Mutant Luciferase Research Articles

Abstract A newly developed procedure is described for the isolation and purification on a large scale of luciferases from two different bioluminescent bacteria, Pf and MAV. In only three major steps luciferase can be obtained in greater than 95% purity. An improved method for the isolation of subunits in milligram quantities by column chromatography in 5 m urea is also described; upon recombination of the subunits with the removal of urea, renatured luciferase with a high specific activity is obtained. These procedures should be generally applicable with luciferases from different bacterial strains, including mutant luciferases. The two luciferases have been used to produce specific antiluciferases, which, by precipitation patterns on Ouchterlony plates, appear distinctively different. Nevertheless, a degree of homology has been demonstrated by cross-reaction in an antibody activity inhibition assay. Interaction between subunits from the different strains has also been demonstrated by inhibition of renaturation (from 5 m urea) of one of the luciferases by a subunit of the other luciferase, indicating that inactive hybrids may be formed.