Development of a cell-based high throughput luciferase enzyme fragment complementation assay to identify nuclear-factor-e2-related transcription factor 2 activators

Assay and Drug Development Technologies
Wensheng XieHu Li

Abstract

Nuclear-factor-E2-related transcription factor 2 (Nrf2) regulates a large panel of Phase II genes and plays an important role in cell survival. Nrf2 activation has been shown as preventing cigarette smoke-induced alveolar enlargement in mice. Therefore, activation of the Nrf2 protein by small-molecule activators represents an attractive therapeutic strategy that is used for chronic obstructive pulmonary disease. In this article, we describe a cell-based luciferase enzyme fragment complementation assay that identifies Nrf2 activators. This assay is based on the interaction of Nrf2 with its nuclear partner MafK or runt-related transcription factor 2 (RunX2) and is dependent on the reconstitution of a "split" luciferase. Firefly luciferase is split into two fragments, which are genetically fused to Nrf2 and MafK or RunX2, respectively. BacMam technology was used to deliver the fusion constructs into cells for expression of the tagged proteins. When the BacMam-transduced cells were treated with Nrf2 activators, the Nrf2 protein was stabilized and translocated into the nucleus where it interacted with MafK or RunX2. The interaction of Nrf2 and MafK or RunX2 brought together the two luciferase fragments that form an active luciferase...Continue Reading

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Citations

Jul 11, 2013·Journal of Medicinal Chemistry·Anthony J WilsonChristopher J Moody
Feb 27, 2013·Molecular Therapy : the Journal of the American Society of Gene Therapy·Kari J AirenneSeppo Ylä-Herttuala
Jul 9, 2014·Analytical and Bioanalytical Chemistry·Taha AzadSaman Hosseinkhani
May 3, 2016·Developmental and Comparative Immunology·Wei QianMeilin Jin
Jul 6, 2013·Analytical Chemistry·Kunka Mohanram RamkumarRamasamy Paulmurugan
Jan 17, 2015·Oncoscience·Madryn C Lake, Eric O Aboagye

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Methods Mentioned

BETA
ubiquitination
Assay

Software Mentioned

GraphPad Prism

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