title: Improved assay sensitivity of an engineered secreted Renilla luciferase.
abstract: We have previously reported the construction of a functional Renilla luciferase enzyme secreted by mammalian cells when fused to the signal peptide of human interleukin-2. The presence of three predicted cysteine residues in the amino acid sequence of Renilla luciferase suggested that its secreted form could contain oxidized sulfhydryls, which might impair enzyme activity. In this work, four secreted Renilla luciferase mutants were constructed to investigate this possibility: three luciferase mutants in which a different cysteine residue was replaced by an alanine residue, and one luciferase mutant in which all three cysteine residues were replaced by alanine residues. Simian cells were transfected with the genes encoding these mutant luciferases, as well as with the original gene construct, and cell culture media were assayed for bioluminescence activity. Only media containing a mutated luciferase with a cysteine to alanine substitution at position 152 in the preprotein showed a marked increase in bioluminescence activity when compared to media containing the original secreted Renilla luciferase. This increase in light emission was due in part to enhanced stability of the mutant enzyme. This new enzyme represents a significant improvement in the sensitivity of the secreted Renilla luciferase assay for monitoring gene expression.
authors.affiliation: Center for Molecular Biology and Gene Therapy, Loma Linda University, CA, USA.
authors.forename: J
authors.initials: J
authors.lastname: Liu
authors.forename: A
authors.initials: A
authors.lastname: Escher
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods; D019556:COS Cells; D003063:Cnidaria / Q000201:enzymology*; D003470:Culture Media; D004795:Enzyme Stability; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism*; D008163:Luminescent Measurements; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D014162:Transfection
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D008156:Luciferases