Construction and characterization of a red-emitting luciferase

Red light is transmitted through live tissue more efficiently than other wavelengths of visible light, thus by red-shifting the emission of bioluminescent reporters, we may enhance their utility for in vivo monitoring of biological processes. Codon changes at positions that may shift the yellow-green emission to red, based on studies of a related luciferase (Luciola cruciata), were introduced into a variant of the North American firefly (Photinus pyralis) luciferase. Clones containing the desired mutation were selected based on the introduction of unique restriction enzyme sites and transfected into NIH 3T3 cells. Expression levels were evaluated using an intensified charge coupled device (ICCD) camera. Upon spectral analysis, ail mutant luciferases demonstrated red-orange emission. Two emission peaks were detected in each spectrum, each clone with different peak heights at 560 nm (yellow-green, wild type) and 610 nm (red, mutant). Sequence analyses of the complete coding regions of several clones confirmed the presence of the target mutations, although sequence variation was observed at several secondary sites, likely resulting from the infidelity of Taq polymerase used in the mutagenesis protocol. A clone that demonstrated a strong 610 nm peak with a minimum shoulder at 560 nm was selected for use in animals. In summary, a redshifted mutant of a well-characterized luciferase reporter gene was generated. Red light from this enzyme may both penetrate mammalian tissues to a greater extent and provide a tool for multicolor biological assays.