Comparative efficiency of subcellular targeting signals for expression of a toxic protein in sugarcane
Mark A. Jackson A B C E , Kerry A. Nutt A D , Rachael Hassall A D and Anne L. Rae A BA Cooperative Research Centre for Sugar Industry Innovation through Biotechnology, The University of Queensland, St Lucia, Qld 4072, Australia.
B CSIRO Plant Industry, Queensland Bioscience Precinct, 306 Carmody Road, St Lucia, Qld 4067, Australia.
C School of Molecular and Microbial Sciences, The University of Queensland, St Lucia, Qld 4072, Australia.
D David North Plant Research Centre, BSES Limited, PO Box 86, Indooroopilly, Qld 4068, Australia.
E Corresponding author. Email: m.jackson1@uq.edu.au
Functional Plant Biology 37(8) 785-793 https://doi.org/10.1071/FP09243
Submitted: 7 October 2009 Accepted: 12 March 2010 Published: 26 July 2010
Abstract
Transgenic sugarcane plants (Saccharum hybrid) have been proposed as a production platform for recombinant proteins, including those providing pathogen resistance as well as high value therapeutic proteins. For the in planta production of proteins that are potentially toxic, a careful consideration of subcellular location is required in order to optimise yield and to avoid detrimental interaction with plant cellular processes. In this study, avidin, a glycoprotein that is potentially toxic to cells because of its high affinity to the co-vitamin biotin, was used to test the effectiveness of a range of targeting signals. Accumulation of avidin was directed to the apoplast, endoplasmic reticulum and to the lytic and delta type vacuoles. Although targeting to the delta vacuole resulted in the highest yields of avidin, these plants developed a biotin deficient phenotype, indicating that this targeting was not fully effective in protecting cellular biotin pools. Similar problems were also observed when avidin was retained in the endoplasmic reticulum. When avidin was targeted to the lytic vacuole using the targeting signal from the sugarcane legumain, plants remained phenotypically normal; however, avidin was predominantly detected as a degraded product due to site-specific limited proteolysis in the vacuole. For avidin and other potentially toxic products, this lytic vacuole targeting signal may be useful if stability within this proteolytic environment can be improved.
Additional keywords: biofactory, endoplasmic reticulum, Saccharum, vacuole.
Acknowledgements
Mark Jackson was supported by a PhD research scholarship from the Cooperative Research Centre for Sugar Industry Innovation through Biotechnology. The authors thank Dr Colleen Murray (HortResearch, NZ) for the potato proteinase inhibitor I sequence and for helpful discussions. We are grateful to Dr Rosanne Casu (CSIRO Plant Industry) for ADF primers sequences and Associate Professor Don Maclean for helpful discussion and critical evaluation of this manuscript.
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