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A. Yu. Khristichenko, A. A. Poloznikov, D. M. Hushpulian, N. A. Smirnova, A. A. Zakhariants, S. V. Kazakov, V. I. Tishkov, I. G. Gazaryan
Quantitative analysis of cell-based
luciferase fusion reporters
Abstract
Cell-based reporters expressing luciferase
fusions with transcription factors or their protein stability domains can be
considered as microbioreactors providing the way to directly monitor the
real-time stability of the luciferase labeled transcription factor or its
domain. To understand principal advantages and/or limitations of these systems
for the applied and fundamental research purposes one needs to develop a
quantitative description of their performance based on the determination of
actual intracellular concentrations of the fusion proteins and rates of their production.
In this work, the experimental data generated by means of luciferase activity
calibration were used to calculate the steady-state intracellular
concentrations of luciferase fusions in SH-SY5Y neuroblastoma cell lines stably
expressing HIF1 ODD-luc and Neh2-luc proteins. For both reporters, the
concentration of fusion proteins was determined as 6080 nM, the values
close to those for Michaelis constants for HIF prolyl hydroxylase (10100
nM HIF) and the dissociation constant for Keap1Nrf2 complex (50 nM),
the parameters controlling rate-limiting steps of HIF1 ODD-luc and Neh2-luc
reporter performance, respectively. New data allowed us to calculate the
production rates and maximum concentrations for the fusion proteins under the
conditions of irreversible activation and protein stabilization. The
quantitative analysis of the Neh2-luc reporter performance employing the newly
generated parameters explains the multi-order shift in the apparent activation
constant versus the real dissociation constant determined for a known Nrf2
displacement activator using fluorescent polarization homogeneous assay with
recombinant Keap1 and labeled Nrf2 peptide.
Key words: Nrf2, Keap1, HIF, prolyl
hydroxylase, bardoxolone, andrographolide, displacement activator.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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