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Date of Award

2014

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Biological Sciences

First Advisor

Craig Vierra

First Committee Member

Geoff Lin-Cereghino

Second Committee Member

Douglas Weiser

Abstract

Spiders produce a diverse number of silk proteins that are well-known for their extraordinary mechanical and biological properties. Dragline silk has been the most prominent focus of research because of its exceptional high tensile strength and extensibility. In our research, we have focused on the characterization of small molecular weight proteins found within dragline and tubuliform silks. Within the black widow spider, Lactrodectus hesperus, these proteins have been named Cysteine-Rich Protein (CRP) and determined to be a family of five individual proteins. The small protein identified within the tubuliform silks has been named Egg Case Protein 3 (ECP-3). In this study, recombinant expression of ECP-3 in the pET-19b-SUMO vector was to facilitate purification and development of an immunological reagent. Using western blot analysis, we have demonstrated that ECP-3 is efficiently expressed in bacteria. We also investigated CRP1 protein and its ability to bind MaSp1 components using pull down assays to determine potential interactions. No substantial biochemical evidence was produced to demonstrate protein-protein interactions between the two. Additionally, we show that using RT-PCR analysis from mRNAs collected from the major ampullate gland that transcript levels for CRP-family members from non-silked and a silked spider are different. CRP2 and CRP4 mRNA levels were shown to increase upon silking. Overall, the major findings of this thesis involved characterizing the ECP-3 protein found within tubuliform silks as well as determining the expression patterns for CRP-family members.

Pages

84

ISBN

9781321285048

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