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A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning

Abstract

The extreme strength and elasticity of spider silks originate from the modular nature of their repetitive proteins. To exploit such materials and mimic spider silks, comprehensive strategies to produce and spin recombinant fibrous proteins are necessary. This protocol describes silk gene design and cloning, protein expression in bacteria, recombinant protein purification and fiber formation. With an improved gene construction and cloning scheme, this technique is adaptable for the production of any repetitive fibrous proteins, and ensures the exact reproduction of native repeat sequences, analogs or chimeric versions. The proteins are solubilized in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 25–30% (wt/vol) for extrusion into fibers. This protocol, routinely used to spin single micrometer-size fibers from several recombinant silk-like proteins from different spider species, is a powerful tool to generate protein libraries with corresponding fibers for structure–function relationship investigations in protein-based biomaterials. This protocol may be completed in 40 d.

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Figure 1: The molecular structure of orb-weaver spiders' silk proteins.
Figure 2: Strategy to build large synthetic spider silk-like tandem repeat sequences from small double-stranded monomer DNAs flanked by compatible but nonregenerable restriction sites.
Figure 3: Strategy to clone the engineered synthetic silk-like sequences in the pET-19b expression vector.
Figure 4: Agarose gel analyses showing the synthetic Flag/MaSp 2 silk DNA multimers at different doubling stages.
Figure 5: Synthetic silk fiber formation by extrusion.
Figure 6: Western blot analysis showing the IMAC purification steps of a chimeric Flag/MaSp 2 recombinant protein.
Figure 7: Native and synthetic spider silk fibers.

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Acknowledgements

These studies were funded by NSF, NIH and DOD grants awarded to the University of Wyoming and CNPQ grants awarded to the Brazilian Agricultural Research Corporation.

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Correspondence to Elibio L Rech or Randolph V Lewis.

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Teulé, F., Cooper, A., Furin, W. et al. A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning. Nat Protoc 4, 341–355 (2009). https://doi.org/10.1038/nprot.2008.250

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