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Biotechnol Prog. 2009 Mar-Apr;25(2):559-69. doi: 10.1002/btpr.144.

Rational design of novel red-shifted BRET pairs: Platforms for real-time single-chain protease biosensors.

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1
Dept. of Molecular Biology and Pharmacology, and Molecular Imaging Center, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

Abstract

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 many 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 Förster 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 ( approximately 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 protease biosensor that is long lived, red-shifted, and utilizes D-luciferin.

PMID:
19330851
PMCID:
PMC2827327
DOI:
10.1002/btpr.144
[Indexed for MEDLINE]
Free PMC Article
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