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.
The vest and scarf were manufactured in cooperation 


Research Article

Heiner Niemann, Institute of Farm Animal Genetics, Germany

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.

Posted 5 October 2014

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Citation: Kuwana Y, Sezutsu H, Nakajima K-i, Tamada Y, Kojima K (2014) High-Toughness Silk Produced by a Transgenic Silkworm Expressing Spider (Araneus ventricosus) Dragline Silk Protein. PLoS ONE 9(8): e105325. doi:10.1371/journal.pone.0105325
Editor: Heiner Niemann, Institute of Farm Animal Genetics, Germany
Received: March 3, 2014; Accepted: July 20, 2014; Published: August 27, 2014
Copyright: © 2014 Kuwana et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: 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. ( und2009.htm). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.

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.

Weaving of a textile using transgenic silks
To investigate the possibilities of transgenic silk weaving, we made a vest and a scarf using transgenic silk from the cocoons of C515-SpA2. Eight pieces of 27-denier raw silk were plied. The twisted yarn was then degummed with a 0.1% alkaline enzyme for 1 h at 60°C. The degummed yarn was dyed by acid dyes (Kayanol Milling Blue GW and Inolar Fast Red MNW) for 20 min at room temperature. The dye temperature was then raised to 90°C over 60 min, and the temperature was held there for 90 min. Acetic acid (5%, based on the fiber weight) was also added to the dye, 10 min after the start of dyeing. At the end of the dyeing process, the yarn was washed with water and the degummed and dyed silks were knitted by a machine (SMS 330 TC4, Stoll Japan).

Results and Discussion
Overview of a transgenic silkworm expressing spider dragline protein Figure 1 provides an overview of our transgenic silkworm, which expresses spider dragline silk protein, and EGFP as a part of the fibroin. First, the partial sequence of the spider (Araneus ventricosus) dragline silk protein gene, SpA, was cloned, and the repeat sequence of SpA (2149 nts) was then subcloned between the N- and C-terminal domains of the Bombyx fibroin H-chain gene [26]. After generating the transgenic silkworm bearing this modified fibroin H-chain gene with piggyBac vector system, the transgenic silkworm expressed the modified protein, HC-SpA, in the silk gland. The HC-SpA protein was then dimerized with the fibroin L-chain, and assembled into an elementary silk unit in the silk gland of the silkworm. Finally, the transgenic silk, which included the spider dragline protein, was spun into a cocoon. Silk fibers were collected as a single fiber and as raw silk from each cocoon and then analyzed. As a control, an EGFP-expressing transgenic silkworm, instead of SpA, was also generated and used.

Cloning of spider dragline protein genes
We first created a cDNA library from the ampullate silk gland of the spider (Araneus ventricosus) and selected one cDNA that corresponded to the dragline silk gene. The cDNA selected corresponded to the longest sequence and was designated “SpA”, AB829892 (DDBJ); its sequence is shown in Figure S3. The SpA gene was 2513 bp long and included one partial open reading frame (ORF) of 803 amino acids and an 82-bp 3′ non-coding region, followed by an 18-nt poly(A) sequence. The ORF contained the highly repeated (18 copies) sequence of a polyalanine block, many “GPGXX” motifs apparent in ADF-3 [4], and a C-terminal non-repeated amino-acid sequence. Collectively, these features indicated that the cloned sequence had the typical characteristics of the 3′ terminal in spider dragline silk proteins, especially the Araneus diadematus fibroin 3 (ADF-3)-like protein [4].

