A photoactivatable green-fluorescent protein from the phylum Ctenophora

Abstract

Genes for the family of green-fluorescent proteins (GFPs) have been found in more than 100 species of animals, with some species containing six or more copies producing a variety of colours. Thus far, however, these species have all been within three phyla: Cnidaria, Arthropoda and Chordata. We have discovered GFP-type fluorescent proteins in the phylum Ctenophora, the comb jellies. The ctenophore proteins share the xYG chromophore motif of all other characterized GFP-type proteins. These proteins exhibit the uncommon property of reversible photoactivation, in which fluorescent emission becomes brighter upon exposure to light, then gradually decays to a non-fluorescent state. In addition to providing potentially useful optical probes with novel properties, finding a fluorescent protein in one of the earliest diverging metazoans adds further support to the possibility that these genes are likely to occur throughout animals.

Figure 1.

In vivo fluorescence of the ctenophore H. beehleri. (a) Live animal photographed in white light. Green tint is due to fluorescent proteins. Scale bar, 1.5 mm. (b) A detailed view using blue-light illumination with yellow long-pass filter showing the fluorescent ectodermal cells. Scale bar, 0.35 mm.

Figure 2.

Unrooted tree of known fluorescent proteins showing the relationships of the major groups of known fluorescent proteins. Major lineages cluster together, although cnidarian (Anthozoan + Hydrozoan) FPs do not form a monophyletic group. See §2 for information about alignment and tree-building. Orange, Anthozoa; green, Hydrozoa; blue, Copepoda; plum, Ctenophora; grey, Chordata.

Figure 3.

(a) Fluorescence absorption and emission spectra before (red line) and after (blue line) photoactivation, along with the fluorescence emission spectrum (green solid line). Arrowhead indicates region of maximum absorbance in active state, with no absorbance before photoactivation. Absorbance values of activated protein (blue) correspond to the excitation spectrum for fluorescence. (b) Time series of photoactivation for HbGFP with two different excitation sources: magenta dashed line, 15.0 mW at 450 nm; blue solid line, 0.3 mW at 460 nm. (c) Time series of fluorescence decay. Solid line shows the data, whereas dashed grey line is an overlay of the fitted exponential decay function. Note that time scales on (b) and (c) are different.

Figure 4.

Native PAGE gel of purified recombinant FPs. (a) White light image of coomassie-blue-stained standards and novel FPs. (b) A blue-illuminated image of the same gel, showing fluorescence emission of native proteins. The native ladder (first column) includes a fluorescent marker that is visible in (b). Lanes 1–5 in both images: native protein ladder, monomeric DsRed, tetrameric DsRed, HbGFP sample 1, HbGFP sample 2. The two H. beehleri samples were from clone HbGFP1 used in this study, but they were expressed and prepared at different times.