Silk is a natural protein produced by members of the class Arachnida (over 30,000 species of spiders) and by several worms. Silk-based materials have been investigated for medical and biotechnological applications for many years. Although silkworm silk has been studied extensively because of ready availability of the protein, lately the advancements in recombinant technology has made production of spider silk proteins increasingly available. Due to the characteristics like biocompatibility, biodegradability and mechanical strength, silk is highly desirable as a biomaterial for medical purpose. Along with this, techniques for functionalization, has further aided in the development of silk into highly sophisticated material for advanced applications. The main objective of this thesis has been to investigate novel strategies for functionalization of the recombinant spider silk protein Masp2. Two distinct approaches were used, chemical modification and genetic fusion. In the first modification, we created an infection responsive silk nanospheres by chemically grafting a thrombin sensitive peptide to the silk protein encapsulating antibiotic. These particles were then evaluated for in vitro infection responsive drug release and antimicrobial activity. From these assessments, we found that these particles can release the drug effectively in the presence of infection providing the evidence that these particles are enzyme responsive and can be used to formulate targeted drug release. In the second modification, spider silk was genetically modified with a heparin binding peptide to create a fusion protein which can prevent both thrombosis and infection simultaneously. This fusion protein was evaluated for its heparin binding ability and anticoagulant properties in its solution form. Furthermore, due to the similarity in structure of HBP with antimicrobial peptides, it is predicted that the fusion protein will also show antimicrobial property. After establishing these properties, next this fusion protein was utilized as a coating for hemodialysis catheter. Deposition of coating was evaluated after which anticoagulant and anti-infective properties of the protein as a coating material was investigated. This thesis provides evidence of successful production of a recombinant silk-based biopolymer that can be chemically and genetically embedded with a various functional motif to create a hybrid product for different applications.

Spider silk has the potential to be a useful biomaterial due to its high tensile strength and elasticity. It is also biocompatible and biodegradable, making it useful for wound dressings and sutures, tissue and bone scaffolds, vessels for drug delivery, and ligament and tendon replacements. In some studies where spider silk has been used to grow cells, the silk has promoted more cell growth than the control. However, it is difficult to obtain the high volume of silk needed for these undertakings on a large scale. Spiders are territorial and cannibalistic, so they cannot be easily farmed. Therefore, spider silk proteins are frequently produced in other organisms. E. coli is often used for spider silk production due to the relative ease of gene manipulation and the cost effectiveness of large-scale fermentation. However, due to the large protein size of the spider silk and the repeating amino acid motifs, there are some challenges with production in E. coli. Metabolic modeling is a way to model the metabolism of an organism and can help overcome some of the difficulties of spider silk production in E. coli by predicting metabolic engineering strategies. In this study, a metabolic modeling tool known as dynamic FBA predicted that ammonium is depleted during cell growth. Laboratory results confirmed that by adding additional ammonium to the medium, the E. coli cells experienced more cell growth and were able to produce more spider silk protein

Spider silks are incredible natural materials that have a wide variety of properties that can rival or outperform even common synthetic materials like Nylon and Kevlar. As nature’s architects, orb-weaving spiders spin seven different silks that are used for very specific roles throughout the spider’s lifecycle. These silks are comprised of proteins called spidroins. Each of these spidroins has evolved to have properties such as strength and/or stretch that make these silks successful and highly adapted in their designated roles in web construction, prey capture and reproduction. This study involves the production of minor ampullate silk by genetically modifying the bacteria Escherichia coli. Minor ampullate is a lesser studied silk that is used for the first spiral of the orb web. This spiral is a template that the spider uses to finish the web and provides stability during the web construction. Minor ampullate silk is strong, however it does not stretch so it may be well-suited for certain applications such as ballistic materials. By producing and purifying different arrangements of minor ampullate silk protein, it is possible to learn how this protein can be expressed without using the spider itself. This investigation sheds light on how deviations in the protein sequence and motif arrangement can produce different properties, which can potentially be used to make new materials.

