Abstract
Genes encoding the heavy and light chains of LO-BM2, a therapeutic IgG antibody, were assembled in the tandem or inverted convergent orientation and expressed in Nicotiana tabacum plants and BY-2 suspension cells. The tandem construct allowed higher expression in both expression systems. A similar degradation pattern was observed for the secreted antibody recovered from the leaf intercellular fluid and BY-2 culture medium. Degradation increased with leaf age or culture time. Antibodies purified from leaf tissues and BY-2 cells were both functional. However, MS analysis of the N-glycosylation showed complex plant-type glycans to be the major type in the antibody purified from plants, whereas, oligomannosidic was the major glycosylation type in that purified from BY-2 cells. LO-BM2 was observed mainly in the endoplasmic reticulum of BY-2 cells while, in leaf cells, it was localized mostly to vesicles resembling prevacuolar compartments. These results and those from endoglycosidase H studies suggest that LO-BM2 is secreted from BY-2 cells more readily than from leaf cells where it accumulates in a post-Golgi compartment.
Similar content being viewed by others
References
An G (1985) High efficiency transformation of cultured tobacco cells. Plant Physiol 79:568–570. doi:10.1104/pp.79.2.568
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. doi:10.1016/0003-2697(76)90527-3
Brodzik R, Glogowska M, Bandurska K, Okulicz M, Deka D, Ko K, van der Linden J, Leusen JHW, Pogrebnyak N, Golovkin M, Steplewski Z, Koprowski H (2006) Plant-derived anti-Lewis Y mAb exhibits biological activities for efficient immunotherapy against human cancer cells. Proc Natl Acad Sci USA 103:8804–8809. doi:10.1073/pnas.0603043103
De Neve M, De Loose M, Jacobs A, Van Houdt H, Kaluza B, Weidle U, Van Montagu M, Depicker A (1993) Assembly of an antibody and its derived antibody fragment in Nicotiana and Arabidopsis. Transgenic Res 2:227–237. doi:10.1007/BF01977353
De Wilde C, De Neve M, De Rycke R, Bruyns AM, De Jaeger G, Van Montagu M, Depicker A, Engler G (1996) Intact antigen-binding MAK33 antibody and F-ab fragment accumulate in intercellular spaces of Arabidopsis thaliana. Plant Sci 114:233–241. doi:10.1016/0168-9452(96)04331-2
Dehoux JP, Hori S, Talpe S, Bazin H, Latinne D, Soares MP, Gianello P (2000) Specific depletion of preformed IgM natural antibodies by administration of anti-mu monoclonal antibody suppresses hyperacute rejection of pig to baboon renal xenografts. Transplantation 70:935–946. doi:10.1097/00007890-200009270-00011
Delannoy M, Alves G, Vertommen D, Ma J, Boutry M, Navarre C (2008) Identification of peptidases in Nicotiana tabacum leaf intercellular fluid. Proteomics 8:2285–2298. doi:10.1002/pmic.200700507
Doran PM (2006) Foreign protein degradation and instability in plants and plant tissue cultures. Trends Biotechnol 24:426–432. doi:10.1016/j.tibtech.2006.06.012
Drake PMW, Chargelegue DM, Vine ND, van Dolleweerd CJ, Obregon P, Ma JKC (2003) Rhizosecretion of a monoclonal antibody protein complex from transgenic tobacco roots. Plant Mol Biol 52:233–241. doi:10.1023/A:1023909331482
During K, Hippe S, Kreuzaler F, Schell J (1990) Synthesis and self-assembly of a functional monoclonal antibody in transgenic Nicotiana tabacum. Plant Mol Biol 15:281–293. doi:10.1007/BF00036914
Fischer R, Liao YC, Drossard J (1999) Affinity-purification of a TMV-specific recombinant full-size antibody from a transgenic tobacco suspension culture. J Immunol Methods 226:1–10. doi:10.1016/S0022-1759(99)00058-7
