**Acknowledgements**

virion), and 'overcoat' overexpression vectors using 2A have also been developed for the related viruses *Pepino mosaic virus* (PepMV) [144] and PlAMV [145]. So far, plant virusexpressed epitopes have been purified mainly as virion-attached surface peptides, but with 2A linkers, it is also possible to produce them mainly as free proteins. For efficient surface display, an optimal ratio of free and fused CP has to be found that maximizes virus-displayed epitopes whilst still enabling efficient encapsidation. The range of 2A-like sequences with different 'cleavage' activities [31, 37] will be useful in the development of further 'overcoat'

Expression vectors using 2A peptides have also been developed based on *Cowpea mosaic virus* (CPMV) [146], *Bean pod mottle virus* (BPMV) [147], and *Wheat streak mosaic virus* (WSMV) [148]. In the case of the CPMV and WSMV vectors, 2A linkers were used to overcome the problem that these viruses encode large polyproteins that are processed into functional subunits by viral proteases. Foreign proteins inserted into the polyprotein open reading frame need to be released and 2A sequences provide an alternative to viral protease cleavage sites. In CPMV, using 2A instead of viral cleavage motifs reduced the number of additional amino acids attached to the over-expressed protein, and as in PVX 'overcoat' vectors, some of the foreign protein is displayed on virus particles, which are very stable and easy to purify. In WSMV, use of FMDV 2A or (and also FMDV 1D/2A) sequences resulted in more efficient release of the foreign protein (GFP) than with viral proteinase sites [148]. WSMV vectors enable protein expression in cereal hosts. In the soybean-infecting BPMV vectors, 2A linkers were used both to enable insertion of foreign genes into the viral polyprotein open reading frame,

and to facilitate simultaneous co-expression of two different foreign proteins.

The first demonstration the 2A was active in plant cells used an artificial polyprotein which comprised two reporter proteins flanking 2A [38]. This co-expression system was soon adopted by plant virologists for use in both rod-shaped and icosahedral virus particles either as highlevel expression systems, or, to produce particles 'decorated' with fluorescent proteins, immunogens, single-chain antibodies etc. [137-147]. Here, plants are used simply as 'bioreac‐ tors' for production of recombinant proteins / virus particles – the plants are not transgenic. In the case of transgenic plants the first reports of the use of 2A to co-express multiple proteins were as a 'proof-of-principle' or research tools [38, 57], but within a few years plants were being genetically engineered to demonstrate how nutritional properties could be improved [105, 149]. Whilst the use of 2A rapidly expanded in the arenas of animal biotechnology and biomedicine (e.g. monoclonal antibody production, cancer gene therapies, production of pluripotent stem cells: reviewed in [25]), progress in transgenic plants was slower-due to a number of reasons, including the 'trickle-down' effects on plant biotechnology from the EU policies concerning genetically-modified plants. Over the past few years, however, the 2A coexpression system has been used in the development of methods to engineer plant genomes [149], the expression of high-value proteins, the improvement of plant tolerance to biotic and abiotic stresses, the improvement of nutritional properties through metabolome engineering

vectors.

180 Biotechnology

**4. Food for thought**

The authors gratefully acknowledge the long-term support of the UK Biotechnology and Biological Sciences Research Council (BBSRC). The University of St Andrews is a charity registered in Scotland no. SCO13532.
