Fig. 1

Design of the split intein-mediated split-Cre recombinase system. a Schematic depicting the autocatalytic split intein-mediated trans-splicing reaction to reconstitute Cre recombinase. Split inteins associate to fuse NCre and CCre with a peptide bond. b Structural comparison of original Cre recombinase-LoxP DNA complex (left, PDB ID: 1NZB [5] with a model for split intein-mediated split-Cre recombinase-LoxP DNA complex (right). The structure of split inteins from IntN (GFL) and IntC (KGCFNKEDGS), NCre and CCre are colored as green, red, yellow and blue respectively. Conserved active site are marked with an asterisk. c Schematic demonstrating the predicted size of the protein when NCre-IntN tagged with HA (49 kD) or IntC-CCre tagged with FLAG (37 kD) are transfected alone. A 67 kD reconstituted product is expected when both NCre-IntN and IntC-CCre are cotransfected. d Western blot analysis showing that when NCre-IntN is transfected alone, a 37 kD protein band was observed when western blotting was performed using a specific antibody against HA. When IntC-CCre was transfected alone, a 49 kD protein band was observed when western blotting was performed using a specific antibody against FLAG. When NCre-IntN and IntC-CCre were cotransfected, a 67 kD protein band with a higher molecular weight was observed when western blotting was performed using a specific antibody against HA or FLAG. These results indicated that the reconstituted 67 kD product comprised of components from both NCre-IntN and IntC-CCre. e Design of the in vitro reporter assay. HEK293T cells were transfected with NCre-IntN or IntC-CCre alone or together with a LoxP-Stop-LoxP-DsRed reporter. f When NCre-IntN or IntC-CCre were transfected alone, no DsRed expression was observed. When NCre-IntN and IntC-CCre were cotransfected along with the reporter in HEK293T cells, we observed strong DsRed expression. Scale bar, 50 μm