Gaussia princeps luciferase

Excerpt

Proteins engineered to generate fluorescent or bioluminescent signals are commonly used for the non-invasive study of a variety of biological processes and diseases in animals and humans (1-3). Usually the enhanced green fluorescent proteins, the variant red or yellow fluorescent proteins, or the bioluminescent luciferase proteins are used as reporter molecules because they produce a photo signal upon stimulation that can be tracked for the study of a dynamic biological system or phenomenon under in vitro and in vivo conditions (4-6). The luciferase family is composed of several different enzymes that catalyze light-producing, oxygen-dependent molecular reactions and have been detected across a diverse group of organisms including bacteria, fungi, insects, and marine animals (7). Among the luciferases, the firefly (Photinus pyralis) luciferase (FLuc) has been extensively studied; it requires ATP and Mg²⁺ as co-factors to function and oxidizes D-luciferin, its substrate, to release energy in the form of photons (8). The sea pansy (Renilla reniformis) is another luciferase (RLuc) that has been extensively investigated; RLuc uses coelenterazine as a substrate and does not require ATP to produce light. However, RLuc has a lower quantum yield compared with FLuc (6% versus 88%) and also shows a low enzymatic efficiency (9). With the use of commercially available molecular kits, the humanized forms of FLuc and RLuc (designated as hFLuc and hRLuc, respectively) were combined as dual reporters to image cultured cells in vivo because both enzymes used different substrates (10, 11). However, these enzymes have very specific requirements to catalyze reactions under in vivo conditions to generate sufficient signal/background ratios for imaging. Tannous et al. decided to explore the possibility of finding a superior luciferase that was more efficient and had a higher quantum yield compared with the FLuc and RLuc enzymes (12).

Gaussia princeps, a marine copepod, naturally secretes a luciferase (GLuc) that was cloned, expressed in Escherichia coli, and used as a detection agent in a DNA hybridization assay (13). Tannous et al. cloned the humanized version of the GLuc enzyme (hGLuc) into a herpes simplex virus-1 (HSV-1) amplicon under a cytomegalovirus (CMV) immediate early (IE) promoter and evaluated the bioluminescence emitted from DNA-transfected and vector-infected mammalian cells (12). The luciferase was also evaluated for imaging cultured cells in vivo after subcutaneous implantation of GLuc-transfected cells into nude mice (12). In another study, GLuc was evaluated for imaging after it was cloned into a lentivirus (LV) vector and transduced into human glioma cells that were implanted into nude mice (14).