PHOTOPROTEINS AS LABELS IN BIOLUMINESCENCE ASSAYS

本提案的目的是证明天然存在的发光蛋白及其突变体可用于设计和开发能够进行小体积分析的高灵敏度发光测定法。 特别是，我们建议制备和表征新的水母发光蛋白，奥贝林，和其他发光蛋白的基因工程变体，使它们能够发射的光在不同的波长比相应的野生型发光蛋白。 这些发光蛋白随后将被用作开发生理学上重要分子的测定和生物药物的高通量筛选的试剂。 通过使用发光蛋白的不同变体，将研究生物分子的阵列检测。 我们小组在本预算期间进行的研究证明了采用重组缀合物在生物分子的高灵敏度检测中的有用性。 我们现在提出设计和开发基于发光蛋白的生物发光的新方法，适用于重要生物分子的微量分析，如维生素、药物、肽等，nL大小的样本。

The objective of this proposal is to demonstrate that naturally occurring photoproteins and their mutants can be used in the design and development of highly sensitive luminescent assays capable of small volume analysis. In particular, we propose to prepare and characterize new genetically engineered variants of aequorin, obelin, and other photoproteins, so that they are capable of emission of light at different wavelengths than those of the corresponding wild-type photoproteins. These photoproteins will be then employed as reagents in the development of assays for physiologically important molecules and for the high-throughput screening of biopharmaceuticals. By using the different variants of the photoproteins, array detection of biomolecules will be investigated. Research performed by our group during the current budget period demonstrated the usefulness of employing recombinant conjugates in the highly sensitive detection of biomolecules. We now propose to design and develop new methods based on the bioluminescence of photoproteins that are suitable for the microanalysis of important biomolecules, such as vitamins, drugs, peptides, etc., in nL-sized samples.