Plasmid-Based Reporter Genes: Assays for Green Fluorescent Protein

Green fluorescent protein (GFP) of the jellyfish Aqueorea victoria is a 238-amino-acid, 28-kDa protein that absorbs light with an excitation maximum of 395 nm and fluoresces with an emission maximum of 509 nm (1 ). GFP owes its unique spectral properties to its chromophore (2 ) that consists of a Ser65, Tyr66, and Gly67 tripeptide (3 ). Autocatalytic cyclization of this tripeptide, induced by oxidation of Tyr66, is a necessary posttranslational step for proper fluorescence (strong reducing agents reversibly convert GFP into a nonfluorescent form) (4 ). This can occur in the absence of any cofactors, making GFP an extremely useful tool for a wide range of applications in a variety of heterologous systems (5 –7 ). GFP activity can be assayed both qualitatively and quantitatively using a variety of techniques, including simple plate counting, fluorescence and confocal microscopy, flow cytometry, and fluorometry. Transcriptional and translational fusions of GFP to a gene or protein of interest can be used as gene expression reporters and subcellular localization tags. GFP is a small protein (28 kDa) compared to other reporters (e.g., β-galactosidase is 465 kDa) and GFP fusions often retain the native protein function (8 –10 ). This makes GFP useful as a generic tag for studying protein synthesis, translocation, and other protein–protein interactions. GFP is also widely used as a reporter in many genetic techniques, including transposon mutagenesis, promoter/enhancer traps, and one-component hybrid systems. GFP can be visualized using microscopy in both live and fixed cells, making it an excellent tool for studying dynamic changes in living cells.