Protein Coexpression Using FMDV 2A: Effect of “Linker” Residues
Figure 6
Efficiency of cleavage in constructs with short 2A sequences. pGFP-2A-CherryFP constructs with F2As 30wt, 20wt, 20mut3, 18wt, 18mut, and T2Awt and T2Amut2 were used to coexpress GFP and CherryFP proteins from a single ORF in transfected HeLa cells. The cells were transfected with 1.5 μg of plasmid DNA, and harvested 30 h after transfection. Cells were lysed in RIPA buffer, and equal amounts of total protein for each transfection were loaded onto 12% SDS-PAGE gel. The proteins were transferred onto a nitrocellulose membrane, blocked in PBS containing 5% milk, and probed with anti-GFP (upper blot) and anti-Cherry (middle blot) antibodies overnight at 4°C. Detection of bound primary antibody was achieved by using respective secondary antibodies, followed by ECL detection. All experiments were done in triplicate.
