Comparative efficiency of subcellular targeting signals for expression of a toxic protein in sugarcane

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.