RECOMBINANT OBELIN AS CALCIUM INDICATOR

重组 OBELIN 作为钙指示剂

• 批准号: 2292009
• 负责人: JOHN R BLINKS
• 金额: $ 2.35万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-03-01 至 1997-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2292009
• 关键词: bioluminescence; calcium indicator; chemical kinetics; gel filtration chromatography; ion exchange chromatography; magnesium; microorganism culture; mutant; photobiology; protein isoforms; protein purification; recombinant proteins; site directed mutagenesis

The Ca2+ ion is one of the most important intracellular messengers in biology. In order to understand the mechanisms by which cells regulate their cytoplasmic Ca2+ concentrations, it is necessary to be able to measure those concentrations from moment to moment in living cells. Although there are now several methods of doing that, none is ideal. The Ca2+-regulated photoproteins, of which aequorin is the best-known example, have been used for this purpose for more than 25 years. These substances, which are responsible for the bioluminescence of a number of marine coelenterates, are self-contained macromolecular "packages" that are readily transferred into other cells, and emit light at a rate that varies with [Ca2+]. Aequorin is the only photoprotein that is currently available in sufficient quantities to be used routinely as a Ca2+ indicator. The photoproteins would be much more widely used but for a number of features of aequorin that might well be improved through application of the methods of modern molecular biology, which can in principle: l. make rare natural photoproteins available in quantity. 2. make available photoproteins that do not occur in nature. 3. lead to the production of transgenic organisms with "built in" Ca2+ indicators. Obelin is a relatively scarce photoprotein that has been reported to respond considerably faster than aequorin to rapid changes in [Ca2+]. Investigators at the Photobiology Laboratory of the Institute of Biophysics at Krasnoyarsk have recently cloned the gene for one of the isospecies of obelin, and expressed it in E. coli. The aim of this proposal is to characterize this recombinant photoprotein as part of a wider effort to "build a better aequorin". The FIRCA would support efforts in Krasnoyarsk to improve existing methods for the producing recombinant obelin, a search for genes coding different isospecies of the protein, and an effort to produce useful mutant forms of obelin by site-directed mutagenesis. The products of this work will be characterized in terms of Ca2+-sensitivity, Mg2+-sensitivity, and kinetics at Friday Harbor.

Ca ~（2+）离子是细胞内最重要的信使之一， 生物学为了了解细胞调节的机制， 他们的细胞质Ca 2+浓度，有必要能够 在活细胞中测量这些浓度。 虽然现在有几种方法可以做到这一点，但没有一种是理想的。的 Ca 2+调节的发光蛋白，其中水母发光蛋白是最著名的例子， 已用于此目的超过25年。这些物质， 这是造成许多海洋生物发光的原因 腔肠动物，是独立的大分子“包装”， 很容易转移到其他细胞中，并以不同的速率发光。 [Ca2+]。水母发光蛋白是目前唯一的发光蛋白 以足够的量常规用作Ca 2+指示剂。的 光蛋白的应用要广泛得多， 的水母发光蛋白，很可能通过应用这些方法得到改善， 现代分子生物学，原则上可以： L.使稀有的天然发光蛋白大量可用。 2.制造自然界中不存在的发光蛋白。 3.导致产生具有“内置”Ca 2+的转基因生物 指标 Obelin是一种相对稀缺的光蛋白，据报道， 比水母发光蛋白对[Ca 2 +]快速变化的反应快得多。 研究所光生物学实验室的研究人员 克拉斯诺亚尔斯克的生物物理学家最近克隆了一种 obelin的同种，并在E.杆菌的目的 建议是将这种重组发光蛋白表征为 更广泛的努力“建立一个更好的水母发光蛋白”。FIRCA将支持 在克拉斯诺亚尔斯克改进现有的生产重组体的方法 obelin，寻找编码不同同种蛋白质的基因， 通过定点突变生产有用的奥贝林突变形式的努力 诱变这项工作的产品将在以下方面的特点 星期五港的Ca 2+敏感性、Mg 2+敏感性和动力学。