Production of Recombinant Spider Silk Proteins for Biomedical Material Applications

Spider silk protein-based materials represent a new generation of biomaterials with tunable mechanical properties for a wide range of biomedical applications. The ability to engineer silk genes and recombinantly produce spider silk proteins is key to generation of tailored silk-based biomaterials. It paves the way for potential uses as biocompatible and high performance tissue scaffolds for tissue regeneration, artificial tendons or ligaments, sutures or nanofiber mats for wound dressings. The dragline and flagelliform web silks from orb weaver spiders are strong and elastic respectively, while being equally exceptionally tough. These properties lay in the presence of key structural amino acid sequences in these highly repetitive silk proteins. Spider silk-like genes are easily engineered for silk protein recombinant production due to the native modular repetitive nature of spider silk proteins. In this study, two roughly 60 kDa chimeric flagelliform-dragline Spider Silk-Like Proteins (SSLPs), differing slightly in their amino acid sequences, were produced in recombinant Escherichia coli clones. The bacterial silk clone cells were harvested following expression of the recombinant silk gene. The total protein recovered through standard bacterial cell lysis methods was heat treated and all Hisitidine-tagged recSSLPs were recovered using immobilized metal (nickel) affinity chromatography (IMAC). SDS-PAGE/Coomassie and Western blot analyses confirmed the presence of the purified SSLPS. All SSLP samples were dialyzed prior to lyophilization. These "dry" SSLPs are used in the electrospinning of nanofiber mats. The biocompatibility of these mats will be evaluated as wound dressings.