Spider silk has extraordinary mechanical properties, displaying high tensile strength, elasticity, and toughness. Given the high performance of natural fibers, one of the long-term goals of the silk community is to manufacture large-scale synthetic spider silk. This process requires vast quantities of recombinant proteins for wet-spinning applications. Attempts to synthesize large amounts of native size recombinant spidroins in diverse cell types have been unsuccessful. In these studies, we design and express recombinant miniature black widow (Latrodectus hesperus) MaSp1 spidroins in bacteria that incorporate the NTD and CTD, along with varying numbers of codon-optimized internal block repeats. Following spidroin overexpression, we perform quantitative analysis of the bacterial proteome to identify proteins associated with spidroin synthesis. Nano-liquid chromatography with tandem mass spectrometry (nLC-MS/MS) reveals a list of molecular targets that are differentially expressed after enforced mini-spidroin production. This list included proteins involved in energy management, proteostasis, translation, cell wall biosynthesis and oxidative stress. Collectively, this study unveils new bacterial genes to target by genetic engineering to overcome bottlenecks that throttle spidroin overexpression in microorganisms.

eDNA microarray technology has generated a tremendous amount of interest among biologists because of its promise to monitor the entire genome on a single chip, thus enabling researchers to have a better picture of the interaction among thousands of genes simultaneously. In the current study, this technology was used to print over 3,000 unknown genes from various silk glands of the black widow spider to profile their expression patterns and to identify novel candidates. Spiders are remarkable creatures because of their ability to make different silks, each with a specific function. Some of these silks have amazing mechanical properties, comparable to those of the finest synthetic materials. Several silk genes have been cloned from various spiders over the last few years, and the contribution of each of those genes in silk production has been identified. However, the majority of cellular and biochemical processes involved in silk manufacture remain a mystery. In our research, we attempt to identify genes that might be involved in silk assembly, on a global scale and investigate more about those genes and their interplay with other key biological molecules involved in silk manufacture. Our study showed that silking spiders for a certain period of time resulted in down-regulation of two important silk genes, ECP-1 and ECP-2. Both these genes are key molecules implicated for their role in maintaining the egg case architecture in the black widow spider.,-and we believe that these genes are also directly or indirectly involved in the manufacture of dragline silk. Microarray analyses also enable the discovery of several other interesting molecules, two of which could be accessory proteins involved in silk formation. Furthermore, in a separate study we also characterized a novel silk glue protein with unique ensemble repeats. In conclusion, we believe that the findings of this study will indeed be significant to silk researchers and material scientists alike and it will enhance our knowledge in understanding the mystery behind silk production.

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.