Though radioactivity is widely used in bioassays due to its high sensitivity and convenience, safety and environmental concerns have accelerated efforts to eliminate its use. Several recent examples of procedures using luminescence have surpassed conventional isotopic methods. Among these, luminescence of firefly luciferase is the most efficient known. The investigators have completed significant new research advancing the knowledge of the enzymatic mechanism of firefly luciferase. The new chemistry provides greater luminescence intensity with simpler reaction kinetics. To gain a broader perspective of properties of beetle luciferases and to develop enzymes better suited to commercial applications, the investigators propose to examine the structure and function of several divergent isozymes by using cDNA clones already isolated coding for these isozymes. Phase I will assess the feasibility of this proposal by expressing and purifying the cloned luciferases in E.coli developing methods for random mutagenesis, and initiating crystallization studies. Phase II will focus on characterizing the recombinant luciferases, examining their relative structural relationships, enhancing their properties by random mutagenesis, and generating protein crystals of the new isozymes. Phase III efforts will use the knowledge acquired on enzyme property enhancement to develop a number of bioassay-related and other commercial applications.
