Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells

Abstract

Spider silks are protein-based “biopolymer” filaments or threads secreted by specialized epithelial cells as concentrated soluble precursors of highly repetitive primary sequences. Spider dragline silk is a flexible, lightweight fiber of extraordinary strength and toughness comparable to that of synthetic high-performance fibers. We sought to “biomimic” the process of spider silk production by expressing in mammalian cells the dragline silk genes ( ADF-3 / MaSpII and MaSpI ) of two spider species. We produced soluble recombinant (rc)–dragline silk proteins with molecular masses of 60 to 140 kilodaltons. We demonstrated the wet spinning of silk monofilaments spun from a concentrated aqueous solution of soluble rc–spider silk protein (ADF-3; 60 kilodaltons) under modest shear and coagulation conditions. The spun fibers were water insoluble with a fine diameter (10 to 40 micrometers) and exhibited toughness and modulus values comparable to those of native dragline silks but with lower tenacity. Dope solutions with rc–silk protein concentrations >20% and postspinning draw were necessary to achieve improved mechanical properties of the spun fibers. Fiber properties correlated with finer fiber diameter and increased birefringence.

Silk-based biomaterials

“…For example, the dragline spider silk from the golden orb weaver N. clavipes displays impressive toughness, and a balance of stiffness, strength and extensibility reflecting the native function of the silk orb web construction [7,13,14]. Transgenic expression of spider silks in plants (tobacco and potato) and mammalian epithelial cells has been reported [15,16] and may point the way toward more substantive production of these proteins in the future.…”

The hidden link between supercontraction and mechanical behavior of spider silks

“…Several studies have investigated spinning of artificial dopes from engineered spider silk proteins (Lazaris et al, 2002;Hardy et al, 2008;Rammensee et al, 2008). 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.…”

Production of renewable polymers from crop plants

“…Although determination of the level of accumulation of such proteins with an unusual amino acid composition is difficult, and is often based on semi-quantitative methods, the expression levels of silk proteins reported in plants are close to the level of 10% and 30% TSP reported in Escherichia coli and Pichia pastoris, respectively (Fahnestock and Bedzyk, 1997;Fahnestock and Irwin, 1997). Synthesis of recombinant silk protein has also been accomplished by secretion from cultured mammalian cells (Lazaris et al, 2002). Expression of collagen, of a synthetic protein made from repeats of a motif found in elastin, and of a chimeric protein made of silk and elastin domains, has also been reported in tobacco or potato (Guda et al, 2000;Ruggiero et al, 2000;Scheller et al, 2004).…”

“…In the spider or silkworm glands, silk proteins are maintained at a concentration of 30% without precipitation or aggregation, and are spun together via an elaborate liquid spinning process, which has not been reproduced in the laboratory (Huang et al, 2007). Recombinant spider silk obtained from mammalian cells has been spun into filaments that show similar toughness to dragline silk but with a lower tenacity (Lazaris et al, 2002). Thus, while processing of fibrous proteins is facilitated by the fact that proteins such as spider fibroins are extremely heat-stable and acid-soluble (Scheller et al, 2001(Scheller et al, , 2004, advances in micro-spinning and other processing technologies are essential to produce good fibers from fibrous proteins.…”