Blueprint for a High-Performance Biomaterial: Full-Length Spider Dragline Silk Genes

Abstract

Spider dragline (major ampullate) silk outperforms virtually all other natural and manmade materials in terms of tensile strength and toughness. For this reason, the mass-production of artificial spider silks through transgenic technologies has been a major goal of biomimetics research. Although all known arthropod silk proteins are extremely large (>200 kiloDaltons), recombinant spider silks have been designed from short and incomplete cDNAs, the only available sequences. Here we describe the first full-length spider silk gene sequences and their flanking regions. These genes encode the MaSp1 and MaSp2 proteins that compose the black widow's high-performance dragline silk. Each gene includes a single enormous exon (>9000 base pairs) that translates into a highly repetitive polypeptide. Patterns of variation among sequence repeats at the amino acid and nucleotide levels indicate that the interaction of selection, intergenic recombination, and intragenic recombination governs the evolution of these highly unusual, modular proteins. Phylogenetic footprinting revealed putative regulatory elements in non-coding flanking sequences. Conservation of both upstream and downstream flanking sequences was especially striking between the two paralogous black widow major ampullate silk genes. Because these genes are co-expressed within the same silk gland, there may have been selection for similarity in regulatory regions. Our new data provide complete templates for synthesis of recombinant silk proteins that significantly improve the degree to which artificial silks mimic natural spider dragline fibers.

The hidden link between supercontraction and mechanical behavior of spider silks

“…Although very few silk genes have been completely cloned (Xia et al, 2004;Ayoub et al, 2007), due to their large size (Xu and Lewis, 1990), the existence of a small number of simple motifs of sequence extensively repeated (Gatesy et al, 2001) has led to the synthesis of artificial analogs that are believed to capture the essential features of the natural proteins, though so far no process has resulted in silk fibers that perfectly mimic the mechanical properties of natural silks.…”

In VitroEvaluation of a Novel Non-Mulberry Silk Scaffold for Use in Tendon Regeneration

“…25,26 Recently, the fibroin fibers of the B. mori mulberry silkworm have been assessed for use in tendon/ ligament regeneration. 28,29,[35][36][37][38]60,61 These silk fibers have also been used as sutures since the late 19 th century 62 and when implanted subcutaneously, B. mori silk fibroin elicits very mild immune responses. 63,64 Indeed, the majority of silk based tissue engineered constructs utilize the fibroin from B. mori, most likely due to its high availability and good mechanical properties.…”

“…Although the chemical composition of silk is thought to vary to match the functional requirement of each species, it largely consists of two main structural proteins, fibroin and sericin. [29][30][31] There is much ongoing research into the effects of various combinations of silk source, processing techniques, structure, and topography, with each combination significantly altering the silk properties and its biocompatibility. 26 However, it is generally believed that the glue-like sericin component is immunogenic, and once the silk has been degummed to remove the sericin, the remaining silk fibroin is highly biocompatible.…”

“…[32][33][34] Studies evaluating silk as a biomaterial for tendon regeneration have largely focused on the mulberry B. mori silk or composites of B. mori silk with either synthetic polymers or collagen. 28,29,[35][36][37][38] However, given that non-mulberry silks contain the cell binding RGD tripeptide motif, [39][40][41] and have been shown to support fibroblast-like and bone marrow-derived mesenchymal stem cell growth in vitro, 42,43 non-mulberry silk-derived scaffolds hold much promise as biomaterials for enhancing tendon tissue regeneration. 44 In this study, in vitro assays were used to assess the cytocompatibility and immunogenicity of a novel knitted, nonmulberry silk fibroin scaffold designed for use in tendon tissue regeneration.…”

The role of terminal domains during storage and assembly of spider silk proteins

“…The large core domain consists of ensemble repeats, each ensemble consisting of 40-200 amino acids ( Figure 2A) which may be repeated up to 100 times in some cases. [15][16][17][18] Some ensembles (in MaSp, MiSp, and FlagSp) comprise distinct amino acid motifs, like b-sheet-crystal forming polyalanine stretches, but the type and arrangement of those small motifs differ significantly between silk types. Therefore, the sequence similarity in the ensemble repeats can be quite low between different silk types.…”