title: Dual detection of peptides in a fluorescence binding assay by employing genetically fused GFP and BFP mutants.
abstract: A competitive fluorescence microplate assay based on a red-shifted green fluorescent protein (rsGFP) and a blue fluorescent protein (BFP) was developed for the detection of two model peptides in the same sample. The assay employed gene fusion to prepare the fluorescently labeled peptide conjugates. Specifically, plasmids were constructed in which the genes encoding for the two small peptides (less than 12 amino acids in length) were fused to either the gene of the rsGFP or the BFP, as desired. The newly constructed plasmids were transformed into E. coli for expression of the fusion proteins. By employing the technique of gene fusion, one-to-one homogeneous populations of peptide-rsGFP or -BFP conjugates were produced. These peptide-GFP mutant conjugates exhibited the same excitation and emission spectral characteristics as the unmodified proteins. The naturally fluorescent proteins act as labels to provide sensitive dual detection of the two selected small peptides in a competitive assay format. To our knowledge, this is the first time that mutants of GFP, such as the rsGFP and BFP, have been used as quantitative labels for the development of a dual-analyte fluorescence immunoassay.
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington 40506-0055, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Lewis
authors.forename: S
authors.initials: S
authors.lastname: Daunert
mesh_terms: D017123:Bacteriophage T7 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001667:Binding, Competitive; D002138:Calibration; D002213:Capsid / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005453:Fluorescence; D015200:Fluoroimmunoassay / Q000379:methods*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D010455:Peptides / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D008164:Luminescent Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins