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A complementary DNA for the Aeguorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabitis elegans) cells. Because exogenous substrates and cofactors are required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.
Light is produced by the bioluminescent jellyfish Aequorea victoria when calcium binds to the photoprotein aequorin (1). Although activation of aequorin in vitro or in heterologous cells produces blue light, the jellyfish produces green light. This light is the result of a second protein in A. victoria that derives its excitation energy from aequorin (2), the green fluorescent protein (GFP).
Purified GFP, a protein of 238 amino acids (3), absorbs blue light (maximally at 395 nm with a minor peak at 470 nm) and emits green light (peak emission at 509 nm with a shoulder at 540 nm) (2, 4). This fluorescence is very stable, and virtually no photobleaching is observed (5). Although the intact protein is needed for fluorescence, the same absorption spectral properties found in the denatured protein are found in a hexapeptide that starts at amino acid 64 (6, 7). The GFP chromophore is derived from the primary amino acid sequence through the cyclization of serine-dehydrotyrosine-glycine within this hexapeptide (7). The mechanisms that produce the dehydrotyrosine and cyclize the polypeptide to form the chromophore are unknown. To determine whether additional factors from A. victoria were needed for the production of the fluorescent protein, we tested GFP fluorescence in heterologous systems. Here, we show that GFP expressed in prokaryotic and eukaryotic cells is capable of producing a strong green fluorescence when excited by blue light. Because this fluorescence requires no additional gene products from A. victoria, chromophore formation is not species-specific and occurs either through the use of ubiquitous cellular components or by autocatalysis.
Expression of GFP in Escherichia coli (8) under the control of the T7 promoter results in a readily detected green fluorescence (9) that is not observed in control bacteria. Upon illumination with a long-wave ultraviolet (UV) source, fluorescent bacteria were detected on plates that contained the inducer isopropyl-beta-D-thiopalactoside (IPTG) (Fig. 1). (Fig. 1 omitted) Because the cells grew well in the continual presence of the inducer, GFP did...