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 proteinis only 16–20 amino acid long. It is responsible for thecleavage of the FMDV polyprotein at its own carboxyl-terminus. Weused the cleavage property of the 2A protein to processartificial polyproteins produced in transgenic plants. In our system, single or multiplecopies of the reporter CAT and GUS genes were fused into a single open readingframe (ORF) with a copy of the FMDV 2A protein gene placed between the reportergenes. Expression of various constructs in transgenic tobacco resulted inconsistent detection of freed CAT and/or GUS proteins, suggesting that FMDV 2Aprotein functioned properly in plant cells. Cleavage efficiencyranged from 80% to 100% depending on the constructs. The variability incleavage efficiency suggested that the contexts flanking a 2Aprotein might modulate its activity. We further expressed constructs wheremultiple copies of the 2A and reporter genes were fused into one ORF. Thepresence of freed GUS protein together with partially processed polyproteinintermediates in the transgenic plants indicated that multiple copies of the 2Aprotein in a single ORF function independently. Our data demonstrate that usingthe FMDV 2A protease as a linker, multiple genes could be easily expressed in asingle ORF.