Cloning, Sequencing, and Characterization of Luciola italica Luciferase

The characteristic yellow-green light of a firefly is the result of a multi-step reaction catalyzed by the luciferase enzyme. This enzyme has many applications in the biomedical field and ongoing work is being done to alter its properties to better fit these applications. The purpose of this project was to clone the Luciola italica luciferase cDNA and to express, purify and fully characterize the corresponding bioluminescence-catalyzing enzyme in hopes of obtaining novel bioluminescent materials. Fireflies were collected in the countryside of Bologna, Italy, flash frozen in liquid nitrogen and total RNA was extracted from the firefly lanterns. The L. italica luciferase cDNA was successfully cloned by RT-PCR using a gene-specific primer set based on the DNA sequence of the Eastern European Luciola mingrelica luciferase gene. The L. italica cDNA was determined to be 1647 base pairs in length with an open reading frame of 548 amino acids. Initial characterization of the enzyme showed that the L. italica protein exhibits bioluminescent activity similar in intensity to the common North American Photinus pyralis luciferase; however it produces light that is slightly red-shifted (having maximum emission at 564 nm). By steady state kinetics analysis, the L. italica Km for LH2 was found to be 0.095 mM, and that of P. pyralis is 0.015 mM. On the converse, both enzymes had similar Km values for Mg-ATP (0.160 mM for P. pyralis and 0.180 mM for L. italica). The L. italica enzyme was found to sustain its light in the visible region for a longer period of time than the P. pyralis enzyme. Phylogenetic analysis showed that the L. italica luciferase gene has 95.8% and 95.6% amino acid sequence identity to the Hotaria unmunsana (Korea) and Hotaria parvula (Japan) luciferase proteins, respectively. The processes that were used to clone the Luciola italica luciferase gene, characterize the protein, and optimize protein growth conditions ar