No description available.

No description available.

Spider silk is known for its extraordinary material properties, being both very strong and extensible. Even though the fibers outperform many synthetic and natural materials, it is impractical to collect industrial amounts of silk from spiders due to their cannibalistic and venomous nature; they cannot be farmed like the commercial silk worm Bombyx mori. Thus, scientists have turned to molecular and engineering techniques to replicate the spider's silk and spinning apparatus. In the current literature there is no detailed protocol on the production of consistent synthetic fibers. To accomplish this, the fibroins and natural spinning apparatus were taken apart and analyzed in order to develop a protocol that biomimics the spider's system. The laboratory procedure, using the natural process as an example, was simplified to: protein production, purification, concentration, fiber spinning, and lastly post spin draw. Large quantities of truncated MaSp I spidroin (spider fibroin) was purified from E. coli and successfully spun into fibers using customized spinning, spooling, and stretching apparatuses. The final fiber products displayed mechanical properties that were comparable to other reported synthetic fibers, but more importantly also displayed low experimental variability between samples. The protocol developed in this study can be further used to characterize other spidroins and silk proteins, and can be further advanced to produce even better fibers with enhanced properties.

Spider silk is one of the most versatile material.s in nature with great mechanical properties, exceeding some of the best man made materials. Native and synthetically produced silk has been used in a wide array of applications throughout the history of mankind including nets, bandages and cloths. It is recognized that spider silk can be a suitable replacement material for many existing materials such as ropes, body armor, parachutes and biodegradable bottles - all of which could show cost and environmental 4 benefits relative to other currently used man made materials. An added advantage to these types of applications is the potential for the products to have intrinsic antimicrobial activity. Studies have demonstrated a level of antimicrobial activity in native silk, a property that may have evolved in order to resist microbial decomposition, to protect developing eggs, and to resist decomposition or destruction by predators, parasites, or fluctuations in the environment. In this study, the novel aqueous glue coating peptides found on the silk fiber of the black widow spider, spider coating peptide 1 and 2, were investigated. Using circular dichroism, it was determined that SCP-1 and SCP-2 display predominantly alpha-helical secondary structures. In temperature gradient studies, SCP-1 is structurally stable at high temperatures while SCP-2 unfolded and lost its alpha-helical structure. The two peptides remained structurally stable both in an acidic and basic environment. This study was the first to characterize the secondary structure of the peptides found coating various silk fibers in Latrodectus hesperus, the black widow spider. The function of the SCPs is unknown but has-been hypothesized to potentially have antimicrobial properties. We investigated this role and found no significant antibacterial activity of the peptides against Escherichia coli and Bacillus subtitlis in growth studies. This study is the first to investigate the functional role of SCPs.

Spider silk from the female black widow spider, Latrodectus hesperus, is made of extraordinary biomolecules of nature. Efforts of the scientific community to commercially synthesize silks have become a collaborating, yet competitive race, to characterize the proteins that contribute to its intriguing biomechanics. Little has been reported on aqueous silk molecules in black widow spider silk, which are quite different from the large water insoluble core fibroins. In this study, a novel, aqueous aggregate gland derived silk factor (AgSF 1) was investigated using proteomics and immunological approaches. Western blot analyses of whole tissue lysates and solublized silk fibers revealed high levels of AgSFl in the aggregate gland, in the web scaffolding junctions, and in wrapping silk. MS/MS analyses of tryptic digest products from solubilized wrapping silk and aggregate gland whole cell lysate also confirmed the presence of AgSFl in these samples. Possible post-translational modifications were also analyzed by two-dimensional gel electrophoresis (2DE) and MS/MS analysis. AgSFl was localized in the web scaffolding junctions and our data supports a role as an adhesive silk protein that serves as a center for connecting scaffolding fibers that functions to reduce the tensile strength of scaffolding fibers, which facilitates capture of aerial insects.

Little is actually known about the transcriptional regulation of spider silk as most studies have focused on the material properties of silks. We isolated and mapped the TuSp1 core promoter from the black widow spider, Latrodectus hesperus. Using a genomic DNA walking strategy, we have isolated an upstream segment (581 bp) of genomic DNA containing the promoter as well as the first exon of the TuSp1 gene. This upstream regulatory element was able to initiate transcription in insect cells when placed upstream the promoterless firefly luciferase reporter gene. Initiation of transcription was orientation dependent, as insertion of this upstream regulatory module in the reverse orientation led to inefficient transcriptional initiation. Only 170 bp of upstream sequence was required for strong transcriptional initiation, showing that core promoter resides within the first 170 bp of upstream 5' -flanking DNA. We also demonstrate the bHLH factor SGSF1 can repress gene transcription of the TuSp1 core promoter, implying SGSF I might participate in the transcriptional regulation of the TuSp1 gene in vivo.

