Expressing multiple genes in a single open reading frame with the 2A region of foot-and-mouth disease virus as a linker

The Food-and-mouth disease virus (FMDV) 2A protein is only 16-20 amino acid long. It is responsible for the 'cleavage' of the FMDV polyprotein at its own carboxyl-terminus. We used the 'cleavage' property of the 2A protein to process artificial polyproteins produced in transgenic plants. In our system, single or multiple copies of the reporter CAT and GUS genes were fused into a single open reading frame (ORF) with a copy of the FMDV 2A protein gene placed between the reporter genes. Expression of various constructs in transgenic tobacco resulted in consistent detection of freed CAT and/or GUS proteins, suggesting that FMDV 2A protein functioned properly in plant cells. 'Cleavage' efficiency ranged from 80% to 100% depending on the constructs. The variability in 'cleavage' efficiency suggested that the contexts flanking a 2A protein might modulate its activity. We further expressed constructs where multiple copies of the 2A and reporter genes were fused into one ORF. The presence of freed GUS protein together with partially processed polyprotein intermediates in the transgenic plants indicated that multiple copies of the 2A protein in a single ORF function independently. Our data demonstrate that using the FMDV 2A protease as a linker, multiple genes could be easily expressed in a single ORF.