In vitro characterization of a cassette to accumulate multiple proteins through synthesis of a self-processing polypeptide.

The strategy for processing the polyprotein encoded by plant potyviruses has been mimicked by constructing an expression cassette based on the nuclear inclusion (Nla) proteinase from tobacco etch virus (TEV). This cassette (pPR01), includes the TEV Nla coding region flanked on each side by its heptapeptide cleavage sequence and cloning sites for the in frame insertion of two different open reading frames. pPR01 allows the synthesis, under the control of a single transcriptional promoter, of two proteins in equimolar amounts as part of a polyprotein which is cleaved into individual mature products by the TEV protease. In in vitro reactions the cassette functioned as expected when several different protein-coding sequences were used. The potential uses of pPR01 are discussed.