Belonging to the diverse order Araneae, the black widow spider Latrodectus 4 hesperus produces high-performance silks with a broad range ofbiological functions and mechanical properties. The cob weaver spider spins different fibers by using seven specialized glands located in its abdomen. Egg case silk originates from the tubuliforrn gland and to date, no proteins that participate in the assembly process of egg case silk proteins have been identified. The goal of this project was the expression, purification, and characterization of such protein products. De novo sequencing of peptides from in-solution tryptic digestion of black widow spider dragline silk, the most studied type of silk, identified a novel cysteine-rich nonfibroin- like peptide that we named cysteine-rich component or CRC- 1. Further analysis of a large pool of nucleic acid sequences deposited in our custom eDNA database revealed 4 additional sequences with similarities to each other at the amino acid level called CRC-2, CRC-3, CRC-4, and CRC-5, suggesting a new family of proteins. Specifically, Q-PCR analysis revealed that the CRC-5 mRNAs were predominantly expressed in the tubuliform and aggregate glands. Since the aggregate gland manufactures a more complex aqueous solution compared to the tubuliforrn gland, we focused these studies on the tubuliform gland and resultant egg case fibers. Westem blot analysis using a cross-reactive polyclonal anti-CRC-1 antiserum conoborated the presence of CRC-5 in the tubulifmm gland and egg case silk, supporting the colocalization ofTuSpl, a tubuliform gland-specific protein, and CRC-5. Thus, we have demonstrated that these two proteins are present within tubuliform silks. In vitro studies suggested that recombinant CRC-5 displayed enzymatic activity similar to a sulfhydryl oxidase. Collectively, our findings provide new insights into novel proteins that have a potential role in the silk assembly and extrusion pathway of egg case silk fibers.

Spider silk is a natural high-performance biopolymer with superior mechanical propetiies. Although these fibers out perfmm several man-made and natural biomaterials, there are cha llenges to be circumvented before commercialization. One of the silkproducing glands warranting further study is the pyrifonn gland, which produces gluelike threads functioning to cement dragline silk to substrates. We focused on the molecular properties of PySp 1, the major component of pyrifonn silk from Latrodectus hersperus, and its putative Oiiholog, PySp2, from Nephi/a clavipes. To date, there are no reports describing the secondary structure of PySp internal block repeats. Moreover, because the PySp C-terminus amino acid residues are distinct from MaSp C-terminus and the morphology of these glands is different, we hypothesized that PySp C-terminal domains form distinct secondary structures. The MaSp C-terminus has been shown to regulate the silk assembly process and whether the PySp C-terminus performs a similar function is unknown. In order to test this supposition, we used the following experimental approaches: I) we developed a series of PySp prokaryotic expression constructs carrying various block repeat modules representative of the internal iterations found within the protein chain; 2) we constructed prokaryotic expression vectors coding for the PySp C-terminal domains; 3) we expressed and purified the PySp C-terminal domains from bacteria; 4) we performed structural analyses of the purified PySp C-terminal domains using cd spectroscopy and atomic force microscopy. After expression and purification of the PySp C-tennini proteins, our studies support that this domain displays a predominantly ~-sheet structure, distinctive from the NMR-determined ahelical nature of MaSp C-tennini. The difference in secondary structure implies the MA and pyriform glands use different biochemical mechanisms during fiber extrusion to control protein folding and assembly. By investigating protein folding and fiber formation for different spider silk types, its characteristics can be customized for spinning different materials for industrial applications.

Spider attachrnentdisc silk fibers are spun into a viscous liquid that rapidly solidifies, gluing dragline silk fibers to substrates for locomotion or web construction. Here we report the identification and artificial spinning of a novel attachment disc glue silk fibroin, Pyriform Spidroin 2 (PySp2), from the golden orb weaver Nephila c/avipes. MS studies support PySp2 is a constituent of the pyriform gland that is spun into attachment discs. Analysis of the PySp2 protein architecture reveals sequence divergence relative to the other silk family members, including the cob weaver glue silk fibroin PySpl. PySp2 contains internal block repeats that consist of two sub-repeat units: one dominated by Ser, Gin and Ala, the other Pro-rich. Artificial spinning of recombinant PySp2 truncations shows that the Ser-Gln-Ala-rich sub-repeat is sufficient for the assembly of polymeric subunits and subsequent fiber formation. These studies support that both orb- and cob-weaving spiders have evolved highly polar block-repeat sequence with the ability to self-assemble into fibers, suggesting a strategy to allow fiber fabrication in the liquid environment of the attachment discs.

Araneoid spiders use specialized sets of abdominal silk glands to produce up to seven different types of silks, each with diverse functional properties. At the time of these studies, fibroin eDNA sequences that encode egg case silk had not been reported in the literature. This study used conventional nucleic acid-nucleic acid screening of a eDNA library to isolate a novel gene, named tubuliform spidroin 1 , from the black widow spider Latrodectus hesperus. TuSp 1 was demonstrated to be selectively expressed in the tubuliform gland (the gland suspected for egg case silk production), and examination of the amino acid sequence revealed highly homogeneous repeats (184 amino acid ensemble repeats), a characteristic feature of fibroin sequences. Analyses of the ensemble repeats within the amino acid sequence of TuSp 1 revealed the lack of long stretches of polyalanine and glycine-alanine sub-repeats, which are commonly found in minor ampullate and major ampullate silks. Polyserine blocks and short polyalanine stretches were highly represented in the TuSp 1 amino acid sequence. Our data support the assertion that TuSp 1 represents the main constituent within egg case silk. This supposition is supported by the observation that the amino acid composition of raw egg case silk was strikingly similar to the amino acid composition predicted from the translated TuSp1 eDNA. Two additional constituents identified in black widow egg case, egg case protein 1 (ECP-1) and egg case protein 2 (ECP-2), were also partially characterized in this study. Using immunohistochemical approaches, we demonstrate that ECPs predominantly localize to the exterior of the large diameter fibers of egg cases. Additionally, these studies revealed smaller amounts of ECPs localized to the interior portion of the fibers. Collectively, these results support TuSp1 as the predominant fibroin within egg sacs as well as reveal a structural role for the ECPs, providing clues regarding the supramolecular structure of egg case fibers.