The Cloning and Characterization of GFP-like Proteins From the Abnormally Pink Pigmented Tissues of Porites lobata

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Bridges, Mary Catherine
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Fluorescent proteins (FPs) form a family of proteins with homologies to green fluorescent protein (GFP), originally isolated from the bioluminescent jellyfish Aequorea victoria; the majority of this family's members have been cloned from coral species, yielding FPs with emission wavelengths along the entire visible spectrum. In compromised tissues of Porites species, a localized visible pink pigmentation exhibiting red fluorescence has been reported; this pathology has been described most recently as an innate immune response. Here we report the cloning, expression, and characterization of a novel red fluorescent protein (tprRFP) and purple chromoprotein (tprCP) from the pink tissue of a Porites lobata specimen. The recombinant tprRFP produces a bright red fluorescence with an Em. Max of 614 nm (Ex. Max: 578 nm), similar to the endogenous red fluorescence of the pigmented coral tissue. This 614 nm emission makes tprRFP the furthest red-shifted, naturally occurring protein isolated. This property, as well as its inherent brightness, photostability, and pH stability characterized in vitro, make it an excellent candidate for deep tissue and cell imaging applications. The non-fluorescent chromoprotein, tprCP, absorbs maximally at 581 nm, resulting in its visible purple coloration, which is visually distinct from the pink of the P. lobata tissue and recombinant tprRFP. We suggest tprRFP is mostly responsible for the visible pink coloration of the coral tissue. The isolation of the tprRFP/CP coding sequences provide the means to more thoroughly investigate the underlying cellular pathology in corals displaying pink tissue pigmentation response as well as evaluate proposed specific roles for these GFP-like proteins in this response.
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