Spider dragline silk is a remarkably strong fiber that makes it attractive for numerous applications. Much has thus been done to make similar fibers by biomimic spinning of recombinant dragline silk proteins. However, success is limited in part due to the inability to successfully express native-sized recombinant silk proteins (250-320 kDa). Here we show that a 284.9 kDa recombinant protein of the spider Nephila clavipes is produced and spun into a fiber displaying mechanical properties comparable to those of the native silk. The native-sized protein, predominantly rich in glycine (44.9%), was favorably expressed in metabolically engineered Escherichia coli within which the glycyl-tRNA pool was elevated. We also found that the recombinant proteins of lower molecular weight versions yielded inferior fiber properties. The results provide insight into evolution of silk protein size related to mechanical performance, and also clarify why spinning lower molecular weight proteins does not recapitulate the properties of native fibers. Furthermore, the silk expression, purification, and spinning platform established here should be useful for sustainable production of natural quality dragline silk, potentially enabling broader applications.metabolic engineering | glycyl-tRNA | silk fiber | Nephila clavipes | spinning S pider dragline silk, used by spiders as the safety line and the web frame, is exceptionally strong and elastic; it is five times stronger by weight than steel, three times tougher than the top quality man-made fiber Kevlar (1, 2). The dragline silk is primarily composed of two proteins, the major ampullate spidroins 1 (MaSp1) and 2 (MaSp2) (3, 4). These spidroins are highly modular, each with a long repetitive sequence that is flanked on both sides by nonrepetitive amino-and carboxy-termini of approximately 100 amino acids (5). The repetitive sequence is rich in glycine and alanine, and characterized by stretches of alanine that are interrupted by glycine-rich repeats (4). The poly alanine regions form hydrophobic crystalline domains that are responsible for the high tensile strength, whereas the glycine-rich regions are hydrophilic and responsible for the links between crystalline domains as well as the elasticity of dragline fiber (6).Due to the unique mechanical properties, spider dragline silk has received much attention as a promising material for numerous industrial applications such as parachute cords, protective clothing, and composite materials in aircrafts. Also, many biomedical applications are envisioned due to its biocompatibility and biodegradability. For example, silk-based materials have been developed as sutures for wounds, coatings for biomedical implants, drug carriers for drug delivery, and scaffolds for cell culture and organ replacement (7-9). Unfortunately, natural spider dragline silk cannot be conveniently obtained by farming spiders because they are highly territorial and aggressive. Thus, many attempts have been made to produce recombinant dragline silk proteins (10-13) followed ...

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Silk fibroin/chitosan blend films were examined through IR spectroscopy to determine the conformational changes of silk fibroin. The effects of the fibroin/chitosan blend ratios (chitosan content) on the physical and mechanical properties were investigated to discover the feasibility of using these films as biomedical materials such as artificial skin and wound dressing. The mechanical properties of the blend films containing 10 -40% chitosan were found to be excellent. The tensile strength, breaking elongation, and Young's modulus were affected by the chitosan contents of the blend films, which were also related to the density and degree of swelling. The coefficient of water vapor permeability of the blend films increased linearly with the chitosan content, and the values of 1000 -2000 g m Ϫ2 day Ϫ1 were comparable to those of commercial wound dressings. Silk fibroin/chitosan blend films had good oxygen and water vapor permeabilities, making them useful as biomaterials. In particular, the blend film containing 40 -50% chitosan showed very high oxygen permeability.

Regenerated silk fibroin (RSF) was prepared by dissolving in a CaCl 2 / ethanol/H 2 O solvent system, freezing, and lyophilization. The effect of freezing temperature, alcohol addition, and molecular weight on the morphological and conformational changes were investigated through scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, circular dichroism spectroscopy, and differential scanning calorimetry analysis. However, the addition of a small amount of methanol induced the morphological change of RSF to a fine-particle aggregate, which resulted from the formation of a ␤-sheet crystalline structure. The lower the freezing temperature was, the more the formation of aggregates was favored, and the finer powder aggregates were formed. As the amount of added hydrophilic alcohol such as methanol and ethanol increased in the silk fibroin solution, a spherical powder form was changed to fine aggregates with the enhancement of thermal stability and crystallinity. On the other hand, RSFs prepared with a hydrophobic alcohol such as 1-butanol or 1-octanol showed a lump-like or sheet-like shape of morphology without any changes in conformational transition. It is concluded that the molecular weight of the silk fibroin and the type and amount of alcohol were determining factors in the morphological features of RSF, especially the size and shape of fibroin particles. A uniform ultrafine powder of RSF with a spherical form (ϳ 1 m) can be obtained when the molecular weight and the alcohol addition to the silk fibroin solution are controlled.