title: Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae.
abstract: Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon-optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half-lives of 40 and 5 min, respectively. The commercial substrate Nano-Glo® is compatible with detection of yNluc bioluminescence in < 50 cells. Using the unstable yNlucPEST to report on the rapid and transient expression of a heat-shock promoter (PCYC1-HSE ), we found a close match between the intensity of the bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C-terminus of a temperature-sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd.
authors: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Sweden.
authors: Anna E
authors: AE
authors: Masser
authors: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Sweden.
authors: Ganapathi
authors: G
authors: Kandasamy
authors: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Sweden.
authors: Jayasankar Mohanakrishnan
authors: JM
authors: Kaimal
authors: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Sweden.
authors: Claes
authors: 0000-0001-8948-0685
authors: C
authors: Andréasson
mesh_terms: D000818:Animals; D003445:Crustacea / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal / Q000502:physiology*; D017930:Genes, Reporter; D008156:Luciferases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D036103:Nanotechnology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D008156:Luciferases
keywords: bioluminescence; gene expression; luciferase; protein stability; reporter