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Expression of recombinant antibody (single chain antibody fragment) in transgenic plant Nicotiana tabacum cv. Xanthi

Abstract

Plants offer an alternative inexpensive and convenient technology for large scale production of recombinant proteins especially recombinant antibodies (plantibodies). In this paper, we describe the expression of a model single chain antibody fragment (B6scFv) in transgenic tobacco. Four different gene constructs of B6scFv with different target signals for expression in different compartments of a tobacco plant cell with and without endoplasmic reticulum (ER) retention signal were used. Agrobacterium mediated plant transformation of B6scFv gene was performed with tobacco leaf explants and the gene in regenerated plants was detected using histochemical GUS assay and PCR. The expression of B6scFv gene was detected by western blotting and the recombinant protein was purified from putative transgenic tobacco plants using metal affinity chromatography. The expression level of recombinant protein was determined by indirect enzyme-linked immunosorbent assay. The highest accumulation of protein was found up to 3.28 % of the total soluble protein (TSP) in plants expressing B6scFv 1003 targeted to the ER, and subsequently expression of 2.9 % of TSP in plants expressing B6scFv 1004 (with target to apoplast with ER retention signal). In contrast, lower expression of 0.78 and 0.58 % of TSP was found in plants expressing antibody fragment in cytosol and apoplast, without ER retention signal. The described method/system could be used in the future for diverse applications including expression of other recombinant molecules in plants for immunomodulation, obtaining pathogen resistance against plant pathogens, altering metabolic pathways and also for the expression of different antibodies of therapeutic and diagnostic uses.

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Abbreviations

ELISA:

Enzyme-linked immunosorbent assay

ER:

Endoplasmic reticulum

GUS:

Beta-glucuronidase

HIV:

Human immunodeficiency virus

scFv:

Single chain variable fragment

rAb:

Recombinant antibody

RBC:

Red blood cells

TSP:

Total soluble protein

YEM:

Yeast extract mannitol

References

  1. 1.

    Giddings G, Allison G, Brooks D, Carter A (2000) Transgenic plants as factories for biopharmaceuticals. Nat Biotechnol 18:1151–1155

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    Goddijn OJM, Pen J (1995) Plants as bioreactors. Trends Biotechnol 13:379–387

    Article  CAS  Google Scholar 

  3. 3.

    Hood EE, Jilka JM (1999) Plant-based production of xenogenic proteins. Curr Opin Biotechnol 10:382–386

    PubMed  Article  CAS  Google Scholar 

  4. 4.

    Moffat AS (1995) Exploring transgenic plants as a new vaccine source. Science 268:658–660

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    Ma JKC, Drake PM, Christou P (2003) The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet 4:794–805

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Pujol M, Ram′ırez NI, Ayala M, Gavilondo JV, Vald′es R, Rodr′ıguez M, Brito J, Padilla S, G′omez L, Reyes B, Peral R, P′erez M, Marcelo JL, Mil′a L, S′anchez RF, P′aez R, Cremata JA, Enr′ıquez G, Mendoza O, Ortega M, Borroto C (2005) An integral approach towards a practical application for a plant-made monoclonal antibody in vaccine purification. Vaccine 23:1833–1837

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Semenyuk EG, Stremovskiy DA, Edelweiss EF, Shirshikova OV, Balandin TG, Buryanov YI, Deyev SM (2007) Expression of single chain antibody-barstar fusion in plants. Biochemie 89(1):31–38

    Article  CAS  Google Scholar 

  8. 8.

    Nejad SM, Michael McLean D, Hirama T, Kurt C, Almquist C, MacKenzie R, Christopher Hall J (2005) Transgenic tobacco plants expressing a dimeric single-chain variable fragment (scFv) antibody against Salmonella enterica serotype Paratyphi B. Transgenic Res 14:785–792

    Article  Google Scholar 

  9. 9.

    Vaquero C, Sack M, Chandler J, Drossard J, Schuster F, Monecke M, Schillberg S, Fischer R (1999) Transient expression of a tumour-specific single chain fragments and chimeric antibody in tobacco leaves. Proc Natl Acad Sci USA 96:11128–11133

    PubMed  Article  CAS  Google Scholar 

  10. 10.

    Hein M, Tang Y, McLeod DA, Janda KD, Hiatt A (1991) Evaluation of immunoglobulins from plant cells. Biotechnol Prog 7:455–461

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Larrick JW, Yu L, Chen J, Jaiswal S, Wycoff K (1998) Production of antibodies in transgenic plants. Res Immunol 149:603–608

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    Ramırez N, Lorenzo D, Palenzuela D, Herrera L, Ayala M, Fuentes A, P′erez M, Gavilondo J, Oramas P (2000) Single-chain antibody fragments specific to the hepatitis B surface antigen, produced in recombinant tobacco cell cultures. Biotechnol Lett 22:1233–1236

    Article  Google Scholar 

  13. 13.

