2A to the Fore – Research, Technology and Applications

The 2A region of the foot-and-mouth disease virus (FMDV) encodes a short sequence that mediates self-processing by a novel translational effect. Translation elongation arrest leads to release of the nascent polypeptide and re-initiation at the next in-frame codon. In this way discrete translation products are derived from a single open read-size of 2A peptides compared to internal promoters or IRES sequences makes them ideal candidates for use in size-restricted viral and nonviral vectors. Additionally, the diversity of the 2A sequence minimizes the chances for homologous recombination which is an important consideration when using retroviral or lentiviral systems. One outstanding question is the effect of the 2A “tag” attached to the C-terminus of the upstream protein. This may interfere with function, or more importantly may present a new epitope that could be subject to immunological surveillance. However, the attachment of extra amino acids is a routine method for labelling transgene products while leaving their function intact (e.g. tags such as the His tag and Myc tag). To our knowledge, the 2A tag does not impair activity and expression - proteins that require authentic termini, or are N-/C- terminally modified, can be introduced as the first or final polyprotein domain, respectively. In any event, strategies have now been devised that allow removal of the 2A linker (see François et al., 2004; Fang et al., 2005). The “unwanted” tag may however stick – antibodies directed against 2A can be used to detect the gene cloned upstream (Ryan and Drew, 1994; de Felipe et al., 2003, 2006). Lastly, the presence of a proline residue at the N-terminus of the downstream protein, as a relict of the 2A self-cleaving process, does not normally interfere with function – it does, however, confer high protein stability (Varshavsky, 1992). Aware of the factors that influence expression levels it is important to empirically design any co-expression cassette to ensure the polyprotein is the most suitable arrangement in respect to desired function. As a form of control of protein biogenesis, 2A sequences are much more wide-spread than was first suspected. To appease different and opposing sensibilities, 2A variants that are not found in mammalian viruses can be used just as effectively for the production of multiple protein products. Although a relative new-kid-on-the-block in terms of co-expression studies, 2A can safely be considered an “established” player. It is clear that assorted 2A-derived proteins with diverse and distinct localized functions may be stably expressed in several different cell types demonstrating the applicability of this technology in biomedicine and biotechnology. The biotechnological applications of 2A are continually updated on www.st-andrews.ac.uk/ryanlab/Index.htm We envisage that 2A technology will become one of the predominant strategies for multigene delivery in the coming years.