We have identified a specific methionine to leucine mutation in Gaussia luciferase that confers stabilized light emission signal at the expense of a drop in specific activity, similar to a recent finding [13]. We have also identified an additional mutation that, in combination, further increases the light emission
Tipifarnib while restoring the specific activity to nearly that of the wild-type enzyme. To our knowledge, this combination of mutations produces the most active GLuc with the longest lasting luminescence signal reported to date. We also show the feasibility of click chemistry conjugation to exposed homopropargylglycine residues incorporated at
methionine sites. While the crystal structure of Gaussia luciferase is currently unknown, our GLuc–PEG conjugation data indicate that these beneficial mutations are on or near the surface of the enzyme. The prolonged emission signal, in combination with the increased production capacity provided by cell-free protein synthesis, should allow this mutated luciferase to be a highly effective enzyme for in vivo and in vitro bioluminescence applications.
