Protein Coexpression Using FMDV 2A: Effect of “Linker” Residues
Figure 2
Effect of various substitutions, introduced in the region immediately upstream of F2A, on cleavage efficiency of [GFP-F2A20-CherryFP] polyproteins. (a) Amino acid sequence comparison (mutated residues are boxed) of parental pGFP-F2A20-Cherry (wt) and three mutant pGFP-F2A20-Cherry constructs (mut1, mut2, and mut3) 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.