Spider silk is a biodegradable and biocompatible natural material that is stronger than steel and more elastic than nylon. These properties make spider silk a desirable material for many commercial products, ranging from textiles to biomedical materials. Due to spiders’ cannibalistic and territorial nature it is impossible to farm them to produce spider silk at a high enough yield to meet product demands. Therefore, a bioengineered synthetic process is necessary to produce spider silk. Synthetic spider silk has been produced in bacteria, goats, yeast, plants, mammalian cells and silkworms, but none of these processes provided a commercially viable yield or were able to express recombinant spider silk proteins (rSSps) that can mechanically imitate the natural spider silks. The overall goal of this research was to increase the yield and mechanical characteristics, e.g. strength and elasticity, to create a commercially viable spider silk. Three different hosts were used: E. coli, alfalfa and an insect cell line. Each host addresses issues with synthetic protein production in both the short-term and long-term scheme. Through this research yields were increased, while the mechanical properties of the synthetic silks were improved and groundwork for future research into the improvement of synthetic spider silk production were identified.

Spider silk has long been a subject of scientific research due to its remarkable mechanical properties. Until recently, there has been no way to effectively obtain spider silk except by harvesting it from individual spiders. With advances in technology, the genes that code for the individual spider silk proteins have been isolated and genetically engineered into other hosts to produce recombinant spider silk proteins (rSSp) of varying sizes, Larger rSSp have correspondingly greater mechanical properties in any resulting materials. Using current production methods, larger rSSp cannot be produced in commercially viable quantities while simultaneously being economically viable. The current production methods have shown that small rSSp are easier to produce and purify in genetically engineered systems while maintaining favorable yields. After the small molecular weight rSSp were expressed and purified, they were polymerized to form larger molecular weight rSSp, while having maintained mechanical properties of similarly sized rSSp from other expression systems. To accomplish this polymerization, two systems were designed that can catalyze this reaction: using a Spy System and an intein system. These two systems require no external cofactors or enzymes and occur spontaneously once initiated. The expression and purification of rSSp from both of these systems has been characterized. The Spy System did not produce high enough quantities of rSSp to be economically viable. Whereas the intein system produced yields of 5 g/L, which is higher than previously reported. The rSSp from the intein system have been made into biomaterials, such as films, hydrogels, and aerogels. The mechanical properties of these biomaterials were comparable to biomaterials from other spider silk protein production. Utilizing the intein system, projected cost estimates for the production of rSSp has been lowered from $350 to $40 per kilogram. This decreased cost of rSSp would allow a wider array of commercial applications.

Spider-silk is known as one of the stronger natural materials, unfortunately it is impossible to farm spiders due to their territorial and cannibalistic nature. To address this issue, researchers have studied spider-silk to discover how it is produced in nature. From their results, spider-silk is composed of large sized proteins produced in two different cell types. Using this knowledge, researchers created transgenic organisms capable of producing spider-silk proteins in large quantities. Using these proteins, several groups have created fibers, films, hydrogels, and adhesives with robust and versatile properties. Wet-spinning is a technique commonly used to create fibers from spider-silk proteins. These fibers unfortunately do not compare to the mechanical properties of natural silk. To address this researchers have used a method known as electrospinning to create spider-silk fibers with substantially smaller diameters. In doing so, these electrospun fibers have increased surface area and enhanced mechanical properties. Using this method, our group has modified the electrospinner to be able to produce continuous fine diameter yarns composed of hundreds of nanofibers with mechanical properties surpassing that of natural silk. Fibers aside, spider-silk proteins can be used to create a variety of different biocompatible materials. To further enhance these materials, our group has utilized a technique traditionally used for observation. This technique employs a high intensity light source to initiate cross-links within the proteins. With this method, our spider-silk protein materials have increased their mechanical properties by a factor of seven. These materials can further be modified through post-treatments, resulting in tunable materials with diverse and robust mechanical properties.

