Skip to main content
SpringerLink
Log in

Apoplastic and cytosolic expression of full‐size antibodies and antibody fragments in Nicotiana tabacum

  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

We compared the expression of a functional recombinant TMV‐specific full‐size antibody (rAb29) in both the apoplast and cytosol of tobacco plants and a single chain antibody fragment (scFv29), derived from rAb29, was expressed in the cytosol. Cloned heavy and light chain cDNAs of full‐size rAb29, which binds to TMV coat protein monomers, were integrated into the plant expression vector pSS. The full‐size rAb29 was expressed in the cytosol and targeted to the apoplast by including the original murine antibody leader sequences. Levels of functional full‐size rAb29 expression were high in the apoplast (up to 8.5 μg per gram leaf tissue), whereas cytosolic expression was low or at the ELISA detection limit. Sequences of the variable domains of rAb29 light and heavy chain were used to generate the single chain antibody scFv29, which was expressed in the periplasmic space of E.coli and showed the same binding specificity as full‐size rAb29. In addition, scFv29 was functionally expressed in the cytosol of tobacco plants and plant derived scFv29 maintained same binding specificity to TMV‐coat protein monomers as rAb29.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Artsaenko O, Peisker M, zur Nieden U, Fiedler U, Weiler EW, Müntz K & Conrad U (1995) Expression of a single-chain Fv antibody against abscisic acid creates a wilty phenotype in transgenic tobacco. The Plant J. 8: 745-750.

    Google Scholar 

  • Benvenuto E, Ordas RJ, Tavazza R, Ancora G, Biocca S, Cattaneo A & Galeffi P (1991) 'Phytoantibodies': a general vector for the expression of immunoglobin domains in transgenic plants. Plant Mol. Biol. 17: 865-874.

    PubMed  Google Scholar 

  • Biocca S, Neuberger MS & Cattaneo A (1990) Expression and targeting of intracellular antibodies in mammalian cells. The EMBO J. 9: 101-108.

    Google Scholar 

  • Biocca S, Pierandrei-Amaldi P, Campioni N & Cattaneo A (1994) Intracellular immunization with cytosolic recombinant antibodies. Biotechnology 12: 396-399.

    PubMed  Google Scholar 

  • Biocca S, Ruberti F, Tafani M, Pierandrei-Amaldi P & Cattaneo A (1995) Redox state of single chain Fv fragments targeted to the endoplasmic reticulum, cytosol and mitochondria. Bio/Technology 13: 1110-1115.

    PubMed  Google Scholar 

  • Bird RE, Hardman KD, Jacobson JW, Johnson S, Kaufman BM, Lee SM, Lee T, Pope SH, Riordan GS & Whitlow M (1988) Singlechain antigen-binding proteins. Science 242: 423-426.

    PubMed  Google Scholar 

  • Bowdish K, Tang Y, Hicks JB & Hilvert D (1991) Yeast expression of a catalytic antibody with chorismate mutase activity. J Biol. Chem. 266: 11901-11908.

    PubMed  Google Scholar 

  • Carlson JR (1988) A new means of inducibly inactivating a cellular protein. Mol. Cell. Biol. 8: 2638-2646.

    PubMed  Google Scholar 

  • Cattaneo A & Biocca S (1997) Assembly and folding of antibodies in natural and artificial environments. In: Cattaneo A and Biocca S (eds.), Intracellular Antibodies: Development and Applications. Springer-Verlag Berlin Heidelberg and Landes Bioscience Georgetown, TX, USA.

  • Chomczynski P (1992) One-hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal. Biochem. 201: 134-139.

    PubMed  Google Scholar 

  • Clackson T, Hoogenboom HR, Griffiths AD & Winter G (1991) Making antibody fragments using phage display libraries. Nature 352: 624-628.

    PubMed  Google Scholar 

  • Collinge DB, Milligan DE, Dow JM, Scofield G & Daniels MJ (1987) Gene expression in Brassica campestris showing a hypersensitive response to the incompatible pathogen Xanthomonas campestris pv. vitians. Plant Mol. Biol. 8: 405-414.

    Google Scholar 

  • Davies J & Riechmann L (1996) Single antibody domains as small recognition units: design and in vitro antigen selection of camalized, human VH domains with improved protein stability. Protein Engineering 9: 531-537.

    PubMed  Google Scholar 

  • De Jaeger G, Buys E, Eeckhout D, De Wilde C, Jacobs A, Kapila J, Angenon G, Van Montagu, M, Gerats T & Depicker A (1999) High-level accumulation of single-chain variable fragments in the cytosol of transgenic Petunia hybrida. Eur. J. Biochem. 259: 426-435.

