Self-cleaving 2A peptide from Thosea asigna virus, codon-optimized for Drosophila melanogaster

Characteristics

Encodes EGRGSLLTCGDVEENPGP (18 amino acids, 54 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, T2A separates 88 ± 3% of a myristoylated GFP-T2A-dsRed-nls product (western blot, Act5c promoter, mean ± SEM, n = 5), versus 96 ± 1% for P2A and 68 ± 3% for E2A. T2A also performs best among five 2A peptides in SL2 cells, and Gal4 keeps its activity with the N-terminal proline.

Applications: Co-expression of myristoylated GFP and nuclear dsRed in Drosophila S2 cells and in larval salivary gland and body wall muscle (UAS constructs, Act5c-Gal4 and how-24B-Gal4). The T2A peptide also releases active Gal4 from mCD8-EGFP-T2A-Gal4 in bursicon neurons, and in-frame T2A-Gal4 fusions in a rickets genomic clone drive Gal4 in rk-expressing cells.

Limitations: The 2A residues remain on the C terminus of the upstream protein. Separation is incomplete, with 88 ± 3% separated in S2 cells, leaving about 12% 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 and varies with experimental context.

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
2. Diao & White (2011). A Novel Approach for Directing Transgene Expression in Drosophila: T2A-Gal4 In-Frame Fusion. Genetics - Diao 2011 T2A Gal4