Characterization of a Putative Peroxidase: Its Implication on the Mechanics of Black Widow Spider Silk
Faculty Mentor Name
Craig Vierra
Research or Creativity Area
Natural Sciences
Abstract
Silk plays a crucial role in nature and biology as a highly specialized protein-based biomaterial that enables organisms such as spiders and certain insects to enhance survival through efficient prey capture, protection of offspring, structural habitat construction, and dispersal, while also providing scientists with a model system for studying the relationship between molecular structure and exceptional mechanical properties in biological materials. Silk’s high strength and flexibility offer the ability for scientists to reform silk and implement its properties to aid in biotechnology. Synthetic fibers fail to fully replicate natural silk properties, suggesting that some steps in the natural silk synthesis are not fully identified. We seek to determine the role of a putative peroxidase in spider silk formation, if it contributes in later stage silk assembly by covalently linking the silk proteins. We hypothesize that the peroxidase is secreted in conjunction with fiber formation, promoting oxidative coupling reactions to link the proteins into a more cohesive network, greater stabilizing the silk which contributes to the properties observed in natural silk fibers. By cloning the peroxidase cDNA via PCR, we inserted its genetic blueprint into a prokaryotic expression vector, then verified its presence via restriction digestion analysis and agarose gel electrophoresis. After recombinant protein expression in bacteria, we utilized mass spectrometry and proteomics to identify its successful expression. Through our advancements, we plan to explore whether this putative peroxidase is utilized in the black widow spider silk assembly process. Therefore, our findings could lead us to observe the change in linking of the web and its individual silk proteins, further opening opportunities to examine biotechnical advances in synthetic spider silk.
Characterization of a Putative Peroxidase: Its Implication on the Mechanics of Black Widow Spider Silk
Silk plays a crucial role in nature and biology as a highly specialized protein-based biomaterial that enables organisms such as spiders and certain insects to enhance survival through efficient prey capture, protection of offspring, structural habitat construction, and dispersal, while also providing scientists with a model system for studying the relationship between molecular structure and exceptional mechanical properties in biological materials. Silk’s high strength and flexibility offer the ability for scientists to reform silk and implement its properties to aid in biotechnology. Synthetic fibers fail to fully replicate natural silk properties, suggesting that some steps in the natural silk synthesis are not fully identified. We seek to determine the role of a putative peroxidase in spider silk formation, if it contributes in later stage silk assembly by covalently linking the silk proteins. We hypothesize that the peroxidase is secreted in conjunction with fiber formation, promoting oxidative coupling reactions to link the proteins into a more cohesive network, greater stabilizing the silk which contributes to the properties observed in natural silk fibers. By cloning the peroxidase cDNA via PCR, we inserted its genetic blueprint into a prokaryotic expression vector, then verified its presence via restriction digestion analysis and agarose gel electrophoresis. After recombinant protein expression in bacteria, we utilized mass spectrometry and proteomics to identify its successful expression. Through our advancements, we plan to explore whether this putative peroxidase is utilized in the black widow spider silk assembly process. Therefore, our findings could lead us to observe the change in linking of the web and its individual silk proteins, further opening opportunities to examine biotechnical advances in synthetic spider silk.
