Jennifer DeAngelis
Connecticut College
Subject Listing - Biology: Molecular Biology
Advisor: Dr. Bruce Branchini
Saturday, Poster Session 6, Presentation Kiosk 7 A, Health & Fitness Center
CLONING AND EXPRESSION OF LUCIOLA ITALICA LUCIFERASE GENE AND PRELIMINARY PROTEIN CHARACTERIZATION
The purpose of this project was to clone the Luciola italica luciferase gene; and to express, purify and characterize the corresponding bioluminescence-catalyzing enzyme. Fireflies were collected in the hills surrounding Bologna, Italy and transported to the laboratory of our Italian collaborator Professor Roda where they were flash frozen in liquid nitrogen. Total RNA was then isolated from the firefly lanterns and the L. italica luciferase cDNA was cloned by RT-PCR using a gene-specific primer set based on the DNA sequence of the Eastern European Luciola mingrelica luciferase. The gene was ligated into the pGEX-6P2 plasmid, which is designed to produce proteins as N-terminal glutathione-S-transferase-fusion products. A highly specific protease can then be used to cleave the protein of interest away from the GST and linker peptide thereby facilitating protein purification. The L. italica gene was sequenced and determined to be 1647 base pairs in length with an open reading frame of 548 amino acids. Prior to expressing the protein, it first was necessary to realign the reading frame of the cDNA in the pGEX-6P-2 plasmid by PCR-based mutagenesis. The L. italica gene was then expressed as the GST-fusion protein in E. coli BL21 cells and purified to homogeneity by affinity chromatography. Initial characterization of the enzyme showed that 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 (maximum emission at 565 nm vs 557 nm). By steady state kinetics analysis, the L. italica Km for LH2 was found to be 0.095mM, approximately 6-fold greater than that of the P. pyralis enzyme. Interestingly, both enzymes had similar Km values for Mg-ATP (0.160 mM). Phylogenetic analysis showed that L. italica luciferase was similar to that of other fireflies in the family Lampyridae, subfamily Luciolinae with 95.8% and 95.6% homology to the Hotaria unmunsana (Korea) and Hotaria parvula (Japan) luciferases, respectively.
Connecticut College
Chemistry Department
Advisor: Dr. Bruce Branchini, Chair and Hans and Ella McCollum '21 Vahlteich Professor of Chemistry, Chemistry, Connecticut College, New London, CT