PHOTOPROTEINS AS LABELS IN BIOLUMINESCENCE ASSAYS

这项建议的目的是证明，自然产生的光蛋白及其突变体可以用于设计和开发能够进行小体积分析的高灵敏度发光分析方法。特别是，我们建议制备和鉴定新的Aequorin，Obelin和其他光蛋白的基因工程变体，使它们能够发射不同波长的光，而不是相应的野生型光蛋白。然后，这些光蛋白质将被用作试剂，用于开发重要生理分子的分析方法，并用于生物制药的高通量筛选。通过使用光蛋白质的不同变体，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表明，在生物分子的高灵敏度检测中使用重组结合物是有用的。我们现在建议设计和开发基于光蛋白的生物发光的新方法，这些方法适合于在NL大小的样品中微量分析重要的生物分子，如维生素、药物、多肽等。

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.