Dual detection of peptides in a fluorescence binding assay by employing genetically fused GFP and BFP mutants

A competitive fluorescence microplate assay based on a red-shifted green fl uorescent protein (rsGFP) and a blue fluorescent protein (BFP) was develope d for the detection of two model peptides in the same sample. The assay emp loyed 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 eith er the gene of the rsGFP or the BFP, as desired, The newly constructed plas mids were transformed into E, coli for expression of the fusion proteins. B y employing the technique of gene fusion, one-to-one homogeneous population s of peptide-rsGFP or -BFP conjugates were produced. These peptide-GFP muta nt conjugates exhibited the same excitation and emission spectral character istics as the unmodified proteins. The naturally fluorescent proteins act a s labels to provide sensitive dual detection of the two selected small pept ides in a competitive assay format, To our knowledge, this is the first tim e that mutants of GFP, such as the rsGFP and BFP, have been used as quantit ative labels for the development of a dual-analyte fluorescence immunoassay .