    PubMed  Google Scholar 

  • Fecker LF, Kaufmann A, Commandeur U, Commandeur J, Koenig R & Burgermeister W (1996) Expression of single-chain antibody fragments (scFv) specific for beet necrotic yellow vein virus coat protein or 25 kDa protein in Escherichia coli and Nicotiana benthamiana. Plant Mol. Biol. 32: 979-986.

    PubMed  Google Scholar 

  • Feinberg AP & Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13.

    Google Scholar 

  • Firek S, Draper J, Owen MRL, Gandecha A, Cockburn B & Whitelam GC (1993) Secretion of a functional single-chain Fv protein in transgenic tobacco plants and cell suspension cultures. Plant Mol. Biol. 23: 861-870.

    PubMed  Google Scholar 

  • Fischer R, Drossard J, Liao YC & Schillberg S (1998) Characterisation and applications of plant-derived recombinant antibodies. In: Cunningham C and Porter AJR (eds.): Methods in biotechnology Vol. 3: Recombinant proteins in plants: Production and Isolation of Clinically useful compounds. Humana press, Totowa, NJ.

    Google Scholar 

  • Franconi R, Tavladoraki P & Benvenuto E (1997) Plantibodies: Immunomodulation and Immunotherapeutic Potential. In: Cattaneo A, Biocca S (eds.): Intracellular Antibodies: Development and Applications. Springer-Verlag Berlin Heidelberg and Landes Bioscience Georgetown, TX, USA.

    Google Scholar 

  • Glockshuber R, Malia M, Pfitzinger I & Plückthun A (1990) A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry 29: 1362-1367.

    PubMed  Google Scholar 

  • Griffiths AD & Duncan AR (1998) Strategies for selection of antibodies by phage display. Curr. Opin. Biotechnol. 9: 102-108.

    PubMed  Google Scholar 

  • Hiatt A, Cafferkey R & Bowdish K (1989) Production of antibodies in transgenic plants. Nature 342: 76-78.

    Article  PubMed  Google Scholar 

  • Höfgen R & Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucl. Acids Res. 16: 9877.

    PubMed  Google Scholar 

  • Horsch RB, Fry JE, Hoffmann NL, Rogers SG & Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227: 1229-1231.

    Google Scholar 

  • Horton RM, Hunt HD, Ho SN, Pullen JK & Pease LR (1989) Engineering hybrid genes without the use of restriction enzymes: Gene splicing by overlap extension. Gene 77: 61-68.

    PubMed  Google Scholar 

  • Knappik A & Plückthun, A. (1995) Engineered turns of a recombinant antibody improve its in vivo folding. Protein Engineering 8: 81-89.

    PubMed  Google Scholar 

  • Koncz C & Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204: 383-396.

    Article  Google Scholar 

  • LaVallie ER, DiBlasio EA, Kovacic S, Grant KL, Schendel PF & McCoy M (1993) A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Bio/Technology 11: 187-193.

    PubMed  Google Scholar 

  • Ma JKC, Lehner T, Stabila P, Fux CI & Hiatt A (1994) Assembly of monoclonal antibodies with IgG1 and IgA heavy chain domains in transgenic tobacco plants. Eur. J. Immunol. 24: 131-138.

    PubMed  Google Scholar 

  • Malby RL, Caldwell JB, Gruen LC, Harley VR, Ivancic N, Kortt AA, Lilley GG, Power BE, Webster RG, Colman PM & Hudson PJ (1993) Recombinant antineuraminidase single chain antibody: expression, characterization, and crystallization in complex with antigen. Proteins 16: 57-63.

    PubMed  Google Scholar 

  • Marasco WA (1995) Intracellular antibodies (intrabodies) as research reagents and therapeutic molecules for gene therapy. Immunotechnology 1: 1-19.

    PubMed  Google Scholar 

  • Martineau P, Jones P & Winter G (1998) Expression of an antibody fragment at high levels in the bacterial cytoplasm. J. Mol. Biol. 280: 117-127.

    PubMed  Google Scholar 

  • Messing J (1983) New M13 vectors for cloning. Meth. Enzymol. 101: 20-78.

    PubMed  Google Scholar 

  • Milenic DE, Yokota T, Filpula DR, Finkelman MA, Dodd SW, Wood JF, Whitlow M, Snoy P & Schlom J (1991) Construction, binding properties, metabolism, and tumor targeting of a singlechain Fv derived from the pancarcinoma monoclonal antibody CC49. Cancer Res. 51: 6363-6371.

