Randolph V Lewis

• PhD
• Professor at Utah State University

Hagfish intermediate filament (HIF) proteins, consisting of α and γ subunits, have been previously recombinantly expressed, purified, and utilized to form dry fibers with impressive mechanical properties. HIFα and HIFγ consist of three protein domains (N-termini, C-termini, and central rod domain). To begin to understand the structure–function rela...

The purpose of this study was to determine a method to purify recombinant hagfish intermediate filament proteins, alpha and gamma, in a scalable manner. The study succeeded by having an increase in protein recovery of up to 35% when comparing centrifuge purification and the developed tangential flow purification. The proteins were approximately the...

Native hagfish intermediate filament proteins have impressive mechanical properties. However, using these native fibres for any application is impractical, necessitating their recombinant production. In the only literature report on the proteins (denoted α and ɣ), heterologous expression levels, using E. coli, were low and no attempts were made to...

Spider silk, which has remarkable mechanical properties, is a natural protein fiber produced by spiders. Spiders cannot be farmed because of their cannibalistic and territorial nature. Hence, large amounts of spider silk cannot be produced from spiders. Genetic engineering is an alternative approach to produce large quantities of spider silk. Our g...

In order to better understand the relationship between flagelliform (Flag) spider silk molecular structural organization and the mechanisms of fiber assembly, the Nephilengys cruentata Flag spidroin analogue rNcFlag2222 is designed and produced. The recombinant proteins are composed by the elastic repetitive glycine‐rich motifs (GPGGX/GGX) and the...

p>In order to better understand the relationship between the elastomeric behavior of Flagelliform (Flag) spider silks and its molecular structure, it was designed and produced the Nephilengys cruentata Flageliform (Flag) spidroin analogue rNcFlag2222. The recombinant proteins are composed by the elastic repetitive glycine-rich motifs (GPGGX/GGX) an...

Spider silks are intriguing biomaterials that have a high potential as innovative biomedical processes and devices. The intent of this study was to evaluate the capacity of recombinant spider silk proteins (rSSps) as a synthetic Bruch’s membrane. Nonporous silk membranes were prepared with comparable thicknesses (<10 µm) to native Bruch’s membrane....

Using transgenic silkworms with their natural spinning apparatus has proven to be a promising way to spin spider silk-like fibers. The challenges are incorporating native-size spider silk proteins and achieving an inheritable transgenic silkworm strain. In this study, a CRISPR/Cas9 initiated fixed-point strategy was used to successfully incorporate...

Compared to silkworm silks, spider silks (SS) are considered stronger, most elastic, and tougher biomaterials. Web-weaving spiders use impressive silks where the proteins in the silks can be described as virtually nondiverged. In the last three decades, several research papers have studied the physical, mechanical, and chemical properties of SS, as...

Although synthetic spider silk has impressive potential as a biomaterial, endotoxin contamination of the spider silk proteins is a concern, regardless of the production method. The purpose of this research was to establish a standardized method to either remove or destroy the endotoxins present in synthetic spider silk proteins, such that the endot...

This work presents electrospun nanofibers from synthetic spider silk protein, and their application as both a mechanical vibration and humidity sensor. Spider silk solution was synthesized from minor ampullate silk protein (MaSp) and then electrospun into nanofibers with a mean diameter of less than 100 nm. Then, mechanical vibrations were detected...

Major ampullate spider silk represents a promising protein-based biomaterial with diverse commercial potential ranging from textiles to medical devices due to its excellent physical and thermal properties. Recent advancements in synthetic biology have facilitated the development of recombinant spider silk proteins from Escherichia coli (E. coli), a...

Although PCR-based techniques have become an essential tool in the field of molecular and genetic research, the amplification of repetitive DNA sequences is limited. This is due to the truncated nature of the amplified sequences, which are also prone to errors during DNA polymerase-based amplification. The complex structure of repetitive DNA can fo...

Freestanding fibrous matrices with proper protein composition and desirable mechanical properties, stability, and biocompatibility are in high demand for tissue engineering. Electrospun (E-spun) collagen–silk composite fibers are promising tissue engineering scaffolds. However, as-spun fibers are mechanically weak and unstable. In this work, we app...

Thermal conductivity, thermal diffusivity and volumetric heat capacity of three spider silks are measured in this paper as a benchmark for further studies. These silks include the major and minor ampullate silks of the Nephila clavipes spider, and a synthetic spider silk fiber made from recombinant dragline silk proteins purified from transgenic go...

