Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research
  • Published:

High-Level Production and Long-Term Storage of Engineered Antibodies in Transgenic Tobacco Seeds

Abstract

We have used transgenic tobacco seeds to produce large amounts of a functionally active engineered antibody. A gene fusion encoding an antigen-binding single chain Fv protein (scFv) that recognizes the hapten oxazolone was constructed and used as a model. After characterization in a bacterial expression system, the scFv gene was cloned into a plant expression cassette conferring seed specific expression, and transferred using Agrobacterium-mediated transformation, into Nicotiana tabacum. The expressed scFv could be detected in developing as well as ripe seeds of regenerated transgenic plants, and the functionally active scFv is stably deposited and accumulates up to 0.67% of the total soluble seed protein. After storage of ripe transgenic tobacco seeds for one year at room temperature there was no loss of scFv protein or its antigen-binding activity.

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

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Bird, K.E., Hardman, K.D., Jacobsen, I.W., Johnsen, S., Kaufman, B., Lee, S.M., Lee, T., Pope, S.H., Riordan, G.S. and Whitlow, M. 1988. Single-chain antigen-binding proteins. Science 242: 423–426.

    Article  CAS  PubMed  Google Scholar 

  2. Whitelam, G.C., Cockburn, B., Gandecha, A.R. and Owen, M.R.L. 1993. Heterologous protein production in transgenic plants. Biotechnology and Genetic Engineering Reviews 11: 1–29.

    Article  CAS  PubMed  Google Scholar 

  3. Tai, M.-S., Mudgett-Hunter, M., Levinson, D., Wu, G.-M., Haber, E., Oppermann, H. and Huston, J.S. 1990. A bifunctional fusion protein containing Fc-binding fragment B of Staphylococcal protein A amino terminal to antidigoxin single-chain Fv. Biochemistry 29: 8024–8030.

    Article  CAS  PubMed  Google Scholar 

  4. Gandecha, A., Owen, M.R.L., Cockburn, B. and Whitelam, G.C. 1994. Antigen detection using recombinant, bifunctional single-chain Fv fusion proteins synthesised in Escherichia coli. Prot. Expression and Purification 5: 385–390.

    Article  CAS  Google Scholar 

  5. Brinkmann, U., Pai, L.H., FitzGerald, D.J., Willingham, M. and Pastan, I. 1991. B3(Fv)-PE38KDEL, a single-chain immunotoxin that causes complete regression of a human carcinoma in mice. Proc. Natl. Acad. Sci. USA 88: 8616–8620.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Yokota, T., Milenic, D.E., Whitlow, M. and Schlom, J. 1992. Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms. Cancer Research 52: 3402–3408.

    CAS  PubMed  Google Scholar 

  7. Wels, W., Harwerth, I.-M., Mueller, M., Groner, B. and Hynes, N.E. 1992. Selective inhibition of tumor cell growth by a recombinant single-chain antibody-toxin specific for the erbB2-receptor. Cancer Research 52: 6310–6317.

    CAS  PubMed  Google Scholar 

  8. Haber, E. 1992. Engineered antibodies as pharmacological tools. Immunological Reviews 130: 189–212.

    Article  CAS  PubMed  Google Scholar 

  9. Artsaenko, O., Peisker, M., zur Nieden, U., Fiedler, U., Weiler, E.W., Müntz, K. and Conrad, U. 1995. Expression of a single-chain Fv antibody against abscisic acid creates a wilty phenotype in transgenic tobacco. Plant. J. Submitted.

  10. Tavladoraki, P., Benvenuto, E., Trinca, S., De Martinis, D., Cattaneo, A. and Galeffi, P. 1993. Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack. Nature 366: 469–472.

    Article  CAS  PubMed  Google Scholar 

  11. Hughes, J. and Qoronfleh, W. 1991. Perspectives in plant genetic engineering and biopharmacy. BioPharm 4: 18–26.

    Google Scholar 

  12. Krebbers, E., Bosch, D. and Vandekerckhove, J. 1992. Prospects and progress in the production of foreign proteins and peptides in plants, p. 315–325. In: Plant Protein Engineering. Shewry, P. R. and Gutteridge, S. (Eds.). University Press, Cambridge.

    Google Scholar 

  13. Vandekerckhove, J., Van Damme, J., Van Lijsebettens, M., Botterman, J., De Block, M., Vandewiele, M., De Clercq, A., Leemans, J., Van Montagu, M. and Krebbers, E. 1989. Enkephalins produced in transgenic plants using modified 2S seed storage proteins. Bio/Technology 7: 929–932.

    CAS  Google Scholar 

  14. Pen, J. and Sijmons, P.C. 1993. Protein production in transgenic crops: analysis of plant molecular farming, p. 238–251. In: Transgenic plants—fundamentals and applications. Hiatt, A. (Ed.). Dekker, New York.

    Google Scholar 

  15. Berek, C. and Milstein, C. 1988. The dynamic nature of the antibody repertoire. Immunological Reviews 105: 5–26.

    Article  CAS  PubMed  Google Scholar 

  16. Chrispeels, M.J. 1991. Sorting of proteins in the secretory system. Ann. Rev. Plant Physiol. Plant Mol. Biol. 42: 35–49.

    Article  Google Scholar 

  17. Bednarek, S.Y. and Raikhel, N.V. 1992. Intracellular trafficking of secretory proteins. Plant Mol. Biol. 20: 133–150.

    Article  CAS  PubMed  Google Scholar 

  18. Clackson, T., Hoogenboom, H.R., Griffith, A.D. and Winter, G. 1991. Making antibody fragments using phage display libraries. Nature 352: 624–628.

    Article  CAS  PubMed  Google Scholar 

  19. Plückthun, A. 1991. Antibody engineering: Advances from the use of E. coli expression systems. Bio/Technology 9: 545–551.

    Google Scholar 

  20. Skerra, A. 1993. Bacterial expression of immunoglobulin fragments. Current Opinion in Immunology 2: 250–262.

    Google Scholar 

  21. Hoogenboom, H.R. and Winter, G. 1992. Bypassing immunization: human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J. Mol. Biol. 227: 381–388.

    Article  CAS  PubMed  Google Scholar 

  22. Munroe, S. and Pelham, H. 1986. An hsp 70-like protein in the E.R.: Identity with the 78 kD glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell 46: 291–300.

    Article  Google Scholar 

  23. Hochuli, E., Döbeli, H. and Schacher, A. 1987. New metal chelate adsorbents selective for proteins and peptide containing neighbouring histidine residues. J. Chromatography 411: 177–184.

    Article  CAS  Google Scholar 

  24. Bäumlein, H., Boerjan, W., Nagy, I., Panitz, R., Inze, D. and Wobus, U. 1991. Upstream sequences regulating legumin gene expression in heterologous transgenic plants. Mol. Gen. Genet. 225: 121–128.

    PubMed  Google Scholar 

  25. Kozak, M. 1984. Compilation and analysis of sequences upstream from the translational start site in eucaryotic mRNA's. Nucleic Acid Res. 12: 857–860.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Saito, K., Kaneko, H., Yamazaki, M., Yoshida, M. and Murakoshi, I. 1990. Stable transfer and expression of chimeric genes in licorice (Glycyrrhiza uralensis) using an Ri plasmid binary vector. Plant Cell Reports 8: 718–721.

    Article  CAS  PubMed  Google Scholar 

  27. Zambryski, 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: 2143–2150.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Conrad, U. and Fiedler, U. 1994. Expression of engineered antibodies in plant cells. Plant Mol. Biol. 26: 1023–1030.

    Article  CAS  PubMed  Google Scholar 

  29. Ma, J.K.-C., Hiatt, A., Hein, M., Vine, N.D., Wang, F., Stabila, P., van Dolleweerd, C., Mostov, K. and Lehner, T. 1995. Generation and assembly of secretory antibodies in plants. Science 268: 716–719.

    Article  CAS  PubMed  Google Scholar 

  30. Töpfer, R., Maas, C., Höricke-Grandpierre, C., Schell, J. and Steinbiss, H.-H. 1993. Expression vectors for high-level expression in dicotyledonous and monocotyledonous plants. Methods in Enzymol. 217: 66–78.

    Article  Google Scholar 

  31. Heim, U., Weber, H., Bäumlein, H. and Wobus, U. 1993. A sucrose-synthase gene of Vicia faba 1: expression pattern in developing seeds in relation to starch synthesis and metabolic regulation. Planta 191: 394–401.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fiedler, U., Conrad, U. High-Level Production and Long-Term Storage of Engineered Antibodies in Transgenic Tobacco Seeds. Nat Biotechnol 13, 1090–1093 (1995). https://doi.org/10.1038/nbt1095-1090

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1095-1090

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing