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Synthesis of a Functional Anti–Phytochrome Single–Chain Fv Protein in Transgenic Tobacco

Bio/Technology volume 10, pages 790–794 (1992)Cite this article

Abstract

We have expressed a synthetic gene that encodes an antigen–binding single–chain Fv protein (scFv) in transgenic tobacco plants. The scFv gene was created by polymerase chain reaction (PCR) amplification of the variable domain coding regions from a mouse monoclonal hybridoma cell line. The monoclonal cell line secretes an IgG1 antibody that binds to the plant regulatory photoreceptor protein, phytochrome. The cloned scFv gene was expressed initially in Escherichia coli and shown to produce a 28 kD, phytochrome–binding scFv protein. Transgenic tobacco plants expressing the scFv gene were also found to produce a functional scFv protein, and seeds from transgenic R1 progeny displayed aberrant phytochrome–dependent germination. The scFv from transgenic tobacco could be isolated, to near homogeneity, by a single phytochrome–Sepharose affinity chromatography step.

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References

  1. Plückthun, A. 1991. Antibody engineering: advances from the use of Escherichia coli expression systems. Bio/Technology 9: 545–551.

    Google Scholar 

  2. Bird, R.E., Hardman, K.D., Jacobson, J.W., Johnson, S., Kaufman, B.M., 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 

  3. Huston, J.S., Levinson, D., Mudgett-Hunter, M., Tai, M., Novotny, J., Margolies, M.N., Ridge, R.J., Bruccoleri, R.K., Haber, E., Crea, R. and Oppermann, H. 1988. Protein engineering of antibody binding sites: recovery of specific activity of an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. USA 85: 5879–5883.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Colcher, D., Bird, R., Roselli, M., Hardman, K.D., Johnson, S., Pope, S., Dodd, S.W., Pantoliano, M.W., Milenic, E. and Schlom, J. 1990. In vivo tumor targeting of a recombinant single-chain antigen-binding protein. J. Natl. Cancer Inst. 82: 1191–1197.

    Article  CAS  PubMed  Google Scholar 

  5. Winter, G. and Milstein, G. 1991. Man-made antibodies. Nature 349: 293–299.

    Article  CAS  PubMed  Google Scholar 

  6. Biocca, S., Neuberger, M.S. and Cattaneo, A. 1990. Expression and targeting of intracellular antibodies in mammalian cells. EMBO J. 9: 101–108.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Chaudhary, V.K., Queen, C., Junghans, R.P., Waldmann, T.A., FitzGerald, D.J. and Pastan, I. 1989. A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin. Nature 399: 394–397.

    Article  Google Scholar 

  8. Holdsworth, M.L. 1987. Characterisation of phytochrome using monoclonal antibodies. PhD thesis. University of Leicester, UK.

  9. Tabor, S. and Richardson, C.C. 1985. A bacteriophage 17 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. USA 82: 1074–1078.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Tang, Y., Hicks, J.B. and Hilvert, D. 1991. In vivo catalysis of a metabolically essential reaction by an antibody. Proc. Natl. Acad. Sci. USA 88: 8784–8786.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Werge, T.M., Biocca, S. and Cattaneo, A. 1990. Intracellular immunization. Cloning and intracellular expression of a monoclonal antibody to the p21ras protein. FEBS Lett. 274: 193–198.

    Article  CAS  PubMed  Google Scholar 

  12. Piccioli, P., Ruberti, F., Biocca, S., Di Luzio, A., Werge, T.M., Bradbury, A. and Cattaneo, A. 1991. Neuroantibodies: molecular cloning of a monoclonal antibody against substance P for expression in the central nervous system. Proc. Natl. Acad. Sci. USA 88: 5611–5615.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Carlson, J.D. 1988. A new means of inducibly inactivating a cellular protein. Mol. Cell. Biol. 8: 2638–2646.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hiatt, A., Cafferkey, R. and Bowdish, K. 1989. Production of antibodies in transgenic plants. Nature 342: 76–78.

    Article  CAS  PubMed  Google Scholar 

  15. Hein, M., Tang, Y., McLeod, D.A., Janda, K.D. and Hiatt, A. 1991. Evaluation of immunoglobulins from plant cells. Biotechnol. Prog. 7: 455–461.

    Article  CAS  PubMed  Google Scholar 

  16. Düring, K., Hippe, S., Kreuzaler, K. and Schell, J. 1990. Synthesis and self assembly of a functional antibody in transgenic Nicotiana tabacum. Plant Mol. Biol. 15: 281–293.

    Article  PubMed  Google Scholar 

  17. Photomorphogenesis in Plants. 1986. Kendrick, R. E. and Kronennberg, G. H. M. (Eds. ). Martinus Nijhoff, Dordrecht.

  18. Smith, H. and Whitelam, G.C. 1990. Phytochrome, a family of photoreceptors with multiple physiological roles. Plant Cell Environ. 13: 695–707.

    Article  CAS  Google Scholar 

  19. Holdsworth, M. 1972. Phytochrome and seed germination. New Phytol. 71: 105–110.

    Article  Google Scholar 

  20. Maniatis, T., Fritsch, E.F. and Sambrook, J. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

  21. Marston, F.A.O. 1987. The purification of eukaryotic polypeptides expressed in Escherichia coli, p 59–88. In: DNA Cloning: A Practical Approach, Vol. 3. Glover, D. M. (Ed.). IRL Press, Oxford, UK.

    Google Scholar 

  22. Oono, Y., Satomi, T. and Uchimiya, H. 1991. Agrobacterium rhizogenes latZ-rolC. gene expression in Escherichia coli: detection of the product in transgenic plants using RolC-specihc antibodies. Gene 104: 95–98.

    Article  CAS  PubMed  Google Scholar 

  23. Van Haute, K.H., Maes, S., Warren, G., Van Montagu, M. and Schell, J. 1983. Intergenic transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. EMBO J. 2: 411–418.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Horsch, R.B., Fry, J.E., Hoffmann, N.I., Eichholtz, D., Rogers, S.C. and Fraley, R.T. 1985. A simple and general method for transferring genes into plants. Science 227: 1229–1231.

    Article  CAS  Google Scholar 

  25. Verwoerd, T.G., Dekker, B.M.M. and Hoekema, A. 1989. A small-scale procedure for the rapid isolation of plant RNAs. Nucl. Acids Res. 17: 2362.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Feinberg, A.P. and Vogelstein, B. 1984. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137: 260–267.

    Google Scholar 

  27. Techniques in Photomorphogenesis. 1984. Smith, H. and Holmes, M. G. (Eds.). Academic Press, London.

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Author notes

  1. Meran Owen: Corresponding author.

Authors and Affiliations

  1. Department of Botany, University of Leicester, University Road, Leicester, LE1 7RH, U.K.

    Meran Owen, Atul Gandecha, Bill Cockburn & Garry Whitelam

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  1. Meran Owen
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  2. Atul Gandecha
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  3. Bill Cockburn
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  4. Garry Whitelam
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Owen, M., Gandecha, A., Cockburn, B. et al. Synthesis of a Functional Anti–Phytochrome Single–Chain Fv Protein in Transgenic Tobacco. Nat Biotechnol 10, 790–794 (1992). https://doi.org/10.1038/nbt0792-790

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  • DOI: https://doi.org/10.1038/nbt0792-790

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