Recombinant Piriform Spider Silk Production

06/25/2020

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Cole Peterson

Cole Peterson

Cole Peterson

Doctoral Dissertation Defense
Department of Biological Engineering

Tuesday, June 30
11:00 AM through WebEx

Co-Advisor - Dr. Ronald Sims

ron.sims@usu.edu

Co-Advisor - Dr. Randy Lewis

randy.lewis@usu.edu

Full Abstract

Spider silks are remarkable biomaterials, possessing a variety of desirable mechanical and material properties. Humans have long-sought to utilize these materials. As spider silks evolved over time to suit specific functions, evolutionary drift was also occurring across species of spiders. Thus, spider silks produced by two widely divergent species are not necessarily equal in terms of mechanical and material properties. One species of spider, the orb weaver Nephila clavipes, has been shown to produce silk with some of the most notable mechanical properties. While most silks from N. clavipes have been well-characterized, some have not been studied due to their relative scarcity in the web. An example of this is piriform silk, which is unique in amino-acid composition as well as macroscopic structure. It is used as a lashing to join other silk strands together, and to anchor the web to a range of substrates. For spider silk to be useable as a commercial product, it must be produced in a cheap and scalable fashion. Spiders themselves do not produce sufficient silk for viable farming. Another approach is to produce the silk as a recombinant protein product using a microbial host. Escherichia coli is an attractive option but requires targeted-optimization to produce recombinant spider silk proteins (rSSps). The goal of this research is to produce recombinant-analogues of piriform spider silk in E. coli, and to scale-up production to produce sufficient recombinant piriform for mechanical studies. Expression in E. coli was improved by engineering targeted metabolic improvements to increase yields of piriform. These systems were scaled and tested in fermentation up to pilot-scale. A range of purification methods were developed to recover piriform silk from microbial lysates. Recombinant produced and purified in this research were tested for material properties using methods shown successful for other recombinant N. clavipes silks.

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