Directed molecular evolution reveals Gaussia luciferase variants with enhanced light output stability

Abstract

Gaussia Luciferase (Gluc) has proven to be a powerful mammalian cell reporter for monitoring numerous biological processes in immunology, virology, oncology, and neuroscience. Current limitations of Gluc as a reporter include its emission of blue light, which is absorbed by mammalian tissues, limiting its use in vivo, and a flash-type bioluminescence reaction, making it unsuited for high-throughput applications. To overcome these limitations, a library of Gluc variants was generated using directed molecular evolution and screened for relative light output, a shift in emission spectrum, and glow-type light emission kinetics. Several variants with a 10-15 nm shift in their light emission peak were found. Further, a Gluc variant that catalyzes a glow-type bioluminescence reaction, suited for high-throughput applications, was also identified. These results indicate that molecular evolution could be used to modulate Gluc bioluminescence reaction characteristics.

Figure 1. Gluc variants and corresponding mutations in the second active domain characterized in this study

Shown, the Gluc signal peptide covering amino acids 1–27, first active domain covering amino acids 27–97, and second active domain covering amino acids 98–168.

Figure 2. Characterization of different Gluc variants

293T cells were transfected with different Gluc variants or wt Gluc and 48 hours later, aliquots of the conditioned medium were assayed for different properties. (a) Relative activity of each Gluc variant normalized to Fluc signal, a marker for transfection efficiency. (b) Enzymatic activity over repeated injections of coelenterazine (diluted in PBS) followed by signal acquisition using a plate luminometer with a built-in injector. (c) Bioluminescence light emission kinetics for each Gluc variant was analyzed by adding coelenterazine (diluted in PBS in the presence or absence of TritonX-100) and acquiring photon counts every 14 seconds over a period of 10 minutes using a luminometer.

Figure 3. Gluc4 variant displays a glow-type reaction suited for high-throughput applications

(a–b) Aliquots from conditioned medium of cells expressing wt Gluc, or Gluc4 were dispensed in 3 different 384-well plates in a high-throughput fashion followed by addition of coelenterazine (diluted in PBS) to all wells and signal acquisition (50 msec/well) using the Flexstation III device. Data presented in (a) as average raw RLUs/column ± SD and in (b) as average %RLU/column ± SD in which the first well was set at 100%. (c) U87 human glioma cells expressing Gluc4 or wt Gluc were plated in a 96-well plate and 12 wells/plate were marked and treated with 1 mM temozolomide. Seventy-two hours later, 10 μl aliquots of conditioned medium were transferred to a new 96-well plate to which 45 μl of coelenterazine was added to all wells simultaneously and signal was acquired using the Dynex luminometer column by column. Scatter plot of each plate in which data are presented as %RLU where the first well of the first column was set at 100%.