Genetically Encoded Reporter for Fluorescence Detection

One drawback of proteins, when chemically labeled with low-molecular-weight fluorescent dyes, is their intracellular visualization, because labeled proteins cannot enter living cells without penetrating the cell membrane.

The identification and cloning [90] of GFP from Aequoria victoria has revolutionized the use of intracellular protein sensoring. GFP can be genetically fused to the target protein, and the fusion protein visualized after expression within the cells. GFP contains an internal chromophore that is responsible for the emission of green light, and is generated upon cyclization and oxidation of a Ser-Tyr-Gly sequence at positions 65-67 within the protein [91].

The color spectrum of fluorescent proteins was expanded with the identification of fluorescent proteins from Anthozoa species. These proteins share the same structure as GFP from Aequoria victoria, but two of them have totally different spectral characteristics, emitting on yellow and red wavelengths [92].

The use of GFP has certain limitations, notably that the protein has a relatively high molecular mass of 248 amino acids, and this can lead to aggregation and subsequent quenching of the fluorescence. One approach to overcome these limitations is to label the fusion proteins with synthetic fluorophores in living cells. The target protein of interest is genetically tagged with a receptor, which binds its synthetic ligand, labeled with a fluorescent dye, inside the cells. This technique was first introduced with the use ofa tetracysteine peptide as the receptor and biarsenical fluorophores as the ligands. This ligand is cell-permeable and nonfluorescent until it binds, with high affinity and specificity, to the tetracystein peptide of the fusion protein [93].

Another example of intracellular fluorophore labeling of fusion proteins is the use of O -alkylguanine-DNA alkyltransferase (AGT). This enzyme binds the substrate O -benzylguanosine, which is then derivatized with fluorescein, thereby enabling intracellular visualization of the tagged fusion protein [94].

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