Cloning, Expression, and Purification of Nanoluc

Abstract

Bioluminescence is a natural phenomenon that occurs in bacteria, insects, fungi and some marine species whereby light is emitted by a living organism. This emitted light is generated by a chemical reaction that occurs when a substrate is reacted upon by a class of enzymes called luciferases. Bioluminescence Resonance Energy Transfer (BRET) is a technique that relies on the use of a luciferase (energy donor) to transfer energy to a nearby fluorescent protein or dye (energy acceptor). If the donor and acceptor molecules are in close proximity and their emission and absorbance spectra overlap, the acceptor absorbs the energy from the donor (luciferase) which results in the emission of light at a longer wavelength (i.e. different color). This spectral shift can be measured and quantified. Because of the widespread applications and utility of luciferase enzymes, many assay systems have been developed that make use of various luciferases as energy donors. One such luciferase is Nanoluc (Nluc), a genetically engineered enzyme from the deep sea shrimp, Oplophorus gracilirostris. In order to explore the use of Nluc as an energy donor in BRET, we cloned the gene for Nluc into the plasmid vector, pET21c(+). The formation of the recombinant DNA was verified by agarose gel electrophoresis. After transformation of the recombinant plasmid into E. coli BL21 cells, the Nluc protein containing a C-terminal His6 tag was over-expressed and purified using affinity chromatography. The purification yielded a relatively pure protein with a molecular weight of 19 kDa as judged by SDS-PAGE. The activity of the protein was assessed by measuring its ability to generate light in the presence of the substrate coelenterazine.