The Relationship between pH and MaSp1 Structure
Poster Number
47
Format
Poster Presentation
Faculty Mentor Name
Craig Vierra
Faculty Mentor Department
Biological Sciences
Abstract/Artist Statement
Spider silk has recently developed into a worthwhile avenue of research for scientists, due in part to the versatility and industrial potential of natural silk. Because of their high tensile strength and extensibility, silk threads are considered some of the strongest biomaterials in existence, exhibiting high mechanical stability and biocompatibility. Prior experimentation concerning the N-terminal region of major ampullate spidroin 1 has shown that it plays a major role in facilitating assembly of the fiber by accelerating and directing assembly in response to the environmental pH. More importantly, the domain contains and codes for an inherent secretion signal, instructing the internal machinery of L. hesperus to transport the spidroins into the glandular lumen and begin fiber secretion. It is believed that fiber aggregation preceding extrusion can be attributed to the concentration of salts in the luminal contents of the major ampullate gland. In order to test this claim, a cDNA sequence responsible for producing MaSp1 was inserted into the plasmid vector pBAD-TOPO and digested to confirm the presence of the insert. The gene was induced in the presence of arabinose to express its MaSp1 protein. The spidroin, which was initially tagged with histidine residues to allow for isolation using a nickel resin, was size fractioned using SDS-PAGE. Finally, a silver stain was performed to allow for protein visualization and removal from the gel to perform an in-gel tryptic digest. Mass spectrometry and atomic force microscopy analysis were utilized as tools to study the relationship between protein structure and pH. The results of this study will help reveal the ideal conditions under which large-scale fiber synthesis can be made possible.
Location
DeRosa University Center, Ballroom
Start Date
20-4-2013 1:00 PM
End Date
20-4-2013 3:00 PM
The Relationship between pH and MaSp1 Structure
DeRosa University Center, Ballroom
Spider silk has recently developed into a worthwhile avenue of research for scientists, due in part to the versatility and industrial potential of natural silk. Because of their high tensile strength and extensibility, silk threads are considered some of the strongest biomaterials in existence, exhibiting high mechanical stability and biocompatibility. Prior experimentation concerning the N-terminal region of major ampullate spidroin 1 has shown that it plays a major role in facilitating assembly of the fiber by accelerating and directing assembly in response to the environmental pH. More importantly, the domain contains and codes for an inherent secretion signal, instructing the internal machinery of L. hesperus to transport the spidroins into the glandular lumen and begin fiber secretion. It is believed that fiber aggregation preceding extrusion can be attributed to the concentration of salts in the luminal contents of the major ampullate gland. In order to test this claim, a cDNA sequence responsible for producing MaSp1 was inserted into the plasmid vector pBAD-TOPO and digested to confirm the presence of the insert. The gene was induced in the presence of arabinose to express its MaSp1 protein. The spidroin, which was initially tagged with histidine residues to allow for isolation using a nickel resin, was size fractioned using SDS-PAGE. Finally, a silver stain was performed to allow for protein visualization and removal from the gel to perform an in-gel tryptic digest. Mass spectrometry and atomic force microscopy analysis were utilized as tools to study the relationship between protein structure and pH. The results of this study will help reveal the ideal conditions under which large-scale fiber synthesis can be made possible.