Floss DM, Falkenburg D, Conrad U (2007) Production of vaccines and therapeutic antibodies for veterinary applications in transgenic plants: an overview. Transgenic Res 16:315–332. doi:10.1007/s11248-007-9095-x
Goderis I, De Bolle MFC, Francois I, Wouters PFJ, Broekaert WF, Cammue BPA (2002) A set of modular plant transformation vectors allowing flexible insertion of up to six expression units. Plant Mol Biol 50:17–27. doi:10.1023/A:1016052416053
Hellwig S, Drossard J, Twyman RM, Fischer R (2004) Plant cell cultures for the production of recombinant proteins. Nat Biotechnol 22:1415–1422. doi:10.1038/nbt1027
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. doi:10.1126/science.227.4691.1229
Horton RM, Cai ZL, Ho SN, Pease LR (1990) Gene-splicing by overlap extension–tailor-made genes using the polymerase chain-reaction. Biotechniques 8:528
Hosy E, Duby G, Very AA, Costa A, Sentenac H, Thibaud JB (2005) A procedure for localisation and electrophysiological characterisation of ion channels heterologously expressed in a plant context. Plant Methods 1:14. doi:10.1186/1746-4811-1-14
Irons SL, Nuttall J, Floss DM, Frigerio L, Kotzer AM, Hawes C (2008) Fluorescent protein fusions to a human immunodeficiency virus monoclonal antibody reveal its intracellular transport through the plant endomembrane system. Plant Biotechnol J 6:649–662. doi:10.1111/j.1467-7652.2008.00348.x
Ko K, Koprowski H (2005) Plant biopharming of monoclonal antibodies. Virus Res 111:93–100. doi:10.1016/j.virusres.2005.03.016
Ko KS, Tekoah Y, Rudd PM, Harvey DJ, Dwek RA, Spitsin S, Hanlon CA, Rupprecht C, Dietzschold B, Golovkin M, Koprowski H (2003) Function and glycosylation of plant-derived antiviral monoclonal antibody. Proc Natl Acad Sci USA 100:8013–8018. doi:10.1073/pnas.0832472100
Kolarich D, Altmann F (2000) N-glycan analysis by matrix-assisted laser desorption/ionization mass spectrometry of electrophoretically separated nonmammalian proteins: application to peanut allergen Ara h 1 and olive pollen allergen Ole e 1. Anal Biochem 285:64–75. doi:10.1006/abio.2000.4737
Lam SK, Tse YC, Robinson DG, Jiang L (2007) Tracking down the elusive early endosome. Trends Plant Sci 12:497–505. doi:10.1016/j.tplants.2007.09.001
Lohaus G, Pennewiss K, Sattelmacher B, Hussmann M, Muehling KH (2001) Is the infiltration-centrifugation technique appropriate for the isolation of apoplastic fluid? a critical evaluation with different plant species. Physiol Plant 111:457–465. doi:10.1034/j.1399-3054.2001.1110405.x
Ma JK, Drake PM, Christou P (2003) The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet 4:794–805. doi:10.1038/nrg1177
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. doi:10.1002/eji.1830240120
Ma JKC, Chikwamba R, Sparrow P, Fischer R, Mahoney R, Twyman RM (2005) Plant-derived pharmaceuticals—the road forward. Trends Plant Sci 10:580–585. doi:10.1016/j.tplants.2005.10.009
Maeda H, Matsushita S, Eda Y, Kimachi K, Tokiyoshi S, Bendig MM (1991) Construction of reshaped human antibodies with HIV-neutralizing activity. Hum Antibodies Hybridomas 2:124–134
Maliga P, Sz-Breznovits A, Marton L (1973) Streptomycin-resistant plants from callus culture of haploid tobacco. Nat New Biol 244:29–30
Moriau L, Michelet B, Bogaerts P, Lambert L, Michel A, Oufattole M, Boutry M (1999) Expression analysis of two gene subfamilies encoding the plasma membrane H+-ATPase in Nicotiana plumbaginifolia reveals the major transport functions of this enzyme. Plant J 19:31–41. doi:10.1046/j.1365-313X.1999.00495.x
Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell-line as the hela-cell in the cell biology of higher-plants. Int Rev Cytol 132:1–30. doi:10.1016/S0074-7696(08)62452-3
