Transgenic Research

, Volume 14, Issue 3, pp 313–324 | Cite as

High yield recombinant silk-like protein production in transgenic plants through protein targeting

  • Jianjun Yang
  • Leslie A. Barr
  • Stephen R. Fahnestock
  • Zhan-Bin Liu


DP1B is a synthetic analogue of spider dragline silk protein. It can be spun to form silk fiber. Previously, it had been expressed in transgenic plants, showing the general feasibility of the plant-based DP1B production. However, success of such a plant-based platform requires a great increase of DP1B productivity in plant cells to reduce production cost. This report describes a protein targeting approach to accumulate DP1B in apoplast, ER lumen, and vacuole in Arabidopsis cells, by utilizing appropriate combinations of sporamin-targeting determinant peptides and ER retention peptide. The approach has dramatically enhanced DP1B accumulation, resulting in high production yield. The accumulation can be as high as 8.5 and 6.7% total soluble protein in leaf tissue by targeting to apoplast and ER lumen, respectively, or as high as 18 and 8.2% total soluble protein in seeds by targeting to ER lumen and vacuole, respectively. However, the vacuole targeting in leaves and the apoplast targeting in seeds have failed to accumulate full length DP1B molecules or any DP1B at all, respectively, suggesting that they may not be suitable for applications in leaf tissues and seeds. Data in this study recommend a combination of seed-specific expression and ER-targeting as one of the best strategies for yield enhancement of plant-based DP1B production.


arabidopsis protein accumulation protein targeting silk protein sporamin 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Arcidiacono, S, Mello, C, Kaplan, D, Cheley, S, Bayley, H 1998Purification and characterization of recombinant spider silk expressed in Escherichia coliAppl Microbiol Biotechnol493138PubMedGoogle Scholar
  2. Barr, LA, Fahnestock, SR, Yang, J 2004Production and purification of recombinant DP1B silk-like protein in plantsMol Breeding13345356Google Scholar
  3. Bechtold, N, Elli, J, Pelletier, G 1993In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plantsC R Acad Sci Paris, Life Sci31611941199Google Scholar
  4. Boevink, P, Martin, B, Oparka, K, Cruz, SS, Hawes, C 1999Transport of virally expressed green fluorescent protein through the secretory pathway in tobacco leaves is inhibited by cold shock and brefeldin APlanta208392400Google Scholar
  5. Caimi, PG, McCole, LM, Klein, TM, Kerr, PS 1996Fructan accumulation and sucrose metabolism in transgenic maize endosperm expressing a Bacillus amyloliquefaciens SacB genePlant Physiol110355363PubMedGoogle Scholar
  6. Cappello, J, Crissman, JW 1990The design and production of bioactive protein polymers for biomedical applicationPolymer Preprints31193194Google Scholar
  7. Cappello, J, Crissman, J, Dorman, M, Mikolajczak, M, Textor, G, Marquet, M, Ferrari, F 1990Genetic engineering of structural protein polymersBiotechnol Prog6198202PubMedGoogle Scholar
  8. Chrispeels, MJ 1991Sorting of proteins in the secretory systemAnnu Rev Plant Physiol Plant Mol Biol422153Google Scholar
  9. Conrad, U, Fiedler, U 1998Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activityPlant Mol Biol38101109PubMedGoogle Scholar
  10. Fahnestock, SR, Bedzyk, LA 1997Production of synthetic spider dragline silk protein in Pichia pastorisAppl Microbiol Biotechnol473339PubMedGoogle Scholar
  11. Fahnestock, SR, Irwin, SL 1997Synthetic spider dragline silk proteins and their production in Escherichia coliAppl Microbiol Biotechnol472332PubMedGoogle Scholar
  12. Fahnestock, SR, Yao, Z, bedzyk, LA 2000Microbial production of spider silk proteinJ Biotechnol74105119PubMedGoogle Scholar
  13. Hattori, T, Nakagawa, T, Maeshima, M, Nakamura, K, Asahi, T 1985Molecular cloning and nucleotide sequence of cDNA for sporamin, the major soluble protein of sweet potato tuberous rootsPlant Mol Biol5313320Google Scholar
  14. Herman, EM, Larkins, BA 1999Protein storage bodies and vacuolesPlant Cell11601613PubMedGoogle Scholar
  15. Kaplan, DL, Mello, CM, Arcidiacono, S, Fossey, S, Senecal, K, Muller, W 1997


