Kinetic characterization and in vitro toxicity evaluation of a luciferase less susceptible to HPV chemical inhibition

Enzyme-based in vitro toxicity assays are often susceptible to inhibition by test compounds. A mutant luciferase selected to be less susceptible to inhibition by chloroform (CNBLuc03-06) and other high production volume (HPV) chemicals, consisting of three point mutations was created and characterized. The mutant luciferase was less inhibited by chloroform, other HPV chemicals and common surfactant release reagents (Triton-X and SDS) compared to the wild-type. Inhibition was shown to be competitive. CNBLuc03-06 was a factor of 1.5–3.2 more active than wild type and exhibited a much higher affinity for ATP. CNBLuc03-06 was more thermostable than wild-type and also more active at pH values higher than 10. Both luciferases exhibited a significant tradeoff between activation and susceptibility to chemical inhibition in the presences of the reducing agent DTT. Inhibition to HPV chemicals was eliminated using an “optimum” formulation of DTT and co-solvent ethanol. The performance of CNBLuc03-06 in cell-based in vitro toxicity assays was shown to be superior to the current commercial formulation.