期刊
BIOTECHNOLOGY PROGRESS
卷 25, 期 2, 页码 559-569出版社
WILEY
DOI: 10.1002/btpr.144
关键词
red-shifted; D-luciferin; BRET; apoptosis
资金
- NIH [P50 CA94056]
Bioluminescence resonance energy transfer (BRET) systems to date have been dominated by use of blue-green Renilla luciferase (Rluc) as the energy donor. Although effective in man-v cases, the expense and unfavorable biochemical attributes of the substrate (phenylcoelenterazine) limit utility of Rluc-based BRET systems. Herein we report a series of novel BRET pairs based on luciferases that utilize D-luciferin, resulting in red-shifted photonic outputs, favorable biochemical attributes, and increased efficacy. We developed a modified. Forster equation to predict optimal BRET luciferase donor-fluorophore pairs and identified tdTomato as the optimal red fluorophore acceptor for click beetle green luciferase (CBG). A prototypical single-chain protease biosensor, capable of reporting on executioner caspase activity in live cells and in real-time, was generated by inserting a DEVD linker between CBG and tdTomato and validated in vitro with recombinant caspases and in cellulo with apoptosis-sensitive and -resistant cell lines. High signal-to-noise ratios (similar to 33) and Z' factors (0.85) were observed in live cell longitudinal studies, sufficient for high-throughput screening. Thus, we illustrate a general methodology for the rational design of new BRET systems and provide a novel single-chain BRET pi-otease biosensor that is long lived, red-shifted, and utilizes D-luciferin. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 559-569, 2009
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