    McGrath, KKaplan, D eds. Protein-Based MaterialBirkhauserBoston, USA104131
    Google Scholar
  16. Lazaris, A, Arcidiacono, S, Huang, Y, Zhou, J-F, Duguay, F, Chretien, N, Welsh, EA, Soares, JW, Karatzas, CN 2002Spider silk fibers spun from soluble recombinant silk produced in mammalian cellsScience295472476PubMedGoogle Scholar
  17. Lewis, RV, Hinman, M, Kothakota, S, Fournier, MJ 1996Expression and purification of a spider silk protein: a new strategy for producing repetitive proteinsProtein Expr Purif7400406PubMedGoogle Scholar
  18. Liu Z-B and Odell JT 1999. Specific gene activation by chimeric Gal4 transcription factors in stable transgenic plants. US patent No: US5968793.Google Scholar
  19. Maeshima, M, Sasaki, T, Asahi, T 1985Characterization of major proteins in sweet potato tuberous rootsPhytochemistry2418991902Google Scholar
  20. Matsuoka, K, Matsumoto, S, Hattori, T, Machida, Y, Nakamura, K 1990Vacuolar targeting and posttranslational processing of the precursor to the sweet potato tuberous root storage protein in heterologous plant cellsJ Biol Chem2651975019757PubMedGoogle Scholar
  21. Matsuoka, K, Nakamura, K 1991Propeptide of a precursor to a plant vacuolar protein required for vacuolar targetingProc Natl Acad Sci USA88834838PubMedGoogle Scholar
  22. Menassa, R, Zhu, H, Karatzas, C, Lazaris, A, Richman, A, Brandle, J 2004Spider dragline silk proteins in transgenic tobacco leaves: accumulation and field productionPlant Biotech J2431438Google Scholar
  23. Moloney, MM, Holbrook, LA 1997Subcellular targeting and purification of recombinant proteins in plant production systemsBiotechnol Genet Eng Rev14321336PubMedGoogle Scholar
  24. Murray, EE, Lotzer, J, Eberle, M 1989Codon usage in plant genesNucleic Acids Res17477498PubMedGoogle Scholar
  25. Prince, JT, McGrath, KP, DiGirolamo, CM, Kaplan, DL 1995Construction, cloning, and expression of synthetic genes encoding spider dragline silkBiochemistry341087910885PubMedGoogle Scholar
  26. Scheller, J, Guhrs, K-H, Grosse, F, Conrad, U 2001Production of spider silk proteins in tobacco and potatoNature Biotechnol19573577Google Scholar
  27. Scheller, J, Henggeller, D, Viviani, A, Conra, U 2004Purification of spider-elastin from transgenic plants and application for human chondrocyte proliferationTransgenic Res135157PubMedGoogle Scholar
  28. Shewry, PR, Napier, JA, Tatham, AS 1995Seed storage proteins: structures and biosynthesisPlant Cell7945956PubMedGoogle Scholar
  29. Yang, J, Fox, GC,Jr, Henry-Smith, TV 2003Intein-mediated assembly of a functional β-glucuronidase in transgenic plantsProc Natl Acad Sci USA10035133518PubMedGoogle Scholar
  30. Ziegler, MT, Thomas, SR, Danna, KJ 2000Accumulation of a thermostable endo-1,4-β-D-glucanase in the apoplast of Arabidopsis thaliana leavesMol Breeding63746Google Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Jianjun Yang
    • 1
  • Leslie A. Barr
    • 1
  • Stephen R. Fahnestock
    • 1
  • Zhan-Bin Liu
    • 2
  1. 1.Central Research and DevelopmentE.I. DuPont de Nemours & Co.WilmingtonUSA
  2. 2.Crop Genetics ResearchE.I. DuPont de Nemours & Co.WilmingtonUSA

Personalised recommendations