Bioluminescent and structural features of native folded Gaussia luciferase

Abstract

The secreted luciferases responsible for light emission of marine copepods have gained popularity for being used in noninvasive imaging of intracellular events. The secreted luciferase of copepod Gaussia princeps is a one-subunit protein catalyzing coelenterazine oxidation to emit blue light. It consists of the N-terminal variable part that bears a signal peptide for secretion and the C-terminal catalytic domain containing ten highly conserved Cys residues supposing the existence of up to five SS bonds. Despite wide application of Gaussia luciferase in biomedical research, its biochemical properties are still insufficiently studied due to the general problem of obtaining the proper folded Cys-rich proteins in bacterial cells. Here we report the properties of the proper folded Gaussia luciferase produced in insect cells using baculovirus expression system. This high purity luciferase reveals the highest activity at 15-20 °C but retains only ~20% activity at 37 °C that may hamper its application for in vivo assays. The maximum of bioluminescent activity of GpLuc is found at NaCl concentrations in the range of 1.0-1.5 M and, furthermore, a high NaCl concentration enhances luciferase stability to thermal denaturation, i.e. Gaussia luciferase displays the features characteristic of halophilic enzymes. The studies on bioluminescence kinetics at different coelenterazine concentrations obviously show a positive cooperativity of Gaussia luciferase with coelenterazine (Hill coefficient - 1.8 ± 0.2; K-2.14 ± 0.17 μM). We suggest this effect to be rather due to the so-called kinetic cooperativity conditioned by conformational changes in response to substrate binding than to the presence of two catalytic sites.

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

“…The presence of intramolecular disulfide bonds necessary for functional structure in CopLucs is supported by the fact that the addition of reducing agents such as dithiothreitol leads to a complete inactivation . 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 .…”

“…Both complementary fragments comprise five cysteines, one of which is unpaired (Cys76 and Cys137 in the first and second fragments of GpLuc, respectively), and consequently, each fragment may contain only two disulfide bonds. 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.…”

“…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.…”

“…The fully active recombinant copepod luciferases with a proper pattern of disulfide bonds were produced as secreted proteins using baculovirus–insect cell expression system. Three isoforms of M. longa luciferase, MLuc164 , MLuc7 and MLuc2 , and Gaussia luciferase were effectively expressed in Sf9 insect cells with their native signal peptides. Further, the luciferases were purified from serum‐free culture medium with a yield of 3–6 mg/L of a high‐purity protein and their properties were studied (see next section).…”

“…Recently, some structural and biochemical properties were reported for GpLuc and two different Metridia longa isoforms, MLuc7 and extremely psychrophilic MLuc2 , produced in insect cells using baculovirus expression system which allowed the proper folding of recombinant luciferases. The properties of these proteins most likely coincide with those of the same luciferases secreted by other eukaryotic cells.…”