Biomolecule structural fluctuations determine function, regulating numerous biological processes My research has shed light on several interesting cases in which structural fluctuations have been identified to assess functional differences. Chapter 2 discusses the effects of structural rearrangement of the β2-β3 loop on the DNA binding affinity of the type 6 human papillomavirus E2 protein. Chapter 3 investigates the effects of phosphorylation on the C-terminal domain of Cdc37, a protein important in the Hsp90 chaperone cycle. Chapter 4 studies the effects on cyclycization on the conformational fluctuations of a γ-AApeptide used for high-throughput libraries. Chapter 5 is a structural study on a mini-fibril of spider dragline silk, in which a native-like ensemble was generated using temperature replica exchange. Chapter 6 investigates the structural features of repetitive motifs found in spider dragline silk when subject to both dope-like and fiber-like conditions. Chapter 7 elucidates conformational differences between the RXRα and the RXRβ ligand-binding domains and seeks to understand the atomic basis for different ligand binding affinities. This body of work has contributed to the understanding of conformational fluctuations and changes that occur in protein-DNA binding systems, drug-binding, regulation of chaperones via post-translations modifications and spider dragline silk.

Spider silks are incredible natural materials that possess desirable combinations of strength, elasticity, weight, and robustness. Other properties such as biocompatibility and biodegradability further increase the worth of these materials. The possibility of farming spiders is impractical due to spiders’ natural behaviors. Modern biotechnologies have allowed for recombinant spider silk proteins (rSSps) to be produced without the use of spiders. However, the features responsible for spider silks impressive properties can cause difficulties with producing silk materials. A recently developed water-based and biomimetic solvation method has provided a solution to such difficulties and has also led to novel silk biomaterials. Most notable among these materials are; coatings, fibers, adhesives, films, foams, hydrogels, aerogels, capsules, and sponges. Many of these material possess specific properties that may be suitable for many commercial, industrial, and biomedical uses. This study has developed numerous spider silk biomaterials, identified their essential properties and features, provided preliminary evidence for various applications, and identified directions for future studies and uses.

Major ampullate spider silk represents a promising biomaterial with diverse commercial potential ranging from textiles to medical devices due to the excellent physical and thermal properties from the protein structure. Recent advancements in synthetic biology have facilitated the development of recombinant spider silk proteins from Escherichia coli (E. coli), alfalfa, and goats. This study specifically investigates the economic feasibility and environmental impact of synthetic spider silk manufacturing. Pilot scale data was used to validate an engineering process model that includes all of the required sub-processing steps for synthetic fiber manufacture: production, harvesting, purification, drying, and spinning. Modeling was constructed modularly to support assessment of alternative protein production methods (alfalfa and goats) as well as alternative down-stream processing technologies. The techno-economic analysis indicates a minimum sale price from pioneer and optimized E. coli plants at $761 kg-1 and $23 kg-1 with greenhouse gas emissions of 572 kg CO2-eq. kg-1 and 55 kg CO2-eq. kg-1, respectively. Spider silk sale price estimates from goat pioneer and optimized results are $730 kg-1 and $54 kg-1, respectively, with pioneer and optimized alfalfa plants are $207 kg-1 and $9.22 kg-1 respectively. Elevated costs and emissions from the pioneer plant can be directly tied to the high material consumption and low protein yield. Decreased production costs associated with the optimized plants include improved protein yield, process optimization, and an Nth plant assumption. Discussion focuses on the commercial potential of spider silk, the production performance requirements for commercialization, and impact of alternative technologies on the sustainability of the system.

In this project, we focus on developing a method to produce synthetic spider silk thin films. Using these films we optimized mechanical properties, lowered cost, and improved the environmental impact using different processing methods. Applications for spider silk films are broad, ranging from physical protection to biocompatible materials. This project was designed to improve mechanical properties and production methods of films made from synthetic forms of MaSp1 and MaSp2 from the dragline silk of Nephila clavipes. We have increased the mechanical stress (200 MPa) to more than 4 times that of similar products with elongations as high as 35%. The films have also been analyzed using NMR, XRD, and AFM or SEM showing that the secondary structure in as-poured films is mainly alpha-helical and after processing this structure turns to an aligned betasheet formation similar to that in spider silk fibers.