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Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco

Pascal M. W. Drake^*,1, Tommaso Barbi^*, Amy Sexton^*, Edward McGowan^*, Johannes Stadlmann, Catherine Navarre, Matthew J. Paul^* and Julian K.-C. Ma^*

^* Molecular Immunology Unit, Department of Cellular and Molecular Medicine, St. George’s Hospital Medical School, University of London, London, UK;

Universitaet fuer Bodenkultur, Institute of Chemistry, Vienna, Austria; and

Institut des Sciences de la Vie, Université Catholique de Louvain, Louvain-la-Neuve, Belgium

¹ Correspondence: Molecular Immunology Unit, Department of Cellular and Molecular Medicine, St. George’s Hospital Medical School, University of London, Cranmer Terrace, London SW17 0RE, UK. E-mail: pdrake{at}sgul.ac.uk

Rhizosecretion is an attractive technology for the production of recombinant proteins from transgenic plants. However, to date, yields of plant-derived recombinant pharmaceuticals by this method have been too low for commercial viability. Studies conducted focused on three transgenic plant lines grown in hydroponic culture medium, two expressing monoclonal antibodies Guy’s 13 and 4E10 and one expressing a small microbicide polypeptide cyanovirin-N. Rhizosecretion rates increased significantly by the addition of the plant growth regulator -naphthalene acetic acid. The maximum rhizosecretion rates achieved were 58 µg/g root dry weight/24 h for Guy’s 13, 10.43 µg/g root dry weight/24 h for 4E10, and 766 µg/g root dry weight/24 h for cyanovirin-N, the highest figures so far reported for a full-length antibody and a recombinant protein, respectively. The plant growth regulators indole-butyric acid, 6-benzylaminopurine, and kinetin were also demonstrated to increase rhizosecretion of Guy’s 13. The effect of the growth regulators differed, as -naphthalene acetic acid and indole-butyric acid increased the root dry weight of hydroponic plants, whereas the cytokinins benzylaminopurine and kinetin increased rhizosecretion without affecting root mass. A comparative glycosylation analysis between MAb Guy’s 13 purified from either hydroponic culture medium or from leaf extracts demonstrated a similar pattern of glycosylation comprising high mannose to complex glycoforms. Analysis of the hydroponic culture medium at harvest revealed significantly lower and less complex levels of proteolytic enzymes, in comparison with leaf extracts, which translated to a higher proportion of intact Guy’s 13 IgG in relation to other IgG products. Hydroponic medium could be added directly to a chromatography column for affinity purification, allowing simple and rapid production of high purity Guy’s 13 antibody. In addition to the attractiveness of controlled cultivation within a contained environment for pharmaceutical-producing plants, this study demonstrates advantages with respect to the quality and downstream purification of recombinant proteins.—Drake, P. M. W., Barbi, T., Sexton, A., McGowan, E., Stadlmann, J., Navarre, C., Paul, M. J., Ma, J. K.-C. Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco.

Key Words: transgenic plants • molecular farming • plant growth regulators • proteases • glycosylation