Development of novel luciferases for real-time monitoring of protein secretion.

了解细胞和组织功能的一个重要方法是测量细胞分泌的蛋白质，传统上这是通过使用抗体的免疫测定技术来完成的。这种方法在测量非常少量的分泌激素或炎症介质时具有显著的灵敏度限制，并且还难以获得随时间的快速重复测量，这阻碍了组织在真实的时间中如何表现的数学建模的发展。自然界中的许多生物，包括萤火虫，使用蛋白质分子来产生光，这一过程称为生物发光，这些动物的相关基因已经被确定并分析了它们的结构。现在已经有多种这样的酶，其中一些已被用于研究细胞内的基因调控。在本申请中，我们现在计划评估一系列酶作为分泌措施的潜力。不同的酶将连接到生物学感兴趣的蛋白质，如激素和炎症信号分子，并靶向细胞的分泌装置。光输出的敏感记录将作为细胞激素分泌的测量，我们将测试不同蛋白质的物理和成像特性，以评估它们在包括血液在内的生物液体中的测量效用。这有望导致一种新的方式，实现快速和高度敏感的评估分泌在真实的时间。

An important way of understanding how cells and tissues function is to measure proteins that are secreted from cells, and this has traditionally been done by immunoassay techniques using antibodies. This approach has significant limitations of sensitivity in measuring very small amounts of secreted hormones or inflammatory mediators, and it is also difficult to obtain rapidly repeated measurements over time, which has hampered the development of mathematical modelling of how tissues behave in real time. A number of organisms in nature, including fireflies, use protein molecules to generate light, a process termed bioluminescence, and the relevant genes from these animals have been identified and their structures analysed. A variety of such luciferases is now available, and some have been used in the study of gene regulation within the cell. In this application we now plan to evaluate a range of luciferases for their potential as measures of secretion. Different luciferases will be linked to proteins of biological interest such as hormones and inflammatory signalling molecules, and targeted to the secretory apparatus of the cell. Sensitive recordings of light output will be made as a measure of hormone secretion from the cell, and we will test the physical and imaging characteristics of the different proteins to assess their utility for measurement in biological fluids including blood. This is expected to lead to a new way of achieving rapid and highly sensitive assessment of secretion in real time.