The processes used to create synthetic spider silk greatly affect the properties of the produced fibers. This paper investigates the effect of process variations during artificial spinning on the thermal and mechanical properties of the produced silk. Property values are also compared to the ones of the natural dragline silk of the Nephila clavipes...

Solid-state NMR and molecular dynamics (MD) simulations are presented to help elucidate the molecular secondary structure of poly(Gly-Gly-X), which is one of the most common structural repetitive motifs found in orb-weaving dragline spider silk proteins. The combination of NMR and computational experiments provides insight into the molecular second...

The production of recombinant spider silk proteins continues to be a key area of interest for a number of research groups. Several key obstacles exist in their production as well as in their formulation into useable products. The original reported method to solubilize recombinant spider silk proteins (rSSp) in an aqueous solution involved using mic...

The mechanical properties and biocompatibility of spider silks have made them one of the most sought after and studied natural biomaterials. A biomimetic process has been developed that uses water to solvate purified recombinant spider silk proteins (rSSps) prior to material formation. The absence of harsh organic solvents increases cost effectiven...

A method of producing synthetic spider silk, including: transforming Escherichia coli with an expression vector; fermenting the transformed E. coli in a culture medium; inducing spider silk protein expression in the cultured E. coli; extracting the spider silk; and purifying the spider silk. Related vectors and genetically modified cells are also d...

Fiber thermal characterization is often accomplished by indirect means, such as embedding the fiber in a matrix, measuring the thermal response of the composite, and relating for the contributions of the fiber and matrix to the overall behavior or measuring bundles of fibers. To improve the accuracy of the composite-based or bundle-based techniques...

Spider silks have unique mechanical properties but current efforts to duplicate those properties with recombinant proteins have been unsuccessful. This study was designed to develop a single process to spin fibers with excellent and consistent mechanical properties. As-spun fibers produced were brittle, but by stretching the fibers the mechanical p...

Methods for the production of synthetic spider silk-like proteins in corn endosperm or plant shoot tissue are provided. The present invention provides further methods for the identification of synthetic spider silk-like proteins in corn endosperm or plant shoot tissue.

Spider silk is a striking and robust natural material that has an unrivaled combination of strength and elasticity. There are two major problems in creating materials from recombinant spider silk proteins (rSSps): expressing sufficient quantities of the large, highly repetitive proteins and solvating the naturally self-assembling proteins once prod...

Biocomposite matrices with high mechanical strength, high stability and the ability to direct matrix-specific stem cell differentiation are essential for the reconstruction of lesioned tissues in tissue engineering and cell therapeutics. Toward this end, we used the electrospinning technique to fabricate well-aligned composite fibers from collagen...

The transient electrothermal technique is a powerful tool to obtain thermal properties of fine fibers. However, the technique suffers from several inherent pitfalls, which affect measurement accuracy, especially with application to coated, nonconductive samples. In this paper, measurement challenges are described and quantified for several associat...

Spider silk has exceptional mechanical and biocompatibility properties. The goal of this study was optimization of the mechanical properties of synthetic spider silk thin films made from synthetic forms of MaSp1 and MaSp2, which compose the dragline silk of Nephila clavipes. We increased the mechanical stress of MaSp1 and 2 films solubilized in bot...

The thermal conductivity and diffusivity of the dragline silk of the Nephila (N.) clavipes spider has been characterized by one research group to be 151-416 W m-1 K-1 and 6.4-12.3 x 10-5 m2 s-1, respectively, for samples with low to high strains (zero to 19.7%). Thermal diffusivity of the dragline silk of a different spider species, Araneus diadema...

Spider silk is well-known for its exceptional mechanical properties, such as strength, elasticity and flexibility. Recently, it has been reported that dragline silk from a Nephila clavipes also has an exceptionally high thermal conductivity, comparable to copper when the fiber is stretched. Synthetic spider silks have been spun from spider silk pro...

Spacer Motif Contributes Tensile Strength to Recombinant Nephila clavipes Flagelliform-like Silk Protein Fibers

Spider silk is a biomaterial with impressive mechanical properties, resulting in various potential applications. Recent research has focused on producing synthetic spider silk fibers with the same mechanical properties as the native fibers. For this study, three proteins based on the Argiope aurantia Major ampullate Spidroin 2 consensus repeat sequ...

Spider silks exhibit excellent strength, stiffness, and toughness simultaneously, a feat unachievable in most synthetic, structural materials. It has recently been reported that the thermal conductivity of dragline silk is comparable to copper, which is uncharacteristically high for a biomaterial. In order to develop a fundamental understanding of...

