Gaussia princeps luciferase: a bioluminescent substrate for oxidative protein folding

Abstract

Gaussia princeps luciferase (GLuc) generates an intense burst of blue light when exposed to coelenterazine in the absence of ATP. Here we show that this 5-disulfide containing enzyme can be used as a facile and convenient substrate for studies of oxidative protein folding. Reduced GLuc (rGLuc), with 10 free cysteine residues, is completely inactive as a luciferase but >60% bioluminescence activity, compared to controls, can be recovered using a range of oxidizing regimens in the absence of the exogenous shuffling activity of protein disulfide isomerase (PDI). The sulfhydryl oxidase QSOX1 can be assayed using rGLuc in a simple bioluminescence plate reader format. Similarly, low concentrations of rGLuc can be oxidized by millimolar levels of dehydroascorbate, hydrogen peroxide or much lower concentrations of sodium tetrathionate. The oxidative refolding of rGLuc in the presence of a range of glutathione redox buffers is only marginally accelerated by micromolar levels of PDI. This modest rate enhancement probably results from a relatively simple disulfide connectivity in native GLuc; reflecting two homologous domains each carrying two disulfide bonds with a single interdomain disulfide. When GLuc is reoxidized under denaturing conditions the resulting scrambled protein (sGLuc) can be used in a sensitive bioluminescence assay for reduced PDI in the absence of added exogenous thiols. Finally, the general facility by which rGLuc can recover bioluminescent activity in vitro provides a sensitive method for the assessment of inhibitors of oxidative protein folding.

Catalytic polymersomes to produce strong and long-lasting bioluminescence

“…To investigate the effects of encapsulation on the enzymatic production of light, we compared the bioluminescence emitted by non-encapsulated (free) enzyme to the one produced by encapsulated enzyme (GLuc Ncomp), at equal final concentration (0.2 µg/mL). 8,9,22 The typical flash-type kinetics of free GLuc was confirmed as after a fast transient burst, the signal quickly faded and was no more detectable after 5 min ( Fig 3A). The advantage of encapsulating GLuc inside polymersomes is immediately observable as the enzyme's kinetics is shifted towards a long-lasting light signal: after 1 min the luminescence signal of GLuc Ncomp overtakes that produced by free enzyme (Fig.…”

Flash properties of Gaussia luciferase are the result of covalent inhibition after a limited number of cycles

“…GLuc has been developed as a tool in several applications ranging from reporter of gene expression, in bacteria and mammals to split‐protein‐complementation assays 5,7–9 . In particular, it has been promoted as a sensitive and useful tool for studying gene expression, systems of secretion 10 or for monitoring disulfide bond formation 11 . Due to the reducing conditions in the cytosol of Escherichia coli and lack of disulfide isomerases, expression here typically gives very low yields of correctly folded protein.…”

Flash properties of Gaussia Luciferase are the result of covalent inhibition after a limited number of cycles

“…Although excellent work has been carried out on in vitro formation of disulfide bonds in GLuc (Yu et al, 2018) and on cell-free expression of correctly folded monomers (Goerke et al, 2008) M CTZ, resulting in a peak in light emission. Irregular spikes between 800 s and 900 s were due to the syringe being taken out for the second injection.…”

“…GLuc has been developed as a tool in several applications ranging from reporter of gene expression, in bacteria and mammals to split-protein-complementation assays (Maguire et al, 2009;Kim et al, 2011;Remy et Michnick, 2006;Markova et al, 2019). In particular, it has been promoted as a sensitive and useful tool for studying gene expression, systems of secretion (Tannous, 2009) or for monitoring disulfide bond formation (Yu et al, 2018). Due to the reducing conditions in the cytosol of E. coli and lack of disulfide isomerases, expression here typically gives very low yields of correctly folded protein.…”