Half-life of RLUC enzyme activity. (A) Time course of in vivo luminescence in E. coli cells expressing wild type RLUC and two P220 mutants. (B) Time course of in vitro luminescence of wild type RLUC and two P220 mutants. Enzyme and substrate concentrations were 10 nM and 2 μM respectively in PBS buffer pH7.2. Absolute activities were normalized for better comparison (first time-point = 100). Results shown are averages from three biological replicates each performed in triplicate. The Coomassie stained SDS-PAGE gels below show that the P220 mutants and wild-type RLUC accumulate to equivalent levels in E. coli. RLU stands for relative luminescence units.