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Investigating recombinant protein exudation from roots of transgenic tobacco

Published online by Cambridge University Press:  07 October 2008

Francesca Pizzuti  and
Lorenza Daroda
Francesca Pizzuti
Affiliation:
ENEA-Casaccia Research Center, BAS-BIOTECGEN Rome, Italy
Lorenza Daroda
Affiliation:
ENEA-Casaccia Research Center, BAS-BIOTECGEN Rome, Italy

Abstract

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It is widely acknowledged that plant-made pharmaceuticals (PMPs) offer numerous benefits, including inexpensive production, biological safety and the facility for production at agricultural scale. At the same time, it is important to minimize any potential risk associated with this new technology, including the potential release of bioactive proteins into the environment. To address this issue, we studied transgenic Nicotiana benthamiana and Nicotiana tabacum plants expressing two recombinant single-chain variable fragment (scFv) antibodies, respectively scFvB9 and scFvH10. ScFvB9 was raised against glycoprotein G1 of Tomato spotted wilt virus (TSWV), and scFvH10 was raised against human tumor-associated antigen tenascin-C. Both antibodies were targeted to the secretory pathway using the N-terminal signal peptide from Phaseolus vulgaris polygalacturonase-inhibiting protein (PGIP), and scFvH10 carried in addition a C-terminal KDEL tetrapeptide for retention in the endoplasmic reticulum (ER). Sterile hydroponic cultures were established, allowing us to investigate whether scFvB9 and scFvH10 were present in root exudates. Intercellular fluids extracted from different plant tissues were analyzed by western blotting revealing the presence of scFvB9. Successful secretion of scFvB9 in hydroponic medium was also demonstrated, whereas no scFvH10 could be detected in the leaf, stem or root apoplast, nor secreted into the hydroponic medium. Our results show that scFvH10 release or diffusion from the roots of transgenic plants was not occurring, suggesting that the KDEL signal might contribute to the environmental biosafety of crops producing PMPs.

Keywords

transgenic tobaccorecombinant antibodyhydroponic cultureKDELrhizosecretionplant molecular farming / biosafety
Type
Research Article
Information
Environmental Biosafety Research , Volume 7 , Issue 4 , October 2008 , pp. 219 - 226
Copyright
© ISBR, EDP Sciences, 2008

