap-55 films activated ERK phosphorylation, confirming apelin-55-mediated GPCR signaling. Similarly, cells adhered to W2Cma2ap-55 fibers, demonstrating their suitability as bioactive scaffolds. Overall, this work establishes chimeric silks as versatile biomaterials. Rational protein design and processing enabled tailoring of mechanical properties (Py2W2, Py2+W2) and the integration of bioactivity (W2Cma2ap-55). These findings highlight the potential of engineered spider silks for advanced biomedical and functional applications.enSpider silksBiomaterialsChimeric silks