Cell-line-dependent sorting of recombinant phytase in cell cultures of Medicago truncatula
Pablo González-Melendi A , Ana Sofia Pires B and Rita Abranches B CA Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid-INIA, Parque Científico y Tecnológico de la U.P.M. Campus de Montegancedo, 28223 Pozuelo de Alarcón (Madrid), Spain.
B Plant Cell Biology Laboratory, Instituto de Tecnologia Quimica e Biologica, ITQB-UNL, Av. Republica, Apartado 127, 2781-901 Oeiras, Portugal.
C Corresponding author. Email: ritaa@itqb.unl.pt
Functional Plant Biology 36(5) 431-441 https://doi.org/10.1071/FP08260
Submitted: 10 October 2008 Accepted: 2 March 2009 Published: 6 May 2009
Abstract
Plant cell cultures as platforms for recombinant protein production are favoured over other systems because they combine the benefits of plants (low cost of production, low biosecurity risk, conserved post-translational modifications) with those of controlled cell cultures. However, many factors that affect the correct synthesis and accumulation of the recombinant product still need to be determined; in particular, the trafficking route of the recombinant proteins is poorly understood. Suspension cell cultures of Medicago truncatula Gaertn. have been shown to offer a viable and highly efficient system for the production of a model glycoprotein – phytase from the fungus Aspergillus niger Tiegh. The present study investigated subcellular protein sorting by immunogold detection of recombinant phytase with an electron microscope in four independent Medicago cell cultures expressing phytase. Two lines contained a C-terminal KDEL targeting signal for retention in the endoplasmic reticulum (ER), and the other two did not and were expected to travel through the secretory route; a high and low expressor were examined for each variant of the protein. A differential subcellular location of phytase was found in the four transgenic lines studied. These differences account not only for the version of the recombinant protein (secreted or retained in the ER), but also for the different expression levels.
Additional keywords: molecular farming, subcellular targeting.
Acknowledgements
We thank MariCarmen Risueño (CIB-CSIC) for the facilities provided in her laboratory, M. Mercedes Lucas (CCMA-CSIC) for the use of TEM facilities and for scientific advice, and José María Seguí and Paul Anderson for comments. We also thank the EM service ‘Luis Bru’ of the Universidad Complutense de Madrid, Fernando Pinto and Sara Paniagua (EM service, CCMA-CSIC) and Carmen Terrón (EM service, CIB-CSIC) for technical support. P. González-Melendi was funded by the program ‘Ramón y Cajal’ from the Spanish Ministry of Education and Science. This work was funded by POCI/BIA-BCM/55762/2004 (FCT Portugal, POCI 2010, FEDER) and the Portuguese–Spanish bilateral action 2005–2006, Acção Integrada Luso-Espanhola E-63/05 (CRUP, Portugal) and Acción Integrada HP2004-0107 (Spanish Ministry of Education and Science, Spain).
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