Preprint

Jahr der Veröffentlichung: 2019

Zeitschrift: BioRxiv

DOI Link: https://doi.org/10.1101/766188

Verlag: Cold Spring Harbor Laboratory

Abstract: 

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) and in vivo in the pest insect Drosophila suzukii. 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. Two of the 2A peptides 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 EGFP-CAAX protein could be bred to homozygosity whereas the DsRed-CAAX construct was lethal in the homozygous state. Our results therefore demonstrate that four different 2A constructs and two novel targeting motifs are functional in D. suzukii, and that 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.

Harvard-Zitierstil: Schwirz, J., Yan, Y., Franta, Z. and Schetelig, M. (2019) Bicistronic expression and differential localization of proteins in insect cells and Drosophila suzukii using picornaviral 2A peptides [Preprint]. BioRxiv, Article 766188. https://doi.org/10.1101/766188

APA-Zitierstil: Schwirz, J., Yan, Y., Franta, Z., & Schetelig, M. (2019). Bicistronic expression and differential localization of proteins in insect cells and Drosophila suzukii using picornaviral 2A peptides. BioRxiv. https://doi.org/10.1101/766188