Bicistronic expression and differential localization of proteins in insect cells and Drosophila suzukii using picornaviral 2A peptides.

Polycistronic expression systems in insects can be used for applications such as recombinant protein production in cells, enhanced transgenesis methods, and the development of novel pest-control strategies based on the sterile insect technique (SIT). Here we tested the performance of four picornaviral 2A self-cleaving peptides (TaV-2A, DrosCV-2A, FMDV 2A1/31 and FMDV 2A1/32) for the co-expression and differential subcellular targeting of two fluorescent marker proteins in cell lines (Anastrepha suspensa AsE01 and Drosophila melanogaster S2 cells). We found that all four 2A peptides showed comparable activity in cell lines, leading to the production of independent upstream and downstream proteins that were directed to the nucleus or membrane by a C-terminal nuclear localization signal (NLS) on the upstream protein and a poly-lysine/CAAX membrane anchor on the downstream protein. TaV-2A and DrosCV-2A were inserted into piggyBac constructs to create transgenic D. suzukii strains, confirming efficient ribosomal skipping in vivo. Interestingly, we found that the EGFP-CAAX protein was distributed homogeneously in the membrane whereas the DsRed-CAAX protein formed clumps and aggregates that induced extensive membrane blebbing. Accordingly, only flies expressing the DsRed-NLS and EGFP-CAAX proteins could be bred to homozygosity whereas expression of EGFP-NLS and DsRed-CAAX was lethal in the homozygous state. Our results therefore demonstrate that the 2A constructs and two novel targeting motifs are functional in D. suzukii, and that the combination of EGFP-NLS and DsRed-CAAX shows dosage-dependent lethality. These molecular elements could be further used to improve expression systems in insects and generate novel pest control strains.