Protein Coexpression Using FMDV 2A: Effect of “Linker” Residues
Figure 5
Effect of “reverse” substitutions, immediately upstream of T2A, on cleavage efficiency of pGFP-T2A-CherryFP constructs. (a) Amino acid substitutions introduced in pGFP-T2A-CherryFP sequence immediately upstream of T2A. Mutated residues are shown in bold, and T2A sequence is underlined. Parental construct pGFP-T2A-CherryFP with -MHSRGSG- linker (wt) and its mutant versions with -MHSRSLE- and -RAKRSLE- substitutions (T2Amut1 and T2Amut2, resp.) were used to coexpress GFP and CherryFP proteins from a single ORF in vitro using coupled transcription/translation rabbit reticulocyte lysate system (b) and in 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 (left blot) and anti-CherryFP (right 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.
