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

描述（申请人提供）：基于遗传编码荧光和发光标记的体内标记和检测技术是现代生物医学科学不可缺少的。我们将从公海和深海中发现的各种生物中克隆负责荧光和发光的蛋白质，并通过在细菌、哺乳动物细胞培养和非洲爪蟾胚胎中表达这些蛋白质来评估这些蛋白质在生物技术应用中的有用性。我们已经做好了实施这样一个项目的准备。我们的两个实验室通过参加由其他机构赞助的研究游船，包括专门为研究海洋荧光和发光的生理学和生态学而装备的探险队，定期接触海洋和深海动物。我们的采样方法包括浮游生物拖曳、蓝水潜水、中水拖网和潜水/ROV操作。一系列潜在的克隆目标已经被收集起来，其中包括从放射虫、水母到海胆和鱼类等具有惊人分类多样性的动物。在即将到来的加勒比海、东太平洋和西太平洋的研究巡航期间，这些收集将进一步扩大。同时，我们在荧光蛋白（Matz）和发光蛋白（Haddock）的克隆、表达和分析方面拥有丰富的经验。保证的结果将是鉴定出一系列新的钙依赖性光蛋白，以及具有生物物理，生化和光学特征的gfp样蛋白，这些特征在彻底采样的珊瑚虫中没有发现（如远红色荧光，真正的单体状态和快速成熟）。然而，我们的最终目标是鉴定出完全新颖的（即与先前克隆的没有同源性）遗传编码的荧光和发光标记，这可能成为新一代体内标记和检测技术的基础。

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.