Purification of Spider Silk-elastin from Transgenic Plants and Application for Human Chondrocyte Proliferation

Abstract

Research on spider silk proteins has led to the possibility of designing genetically engineered silks according to defined material properties. Here we show the efficient and stable production of spider silk-elastin fusion proteins in transgenic tobacco and potato plants by retention in the ER. The proteins were purified by a simple method, using heat treatment and ‘inverse transition cycling’. Laboratory scale extraction of 1 kg tobacco leaf material leads to a yield of 80 mg pure recombinant spider silk-elastin protein. As a possible application, as well as to demonstrate biocompatibility, the growth of anchorage-dependent mammalian cells on spider silk-elastin coated culture plates was compared with conventional coatings such as collagen, fibronectin and poly-D-lysine. The anchorage-dependent chondrocytes showed similar growth behaviour and a rounded phenotype on collagen and on spider silk-elastin coated plates and the proliferation was remarkably superior to untreated polystyrene plates.

This is a preview of subscription content, access via your institution.

References

  1. Artsaenko O, Kettig B, Fiedler U, Conrad U and Düring K (1998) Potato tubers as a biofactory for recombinant antibodies. Mol Breeding 4: 313–319.

    Google Scholar 

  2. Betre H, Setton L A, Meyer DE and Chilkoti A (2002) Characterization of a genetically engineered elastin-like polypeptide for cartilaginous tissue repair. Biomacromolecules 3: 910–916.

    Google Scholar 

  3. Bevan M (1984) Binary Agrobacterium vectors for plant transformation.Nucl Acids Res 12: 8711–8721.

    Google Scholar 

  4. Conrad U, Fiedler U, Artsaenko O and Phillips J (1997) Singlechain Fv antibodies expressed in plants. In: Cunningham C and Porter S (eds.), Methods in Biotechnology-Recombinant Proteins from Plants: Production and Isolation of Clinically Useful Compounds. (pp. 103–127) Humana Press, Totowa.

    Google Scholar 

  5. Daniell H, Streatfield SJ and Wycoff K (2001) Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 6: 219–226.

    Google Scholar 

  6. Dunham BP and Koch RJ (1998) Basic fibroblast growth factor and insulinlike growth factor I support the growth of human septal chondrocytes in a serum-free environment. Arch Otolaryngol Head Neck Surg 124: 1325–1330.

    Google Scholar 

  7. Gosline JM, Guerette PA, Ortlepp CS and Savage KN (1999) The mechanical design of spider silks: from fibroin sequence to mechanical function J Exp Biol 202: 3295–3303.

    Google Scholar 

  8. Hayashi CY and Lewis RV (2000) Molecular architecture and evolution of a modular spider silk protein gene. Science 287: 1477–1479.

    Google Scholar 

  9. Hinman MB and Lewis RV (1992) Isolation of a clone encoding a second dragline silk fibroin. Nephila clavipes dragline silk is a two-protein fiber. J Biol Chem 267: 19320–19324.

    Google Scholar 

  10. Hinman MB, Jones AJ and Lewis RV (2000) Synthetic spider silk: a modular fiber Trends Biotechnol 18: 374–379.

    Google Scholar 

  11. Larrick JW and Thomas DW (2001) Producing proteins in transgenic plants and animals. Current Opinion Biotech 12: 411–418.

    Google Scholar 

  12. McPherson JM, Yaeger PC, Brown ME, Hanlon JG and Binette F (2000) Chondrocyte media formulations and culture procedures.United States Patent 6,150,163.

  13. Meyer DE and Chilkoti A (1999) Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat Biotech 17: 1112–1115.

    Google Scholar 

  14. Munro S and Pelham HR (1987) A C-terminal signal prevents secretion of luminal ER proteins. Cell 48: 899–907.

    Google Scholar 

  15. Richter LJ, Thanavala Y, Arntzen CJ and Mason HS (2001) Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nat Biotech 18: 1167–1171.

    Google Scholar 

  16. Rosenbloom J, Abrams WR and Mecham R (1993) Extracellular matrix 4: the elastic fiber. Faseb J 7: 1208–1218.

    Google Scholar 

  17. Scheller J, Gührs KH, Grosse F and Conrad U (2001) Production of spider silk proteins in tobacco and potato. Nat Biotech 19: 573–577.

    Google Scholar 

  18. Tirrell DA (1996) Putting a new spin on spider silk. Science 271: 39–40.

    Google Scholar 

  19. Urry DW (1988) Entropic elastic processes in protein mechanisms II. Simple (passive) and coupled (active) development of elastic forces. J Protein Chem 7: 1–34.

    Google Scholar 

  20. Vollrath F (2000) Strength and structure of spiders' silks. J Biotechnol 74: 67–83.

    Google Scholar 

  21. Vollrath F and Knight DP (2001) Liquid crystalline spinning of spider silk. Nature 410: 541–548.

    Google Scholar 

  22. Xiang C, Han P, Lutziger I, Wang K and Oliver DJ (1999) A mini binary vector series for plant transformation. Plant Mol Biol 40: 711–717.

    Google Scholar 

  23. Zambrinski P, Joos H, Gentello J, Leemans J, Van Montagu M and Schell J (1983) Ti-plasmid vector for introduction of DNA into plant cells without altering their normal regeneration capacity. EMBO J 2: 2143–2121.

    Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to Udo Conrad.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Scheller, J., Henggeler, D., Viviani, A. et al. Purification of Spider Silk-elastin from Transgenic Plants and Application for Human Chondrocyte Proliferation. Transgenic Res 13, 51–57 (2004). https://doi.org/10.1023/B:TRAG.0000017175.78809.7a

Download citation

  • chondrocytes
  • elastin
  • spider silk
  • transgenic tobacco