    PubMed  Google Scholar 

  • Owen M, Gandecha A, Cockburn B & Whitelam G (1992) Synthesis of a functional anti-phytochrome single-chain Fv protein in transgenic tobacco. Bio/Technology 10: 790-794.

    Article  PubMed  Google Scholar 

  • Patten PA, Howard RJ & Stemmer WPC (1997) Applications of DNA shuffling to pharmaceuticals and vaccines. Current Opinion Biotechnology 8: 724-733.

    Google Scholar 

  • Proba K, Wörn A, Honegger A & Plückthun A (1998) Antibody scFv fragments without disulfide bonds made by molecular evolution. J. Mol. Biol. 275: 245-253.

    PubMed  Google Scholar 

  • Sawyer JR & Blattner FR (1991) Rapid detection of antigen binding by antibody fragments expressed in the periplasm of Escherichia coli. Protein Engineering 4: 947-953.

    PubMed  Google Scholar 

  • Schouten A, Roosien J, de Boer JM, Wilmink A, Rosso MN, Bosch D, Stiekema WJ, Gommers FJ, Bakker J & Schots A (1997) Improving scFv antibody expression levels in the plant cytosol. FEBS L. 415: 235-241.

    Google Scholar 

  • Schouten A, Roosien J, van Engelen FA, de Jong GA, Borst-Vrenssen AW, Zilverentant JF, Bosch D, Stiekema WJ, Gommers FJ, Schots A & Bakker J (1996) The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco. Plant Mol. Biol. 30: 781-793.

    PubMed  Google Scholar 

  • Smith DB & Johnson KS (1988) Single-step purification of polypeptides expressed in Escherichia coli fusions with glutathione S-transferase. Gene 67: 31-40.

    Google Scholar 

  • Stieger M, Neuhaus G, Momma T, Schell J & Kreuzaler F (1991) Self assembly of immunoglobulins in the cytoplasm of the alga Acetabularia mediterranea Plant. Science 73: 181-190.

    Google Scholar 

  • Tang Y, Hicks JB & Hilvert D (1991) In vivo catalysis of a metabolically essential reaction by an antibody. Proc. Natl. Acad. Sci. 88: 8784-8786.

    PubMed  Google Scholar 

  • Tavladoraki P, Benvenuto E, Trinca S, De Martinis D & Galeffi P (1993) Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack. Nature 366: 469-472.

    Article  PubMed  Google Scholar 

  • Virca GD, NorthemannW, Shiels BR, Widera G & Broome S (1990) Simplified Northern blot hybridisation using 5% sodium dodecyl sulfate. Biotechniques 8: 370-371.

    PubMed  Google Scholar 

  • Voss A, Niersbach M, Hain R, Hirsch HJ, Liao YC, Kreuzaler F & Fischer R (1995) Reduced virus infectivity in N. tabacum secreting a TMV-specific full-size antibody. Molecular Breeding 1: 39-50.

    Google Scholar 

  • Ward ES, Güssow D, Griffiths AD, Jones PT & Winter G (1989) Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 341: 544-546.

    PubMed  Google Scholar 

  • Willoughby EW & Lambert A (1983) A sensitive silver stain for proteins in agarose gels. Anal. Biochem. 130: 353-358.

    PubMed  Google Scholar 

  • Zimmermann S, Schillberg S, Liao YC & Fischer R (1998) Intracellular expression of a TMV-specific single chain Fv fragment leads to improved virus resistance in Nicotiana tabacum. Molecular Breeding 4: 369-379.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Auf dem Aberg 1, D‐57392, Schmallenberg, Germany

    Stefan Schillberg

  2. Institut für Biologie I (Botanik/Molekulargenetik), RWTH Aachen, Worringerweg 1, D‐52074, Aachen, Germany

    Sabine Zimmermann & Andreas Voss

  3. Fraunhofer Abteilung für Molekulare Biotechnologie, IUCT, Grafschaft, Auf dem Aberg 1, D‐57392, Schmallenberg, Germany

    Rainer Fischer

Authors
  1. Stefan Schillberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sabine Zimmermann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andreas Voss
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rainer Fischer
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schillberg, S., Zimmermann, S., Voss, A. et al. Apoplastic and cytosolic expression of full‐size antibodies and antibody fragments in Nicotiana tabacum. Transgenic Res 8, 255–263 (1999). https://doi.org/10.1023/A:1008937011213

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1008937011213

Search

Navigation