Beyond GFP and aequorin: Ocean-wide study of fluorescent and luminous proteins

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DESCRIPTION (provided by applicant): In vivo labeling and detection technologies based on genetically encoded fluorescent and luminescent markers is indispensable for modem biomedical science. We will clone proteins responsible for fluorescence and luminescence from a wide variety of organisms found in the open ocean and deep sea, and assess the usefulness of these proteins for biotechnology applications by expressing them in bacteria, mammalian cell culture and Xenopus laevis embryos. We are well poised to carry out such a project. Both our laboratories have regular access to oceanic and deep-sea animals through participation in research cruises sponsored by other agencies, including expeditions specifically equipped for studying physiology and ecology of oceanic fluorescence and luminescence. Our sampling methods include plankton towing, blue water SCUBA diving, midwater trawling and submersible/ROV operations. A collection of potential cloning targets has been already assembled that includes spectacular taxonomic diversity of animals, ranging from radiolarians and jellyfish to sea urchins and fish. This collection will be further augmented during upcoming research cruises in Caribbean, Eastern and Western Pacific. In the same time, we possess extensive experience in cloning, expression and analysis of fluorescent (Matz) and luminescent (Haddock) proteins. The guaranteed result will be identification of a series of novel calcium-dependent photoproteins, as well as GFP-like proteins possessing combinations of biophysical, biochemical and optical features that are not found in thoroughly sampled Anthozoa (such as far-red fluorescence, true monomeric state and rapid maturation). Our ultimate goal, however, is to identify completely novel (i.e., sharing no homology with the previously cloned ones) genetically encoded fluorescent and luminescent markers which may become a basis for the new generation of in vivo labeling and detection technologies.