Prion diseases are fatal neurodegenerative disorders characterized by misfolding of the cellular prion protein (PrP(c)) into the disease-associated isoform (PrP(Sc)) that has increased β-sheet content and partial resistance to proteolytic digestion. Prion diseases from different mammalian species have varying propensities for transmission upon expo...

Flagelliform spider silk is the most extensible silk fiber produced by orb weaver spiders, though not as strong as the dragline silk of the spider. The motifs found in the core of the Nephila clavipes flagelliform Flag protein are: GGX, spacer, and GPGGX. Flag does not contain the polyalanine motif known to provide the strength of dragline silk. To...

Spider silk has biocompatibility and biodegradability properties and is known for the mechanical, physical and chemical properties that make it a promising building block in the development of novel biofibers. Its unique properties partially result from the repetitive polypeptide sequences that compose the silk proteins. The strength is related to...

Dragline silk from orb-weaving spiders is a co-polymer of two large proteins, major ampullate spidroin 1 (MaSp1) and 2 (MaSp2). The ratio of these proteins is known to have a large variation across different species of orb-weaving spiders. NMR results from gland material of two different species of spiders, N. clavipes and A. aurantia, indicates th...

Spider silks have been shown to have impressive mechanical properties. In order to assess the effect of extension rate, both quasi-static and high-rate tensile properties were determined for single fibers of major (MA) and minor (MI) ampullate single silk from the orb weaving spider Nephila clavipes . Low rate tests have been performed using a DMA...

The two Flag/MaSp 2 silk proteins produced recombinantly were based on the basic consensus repeat of the dragline silk spidroin 2 protein (MaSp 2) from the Nephila clavipes orb weaving spider. However, the proline-containing pentapeptides juxtaposed to the polyalanine segments resembled those found in the flagelliform silk protein (Flag) composing...

Regulations for the disposal of genetically engineered animals are strict due to concern for their inappropriate introduction into the food chain, and of the possible public health and environmental impacts of these organisms. Nontransgenic animals that give birth to transgenic offspring are treated as if they are transgenic due to concern of fetal...

The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors t...

As a promising biomaterial with numerous potential applications, various types of synthetic spider silk fibers have been produced and studied in an effort to produce man-made fibers with mechanical and physical properties comparable to those of native spider silk. In this study, two recombinant proteins based on Nephila clavipes Major ampullate Spi...

Spider silk has been evolutionarily optimized for contextual mechanical performance over the last 400 Ma. Despite precisely balanced mechanical properties, which have yet to be reproduced, the underlying molecular architecture of major ampullate spider silk can be simplified being viewed as a versatile block copolymer. Four primary amino acid motif...

Orb-weaving spider silk fibers are assembled from very large, highly repetitive proteins. The repeated segments contain, in turn, short, simple, and repetitive amino acid motifs that account for the physical and mechanical properties of the assembled fiber. Of the six orb-weaver silk fibroins, the piriform silk that makes the attachment discs, whic...

Two-dimensional homo- and heteronuclear solid-state MAS NMR experiments on (13)C/(15)N-proline labeled Argiope aurantia dragline silk provide evidence for an elastin-like beta-turn structure for the repetitive Gly-Pro-Gly-X-X motif prevalent in major ampullate spidroin 2 (MaSp2).

Major ampullate (dragline) spider silk is a coveted biopolymer due to its combination of strength and extensibility. The dragline silk of different spiders have distinct mechanical properties that can be qualitatively correlated to the protein sequence. This study uses amino acid analysis and carbon-13 solid-state NMR to compare the molecular compo...

Two unique spidroins are present in the silk of the Amazon mygalomorph spider - Avicularia juruensis (Theraphosidae), and for the first time the presence and expression of a major ampullate spidroin 2-like in Mygalomorphae are demonstrated. Molecular analysis showed the presence of (GA)(n,) poly-A and GPGXX motifs in the amino acid sequence of Spid...

Synthetic spider silk holds great potential for use in various applications spanning medical uses to ultra lightweight armor; however, producing synthetic fibers with mechanical properties comparable to natural spider silk has eluded the scientific community. Natural dragline spider silks are commonly made from proteins that contain highly repetiti...

Spider silks have the potential to provide new bio-based materials for numerous military applications ranging from protective clothing to parachute cords to composite materials in aircraft. Specific amino acid motifs have been identified which have been conserved for over 125 million years in all spiders using their silk to physically trap their pr...

