Dual-function reporter protein for analysis of gene expression in living cells.
The firefly luciferase (Luc) protein and the jellyfish green fluorescent protein (GFP) are two commonly used molecular reporters that can be detected noninvasively in living cells. The properties that make GFP or Luc useful for a particular experimental application are quite distinct. A recombinant protein with both fluorescent and bioluminescent characteristics might take advantage of the strengths of both reporters. An expression vector encoding a chimeric protein in which GFP was tethered to Luc through a 19-amino acid linker was prepared and characterized. Western blotting with antibodies specific for either GFP or Luc showed that a protein of appropriate size was expressed in transfected cells. Fluorescence microscopy revealed bright green fluorescence from transfected cells, indicating proper formation of the GFP chromophore. Luc enzymatic activity in protein extracts from transfected cells showed that Luc was fully functional. The treatment of living cell cultures stably expressing the GFP-Luc fusion protein with the protein translation-inhibitor cycloheximide (Chx) was used to show that the half-life for Luc protein activity was approximately 2 h at 37 degrees C. The utility of this dual-function reporter protein was shown by the identification of single living cells expressing the chimeric protein within a population by fluorescence microscopy, followed by quantification of Luc activity from the same living cells.
Day
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RN
University of Virginia Health Sciences Center, Charlottesville, USA.
Kawecki
M
M
Berry
D
D
D002460:Cell Line; D004795:Enzyme Stability; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases