Self-cleaving 2A peptide from equine rhinitis A virus, codon-optimized for Drosophila melanogaster

Characteristics

Encodes QCTNYALLKLAGDVESNPGP (20 amino acids, 60 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, E2A separates 68 ± 3% of a myristoylated GFP-E2A-dsRed-nls product (western blot, Act5c promoter, mean ± SEM, n = 5), lower than P2A (96 ± 1%) and T2A (88 ± 3%). In flies, E2A gives more perinuclear GFP/RFP puncta in salivary gland and more and larger aggregates in muscle than P2A or T2A.

Applications: Tested as the linker in myristoylated GFP-E2A-dsRed-nls separation assays in Drosophila S2 cells (Act5c promoter) and in transgenic flies (UAS constructs integrated at one attP site), imaged in larval salivary gland and body wall muscle. No application beyond these separation assays is reported in the cited paper.

Limitations: The 2A residues remain on the C terminus of the upstream protein. Separation is incomplete, with 68 ± 3% separated in S2 cells, leaving about 32% as uncleaved fusion, far more than P2A or T2A. The N-terminal proline on the downstream protein could limit its perdurance. Efficiency is measured with one reporter pair.

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