Journal
BIOTECHNOLOGY JOURNAL
Volume 10, Issue 11, Pages 1792-1802Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/biot.201500042
Keywords
Potyvirus vector; Recombinant protein; Signal peptides; Subcellular localization; Tobacco etch virus
Funding
- Ministerio de Economia y Competitividad (MINECO, Spain) [BIO2011-26741, BIO2014-54269-R]
- Ministerio de Educacion, Cultura y Deporte (Spain) [AP2012-3751]
- MINECO [BIO2011-25018]
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Plant virus-based expression systems allow quick and efficient production of recombinant proteins in plant biofactories. Among them, a system derived from tobacco etch virus (TEV; genus potyvirus) permits coexpression of equimolar amounts of several recombinant proteins. This work analyzed how to target recombinant proteins to different subcellular localizations in the plant cell using this system. We constructed TEV clones in which green fluorescent protein (GFP), with a chloroplast transit peptide (cTP), a nuclear localization signal (NLS) or a mitochondrial targeting peptide (mTP) was expressed either as the most amino-terminal product or embedded in the viral polyprotein. Results showed that cTP and mTP mediated efficient translocation of GFP to the corresponding organelle only when present at the amino terminus of the viral polyprotein. In contrast, the NLS worked efficiently at both positions. Viruses expressing GFP in the amino terminus of the viral polyprotein produced milder symptoms. Untagged GFPs and cTP and NLS tagged amino-terminal GFPs accumulated to higher amounts in infected tissues. Finally, viral progeny from clones with internal GFPs maintained the extra gene better. These observations will help in the design of potyvirus-based vectors able to coexpress several proteins while targeting different subcellular localizations, as required in plant metabolic engineering.
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