Investigating recombinant protein exudation from roots of transgenic tobacco

Issue		Environ. Biosafety Res. Volume 7, Number 4, October-December 2008


Page(s)		219 - 226
DOI		10.1051/ebr:2008020
Published online		07 October 2008

Environ. Biosafety Res. 7 (2008) 219-226
DOI: 10.1051/ebr:2008020

Investigating recombinant protein exudation from roots of transgenic tobacco

Francesca Pizzuti and Lorenza Daroda

ENEA-Casaccia Research Center, BAS-BIOTECGEN Rome, Italy

Received January 24, 2008; accepted August 24, 2008; published online 18 December 2008

Abstract - 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.

Key words: transgenic tobacco / recombinant antibody / hydroponic culture / KDEL / rhizosecretion / plant molecular farming / biosafety

Corresponding author: lorenza.daroda@casaccia.enea.it

© ISBR, EDP Sciences 2008

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