Abstract
BackgroundMembers of the green fluorescent protein (GFP) family share sequence similarity and the 11-stranded β-barrel fold. Fluorescence or bright coloration, observed in many members of this family, is enabled by the intrinsic properties of the polypeptide chain itself, without the requirement for cofactors. Amino acid sequence of fluorescent proteins can be altered by genetic engineering to produce variants with different spectral properties, suitable for direct visualization of molecular and cellular processes. Naturally occurring GFP-like proteins include fluorescent proteins from cnidarians of the Hydrozoa and Anthozoa classes, and from copepods of the Pontellidae family, as well as non-fluorescent proteins from Anthozoa. Recently, an mRNA encoding a fluorescent GFP-like protein AmphiGFP, related to GFP from Pontellidae, has been isolated from the lancelet Branchiostoma floridae, a cephalochordate (Deheyn et al., Biol Bull, 2007 213:95).ResultsWe report that the nearly-completely sequenced genome of Branchiostoma floridae encodes at least 12 GFP-like proteins. The evidence for expression of six of these genes can be found in the EST databases. Phylogenetic analysis suggests that a gene encoding a GFP-like protein was present in the common ancestor of Cnidaria and Bilateria. We synthesized and expressed two of the lancelet GFP-like proteins in mammalian cells and in bacteria. One protein, which we called LanFP1, exhibits bright green fluorescence in both systems. The other protein, LanFP2, is identical to AmphiGFP in amino acid sequence and is moderately fluorescent. Live imaging of the adult animals revealed bright green fluorescence at the anterior end and in the basal region of the oral cirri, as well as weaker green signals throughout the body of the animal. In addition, red fluorescence was observed in oral cirri, extending to the tips.ConclusionGFP-like proteins may have been present in the primitive Metazoa. Their evolutionary history includes losses in several metazoan lineages and expansion in cephalochordates that resulted in the largest repertoire of GFP-like proteins known thus far in a single organism. Lancelet expresses several of its GFP-like proteins, which appear to have distinct spectral properties and perhaps diverse functions.ReviewersThis article was reviewed by Shamil Sunyaev, Mikhail Matz (nominated by I. King Jordan) and L. Aravind.
Highlights
Members of the green fluorescent protein (GFP) family share sequence similarity and the 11-stranded β-barrel fold
We identified a family of genes encoding GFP-like proteins in lancelet B. floridae and expressed two of them in bacterial and mammalian cell cultures
LanFP1 and LanFP2, exhibit green fluorescence of different brightness and distinct spectral properties when expressed in mammalian cells, and they, along with at least four other GFP-like proteins, appear to be expressed at various stages of lancelet development as judged from the analysis of the EST libraries
Summary
Members of the green fluorescent protein (GFP) family share sequence similarity and the 11-stranded β-barrel fold. GFPs proved to be extraordinarily amenable to genetic manipulation: some of the useful traits of the engineered GFP derivatives include shifts in the maxima of excitation and/or emission; timed responses, such as kindling or color change after excitation; photoactivation; assembly of functional monomers from the fragments of the molecule; and others [3,4,5]. With all this knowledge about structure-function relationships in the GFP family, there is a considerable interest in naturally occurring GFP-like proteins with novel properties. The lack of discernible sequence similarity between G2 domains and GFPs, leaves open the evolutionary questions, i.e., whether the two β-barrels descend from the common ancestral gene, and, if such common ancestor existed, what was its function and in which organism did it reside
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