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J Biol Eng. 2013 Sep 8;7(1):20. doi: 10.1186/1754-1611-7-20.

Targeting a heterologous protein to multiple plant organelles via rationally designed 5' mRNA tags.

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Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA.



Plant bioengineers require simple genetic devices for predictable localization of heterologous proteins to multiple subcellular compartments.


We designed novel hybrid signal sequences for multiple-compartment localization and characterize their function when fused to GFP in Nicotiana benthamiana leaf tissue. TriTag-1 and TriTag-2 use alternative splicing to generate differentially localized GFP isoforms, localizing it to the chloroplasts, peroxisomes and cytosol. TriTag-1 shows a bias for targeting the chloroplast envelope while TriTag-2 preferentially targets the peroxisomes. TriTag-3 embeds a conserved peroxisomal targeting signal within a chloroplast transit peptide, directing GFP to the chloroplasts and peroxisomes.


Our novel signal sequences can reduce the number of cloning steps and the amount of genetic material required to target a heterologous protein to multiple locations in plant cells. This work harnesses alternative splicing and signal embedding for engineering plants to express multi-functional proteins from single genetic constructs.

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