Fig. 5.
Analysis of alanine substitutions at conserved cysteines in the Bvi IcmO intein. a Double mutations were made in the MVP precursor substituting alanine for AsnG:7 and either CysF:4 or Cys+1. The AsnG:7 substitution prevents on-pathway BIG resolution. MVP precursors with the indicated residues mutated to alanine were expressed at 30 °C for 2 h or 15 °C overnight. Left panel SDS-PAGE stained with Simply Blue Safe Stain. Right panel Western Blots were probed using IR-dye tagged secondary antibodies with M shown in green and P shown in red. Yellow bands indicate an equal reaction with both secondary antibodies. b The forward and reverse reaction scheme for MVP precursor to BIG. The red ‘X’ indicates that this reaction was not detected