Navarre C, Delannoy M, Lefebvre B, Nader J, Vanham D, Boutry M (2006) Expression and secretion of recombinant outer-surface protein A from the Lyme disease agent, Borrelia burgdorferi, in Nicotiana tabacum suspension cells. Transgenic Res 15:325–335. doi:10.1007/s11248-006-0002-7
Ramessar K, Rademacher T, Sack M, Stadlmann J, Platis D, Stiegler G, Labrou N, Altmann F, Ma J, Stoger E, Capell T, Christou P (2008) Cost-effective production of a vaginal protein microbicide to prevent HIV transmission. Proc Natl Acad Sci USA 105:3727–3732. doi:10.1073/pnas.0708841104
Schillberg S, Fischer R, Emans N (2003) Molecular farming of recombinant antibodies in plants. Cell Mol Life Sci 60:433–445. doi:10.1007/s000180300037
Sharp JM, Doran PM (1999) Effect of bacitracin on growth and monoclonal antibody production by tobacco hairy roots and cell suspensions. Biotechnol Bioprocess Eng 4:253–258. doi:10.1007/BF02933748
Sharp JM, Doran PM (2001) Characterization of monoclonal antibody fragments produced by plant cells. Biotechnol Bioeng 73:338–346. doi:10.1002/bit.1067
Stevens LH, Stoopen GM, Elbers IJW, Molthoff JW, Bakker HAC, Lommen A, Bosch D, Jordi W (2000) Effect of climate conditions and plant developmental stage on the stability of antibodies expressed in transgenic tobacco. Plant Physiol 124:173–182. doi:10.1104/pp.124.1.173
Terry ME, Bonner BA (1980) An examination of centrifugation as a method of extracting an extracellular solution from peas, and its use for the study of indoleacetic acid-induced growth. Plant Physiol 66:321–325. doi:10.1104/pp.66.2.321
van der Fits L, Deakin EA, Hoge JHC, Memelink J (2000) The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation. Plant Mol Biol 43:495–502. doi:10.1023/A:1006440221718
Van Engelen FA, Schouten A, Molthoff JW, Roosien J, Salinas J, Dirkse WG, Schots A, Bakker J, Gommers FJ, Jongsma MA, Bosch D, Stiekema WJ (1994) Coordinate expression of antibody subunit genes yields high-levels of functional antibodies in roots of transgenic tobacco. Plant Mol Biol 26:1701–1710. doi:10.1007/BF00019485
Voss A, Niersbach M, Hain R, Hirsch HJ, Liao YC, Kreuzaler F, Fischer R (1995) Reduced virus infectivity in Nicotiana-Tabacum secreting a Tmv-specific full-size antibody. Mol Breed 1:39–50. doi:10.1007/BF01682088
Wessel D, Flugge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138:141–143. doi:10.1016/0003-2697(84)90782-6
Wongsamuth R, Doran PM (1997) Production of monoclonal antibodies by tobacco hairy roots. Biotechnol Bioeng 54:401–415. doi:10.1002/(SICI)1097-0290(19970605)54:5<401::AID-BIT1>3.0.CO;2-I
Zhao RM, Moriau L, Boutry M (1999) Expression analysis of the plasma membrane H+-ATPase pma4 transcription promoter from Nicotiana plumbaginifolia activated by the CaMV 35S promoter enhancer. Plant Sci 149:157–165. doi:10.1016/S0168-9452(99)00155-7
Acknowledgments
This work was supported financially by a grant from the European community (PHARMA-PLANTA integrated Project), the Région Wallonne (DGTRE/SUBCELL), the Inter-university Attraction Poles Program-Belgian Science Policy, and the Belgian fund for scientific research. BDM is the recipient of a fellowship from the Fonds pour la Formation à la Recherche dans l’Industrie et dans l’Agriculture (Belgium).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
De Muynck, B., Navarre, C., Nizet, Y. et al. Different subcellular localization and glycosylation for a functional antibody expressed in Nicotiana tabacum plants and suspension cells. Transgenic Res 18, 467–482 (2009). https://doi.org/10.1007/s11248-008-9240-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-008-9240-1