(694e) A Modified Gaussia Luciferase Demonstrates Prolonged and Intense Bioluminescence

Luciferase bioluminescence has provided valuable molecular signals for both in vivo and in vitro applications in fields such as cancer diagnostics. Luciferase from Gaussia princeps (GLuc) is a small, secreted luciferase that has a much brighter signal compared to firefly luciferase from Photinus pyralis (FLuc) and Renilla reniformis (RLuc). However, the utility of GLuc has been limited by: difficulties in production due to its multiple disulfide bonds, burst kinetics that rapidly decay within seconds, and its blue emission peak (480 nm) which is attenuated by tissues and hemoglobin.

Our lab has successfully produced bioactive GLuc at high yields by utilizing a modified cell-free protein expression system. We have also addressed the burst kinetics of GLuc by identifying point mutations that allow GLuc to exhibit longer lasting glow kinetics similar to FLuc without showing a change in the native specific activity or emission spectrum. We have further used the cell-free synthesis platform to insert non-natural amino acids that allow for covalent attachment of active GLuc to antibody fragments that can recognize cell surface markers such as those unique for certain tumors.

The longer lasting signal of our mutated GLuc, combined with the high production and non-natural amino acid coupling capabilities of our cell-free protein synthesis platform, provides a valuable tool for in vitro studies and diagnostics.