The various silks that make up the web of the orb web spiders have been studied extensively. However, success in prey capture depends as much on the web glue as on the fibers. Spider silk glue, which is considered one of the strongest and most effective biological glues, is an aqueous solution secreted from the orb weaving spider's aggregate glands...

The extreme strength and elasticity of spider silks originate from the modular nature of their repetitive proteins. To exploit such materials and mimic spider silks, comprehensive strategies to produce and spin recombinant fibrous proteins are necessary. This protocol describes silk gene design and cloning, protein expression in bacteria, recombina...

Ante-mortem assays exist for some Transmission Spongiform Encephalopathies (TSE). These assays facilitate our understanding of disease pathology and epidemiology; however, the limitations of these ante-mortem assays include the inability to quantify protein amount, poor sensitivity, and/or limited robustness. Here, we utilize a bioinformatics appro...

Although spider silk displays an amazing combination of strength and extensibility unrivalled by most synthetic biomaterials, its molecular architecture is relatively simplistic. Four primary amino acid block motifs (An, (GA)n, GPGXX, GGX) have been correlated with mechanical functions. Recent genetic engineering to control the mechanical behavior...

Solid-state two-dimensional refocused INADEQUATE MAS NMR experiments resolve distinct helical and beta-sheet conformational environments for both alanine and glycine in Nephila clavipes dragline silk fibers; the fraction of alanine and glycine in beta-sheet structures is determined to be 82% +/- 4% and 28% +/- 5%, respectively.

The protein compositions of selected high-performance biological materials such as silks, collagen and mussel byssus threads are reviewed. The possible roles of key amino structural motifs present in these fibrous proteins are outlined. Experimental investigation of structure/function relationships in fibrous proteins is discussed. More specificall...

All characterized major ampullate silks from orb-web weaving spiders are composites of primarily two different proteins: MaSp1 and MaSp2. The conserved association of MaSp1 and MaSp2 in these spider species, the highly conserved amino acid motifs, and variable ratios of MaSp1 to MaSp2 demonstrate the importance of both MaSp1 and MaSp2 to the streng...

The bio-bar code assay is an assay for ultrasensitive detection of proteins. The main technical hurdle in bio-bar code assay development is achieving a dose-dependent, reproducible signal with low background. We report on a magnetic bead ELISA screening mechanism for characterizing antibody pairs that are effective for use in the bio-bar code assay...

Two-dimensional (2D) (13)C-(13)C NMR correlation spectra were collected on (13)C-enriched dragline silk fibers produced from Nephila clavipes spiders. The 2D NMR spectra were acquired under fast magic-angle spinning (MAS) and dipolar-assisted rotational resonance (DARR) recoupling to enhance magnetization transfer between (13)C spins. Spectra obtai...

Spiders have evolved a complex system of silk producing glands. Each of the glands produces silk with strength and elasticity tailored to its biological purpose. Sequence analysis of the major ampullate silk reveals four highly conserved concatenated blocks of amino acids: (GA) n , A n , GPGXX, and GGX. While the GPGXX motif, which has been hypothe...

Silks spun from the major (Ma) and minor (Mi) ampullate glands by the spider Nephila clavipes respond to water differently. Specifically, Ma silk supercontracts (shrinks 40-50% in length) while Mi silk does not contract at all when hydrated with water. In the present study, 1H --> 13C cross polarization magic angle spinning (CP-MAS), 13C MAS NMR co...

Spiders spin a diverse array of silk fibers that are predominately composed of repetitive proteins (spidroins) encoded by a gene family. Characterization of this gene family has focused on spidroins synthesized by the Araneomorphae (true spiders), whereas only a single sequence is known from the Mygalomorphae (tarantulas and their kin). To better u...

Even before material science was a recognized discipline, the amazing mechanical properties of spider silk were documented and became the object of much study. In addition to the exceptional material properties of spider silk and the reported low immunogenicity, its concatenated amino acid motif arrangement facilitates a distinct possibility of man...

Spiders produce up to six different kinds of silk, each one for a specific biological function. Spider silks are also known for their unique mechanical properties. The possibility of producing new materials with similar properties motivated research on these silk proteins (spidroins). Using expression sequence tags, we identified four spidroins pro...

Bacteria were genetically engineered to produce two spider silk protein variants composed of basic repeat units combining a flagelliform elastic motif ([GPGGX]4) and a major ampullate silk strength motif ([linker/poly-alanine]. The secondary structures of the pure recombinant proteins in solution were determined by circular dichroism. The data pres...

