Scientists report on the development of engineered silkworms that express a synthetic spider silk protein and stably produce chimeric silk fibers that are stronger than parental silkworm silk fibers and as tough as spider dragline silk. The team, from the University of Wyoming, Laramie, Zhejiang University in China, and the University of Notre Dame in Illinois’s Eck Institute for Global Health, suggest their approach could provide the foundation for an effective biotechnological approach for producing spider silk fibers on a large scale for a range of biomedical applications.
The University of Wyoming’s Donald L. Jarvis, Ph.D., and colleagues, report their work in PNAS, in a paper titled “Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties.”
Farmed silkworms are the current source of silk used in sutures, and silk also has major potential as a biomaterial for applications in wound dressings, artificial ligaments, tendons, tissue scaffolds, and microcapsules, the authors write. However, while spider silk is much stronger than silkworm silk and has properties that make it ideal for a range of uses, spider territorialism and cannibalism means they can’t be farmed.
To try and get around this problem recombinant spider silk proteins have been produced in hosts such as bacteria, yeast, baculovirus/insect systems, mammalian cells, and transgenic plants and animals. However, the team continues, these approaches are expensive to scale up, result in low yields, and aren’t equipped naturally to spin silk fibers.
An alternative approach would be to use silkworms as hosts for spider silk production. Theoretically they represent the perfect host for spider silk because transgenic silkworms can be produced using piggyBac vectors. Recombinant protein production can be targeted to the silk gland using tissue-specific promoters, and the silk gland naturally produces silk fibers.
In their attempt to generate transgenic silkworms that produced spider-silk proteins in their silk, the team designed a piggyBac vector with specific features. The vector encoded the synthetic spider silk protein A2S814, which exhibits both elastic and strength motifs, along with the silkmoth Bombyx mori fibroin heavy chain (fhc) protein, to target spider silk protein production specifically to the silkworm’s posterior silk gland, and an fhc enhancer to increase expression levels. The synthetic spider silk sequence was flanked by the N- and C-terminal domains of the B. mori fhc protein. Two versions of the overall construct were generated, one of which included an enhanced GFP (EGFP) tag.
Following initial ex vivo tests to confirm that the chimeric fusion protein was expressed in posterior silk glands, the piggyBac vector was mixed with a plasmid encoding the piggyBac transposase and injected into eggs isolated from a strain of the B. mori silk moth that has a melaninization deficiency to facilitate detection of the EGFP-tagged chimeric silkworm/spider silk protein in transformed larvae.
Resulting transgenic silkworm larva were designated spider 6, or spider 6-GFP, dependent on whether they carried the EGFP tag or not. Visual and fluorescence microscopic examination showed that the spider 6-GFP transformants exhibited EGFP fluorescence in cocoons and silk glands, and a proportion of the silk fibers appeared to contain integrated EGFP signals.
Further analayses of silk extracted from the cocoons confirmed that at least some of the chimeric silkworm/spidersilk proteins expressed by the transgenic larvae were stably incorporated into the composite silk fibers. Importantly, the team states, although the mechanical properties of the composite silks was variable, fibers containing either EGFP-tagged or untagged chimeric silkworm/spider silk proteins were generally much tougher than parental silkworm fibers and as tough as native dragline spider silk fibers tested under the same conditions.
The composite fibers were also on average stronger than the parental fibers (though not as strong as native dragline spider silk) and more extensible than parental silkworm and native dragline spider silk. “Thus, these results demonstrate that the incorporation of chimeric silkworm/spider silk proteins can significantly improve the mechanical properties of composite silk fibers produced using the transgenic silkworm platform.”