Transgenic silkworms
We generated transgenic silkworms using a Japanese commercial silkworm strain, C515. Generally, the w1-pnd experimental strain of silkworm has been used for transgenesis. Since the experimental strains were generated without concern for silk production, the physical properties of the silk fibers could not be properly analyzed. The phenotype of C515 is unsuitable for transgenesis, but its silk is ideal for fiber analysis. We generated three transgenic silkworm strains in this study using C515; the transgenic efficiencies were 16.7% (C515-SpA1), 22.6% (C515-SpA2), and 20.0% (C515-EGFP), which were almost identical to other transgenic silkworms generated to date (average of all germline transformation efficiencies: 15.0±10.7% (s.d.)). The transgenes in each strain were detected by Southern hybridization (Fig. S4), which identified three insertions in each transgenic line. These results indicate that the C515 strain is a good host for transgenesis, and, in fact, many silkworm strains without w1-pnd phenotypes could potentially host transgenesis. These transgenic silkworm strains were maintained for over 15 generations by sib-mating and sequential analysis of transgenes. We obtained one race for each transgenic strain. The three strains specified above and the F1 cross of C515-SpA1 and C515-SpA2 were used for further analysis.
Figure 3 shows the cocoons of transgenic and parental C515 silkworms, along with w1-pnd. A cocoon of the C515-EGFP transgenic silkworm showed green fluorescence under fluorescence microscopy, while the other transgenic silkworm cocoons did not. Green fluorescence of C515-EGFP cocoons indicated that the EGFP protein formed its natural “beta-barrel structure” in the spun silk fiber. Hence, the HC-SpA protein expressed in the silk was also expected to be expressed in the silk fibroin and form its natural structure in the cocoons. Additionally, the weights of the C515-SpAs and C515-EGFP cocoon shells were almost the same as that of the C515 shell, indicating that the expression of the fusion protein did not affect the silk productivity of the transgenic silkworms. On the other hand, the cocoon of w1-pnd was about one-third the weight of the C515 cocoon. This result clearly indicates that the C515 strain is more suitable for silk production and that the silk from the C515 strain was more suitable for precise tensile analysis.

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.

Spider silk protein expression in a transgenic cocoon
To identify the expression of HC-SpA and HC-EGFP proteins in fibroin, SDS-PAGE analysis and Western blot analysis were carried out using degummed fibroin proteins (Figure 4A). All samples contained the fibroin L-chain protein (27 kDa), fhx/P25 protein (ca. 30 kDa), and fibroin H-chain protein (300 kDa). Additionally, specific proteins having a molecular weight around 100 kDa in were also present C515-SpA1 and C515-SpA2. These proteins were identified as the fusion protein HC-SpA, after detection by anti 6xHis-tag antiserum and anti-SpA-specific peptide antibodies (Figure 4 B). Also, the ca. 60-kDa protein detected in C515-EGFP silk was also identified as a fusion protein, HC-EGFP, by comparison with our previous work [20] and its detection using only anti 6xHis-tag antiserum (Figure 4 B).

Weaving using C515-SpA2 silk
Because cocoons from transgenic silkworms produced in this study could be pulled into raw silk, mass production of raw silk was performed with the intent of machine knitting a vest and scarf. Figure 6 shows pictures of the raw silk yarn and the resultant woven vest and scarf, using C515-SpA2 silk. The weakest silk among the HC-SpA expressing transgenic silkworms was used for these articles of clothing. That it could be used with a knitting machine clearly demonstrates the strength and toughness of our transgenic silk for reeling. Moreover, the fabric was also dyed and sewn by a machine in an industrial setting. This demonstrates the use of the transgenic silks produced in this study as a strong, high-toughness raw silk in the textile industry.

In this study, we cloned the spider (Araneus ventricosus) dragline protein gene, SpA, and generated a transgenic silkworm, which expressed the fusion protein of fibroin H-chain and SpA, HC-SpA, in cocoon silk. The raw silk prepared with transgenic cocoon-expressed spider-dragline silk protein showed improved breaking strain, breaking stress, and high toughness, nearly reaching that of spider dragline silk. We also demonstrated that resultant transgenic cocoons could be used directly in the textile industry.
Spider silk proteins can be used not only as fibers, but also as biomaterials for biomedical applications. In our group, silk fibroins have been used for regenerative medicine [31-34]. Silk containing the spider silk protein offers wide possibilities for biomedical applications, such as artificial tendons and ligaments, due to the ability to use transgenic technologies to control the tensile properties.
Yoshihiko Kuwana, Affiliation: Silk Materials Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan XHideki Sezutsu, Affiliation: Transgenic Silkworm Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan XKen-ichi Nakajima, Affiliation: Transgenic Silkworm Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan XYasushi Tamada, Affiliations: Silk Materials Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan, Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano, Japan XKatsura Kojima mail * E-mail: Silk Materials Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan X
Published: August 27, 2014

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