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

超越 GFP 和水母发光蛋白：荧光和发光蛋白的全海洋研究

• 批准号: 7133657
• 负责人: MIKHAIL V MATZ
• 金额: $ 27.62万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7133657
• 关键词: Xenopus; animal extract; aquatic organism; cell line; chemical stability; embryo /fetus; expression cloning; fluorescence; fluorescent dye /probe; genetic library; genetic screening; luminescence; mass screening; microorganism culture; protein purification; protein quantitation /detection; protein structure function; proteomics; transfection /expression vector

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.

描述(申请人提供)：基于基因编码的荧光和发光标记的活体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哇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效性。我们已经做好了实施这样一个项目的准备。我们的两个实验室都可以通过参加由其他机构赞助的研究游轮定期接触海洋和深海动物，包括专门为研究海洋荧光和发光的生理学和生态学而配备的探险队。我们的采样方法包括浮游生物拖曳、蓝水潜水、中水拖网和潜水器/ROV作业。潜在克隆目标的集合已经组装完毕，其中包括令人惊叹的分类多样性的动物，从放射虫和水母到海胆和鱼。在即将在加勒比海、东太平洋和西太平洋进行的研究巡航期间，这一收藏将进一步扩大。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一系列新的钙依赖的光蛋白，以及具有生物物理、生化和光学特征(如远红荧光、真实单体状态和快速成熟)的组合的GFP样蛋白。然而，我们的最终目标是识别完全新颖的(即与以前克隆的没有同源性的)基因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。