New Cell Biology Assays Using a Stabilized Luciferase Exhibiting Red Luminescence

Abstract

Bioluminescence is the light produced in certain organisms as a result of luciferase enzyme mediated oxidation of a luciferin substrate. This oxidation is linearly dependent on the presence of adenosine tri‐phosphate (ATP). We have recently developed a codon‐optimized luciferase based upon the gene sequence of the natural luciferase isolated from Luciola cruciata (Japanese firefly). This new gene codes for a modified amino acid sequence which exhibits improved expression levels in mammalian cells, red‐shifted emission wavelengths (560 to 619 nm) as well as improved thermal stability, while still utilizing D‐luciferin and ATP as substrates.This new recombinant enzyme was purified to 98% homogeneity from both transfected E. coli bacteria and HEK 293 mammalian cells using Ni+2‐affinity chromatography as the His6‐tagged protein. Enzymes isolated from either source were found to have equivalent activity in ATP dependent assays. The purified recombinant luciferase was used to develop a number of ultrasensitive biological assays useful in measuring cell number, cell viability as well as the cytotoxicity effects of several chemotherapeutic drugs (Etoposide and Doxorubicin) when added to murine NIH3T3 cells in culture. We also utilized the purified enzyme to sensitively detect bacterial cell number down to 1 × 102 cells and adapted the methods for use in high‐throughput antibiotic screening protocols. By cloning the luciferase downstream of TFkB responsive elements, the activation of TF‐κB (with stimulation by TNFα) in human HEK 293 cells could be quantitated by directly monitoring the increase in luminescence from activation of luciferase expression. Finally, since this new luciferase has a red‐shift in light emission, it was utilized in multiplexed assay conditions with other green (firefly) or blue‐emitting (Renilla) luciferases in a number of coupled biological assays. Moreover, application of this new luciferase for use in in vivo imaging was explored.