Expression, purification, and refolding of active Nrf2 transcription factor fused to protein transduction TAT tag
Expression, purification, and refolding of active Nrf2 transcription factor fused to protein transduction TAT tag
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DOI：
10.1016/j.pep.2010.06.017
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发表时间：
2010-12-01
影响因子：
1.6
通讯作者：
Burgess, Richard R.
中科院分区：
文献类型：
--
作者：
Chumanov, Robert S.;Burgess, Richard R.
核因子红细胞2相关因子2（Nrf 2）是一种转录因子，负责激活保护细胞免受外源性物质和氧化应激的多种基因。Nrf 2依赖性转录受Nrf 2与其抑制剂Kelch ECH相关蛋白1（Keap 1）的细胞质相互作用的严格控制。Keap 1介导的抑制作用可以通过添加外源性物质或通过过表达Nrf 2蛋白来克服。过表达的Nrf 2消除Keap 1抑制，易位到细胞核中并激活抗氧化反应元件（ARE）依赖性基因转录，保护细胞免受氧化应激。我们表达并纯化了与来自HIV-1的转录反式激活蛋白的蛋白转导结构域（达特）融合的重组小鼠Nrf 2蛋白。全长TAT-Nrf 2在大肠杆菌中以不溶性包涵体形式表达，并使用变性尺寸排阻色谱法纯化至均一。通过使用基于光散射的复性分析和分析尺寸排阻色谱法确定最佳复性条件。结果表明，TAT-Nrf 2可在体外培养的人神经母细胞瘤细胞中表达。转导的TAT-Nrf 2激活ARE依赖性基因的转录，并对过氧化氢暴露产生的细胞内活性氧（ROS）提供保护。(C)2010年由Elsevier Inc.出版
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor responsible for activation of diverse genes that protect the cell against xenobiotics and oxidative stress. The Nrf2-dependent transcription is tightly controlled by cytoplasmic interaction of Nrf2 with its inhibitor, Kelch ECH-associating protein 1 (Keap1). The Keap1-mediated inhibition can be overcome by addition of xenobiotics or by overexpression of Nrf2 protein. The overexpressed Nrf2 overwhelms the Keap1 inhibition, translocates into the nucleus and activates antioxidant response element (ARE)-dependent gene transcription that protects the cell against oxidative stress. We expressed and purified recombinant mouse Nrf2 protein fused to a protein transduction domain (TAT), derived from transactivator of transcription protein from HIV-1. Full-length TAT-Nrf2 was expressed in Escherichia coli in insoluble inclusion bodies and purified to homogeneity using denaturing size exclusion chromatography. Optimal refolding conditions were determined through the use of a light scattering-based refolding assay and analytical size exclusion chromatography. The results demonstrate that the refolded TAT-Nrf2 could transduce into cultured human neuroblastoma cells. The transduced TAT-Nrf2 activated transcription of ARE-dependent genes and conferred protection against intracellular reactive oxygen species (ROS) generated by hydrogen peroxide exposure. (C) 2010 Published by Elsevier Inc.