• Cloning, Over-expression and Purification of Nanoluc

      DuPlain, Holly; Parks, Jasmine; Blocker, Brittany; College of Science and Mathematics; Spencer, Angie (2015-11-13)
      Bioluminescence is a natural phenomenon that occurs in bacteria, insects, fungi and some marine species whereby a living organism emits light via a chemical reaction catalyzed by enzymes called luciferases. Luciferases are utilized in various applications including Bioluminescence Resonance Energy Transfer (BRET). BRET uses a luciferase (energy donor) to transfer energy to a fluorescent protein or dye (energy acceptor). If the donor and acceptor are close together and their emission and absorbance spectra overlap, the acceptor absorbs the energy from the donor and light is emitted at a longer wavelength. This spectral shift is measured. One such luciferase is Nanoluc (Nluc), a genetically engineered enzyme from the deep sea shrimp Oplophorus gracilirostris. To explore the use of Nluc as a BRET energy donor, the Nluc gene was cloned into the plasmid vector pET21c(+). Recombinant DNA formation was verified by agarose gel electrophoresis. After transformation of the recombinant plasmid into E. coli BL21 cells, C-terminal His6 tagged Nluc protein was over-expressed and purified using affinity chromatography. Purification yielded a relatively pure protein with a molecular weight of 19 kDa as judged by SDS-PAGE. Protein activity was assessed by measuring its ability to generate light in the presence of the substrate coelenterazine. Begin Time: 08:25 End Time: 31:16