Previous article Next article Contents  

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


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 60–80 nM, the values close to those for Michaelis constants for HIF prolyl hydroxylase (10–100 nM HIF) and the dissociation constant for Keap1–Nrf2 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.
Moscow University Chemistry Bulletin.
2019, Vol. 60, No. 4, P. 240

Copyright (C) Chemistry Dept., Moscow State University, 2002
   Editorial board
   Tables of Contents

The site is supported by Russian Foundation for Basic Research
  The using of published on this page materials is not allowed without special permission
Copyright (C) Chemisty Department of Moscow State University
Web-Editor: B.I.Pokrovskii
Web-design: Copyright (C) MIG and VVM