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

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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.