The novel extremely psychrophilic luciferase from Metridia longa : Properties of a high-purity protein produced in insect cells

Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications

“…). This MLuc2 luciferase works with a maximum efficiency at low temperature around 5°C . The presence of the luciferase isoforms with different temperature optima in copepod M. longa seems to support their bioluminescent function during diel vertical migration when the temperature can vary from 24°C on the sea surface to negative values at the bottom.…”

“…Since GpLuc nonetheless contains five disulfide bonds , the fifth S‐S bond is most likely formed by these unpaired cysteines, thereby joining together these two domains of luciferase. Since it is evident that this interdomain disulfide bond does not appear at complementation, we may reasonably suggest that this S‐S bond is important rather for rigidity of protein conformation than for bioluminescence itself.…”

“…Also, no free SH groups were found in the investigated natively folded luciferases: MLuc7 isoform of M. longa and GpLuc at denaturing conditions; that is, these luciferases with ten conserved cysteines contain five intramolecular S‐S bonds per molecule. Meanwhile, the extremely psychrophilic MLuc2 isoform with a more flexible structure revealed only four S‐S bonds . Thus, the fifth disulfide bond is not crucial for the bioluminescent function of the copepod luciferase and obviously the third conservative cysteine (Cys76 in GpLuc) is involved in this bond formation.…”

“…Substantial differences in sequences of isoforms of one copepod species along with their greater similarity to the isoforms of other species allow postulating the existence of several paralogous genes for a luciferase within copepod genome. For instance, the cold‐active isoform MLuc2 of M. longa luciferase shows maximum sequence identity of 91% to MoLuc2 isoform from Metridia okhotens is, but not to other M. longa isoforms identity to which constitutes 54–64% (Fig. ).…”

“…The copepod luciferases were extensively secreted into the culture medium as natively folded proteins, when expressed in mammalian and insect cells with their native signal peptides. But the presence of up to five disulfide bonds per molecule of CopLucs makes obtaining of the correctly folded luciferases difficult under the expression in cytoplasm of prokaryotic and eukaryotic cells where the reductive environment hampers the formation of disulfide bonds. The bioluminescent activity in mammalian cells expressing Gaussia luciferase without signal peptide in cytoplasm was very low as compared to the activity in culture medium from the same type of cells secreting GpLuc which was expressed with signal peptide and maturated at oxidizing conditions of the endoplasmic reticulum during secretion.…”