    Brar HK, Bhattacharyya MK (2012) Expression of a single-chain variable-fragment against a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in transgenic soybean plants. MPMI 25:817–824

    PubMed  Article  CAS  Google Scholar 

  14. 14.

    Ayala M, Fernández-de-Cossío ME, Canaán-Haden L, Balint RF, Larrick JW, Gavilondo V (1995) Variable region sequence modulates periplasmic export of a single chain Fv antibody fragment in E. coli. Biotechniques 18:832–842

    PubMed  CAS  Google Scholar 

  15. 15.

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

    PubMed  Article  CAS  Google Scholar 

  16. 16.

    Franconi R, Tavladoraki P, Benvenuto E (1997) Plantibodies: immunomodulation and immunotherapeutic potential. In: Cattaneo A, Biocca S (eds) Intracellular antibodies, development and applications. Springer/Landes Bioscience, Berlin/Georgetown

    Google Scholar 

  17. 17.

    Schillberg S, Zimmermann S, Voss A, Fischer R (1999) Apoplastic and cytosolic expression of full-size antibodies and antibody fragments in Nicotiana tabacum. Transgenic Res 8:255–263

    PubMed  Article  CAS  Google Scholar 

  18. 18.

    Schillberg S, Fischer R, Emans N (2003) Molecular farming of recombinant antibodies in plants. Cell Mol Life Sci 60(3):433–445

    PubMed  Article  CAS  Google Scholar 

  19. 19.

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

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    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. Mol Breed 4:369–379

    Article  CAS  Google Scholar 

  21. 21.

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

    PubMed  Article  CAS  Google Scholar 

  22. 22.

    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

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Conrad U, Fiedler U (1998) Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity. Plant Mol Biol 38:101–109

    PubMed  Article  CAS  Google Scholar 

  24. 24.

    Conrad U, Fiedler U, Artsaenko O, Phillips J (1998) High level and stable accumulation of single chain Fv antibodies in plant storage organs. Plant Physiol 152:708–711

    Article  CAS  Google Scholar 

  25. 25.

    Fiedler U, Phillips J, Artsaenko O, Conrad U (1997) Optimization of scFv antibody production in transgenic plants. Immunotechnology 3:205–216

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    Fischer R, Liao YC, Hoffmann K, Schillberg S, Emans N (1999) Molecular farming of recombinant antibodies in plants. Biol Chem 380:825–839

    PubMed  Article  CAS  Google Scholar 

  27. 27.

    Gupta A, Chaudhary VK (2003) Whole-blood agglutination assay for on-site detection of human immunodeficiency virus infection. J Clin Microbiol 7:2814–2821

    Article  Google Scholar 

  28. 28.

    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor, New York

    Google Scholar 

  29. 29.

    Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) ClustalW2 and ClustalX version 2. Bioinformatics 23(21):2947–2948

    PubMed  Article  CAS  Google Scholar 

  30. 30.

    Rozenand S, Skaletsky HJ (2000) Primer 3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, pp 365–386

    Google Scholar 

  31. 31.

    Murashige T, Skoog F (1962) A revised medium for the rapid growth and bioassay with tobacco tissue culture. Plant Physiol 15:473–497

    Article  CAS  Google Scholar 

  32. 32.

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

    Article  CAS  Google Scholar 

  33. 33.

    Dobhal S, Pandey D, Kumar A, Agrawal S (2010) Studies on plant regeneration and transformation efficiency of Agrobacterium mediated transformation using neomycin phosphotransferase II (nptII) and glucuronidase (GUS) as a reporter gene. Afr J Biotechnol 9(41):6853–6859

    CAS  Google Scholar 

  34. 34.

    Jefferson RA (1987) Assaying chimeric genes in plants. The GUS gene fusion system. Plant Mol Biol Rep 5:387–405

    Article  CAS  Google Scholar 

  35. 35.

    Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissues. Focus 12:13–15

    Google Scholar 

  36. 36.

    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    PubMed  Article  CAS  Google Scholar 

  37. 37.

    Ehsani P, Meunier A, Nato F, Jafari A, Nato A, Lafaye P (2003) Expression of anti human IL-4 and IL-6 scFvs in transgenic tobacco plants. Plant Mol Biol 52(1):17–29

    PubMed  Article  CAS  Google Scholar 

  38. 38.

    Engvall E, Pediman P (1971) Enzyme linked immunosorbent assay (ELISA) quantitative assay of immunoglobin G. Immunochemistry 8:871–874

    PubMed  Article  CAS  Google Scholar 

  39. 39.

    Clark MF, Adams AN (1977) Characteristics of micro plate method of enzyme linked immunosorbent assay for detection of plant viruses. J Gen Virol 34:475–483

    PubMed  Article  CAS  Google Scholar 

  40. 40.