The major ampullate fiber of both Nephila clavipes and Argiope aurantia is composed of two different proteins, MaSp1 and MaSp2. Each of these proteins has a highly conserved pattern of silk-associated amino acid motifs. The GPGXX motif is the only source of proline and is unique to MaSp2. On the basis of the percent of proline, Nephila clavipes maj...

Interferon-stimulated gene (ISG) 15 mediates antiviral responses and also is upregulated within the endometrium in response to the developing embryo during early pregnancy. Structurally, ISG15 resembles two ubiquitin domains (30% identical) that are separated by a hinge region. Recombinant (r) bovISG15 is not stable in solution. It was hypothesized...

Spider silks have been noted for their extraordinary mechanical properties for centuries. Recent research has provided information that helps explain these properties on a molecular basis. This article describes the biological and mechanical properties of spider silks. Three different spider silks are discussed with particular emphasis on the prote...

The past 15 years has seen a tremendous increase in information about many aspects of spider silk. This includes protein sequences, biophysical studies of the proteins in the fiber, the fiber-spinning process, and evolutionary studies. Despite this progress there are still several key connections needed for a full understanding of spider silk. Thes...

This project is designed to create new proteins based on naturally occurring spider silk proteins with the goal of controlling elasticity and tensile strength in fibers spun from the proteins. The new genes have been constructed in year 1, the proteins have been expressed and purified in this, year 2, and characterized to insure they are correct. W...

Silk fibers possess impressive mechanical properties, dependant, in part, on the crystalline β-sheets silk II conformation. The transition to silk II from soluble silk I-like conformation in silk glands, is thought to originate in the spinning ducts immediately before the silk is drawn down into a fiber. However the assembly process of these silk m...

Orb–web weavers can produce up to six different types of silk and a glue for various functions. Tubuliform silk is unique among them due to its distinct amino acid composition, specific time of production, and atypical mechanical properties. To study the protein composing this silk, tubuliform gland cDNA libraries were constructed from three orb–we...

Major ampullate silk, also known as dragline silk, is one of the strongest biomaterials known. This silk is composed of two proteins, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2). Only partial cDNA sequences have been obtained for these proteins, and these sequences are toward the C-terminus. Thus, the N-terminal domain...

As a result of hundreds of millions of years of evolution, orb-web-weaving spiders have developed the use of seven different silks produced by different abdominal glands for various functions. Tubuliform silk (eggcase silk) is unique among these spider silks due to its high serine and very low glycine content. In addition, tubuliform silk is the on...

Spiders have evolved to produce up to seven different silks; for each silk the balance between strength and elasticity is optimized according to the silk's specific use. Amino acid motifs have been identified and predicted functions have been assigned to these motifs. In addition to understanding the underlying molecular basis for structure/functio...

Introduction Historical Outline Biological Aspects Mechanical Properties Protein Sequences: Conservation, Divergence, and Convergence Major Ampullate Silk Proteins Minor Ampullate Silk Proteins Flagelliform Silk Proteins Sequence Comparisons Between Spider Species Biophysical Studies Protein Structure–Function Relationships Expression of...

Araneoid spiders use specialized abdominal glands to produce up to seven different protein-based silks/glues that have diverse physical properties. The fibroin sequences that encode aciniform fibers (wrapping silk) and the mechanical properties of these fibers have not been characterized previously. To gain a better understanding of the molecular r...

The addition of water to spider dragline silk results in fiber contraction to 50% its initial length and significant changes to the mechanical properties of the silk. This event has been termed supercontraction. A decrease in strength and increase in elasticity have been reported when the silk is in contact with water. Two-dimensional wide-line sep...

Compared to other arthropods, spiders are unique in their use of silk throughout their life span and the extraordinary mechanical properties of the silk threads they produce. Studies on orb-weaving spider silk proteins have shown that silk proteins are composed of highly repetitive regions, characterized by alanine and glycine-rich units. We have i...

Major ampullate (dragline) silk is the main web component as well as the silk that spiders use for a lifeline when they fall. This silk has a breaking stress of 4.6 GPa, which is similar to that of Kevlar. The majority of the previous mechanical testing studies involved the major ampullate silk from orb-weaving spiders. To date, there have been no...

Spider silk, one of nature's greatest accomplishments, has a combination of strength and elasticity that is unrivaled. Spiders produce up to 7 different silks; each one with a unique combination of tensile strength and elasticity that allows the spiders' web to hold prey while being resilient enough not to break upon impact. In an attempt to determ...