Team:Ionis Paris/GLuc

The Gaussia Luciferase

A bit of chemistry

The Gaussia luciferase (GLuc) was identified from the marine copepod Gaussia princeps. Discovered relatively recently, this luciferase has been attracting attention as a potential reporter protein [1] and as a useful tool for bio-imaging applications. However, its biophysical properties have yet to be fully characterized.[2]

This luciferase is coelenterazine-dependent. It catalyzes the oxidation of its substrate, coelenterazine, into coelenteramide to emit blue light with a peak emission at 488 nm. This reaction does not require ATP or other co-factors for activity and follows a simple equation[2][3].

GLuc shows the strongest bioluminescence intensity (over 200 fold more than the firefly Photinus pyralis and the Renilla reniformis luciferase bioluminescence activity). Moreover, this luciferase shows narrow substrate specificity and is highly specific for coelenterazine. [4] Among all other luciferases, GLuc has the highest ability to catalyze the oxidation of coelenterazine for the luminescence reaction and this catalytic activity was found to be highly stable under a wide range of temperatures and in an acidic environment. However, its activity is salt dependent (with the highest activity at 50mM NaCl), strongly inhibited by heavy metal ions (Cu2+) and stimulated by monovalent ions (Cl−, I−, Br−).[2][4]


GLuc

Protein structure

GLuc's sequence was first reported in 1999 by isolated the cDNA from G. princeps and cloning it in E.coli cells. This protein is composed of 185 amino acid residues including a putative signal peptide sequence for secretion. [5] With a molecular mass of 19.9 kDa and a short coding sequence (555 bp), it is the smallest known luciferase.

This protein is composed of two domains (Domain 1: residues 27–97; Domain 2: 98–168) linked together and preceded by a pre-sequence (residues 1 – 26). GLuc is predicted to be a helical protein, and sequence alignment predicts that each domain contains two intra-domain disulfide bonds and an inter-domain disulfide bond between cysteines C59 and C120.

The highly conserved regions comprised of residues 52–77 in Domain 1 and 123–148 in Domain 2 seems to overlap with the active site. [2][4] Gaussia luciferase possesses a natural secretory signal (residues 1-16) composed of an N-terminal basic residue linked to a stretch of amino acids containing a hydrophobic core. [5] Upon expression, the luciferase is secreted into the cell medium. Therefore, lysing cells in order to assay GLuc activity is not necessary. [2]


References (Links are provided when available):

  1. Wille, T., Blank, K., Schmidt, C., Vogt, V., and Gerlach, R.G. (2012). Gaussia princeps Luciferase as a Reporter for Transcriptional Activity, Protein Secretion, and Protein-Protein Interactions in Salmonella enterica Serovar Typhimurium. Applied and Environmental Microbiology 78, 250–257

  2. Wu, N., Rathnayaka, T., and Kuroda, Y. (2015). Bacterial expression and re-engineering of Gaussia princeps luciferase and its use as a reporter protein. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1854, 1392–1399.

  3. Inouye, S., Sahara-Miura, Y., Sato, J., Iimori, R., Yoshida, S., and Hosoya, T. (2013). Expression, purification and luminescence properties of coelenterazine-utilizing luciferases from Renilla, Oplophorus and Gaussia: Comparison of substrate specificity for C2-modified coelenterazines. Protein Expression and Purification 88, 150–156.

  4. S. Inouye, Y. Sahara, Identification of two catalytic domains in a luciferase secreted by the copepod Gaussia princeps, Biochem. Biophys. Res. Commun. 365 (2008) 96–101.