Potato modified to synthesise fusion spider silk fragment/ elastin protein

Potatoes pCB301-Kan-SO1-100xELP produce a fusion protein consisting of the repetitive part of the spider silk protein MaSPI and an artificial elastin which is mainly observed in tubers. Female golden silk orbweaver spiders Nephila clavipes incorporate MaSpI protein within the very strong and elastic silk of the main threads of the spider net which is of high industrial interest. The artificial elastin 100xELP has high similarity to human elastin and consists of oligomeric repeats of the pentapeptide Val-Pro-Gly-Xaa-Gly (Xaa - any amino acid but proline). 100xELP exhibits a good ability to be solved in aqueous solution depending on salt concentration and temperature. It can undergo an inverse transition, i.e. the protein is soluble in an aqueous solution below its transition temperature but insoluble above this critical value. This characteristic is delegated to its fusion partner SO1 resulting in easy isolation and purification of spider silk protein from the biomass of potato tubers.

pCB301-Kan-SO1-100xELP (derivate of pCB301-Kan from pBIN19)

The introduced recombinant DNA includes two gene cassettes.

First, the fusion protein consisting of the synthetic spider silk protein SO1 and the elastin-like protein 100xELP carries the signal peptide LeB4 for translocation to the endoplasmic reticulum (ER) at the N-terminus and the KDEL signal peptide for retention in the ER at the C-terminus. The expression of this construct is controlled by the CaMV 35S promoter and -terminator.  The coding sequence SO1 represents the repetitive part of the spider silk protein MaSpI; approx. 180 bp of the MaSpI gene was removed form the 3´-end. The c-myc tag serves as immunochemical evidence of the fusion protein in Western blot analyses using antibodies.

Second, a nptII gene cassette consisting of the nos promoter, the nptII gene and nos terminator serves as a selection marker system via aminoglycosid antibiotics.