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. 2014 Aug 27;9(8):e105325.
doi: 10.1371/journal.pone.0105325. eCollection 2014.

High-toughness Silk Produced by a Transgenic Silkworm Expressing Spider (Araneus Ventricosus) Dragline Silk Protein

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Free PMC article

High-toughness Silk Produced by a Transgenic Silkworm Expressing Spider (Araneus Ventricosus) Dragline Silk Protein

Yoshihiko Kuwana et al. PLoS One. .
Free PMC article

Abstract

Spider dragline silk is a natural fiber that has excellent tensile properties; however, it is difficult to produce artificially as a long, strong fiber. Here, the spider (Araneus ventricosus) dragline protein gene was cloned and a transgenic silkworm was generated, that expressed the fusion protein of the fibroin heavy chain and spider dragline protein in cocoon silk. The spider silk protein content ranged from 0.37 to 0.61% w/w (1.4-2.4 mol%) native silkworm fibroin. Using a good silk-producing strain, C515, as the transgenic silkworm can make the raw silk from its cocoons for the first time. The tensile characteristics (toughness) of the raw silk improved by 53% after the introduction of spider dragline silk protein; the improvement depended on the quantity of the expressed spider dragline protein. To demonstrate the commercial feasibility for machine reeling, weaving, and sewing, we used the transgenic spider silk to weave a vest and scarf; this was the first application of spider silk fibers from transgenic silkworms.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Overview of the strategy used in this study.
We cloned a partial sequence spider (Araneus ventricosus) dragline silk. The spider dragline protein (SpA) gene or enhanced green fluorescent protein (EGFP) gene were fused between the N- and C-terminal domains of the fibroin H-chain protein gene and the transgenic silkworm expressing these modified proteins, respectively. The transgenic silkworms expressed spider dragline protein or EGFP as a part of the silk fibroin proteins. The elementary units, consisting of fib-H, fib-L, and fhx/P25, were secreted into the lumen of the silk gland, and the silk was spun into a cocoon. The single cocoon silk and reeled raw silk were then prepared and their tensile properties analyzed. The raw silk was woven into yarn by an industrial machine to demonstrate its suitability for commercial applications.
Figure 2
Figure 2. Dose dependence of tensile properties in C515-SpA silks (A) and representative stress–strain curves (B).
(A) Breaking stress (left) and toughness (right) are plotted as a function of the amount of HC-SpA protein (%) in total fibroin proteins. The amounts of HC-SpA proteins in total fibroin proteins, estimated by SDS-PAGE using densitometry, were 0.37 (±0.08 s.d.), 0.49 (±0.08 s.d.), and 0.61% (±0.07 s.d.) w/w in C515-SpA1, C515-SpA2, and C515-SpA1×2, respectively (n = 21). The p-values of a one-tailed t-test indicated significant differences in the amounts of each transgenic strain (p-values: * 3.1×10−6, ** 7.8×10−6, *** 2.4×10−13). The broken lines are the regression lines obtained by the least-squares method. The breaking stress and toughness were high for raw silk; the highest values were found for the C515-SpA1×2 silks, which contained the most HC-SpA protein. The breaking stress and toughness were proportional to the HC-SpA content for both cocoon fiber and raw silk. (B) Typical strain–stress curves with average breaking points are shown for each strain. Circles, rectangles, and diamonds show the average strain and stress values. Error bars correspond to the standard deviations. All values are shown in Tables S1–S3. There were clear differences between the cocoon fibers and the raw silks; cocoon fibers were more elastic with low breaking stress, while raw silks had low elasticity and high breaking stress. For the raw silks, the C515-EGFP silks showed high elongation and low breaking stress, compared with those of C515. C515-SpA1×2 silks showed the highest breaking stresses, elongation, and toughness (shaded area).
Figure 3
Figure 3. Comparison of the cocoons in a commercial (C515) and an experimental strain (w1-pnd).
Schematic diagrams show the expressed silk elementary units and average cocoon shell weights of the C515 and transgenic cocoons, along with the w1-pnd cocoon, which is used widely as a host for the transgenic silkworm. The C515 and transgenic cocoons had comparable shapes and weights, while the w1-pnd cocoon was small and light. Raw silk could not be obtained from w1-pnd cocoons, because it was difficult to reel from the cocoons. The C515-EGFP cocoon showed green fluorescence, derived from the expressed modified protein HC-EGFP, indicating that the expressed modified protein formed its native tertiary structure. The HC-SpA protein expressed in the silks also formed its native structure, and contributed to high tensile properties.
Figure 4
Figure 4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting assay of degummed silk proteins.
(A) The fibroin proteins of the cocoons of C515 and three transgenic silkworms prepared in this study were analyzed. The cocoons were urea-degummed, and the resultant fibroin fibers were dissolved in 9-M aqueous lithium bromide solution, dialyzed against deionized water, and applied to SDS-PAGE. Fibroin H-chain protein, fibroin L-chain proteins, fhx/P25, and the fusion proteins, HC-SpA and HC-EGFP, were observed. (B) Western blotting analysis of fibroin proteins was performed using anti-6xHis tag antiserum (left) and anti-SpA protein specific peptide antibody (right). Fibroin proteins, such as C515, were not detected by these antibodies. HC-SpA protein, which contains 6xHis-tag and SpA proteins, could be detected with anti-6xHis tag antiserum and anti-SpA peptide antibodies. HC-EGFP protein, which contains EGFP and 6xHis tag, could only be detected using anti 6xHis-tag antiserum. Small amounts of degraded protein, which were not identified by SDS-PAGE, were present.
Figure 5
Figure 5. Tensile properties of the cocoon fiber and raw silk.
The tensile properties are shown for cocoon silk (open box) and raw silk (filled box): breaking stress (A), breaking strain (B), Young's modulus (C), and toughness (D) for wild-type silk (C515), HC-SpA-expressed silk (C515-SpA1×2: F1 breed of C515-SpA1 and C515-SpA2), and HC-EGFP-expressed silk (C515-EGFP). The average values and their standard deviations (s.d.) are shown; detailed data and p-values are given in Tables S1–S3. For cocoon silk (open boxes), HC-SpA-containing cocoon silk (C515-SpA1×2) showed improvement in all properties, while C515-EGFP-containing silk showed only improved elongation. This indicated that expressed HC-SpA protein specifically improved the tensile properties of silk. This improvement was also evident, and more apparent, with raw silk as a consequence of the high speeds and high tension used in the reeling process. The breaking stress and breaking strain of C515-SpA1×2 raw silk improved by 13.5% and 42.5%, respectively, over that of C515 and resulted in an increase in toughness of 53.2%. In the HC-EGFP-containing raw silk, the breaking strain increased; however, the breaking stress decreased relative to C515. This means that the incorporation of HC-EGFP protein in silk weakens the raw silk and renders it more elastic. An asterisk “*” indicates a significant difference from C515 (i.e., p<0.01). The number of fiber specimens is shown in the breaking stress graph.
Figure 6
Figure 6. Raw silks and a woven vest and scarf knitted from transgenic spider silks.
(A) Reeled raw silks of C515, C515-SpA1, and C515-SpA2 are shown. (B) A vest and scarf made by a knitting machine using C515-SpA2 to demonstrate the commercial possibilities of transgenic spider silk. Cocoons of C515-SpA2 were reeled by a reeling machine, woven, dyed, and knitted.

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MeSH terms

Grant support

This Study was supported by MAFF Research projects of NIAS and by Research and Development Projects for Application in Promoting New Policy of Agriculture, Forestry, and Fisheries grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan. (http://www.s.affrc.go.jp/docs/research_fund2009.htm). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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