Quantitative analysis of cell-based luciferase fusion reporters

Khristichenko, A.Y.; Poloznikov, A.A.; Hushpulian, D.M.; Smirnova, N.A.; Zakhariants, A.A.; Kazakov, S.V.; Tishkov, V.I.; Gazaryan, I.G.

Авторы: Khristichenko A.Yu, Poloznikov A.A., Hushpulian D.M., Smirnova N.A., Zakhariants A.A., Kazakov S.V., Tishkov V.I., Gazaryan I.G.
Журнал: Moscow University Chemistry Bulletin
Том: 74
Номер: 4
Год издания: 2019
Издательство: Allerton Press Inc.
Местоположение издательства: United States
Первая страница: 180
Последняя страница: 185
DOI: 10.3103/S0027131419040047
Аннотация: Cell-based reporters expressing luciferase fusions with transcription factors or their protein stability domains can be considered as microbioreactors providing a way to directly monitor the stability of the luciferase labeled transcription factor or its domain in real-time. To understand principal advantages and/or limitations of these systems for the purposes of applied and fundamental research 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.
Добавил в систему: Тишков Владимир Иванович

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