References

Arcalis E, Marcel S, Altmann F, Kolarich D, Drakakaki G, Fischer R, Christou P, Stoger E (2004) Unexpected deposition patterns of recombinant proteins in post-endoplasmic reticulum compartments of wheat endosperm. Plant Physiol. 136: 3457–3466 CrossRef
Boehm R (2007) Bioproduction of therapeutic proteins in the 21st century and the role of plants and plant cells as production platforms. Ann. N. Y. Acad. Sci. 1102: 121–134
Bradford, MM (1976) A rapid and sensitive method for the quantification of micrograms quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72: 248–254 CrossRef
Commandeur, U, Twyman, M, Fisher, R (2003) The biosafety of molecular farming in plants. AgBiotechNet 5: 1–9
Drakakaki G, Marcel S, Arcalis E, Altmann F, Gonzalez-Melendi P, Fischer R, Christou P, Stoger E (2006) The intracellular fate of a recombinant protein is tissue dependent. Plant Physiol. 141: 578–586 CrossRef
Elbehri A (2005) Biopharming and the food system: examining the potential benefits and risks. Agbioforum 8: 18–25
Faye, L, Boulaflous, A, Benchabane, M, Gomord, V, Michaud, D (2005) Protein modifications in the plant secretory pathway: current status and practical implications in molecular pharming. Vaccine 23: 1770–1778 CrossRef
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
Franconi R, Roggero P, Pirazzi P, Arias FJ, Desiderio A, Bitti O, Pashkoulov D, Mattei B, Bracci L, Masenga V, Milne RG, Benvenuto E (1999) Functional expression in bacteria and plants of an scFv antibody fragment against tospoviruses. Immunotechnology 4: 189-201
Jurgens G (2004) Membrane traffiking in plants. Annu. Rev. Cell Dev. Biol. 20: 481–504
Kermode AR (2006) Plants as factories for the production of biopharmaceutical and bioindustrial proteins: lessons from cell biology. Can. J. Bot. 84: 679–694
Komarnytsky S, Borisjuk N, Yakoby N, Garvey A, Raskin I (2006) Cosecretion of protease inhibitor stabilize antibodies produced by plant roots. Plant Physiol. 141: 1185–1193 CrossRef
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685
Liénard D, Sourrouille C, Gomord V, Faye L (2007) Pharming and transgenic plants. Biotechnol. Annu. Rev. 13: 115–147
Lilley AK, Bailey MJ, Cartwright C, Turner SL, Hirsch PR (2006) Life in earth: the impact of GM plants on soil ecology? Trends Biotechnol. 24: 9–14
Munro, S, Pelham, HR (1987) A C-terminal signal prevents secretion of luminal ER proteins. Cell 48: 899–907 CrossRef
Murashige, T, Skoog, F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15: 473–497 CrossRef
Petruccelli S, Otegui MS, Lareu F, Tran Dinh O, Fitchette AC, Circosta A, Rumbo M, Bardor M, Carcamo R, Gomord V, Beachy RN (2006) A KDEL-tagged monoclonal antibody is efficiently retained in the endoplasmic reticulum in leaves, but is both partially secreted and sorted to protein storage vacuoles in seed. Plant Biotechnol. J. 4: 511–527
Pujol M, Gavilondo J, Ayala M, Rodrìguez M, Gonzàlez EM, Pèrez L (2007) Fighting cancer with plant-expressed pharmaceuticals. Trends Biotechnol. 25: 455–459 CrossRef
Ramessar K, Peremerti A, Gomez-Galera S, Naqvi S, Moralejo M, Munoz P, Capell T, Christou P (2007) Biosafety and risk assessment framework for selectable marker genes in transgenic crop plants: a case of the science not supporting the politics. Transgenic Res. 16: 261–280
Sabharwal N, Icoz I, Saxena D, Stotzky G (2007) Release of the recombinant proteins, human serum albumin, beta-glucuronidase, glycoprotein B from human cytomegalovirus, and green fluorescent protein, in root exudates from transgenic tobacco and their effects on microbes and enzymatic activities in soil. Plant Physiol. Biochem. 45: 464–469
Saint-Jore-Dupas C, Faye L, Gomord V (2007) From planta to pharma with glycosylation in the toolbox. Trends Biotechnol. 25: 317–323
Sanvido, O, Romeis, J, Bigler, F (2007) Ecological impacts of genetically modified crops: ten years of field research and commercial cultivation. Adv. Biochem. Eng. Biotechnol. 107: 235–278
Saxena D, Flores S, Stotzky G (2002) Bt toxin is released in root exudates from 12 transgenic corn hybrids representing three transformation events. Soil Biol. Biochem. 34: 133–137
Saxena D, Stewart CN, Altosaar I, Shu Q, Stotzky G (2004) Larvicidal Cry proteins from Bacillus thuringensis are released in root exudates of transgenic B. thuringensis corn, potato and rice but not of B. thuringensis canola, cotton and tobacco. Plant Physiol. Biochem. 42: 383–387
Sparrow PAC, Irwin JA, Dale PJ, Twyman RM, Ma J-KC (2007) Pharma-Planta: road testing the developing regulatory guidelines for plant-made pharmaceuticals. Transgenic Res. 16: 147–161
Spök A (2007) Molecular farming on the rise-GMO regulators still walking a tightrope. Trends Biotechnol. 25: 74–82 CrossRef
Streatfield SJ (2007) Approaches to achieve high-level heterologous protein production in plants. Plant Biotechnol. J. 5: 2–15
Toubart, P, Desiderio, A, Salvi, G, Cervone, F, Daroda, L, De Lorenzo, G (1992) Cloning and characterization of the gene encoding the endopolygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris L. Plant J. 2: 367–373
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc. Natl. Acad. Sci. USA 76: 4350–4353
Twyman, RM, Schillberg, S, Fischer, R (2005) Transgenic plants in the biopharmaceutical market. Expert Opin. Emerg. Drugs 10: 185–218 CrossRef
Van Droogenbroeck B, Cao J, Stadlmann J, Altmann F, Colanesi S, Hillmer S, Robinson DG, Van Lerberge E, Terryn N, van Montagu M, Liang M, Depicker A, De Jaeger G (2007) Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds. Proc. Natl. Acad. Sci. USA 104: 1430–1435
van Esse HP, Thomma BPHJ, van't Klooster JW, de Wit PJGM (2006) Affinity-tags are removed from Cladosporium fulvum effector proteins expressed in the tomato leaf apoplast. J. Exp. Bot. 57: 599–608
Vitale A, Pedrazzini E (2005) Recombinant pharmaceuticals from plants: the plant endomembrane system as bioreactor. Mol. Interv. 5: 216-225
Widmer (2007) Assessing effects of transgenic crops on soil microbial communities. Adv. Biochem. Eng. Biotechnol. 107: 207–234
Wolt JD, Karaman S, Wang K (2007) Risk assessment for plant-made pharmaceuticals. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources 2: 1–9