Protein Coexpression Using FMDV 2A: Effect of “Linker” Residues
Figure 3
Effect of -RAKRSLE- to -SGSRGAC- substitution immediately upstream of F2A on cleavage efficiency of pGFP-F2A-Cherry constructs with F2As of 25–18aa. Parental pGFP-F2A-Cherry constructs and their mutant versions (mut or Fmut) (mutated residues are boxed, (a)) were used to coexpress GFP and CherryFP proteins from a single ORF in vitro using coupled transcription/translation rabbit reticulocyte lysate system (b) and transfected HeLa cells (c). For TnT, reticulocyte lysates were programmed with 20 ng of plasmid DNA, and translation products were resolved by the 12% SDS-PAGE. For in vivo studies, HeLa 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-CherryFP (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.
