Self-processing 2A-polyproteins - a system for co-ordinate expression of multiple proteins in transgenic plants

Achieving co-ordinate, high-level and stable expression of multiple transge nes in plants is currently difficult. Expression levels are notoriously var iable and influenced by factors that act independently on transgenes at dif ferent genetic loci. Instability of expression due to loss, re-arrangement or silencing of transgenes may occur, and is exacerbated by increasing numb ers of transgenic loci and repeated use of homologous sequences. Even linki ng two or more genes within a T-DNA does not necessarily result in co-ordin ate expression. Linking proteins in a single open reading frame - a polypro tein - is a strategy for co-ordinate expression used by many viruses. After translation, polyproteins are processed into constituent polypeptides, usu ally by proteinases encoded within the polyprotein itself. However, in foot -and-mouth disease virus (FMDV), a sequence (2A) of just 16-20 amino acids appears to have the unique capability to mediate cleavage at its own C-term inus by an apparently enzyme-independent, novel type of reaction. This sequ ence can also mediate cleavage in a heterologous protein context in a range of eukaryotic expression systems. We have constructed a plasmid in which t he 2A sequence is inserted between the reporter genes chloramphenicol acety ltransferase (CAT) and P-glucuronidase (GUS), maintaining a single open rea ding frame. Here we report that expression of this construct in wheatgerm l ysate and transgenic plants results in efficient cleavage of the polyprotei n and co-ordinate expression of active CAT and GUS. Self-processing polypro teins using the FMDV 2A sequence could therefore provide a system for ensur ing coordinated, stable expression of multiple introduced proteins in plant cells.