    Khoudi H, Laberge S, Ferullo JM, Bazin R, Darveau A, Castonguay Y, Allard G, Lemieux R, Vezina LP (1999) Production of a diagnostic monoclonal antibody in perennial alfalfa plants. Biotech Bioeng 64:135–143

    Article  CAS  Google Scholar 

  41. 41.

    Torres E, Vaquero C, Nicholson L, Sack M, Stoger E, Drossard J, Christou P, Fischer R, Perrin Y (1999) Rice cell culture as an alternative production system for functional diagnostic and therapeutic antibodies. Transgenic Res 8:441–449

    PubMed  Article  CAS  Google Scholar 

  42. 42.

    Perrin Y, Vaquero C, Gerrard I, Sack M, Drossard J, Stoger E, Christou P, Fischer R (2000) Transgenic pea seeds as bioreactors for the production of a single-chain Fv fragment (scFV) antibody used in cancer diagnosis and therapy. Mol Breed 6:345–352

    Article  CAS  Google Scholar 

  43. 43.

    Stoger E, Vaquero C, Torres E, Sack M, Nicholson L, Drossard J, Williams S, Keen D, Perrin Y, Christou P, Fischer R (2000) Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies. Plant Mol Biol 42:583–590

    PubMed  Article  CAS  Google Scholar 

  44. 44.

    Fischer R, Emans N (2000) Molecular farming of pharmaceutical proteins. Transgenic Res 9:279–299

    PubMed  Article  CAS  Google Scholar 

  45. 45.

    Teli NP, Timako MP (2004) Recent developments in the use of transgenic plants for the production of human therapeutics and biopharmaceuticals.  Plant Cell Tissue Organ 79:125–145

    Google Scholar 

  46. 46.

    Schouten A, Roosien J, van Engelen FA, de Jong GAM, Borst- Vrenssen, AWM, 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 cytosol and the secretory pathway in transgenic tobacco. Plant Mol Biol 30:781–793

  47. 47.

    Schellar J, Leps M, Conrad U (2006) Forcing single-chain variable fragment production in tobacco seeds by fusion to elastin-like polypeptides. Plant Biotechnol 4(2):243–249

    Google Scholar 

  48. 48.

    Sarala K (2001) Towards Agrobacterium transformation of FCV tobacco. Tob Res 27(1):88–90

    Google Scholar 

  49. 49.

    Li D, Leary JO, Huang Y, Huner NPA, Jevnikar AM, Ma S (2006) Expression of cholera toxin B subunit and the B chain of human insulin as a fusion protein in transgenic tobacco plants. Plant Cell Rep 25:417–424

    PubMed  Article  Google Scholar 

  50. 50.

    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  Article  CAS  Google Scholar 

  51. 51.

    Ramírez N, Ayala M, Lorenzo D, Palenzuela D, Herrera L, Doreste V, Falcón V, Gavilondo J, Oramas P (2002) Expression of single-chain antibody fragments (scFv) against the hepatitis B antigen (HBsAg) in transgenic tobacco plants. Transgenic Res 11:61–64

    PubMed  Article  Google Scholar 

  52. 52.

    Pelham HRB (1990) The retention signal for soluble proteins of the endoplasmic reticulum. Trends Biochem Sci 15:483–486

    PubMed  Article  Google Scholar 

  53. 53.

    Ituriaga G, Jefferson RA, Bevan M (1989) Endoplasmic reticulum targeting and glycosylation of hybrid proteins in transgenic tobacco. Plant Cell 1:381–390

    Google Scholar 

  54. 54.

    Bruyns AM, De Jaeger G, De Neve M, De Wilde C, Van Montagu M, Depicker A (1996) Bacterial and plant-produced scFv proteins have similar antigen-binding properties. FEBS Lett 386:5–10

    PubMed  Article  CAS  Google Scholar 

  55. 55.

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

    PubMed  CAS  Google Scholar 

  56. 56.

    Biocca S, Pierandreiamaldi P, Campioni N, Cattaneo A (1994) Intracellular immunization with cytosolic recombinant antibodies. Biotechnology 12:396–399

    PubMed  Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by Department of Biotechnology, Govt. of India. We thanks to Dr. A. Tyagi, Department of Plant Molecular Biology, UDSC, New Delhi for providing Agrobacterium strains, and Central Tobacco Research Institute, Rajamundry, Andra Pradesh (ICAR) for providing tobacco seeds. The authors also thank to Dr. M. K. Saxena and Dr. V. Umapathi for their valuable guidance.

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Correspondence to S. Dobhal.

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Dobhal, S., Chaudhary, V.K., Singh, A. et al. Expression of recombinant antibody (single chain antibody fragment) in transgenic plant Nicotiana tabacum cv. Xanthi. Mol Biol Rep 40, 7027–7037 (2013). https://doi.org/10.1007/s11033-013-2822-x

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Keywords

  • Recombinant antibody
  • B6scFv
  • Transgenic plants
  • Targeting
  • Molecular farming
  • Tobacco plant