Self-cleaving 2A peptide from porcine teschovirus-1, codon-optimized for Drosophila melanogaster

Characteristics

Encodes ATNFSLLKQAGDVEENPGP (19 amino acids, 57 nucleotides). A 2A peptide makes the ribosome skip formation of the last peptide bond of the 2A sequence, so the upstream protein is released with the 2A residues at its C terminus and the downstream protein starts with a proline. In Drosophila S2 cells, P2A separates 96 ± 1% of a myristoylated GFP-P2A-dsRed-nls product (western blot, Act5c promoter, mean ± SEM, n = 5), the highest of four 2A peptides and not different from T2A (88 ± 3%). Proteins before and after P2A are produced at similar levels (normalized intensity 98 ± 2 vs 93 ± 7, n = 2).

Applications: Co-expression of tdTomato and GCaMP5G (tdTomato-P2A-GCaMP5G, UAS, n-syb-Gal4) in Drosophila larval motoneurons for ratiometric calcium imaging at the neuromuscular junction, in dissected larvae and through the cuticle of intact larvae. Also validated as myristoylated GFP-P2A-dsRed-nls in S2 cells and in larval salivary gland and muscle.

Limitations: The 2A residues remain on the C terminus of the upstream protein. Separation is incomplete, with 96 ± 1% separated in S2 cells, leaving about 4% as uncleaved fusion. The N-terminal proline on the downstream protein could limit its perdurance. Efficiency is measured with one reporter pair in S2 cells; separation in other Drosophila cell types and stages is not established.

References

1. Daniels et al. (2014). Expression of Multiple Transgenes from a Single Construct Using Viral 2A Peptides in Drosophila. PLoS ONE - Daniels 2014 2A Peptides