FIG 3.

Detection of C-terminal HiBiT^opt tags. (A) Representation of the HiBiT^opt modular cassette. The protein of interest fused at the C terminus to HiBiT^opt through the GS linker is indicated. The positions of the restriction sites used (SacI, XhoI, and BamHI) are marked, and the cwp2 ribosomal binding site (rbs) is represented. (B) The proteins of interest were C-terminally fused to a HiBiT protein tag, and their expression was induced with 50-ng/ml ATc for 45 min. Optical density-normalized luciferase activity (RLU/OD) right before induction (T0), after 45 min of induction (T1), and after subsequent lysis of T1 samples (Lysed) is shown. HiBiT^opt-tagged sortase and HupA proteins were used as extracellular and intracellular controls, respectively. TcdC-HiBiT^opt-associated luciferase activity is also displayed. The averages for biological quadruplicate measurements are shown, with error bars indicating the standard deviation from the mean. *, P < 0.001 by two-way ANOVA. (C) Blot detection of HiBiT^opt-tagged proteins resolved on a 12% SDS-PAGE gel. Sample volumes were normalized for the optical density of the cultures from which they were derived. Expression of HiBiT^opt-fused proteins was observed at 0 min (T0) and 45 min (T1) after induction.